Microbe Pundit

Communicating with electricity | Lab Rat, Scientific American Blog Network

2011-07-12T15:37:00.001+10:00

Communicating with electricity | Lab Rat, Scientific American Blog Network

Electricity is usually thought of as a very human thing. Animals and plants in nature may be capable of extraordinary feats of engineering, but there are still a few developments that humans claim as uniquely their own; fire, the wheel, and electricity.

For bacteria, on the other hand, the ability to push electrons down a small cable is just one more way to live, breath and communicate in a world full of niches to exploit.

Ask not what your colony can do for you, but what you can do for your colony.

2007-09-03T20:43:00.001+10:00

The stone monument for microorganisms who gave their lives as research specimens. It was constructed in the precincts of the Manshuin temple in north portion of the city of Kyoto.

Genetics sheds light on frog fungus.

2007-08-08T06:44:00.000+10:00

Genes shine light on mystery frog fungus

By ABC Science Online's Dani Cooper

Posted Tue Aug 7, 2007 6:31pm AEST

Scientists have uncovered genetic markers of a deadly fungus that is wiping out frog populations worldwide.

Researchers will now use them to pinpoint where on the globe the killer micro-organism originated.

Lead researcher Dr Jess Morgan, an Australian scientist from the Queensland Department of Primary Industries, says evidence has emerged that the frog-killing fungus Batrachochytrium dendrobatidis reproduces sexually and may be creating resistant spores, which can survive for a decade.

The international research findings, published in the journal Proceedings of the National Academy of Sciences, suggests the pathogen will be harder to eliminate.

Dr Morgan, who was a post-doctoral fellow at the University of California, Berkeley at the time of the study, says little is known about the fungus.

She says it was only identified in 1998 after a wave of frog population extinctions worldwide from chytridiomycosis, a disease caused by the fungus.

Scientists believe the fungus kills by attacking the frog's ability to absorb water through its skin, causing it to dehydrate to death.

But they still do not know exactly how the pathogen has spread around the globe.

In the paper, Dr Morgan says the team used genetic analysis of a well-studied population of mountain yellow-leg frogs in California's Sierra Nevada to determine whether the fungus was endemic or had been recently introduced.

Dr Morgan says of six sites studied, four were dominated by a single genetic make-up or genotype, suggesting the fungus had been recently introduced and spread through clonal reproduction.

But she says at two sites evidence of recombination was found with multiple genotypes present.

This indicates for the first time that the fungus reproduces sexually and may be producing resistant spores.

Dr Morgan says the presence of resistant spores helps explain the global spread of the disease and means the fungus can survive for long periods in areas where the frog population has been vastly reduced.

But it also means any attempts to reintroduce frog populations at sites of local extinction are likely to fail as the spores will re-infect the frogs.

Reintroduction

Dr Morgan says of 10 attempts at reintroducing frogs in the Sierra Nevada during the past four years, seven have failed and three are ongoing.

She says resistant spores help spread the fungus as they are easily transported in dirt on tyres and shoes, and can hitchhike on birds and other wildlife.

Dr Morgan says during the study researchers isolated 15 marker genes for the fungus, which will now be used in a worldwide hunt to track the geographic origin of the killer fungus.

"The next thing in terms of genetics is to find out where this is coming from," she said.

"The area which is most likely the origin will not be suffering a decline in frog population. We are looking for a healthy population of frogs.

"If we can look at the frogs and find out how they are living with the disease then maybe we can [help] our frogs."

Dr Morgan says the study also found some frogs within one species are resistant to the disease and could survive a mass mortality.

"It could be the frogs and the fungus are evolving to be able to live together," she said.

But she says more research is needed on the factors, either physical or environmental, behind this phenomenon.

New biocontrol method to manage mycotoxins in Africa

2007-08-03T05:35:00.000+10:00

NIGERIA: New hope for improved food safety in sub-Saharan Africa
01.aug.07 Via Agnet
Cross posting
Consultative Group on International Agricultural Research
Ranajit Bandyopadhyay

Scientists at the International Institute of Tropical Agriculture (IITA) have developed a safe and effective method for biological control of aflatoxins. These are toxic chemicals of fungal origin, which contaminate maize and other major food crops, posing a chronic threat to human health in sub-Saharan Africa.
With the new method, strains of the fungi that produce aflatoxin are overwhelmed through the introduction of related but entirely harmless strains. These were identified and tested through several years of meticulous research supported by the German Agency for Technical Cooperation (GTZ) and carried out in collaboration with the Agriculture Research Service of the US Department of Agriculture, University of Arizona in the USA, University of Bonn in Germany and University of Ibadan in Nigeria.

Researchers found that inoculum containing the beneficial strains can be produced most efficiently on sorghum grain, resulting in a dry formulation, which can then be broadcast on moist soil in farmers’ maize fields. Laboratory and field tests have demonstrated that the harmless strains spread quickly from the soil to maize plants, where they reduce aflatoxin-producing strains on maize grain by 91 to almost 100 percent. A single application is sufficient to control the problem in the crop treated, though a few additional applications may be required to achieve long-term control in farmers´ fields. Large-scale testing of the new method is currently under way in Nigeria.

An insidious threat
Aflatoxins are produced by various fungi, such as Apergillus flavus and A. parasiticus, which grow as molds on staple grains and root crops both before harvest and in storage. Contamination of maize causes particular concern, because it is sub-Saharan Africa’s most important cereal.
The toxins are especially damaging to children. Continuous exposure has been shown to stunt growth and even contribute to infant mortality when coinciding with kwashiorkor, a form of malnutrition caused by dietary deficiency of protein and other nutrients. The insidious combination of impaired development and undernourishment accounts for about half of the 4.5 million deaths of children under the age of 5 occurring annually in sub-Saharan Africa.
Aflatoxins are also believed to affect the human immune system, making people more vulnerable to infectious diseases, such as malaria and HIV/AIDS. In addition, the toxins are linked to liver disorders and can act in synergy with the Hepatitis B virus to cause hepatocellular carcinoma. This is the most common cancer in sub-Saharan Africa, accounting for as many as 10 percent of adult male deaths in parts of West Africa.
Aflatoxins further damage the well-being of Africa’s rural families by limiting exports of maize and groundnut in particular. Grain-importing countries maintain high food quality standards, with especially strict controls on aflatoxin content. African food and feed products showing levels of contamination above the acceptable limits cannot penetrate major grain markets, resulting in significant loss of agricultural income.
Except when people die of acute poisoning, as happened in Kenya during 2004-2006, aflatoxins seldom receive adequate attention in the region, even though they clearly have serious consequences and are quite widespread. In Benin and Togo, for example, researchers found that aflatoxin levels are about five times the safe limit of 20 parts per billion in up to 30 percent of household grain stores. According to other results from a study carried out in those countries and Nigeria, 99 percent of blood samples collected randomly from children contained aflatoxins.

In pursuit of a biocontrol strategy
In an effort to reduce aflatoxin contamination, researchers at IITA and elsewhere have deployed various methods, involving, for example, modifications in grain drying, storage and food preparation practices. To complement strategies that have already proved effective, scientists have also actively pursued in recent years the option of biological control, building on the Institute’s long and extraordinary record of success in using this approach to combat major pests such as the mango and cassava mealybugs, cassava green mite, desert locust and banana nematodes and weevils.
The biocontrol strategy that appears to be effective against aflatoxin employs a mechanism that researchers refer to as “competitive exclusion.” This is made possible by the presence in nature A. flavus populations, not only of “toxigenic” strains, which produce copious amounts of aflatoxin, but also “atoxigenic” strains, which lack this capacity. In order for the strategy to work, researchers must identify and successfully introduce harmless strains that show a large competitive advantage over the dangerous ones.
In the resulting biological struggle, explains IITA plant pathologist Ranajit Bandyopadhyay, “the good strains of A. flavus virtually eliminate their highly toxic relatives and ensure that the ‘bad guys’ cannot re-emerge.”
Such a strategy has proved effective for controlling aflatoxin on cottonseed, groundnut and maize in the USA and on groundnut in Australia.
The challenge for scientists at IITA was to identify entirely safe atoxigenic strains of A. flavus that are indigenous to Africa and serve effectively as biocontrol agents. For this purpose, they first collected more than 4,200 samples of the fungus from different ecological zones of Nigeria. In these they identified 2,127 distinct strains, of which 1,000 proved to be atoxigenic. Only 26, though, were selected for further testing.
This involved an extremely laborious series of procedures, one of them, for example, involving.more than 30,000 crosses between different strains. The purpose was to group the selected strains according to “vegetative compatibility” and make sure they belong to groups consisting only of atoxigenic strains that cannot cross with toxigenic strains in nature. This procedure ensured that release of the biocontrol agents in the field would be absolutely safe, leading to a drastic reduction, rather than an inadvertent boost, in aflatoxin levels.

Proven effectiveness
From the 14 unique atoxigenic groups identified, one strain each from eight groups was chosen for further evaluation for ability to compete with toxigenic strains. When maize grains were inoculated in the laboratory and field with both a highly toxigenic A. flavus strain as well as atoxigenic strains, one of the latter reduced aflatoxin levels by 91 percent and two others by nearly100 percent.
In experimental field plots, various atoxigenic strains proved capable of spreading quickly from the soil on which they were released to maize plants. One especially promising strain was found on nearly 99 percent of the maize grains analyzed.
The eight promising strains were further screened for their ability to get established rapidly after release, continue spreading and survive in plant debris. Strains showing these abilities can achieve effective biocontrol of aflatoxin-producing strains over multiple years, with only a few additional applications after initial release.
Having identified several excellent candidate strains to serve as agents of biocontrol, IITA researchers are further testing these in large-scale trials at various locations in Nigeria. To permit wide release of these biocontrol agents in the fields, researchers have developed safe, efficient and effective methods for producing and applying inoculum. IITA now seeks further support to disseminate the biocontrol technology as part of a “basket” of simple aflatoxin management practices that can reduce aflatoxin levels in Africa’s food and improve the health of its people.

Introducing the technology Pundits.

2007-08-02T14:29:00.001+10:00

Image courtesy of Kamat.com

The image shows some of the 16 famous Brahmin Pundit brothers.

(Pundit is pandit in Hindi, but in ancient Sanskit was pandita, meaning learned, skilled)

Two of these brothers, twins Microbe Pundit and Gmo Pundit, run well known weblogs which have rich collections of articles relating to the modern challenges of biotechnological innovation.

In the photo, Gmo Pundit appears on the high ground to the extreme right.

Microbe Pundit sits right in the middle

In the Pundits' blogs, hyper-linked tags classify the articles in the blogs into different categories, making them easier to search for particular items.

(For instance the current article is tagged Information retrieval - see link below).

Microbe Pundit's blog collections about using new biotechnology for commercial innovation are thus easily accessible via Microbe Pundit's Discovery tab, and by his Innovation tab.

On the other hand, Gmo Pundit's Agric. Innovation tab is useful for finding news and analysis about commercialisation of agricultural technology.

Rich pickings for proposals in dairy microbe biotechnology

2007-07-27T17:19:00.000+10:00

This is what a quick literature search with PubMed can reveal:

J Bacteriol. 2007 Apr;189(8):3256-70. Epub 2007 Feb 16.
Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363.
Wegmann U, O'Connell-Motherway M, Zomer A, Buist G, Shearman C, Canchaya C, Ventura M, Goesmann A, Gasson MJ, Kuipers OP, van Sinderen D, Kok J.
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.

Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in
comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.
PMID 17307855 [PubMed - indexed for MEDLINE]

Mol Microbiol. 1998 Aug;29(4):1029-38.
Comment in:
Mol Microbiol. 1999 Mar;31(5):1598-600.
Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147.
Dougherty BA, Hill C, Weidman JF, Richardson DR, Venter JC, Ross RP. The Institute for Genomic Research, Rockville, MD, USA.

The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60,232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty-four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16-gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large gram-positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel
dairy industry.
PMID 9767571 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2004 Jan;70(1):34-42.
Variable bacteriocin production in the commercial starter Lactococcus lactis DPC4275 is linked to the formation of the cointegrate plasmid pMRC02.
Trotter M, McAuliffe OE, Fitzgerald GF, Hill C, Ross RP, Coffey A.
Department of Microbiology, University College Cork, Ireland.

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin
3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.
PMID 14711623 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 1996 Feb;62(2):612-9.
An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.
Ryan MP, Rea MC, Hill C, Ross RP.
National Dairy Products Research Centre, Moorepark, Fermoy, County Cork, Republic
of Ireland.

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147.
The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials.
Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products.
PMID 8593062 [PubMed - indexed for MEDLINE]

J Appl Microbiol. 2003;95(6):1235-41.
A lacticin 481-producing adjunct culture increases starter lysis while inhibiting nonstarter lactic acid bacteria proliferation during Cheddar cheese ripening.
O'Sullivan L, Ross RP, Hill C.
Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, County Cork, Ireland.

AIMS: The main aim of this study was to exploit a lacticin 481 producing strain, Lactococcus lactis CNRZ481, as an adjunct for Cheddar cheese manufacture, to increase starter cell lysis and control nonstarter lactic acid bacteria (NSLAB) proliferation in cheese. METHODS AND RESULTS: Lactococcus lactis CNRZ481 was exploited as an adjunct to L. lactis HP for the manufacture of Cheddar cheese at pilot scale (450 l). In these trials, inclusion of the adjunct strain did not compromise acid production by L. lactis HP and cheese was successfully manufactured within 5 h. Experimental cheese exhibited levels of lactate dehydrogenase (LDH) up to five-fold higher than control cheese and a significant reduction in NSLAB growth was also observed throughout the ripening period.
CONCLUSIONS: The aims of the study were accomplished as (i) greater enzyme release was achieved through lacticin 481-induced lysis which was associated with an improved flavoured cheese as assessed by a commercial grader and (ii) NSLAB growth was controlled, thus reducing the risk of off-flavour development.
SIGNIFICANCE AND IMPACT OF THE STUDY: The use of lacticin 481-producing adjuncts for cheese manufacture may prove beneficial for manufacturers who aim to achieve faster ripening through premature and elevated intracellular enzyme release while minimizing inconsistencies in cheese quality because of NSLAB activity.
PMID 14632996 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2001 Jun;67(6):2699-704.
Strategy for manipulation of cheese flora using combinations of lacticin 3147-producing and -resistant cultures.
Ryan MP, Ross RP, Hill C.
Dairy Products Research Centre, Fermoy, County Cork, Ireland.

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A Lactobacillus paracasei subsp. paracasei strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either Lactococcus lactis DPC3147 (a natural producer) or L. lactis DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening,
with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10(7) CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100- to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora
in the test cheeses during ripening.
PMID 11375183 [PubMed - indexed for MEDLINE]

J Food Prot. 2001 Jan;64(1):81-6.
The effects of cultivating lactic starter cultures with bacteriocin-producing lactic acid bacteria.
Oumer A, Garde S, Gaya P, Medina M, Nuñez M.
Departamento de Tecnologiá de Alimentos, INIA, Madrid, Spain.

The effects of bacteriocins produced by six strains of lactic acid bacteria on 9 mesophilic and 11 thermophilic commercial starter cultures were investigated in mixed cultures of commercial starters with bacteriocin-producing strains in milk.
The bacteriocins produced by the test organisms were nisin A, nisin Z, lacticin 481, enterocin AS-48, a novel enterocin, and a novel plantaricin. Mesophilic commercial starters were in most cases tolerant of bacteriocins, with only two of the starters being partially inhibited, one by four and the other by two bacteriocins. The aminopeptidase activities of mesophilic starters were generally low, and only one of the combinations of mesophilic starter-bacteriocin producer gave double the aminopeptidase activity of the starter culture without the bacteriocin producer. Thermophilic commercial starters were more sensitive to bacteriocins than mesophilic starters, with six thermophilic starters being partially inhibited by at least one of the bacteriocins. Their aminopeptidase activities were generally higher than those of the mesophilic starters. The aminopeptidase activities of seven thermophilic starters were increased in the presence of bacteriocins, by factors of up to 9.0 as compared with the corresponding starter cultures alone. Bacteriocin-producing strains may be used as adjunct cultures to mesophilic starters for the inhibition of pathogens in soft and semihard cheeses, because mesophilic starters are rather tolerant of bacteriocins. Bacteriocin producers may also be used as adjunct cultures to thermophilic starters of high aminopeptidase activity, more sensitive to lysis by bacteriocins than mesophilic starters, for the acceleration of ripening in semihard and hard cheeses.
PMID 11198445 [PubMed - indexed for MEDLINE]

J Food Prot. 2005 May;68(5):1026-33.
Effect of milk inoculation with bacteriocin-producing lactic acid bacteria on a Lactobacillus helveticus adjunct cheese culture.
Avila M, Garde S, Medina M, Nuñez M.
Departamento de Tecnología de Alimentos, INIA, Carretera de La Coruña Km 7,
Madrid, 28040 Spain.

The effect of eight strains of lactic acid bacteria (two strains of Enterococcus, one strain of Lactobacillus, and five strains of Lactococcus, which produce enterocin AS-48, enterocin 607, nisin A, nisin Z, plantaricin 684, lacticin 481, or nisin Z plus lacticin 481) on acid production and proteolytic activity of Lactobacillus helveticus LH 92 (a highly peptidolytic strain used as an adjunct in cheese making) was evaluated in mixed cultures in milk. Acid production by mixed cultures depended on the sensitivity of L. helveticus LH 92 to the different bacteriocins and on the acidification rates of bacteriocin-producing strains. Proteolysis values of mixed cultures were, in all cases, lower than those of L. helveticus LH 92 single culture (control). Cell-free aminopeptidase activity values after 9 h of incubation did not increase in the presence of enterocin producers or the nisin A producer, whereas in the presence of the nisin Z producer, cell-free aminopeptidase activity was, at most, 3.7-fold greater than the control value. In mixed cultures with the plantaricin producer, a progressive lysis of L. helveticus LH 92 took place, with cell-free aminopeptidase activity values after 9 h being, at most, 10.5-fold greater than the control value. The highest cell-free aminopeptidase activity values after 9 h were recorded in the presence of lacticin 481 producers, with the values being, at most, 25.1-fold greater than the control value. L. helveticus LH 92 was extremely sensitive to small variations in the concentration of the inoculum of the nisin Z plus lacticin 481 producer, with there being a narrow optimum for the release of intracellular aminopeptidases. Plantaricin and lacticin 481 producers seemed the most promising strains to be combined with L. helveticus LH 92 as lactic cultures
for cheese manufacture,because of the accelerated release of intracellular
aminopeptidases.
PMID 15895737 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 2006 Dec 1;112(3):230-5. Epub 2006 Jun 9.
Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation.
Topisirovic L, Kojic M, Fira D, Golic N, Strahinic I, Lozo J.
Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a,
11010 Belgrade, Serbia and Montenegro. lab6@eunet.yu

Autochthonous strains of lactic acid bacteria (LAB) have been isolated from traditionally homemade cheeses collected from specific ecological localities across Serbia and Montenegro. Genetic and biochemical analysis of this LAB revealed that they produce bacteriocins, proteinases and exopolysaccharides. LAB produces a variety of antimicrobial substances with potential importance for food fermentation and preservation. Apart from the metabolic end products, some strains also secrete antimicrobial substances known as bacteriocins. Among the
natural isolates of LAB from homemade cheeses, bacteriocin producers were found in both lactococci and lactobacilli. Lactococcus lactis subsp. lactis BGMN1-5 was found to produce three narrow spectrum class II heat-stable bacteriocins. In addition to bacteriocin production, BGMN1-5 synthesized a cell envelope-associated proteinase (CEP) and shows an aggregation phenotype. Another isolate, L. lactis subsp. lactis BGSM1-19 produces low molecular mass (7 kDa) bacteriocin SM19 that showed antimicrobial activity against Staphylococcus aureus, Micrococcus flavus and partially against Salmonella paratyphi. Production of bacteriocin reaches a plateau after 8 h of BGSM1-19 growth. Bacteriocin SM19 retained activity within the wide pH range from 1 to 12 and after the treatment at 100 degrees C for 15 min. Among collection of lactobacilli, the isolate Lactobacillus paracasei subsp. paracasei BGSJ2-8 produces heat-stable bacteriocin SJ (approx. 5 kDa) polypeptide. It retained activity after treatment for 1 h at 100 degrees C, and in the pH range from 2 to 11. In addition to isolates from cheeses, bacteriocin-producing human oral lactobacilli were detected. Most of them showed antimicrobial activity against streptococci, staphylococci and micrococci, but not against Candida. Isolate BGHO1 that showed the highest antimicrobial activity was determined as Interestingly, L. paracasei. Lactobacillus helveticus BGRA43, which was isolated from the human intestine showed strong activity against Clostridium sporogenes, but it was not possible to detect any bacteriocin production in this isolate by using standard procedures.
Further analysis of antimicrobial activity revealed that BGRA43 has a relatively broad spectrum. Lactobacilli resistant to nisin were also detected among natural isolates. They produce bacteriocins, which have no activity against nisin producing lactococci.
PMID 16764959 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 1999 Dec 15;53(2-3):141-52.
Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product.
Olasupo NA, Schillinger U, Narbad A, Dodd H, Holzapfel WH.
Department of Botany and Microbiology, Faculty of Science, Lagos State University
Ojo, Nigeria.

Screening for bacteriocin production of 500 strains of lactic acid bacteria (LAB) from various African fermented foods resulted in the detection of a bacteriocin producing Lactococcus lactis (BFE 1500) isolated from a dairy product called wara. The bacteriocin inhibited not only the closely related LAB, but also strains of Listeria monocytogenes, Listeria innocua, Clostridium butyricum, Clostridium perfringens, Bacillis cereus and Staphylococcus aureus. It was heat
stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10), but highest activity was observed in the lower pH range. The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K, but not by other proteases. Growth kinetic assay indicated stronger growth inhibition by the bacteriocin produced by Lc. lactis BFE 1500 on L. monocytogenes WS 2250 and B. cereus DSM 2301 than with the nisin A producing strain DSM 20729.
Polymerase chain reaction indicated the presence of the nisin operon in strain BFE 1500 and sequencing of its structural gene showed that Lc. lactis BFE 1500 produced the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The genetic determinants for bacteriocin production in strain BFE 1500 are located on a conjugative transposon. The ability of the bacteriocin produced by Lc. lactis BFE 1500 to
inhibit a wide range of food-borne pathogens is of special interest for food safety, especially in the African environment with perennial problems of poor food hygiene.
PMID 10634705 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 2003 Mar 15;81(2):137-45.
Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham, a traditional Thai fermented sausage.
Noonpakdee W, Santivarangkna C, Jumriangrit P, Sonomoto K, Panyim S.
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok
10400, Thailand. scwnp@mahidol.ac.th

A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10). The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to
that of nisin. The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the fermented product.
PMID 12457588 [PubMed - indexed for MEDLINE]

J Basic Microbiol. 1997;37(3):187-96.
Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables.
Franz CM, Du Toit M, von Holy A, Schillinger U, Holzapfel WH.
Bundesforschungsanstalt für Ernährung, Institut für Hygiene and Toxikologie,
Karlsruhe, Germany.

Four bacteriocin producing lactic acid bacteria isolated from vegetables were identified as Lactococcus lactis strains on the basis of physiological and biochemical characteristics, carbohydrate fermentation patterns and analysis of total soluble protein pattern by SDS PAGE. The bacteriocins had a wide spectrum of activity as antagonism was detected not only towards a variety of lactic acid bacteria, but also to Staphylococcus aureus and Listeria monocytogenes. These bacteriocins were resistant to heating at 121 degree C for 15 minutes and showed highest activity at low pH (less than 5.0). They were inactivated by the proteolytic enzymes alpha-chymotrypsin and proteinase K, but not by lipase, alpha-amylase, catalase or lysozyme. These bacteriocinogenic Lactococcus strains were all immune to the bacteriocins produced as well as to commercial nisin. Bacteriocin producer culture supernatants showed a high degree (70 or 100%) of cross-reactivity in the nisin ELISA, suggesting similarity of the produced bacteriocins to nisin. The potential application of bacteriocin producing lactococci of vegetable origin for safety assurance of vegetable foods and controlling vegetable fermentations is
discussed.
PMID 9265741 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 1992 Jun;16(2):141-51.
Identification and characterization of two bacteriocin-producing strains of Lactococcus lactis isolated from vegetables.
Uhlman L, Schillinger U, Rupnow JR, Holzapfel WH.
Federal Research Centre for Nutrition, Institute of Hygiene and Toxicology, Karlsruhe, Germany.

Isolated from mixed salad and fermented carrots, 123 strains of lactic acid bacteria were screened for bacteriocin production. Two strains, D53 and 23, identified as Lactococcus lactis by DNA-DNA hybridizations, produced heat stable bacteriocins which were resistant to trypsin and pepsin, but were inactivated by alpha-chymotrypsin and proteinase K. The bacteriocins were active from pH 2 to 9 and inhibited species of Listeria, Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Carnobacterium, Bacillus and Staphylococcus. Strain D53 produced bacteriocin at pH values of 4.5-8.0 and from 10 to 37 degrees C.
PMID 1445757 [PubMed - indexed for MEDLINE]

J Appl Microbiol. 2000 Apr;88(4):563-71.
Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi.
Choi HJ, Cheigh CI, Kim SB, Pyun YR.
Department of Biotechnology and Bioproducts Research Center, Yonsei University,
Seoul, Korea.

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin.
Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion
exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.
PMID 10792514 [PubMed - indexed for MEDLINE]

Curr Microbiol. 2003 May;46(5):385-8.
Identification and characteristics of nisin Z-producing Lactococcus lactis subsp.
lactis isolated from Kimchi.
Park SH, Itoh K, Kikuchi E, Niwa H, Fujisawa T.
Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi.
The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was
stable against heat and pH, but it was lost at 100 degrees C for 1 h and at 121 degrees C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or beta-glucosidase. There were vsome differences in characteristics from those of nisins described previously.
PMID 12732968 [PubMed - indexed for MEDLINE]

J Appl Microbiol. 2004;97(3):621-8.
Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese.
Ghrairi T, Manai M, Berjeaud JM, Frère J.
Laboratoire de Biochimie et Biologie Moléculaire, Faculté des Sciences de Tunis,
Campus universitaire, Tunis, Tunisia.

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as
demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III
bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety. Copyright 2004 The Society for Applied Microbiology
PMID 15281944 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 2006 Mar 15;107(2):138-47. Epub 2005 Nov 8.
Streptococcus macedonicus ACA-DC 198 produces the lantibiotic, macedocin, at temperature and pH conditions that prevail during cheese manufacture.
Van den Berghe E, Skourtas G, Tsakalidou E, De Vuyst L.
Research Group of Industrial Microbiology, Fermentation Technology and Downstream
Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit
Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium.

Streptococcus macedonicus ACA-DC 198, a natural cheese isolate, produces the anticlostridial bacteriocin, macedocin. Bacteriocin activity was detected from the mid-exponential growth phase and remained constant during the stationary phase. A secondary model was setup to describe the influence of temperature (20-45 degrees C) and pH (5.1-6.9) on cell growth of and bacteriocin production by S. macedonicus ACA-DC 198 during in vitro laboratory fermentations. The optimum temperature for bacteriocin production (20-25 degrees C) was markedly lower than the optimum growth temperature (42.3 degrees C). In contrast, the
specific macedocin production was maximal around pH 6.0, whereas growth was optimal at pH 6.4. Consequently, the maximum bacteriocin activity was reached between pH 6.0 and 6.5.
PMID 16288813 [PubMed - indexed for MEDLINE]

J Food Prot. 2004 Dec;67(12):2727-34.
Characterization and antimicrobial activity of bacteriocin 217 produced by natural isolate Lactobacillus paracasei subsp. paracasei BGBUK2-16.
Lozo J, Vukasinovic M, Strahinic I, Topisirovic L.
Institute of Molecular Genetics and Genetic Engineering, 11010 Belgrade, Serbia
and Montenegro.

The strain Lactobacillus paracasei subsp. paracasei BGBUK2-16. which was isolated from traditionally homemade white-pickled cheese, produces bacteriocin 217 (Bac217; approximately 7 kDa). The onset of Bac217 biosynthesis was observed in the logarithmic phase of growth, and the production plateau was reached after 9 or 12 h of incubation at 37 and 30 degrees C, respectively, when culture entered the early stationary phase. Biochemical characterization showed that Bac217 retained antimicrobial activity within the range of pH 3 to 12 or after treatment at 100 degrees C for 15 min. Bac217 antimicrobial activity also remained unchanged after storage at 4 degrees C for 6 months or -20 degrees C for up to 12 months. However, Bac217 activity was completely lost after treatment with different proteolytic enzymes. BGBUK2-16 contains only one plasmid about 80 kb in size. Plasmid curing indicated that genes coding for Bac217 synthesis and immunity seem to be located on this plasmid. Bac217 exhibited antimicrobial activity against some pathogenic bacteria, such as Staphylococcus aureus and
Bacillus cereus. Interestingly, Bac217 showed activity against Salmonella sp. and Pseudomonas aeruginosa ATCC27853. The inhibitory effect of BGBUK2-16 on the growth of S. aureus in mixed culture was observed. S. aureus treatment with Bac217 led to a considerable decrease (CFU/ml) within a short period of time. The mode of Bac217 action on S. aureus was identified as bactericidal. It should be noted that the strain BGBUK2-16 was shown to be resistant to bacteriocin nisin, which is otherwise widely used as a food additive for fermented dairy products.
PMID 15633678 [PubMed - indexed for MEDLINE]

J Dairy Sci. 2006 Aug;89(8):2882-93.
Effect of high-pressure treatment and a bacteriocin-producing lactic culture on the proteolysis, texture, and taste of Hispánico cheese.
Avila M, Garde S, Gaya P, Medina M, Nuñez M.
Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y
Tecnología Agraria y Alimentaria (INIA) Madrid, 28040 Spain.

The effects of high-pressure treatment, by itself or in combination with a bacteriocin-producing culture added to milk, on the proteolysis, texture, and taste of Hispánico cheese were investigated. Two vats of cheese were manufactured from a mixture of cow and ewe milk. Milk in one vat was inoculated with 0.5% Lactococcus lactis ssp. lactis INIA 415, a nisin Z and lacticin 481 producer; 0.5% L. lactis ssp. lactis INIA 415-2, a bacteriocin-nonproducing mutant; and 2% of a commercial Streptococcus thermophilus culture. Milk in the other vat was inoculated with 1% L. lactis ssp. lactis INIA 415-2 and 2% S. thermophilus culture. After ripening for 15 d at 12 degrees C, half of the cheeses from each vat were treated at 400 MPa for 5 min at 10 degrees C. Ripening of high-pressure-treated and untreated cheeses continued at 12 degrees C until d 50.
High-pressure treatment of cheese made from milk without the bacteriocin producer accelerated casein degradation and increased the free AA content, but it did not significantly influence the taste quality or taste intensity of the cheese.
Addition of the bacteriocin producer to milk lowered the ratio of hydrophobic peptides to hydrophilic peptides, increased the free AA content, and enhanced the taste intensity. The combination of milk inoculation with the bacteriocin producer and high-pressure treatment of the cheese resulted in higher levels of both hydrophobic and hydrophilic peptides but had no significant effect on the free AA content, taste quality, or taste intensity.
PMID 16840604 [PubMed - indexed for MEDLINE]

J Appl Microbiol. 2006;100(1):135-43.
Evaluation of live-culture-producing lacticin 3147 as a treatment for the control of Listeria monocytogenes on the surface of smear-ripened cheese.
O'Sullivan L, O'connor EB, Ross RP, Hill C.
Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland.

AIMS: A live Lactococcus lactis culture, producing the two-component broad spectrum bacteriocin lacticin 3147, was assessed for ability to inhibit the food pathogen Listeria monocytogenes on the surface of smear-ripened cheese. METHODS AND RESULTS: In initial experiments, the addition of Listeria to a lacticin 3147-containing fermentate produced with L. lactis DPC4275 (a transconjugant strain derived from L. lactis DPC3147) resulted in at least a 4 log reduction of the pathogen in 30 min. Two separate trials were performed in order to assess the most suitable method for application of the potential protective culture to smear-ripened cheese. In the initial trial, the L. lactis was sprayed onto the surface of the cheese either before or after Listeria was deliberately applied.
Application of the culture following Listeria challenge, yielded up to a 1000-fold reduction of the pathogen in contrast to the pretreatment where Listeria numbers were unaffected. In a further trial, three applications of the live lacticin 3147-producing culture was used on a cheese surface containing Listeria. Listeria numbers were found to be up to 100-fold lower than in the cheese treated with L. lactis DPC4268 (control). CONCLUSION: While application of the live lacticin 3147 producer did not give complete elimination of the pathogen the results nonetheless demonstrate the potential of the bioprotectant for improving the safety of smear-ripened cheeses and particularly those that contain low level contamination with Listeria. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of lacticin 3147 as a live-culture can serve as a bioprotectant for the control of L. monocytogenes on the surface of smear-ripened cheese.
PMID 16405693 [PubMed - indexed for MEDLINE]

FEMS Microbiol Lett. 1993 Sep 15;112(3):313-8.
Conjugal transfer of the determinants for bacteriocin (lacticin 481) production and immunity in Lactococcus lactis subsp. lactis CNRZ 481.
Piard JC, Delorme C, Novel M, Desmazeaud M, Novel G.
INRA, Station de Recherches Laitières, Jouy-en-Josas, France.

The lacticin 481-producer (Lct+), L. lactis subsp. lactis (L. lactis) CNRZ 481 harbours 5 plasmids of 6.5, 7.5, 20, 37 and 69 kb. Novobiocin treatment of L. lactis 481 led to the appearance of lacticin 481 deficient variants which had all lost the 69 kb plasmid. Conjugal transfer of the lacticin 481 structural gene (lct) into the plasmid free strain L. lactis IL1441 yielded Lct+ transconjugants at a 10(-4) frequency, which carried a plasmid with an apparent size of 120-130
kb. Southern hybridization analyses showed that the lct gene was located on the 69 kb plasmid in L. lactis 481 and on the 120-130 kb plasmid in the transconjugants. The lct gene was in higher copy number in transconjugants than in the parental strain resulting in two-fold higher lacticin 481 production in the former strain.
PMID 8224796 [PubMed - indexed for MEDLINE]

Mol Nutr Food Res. 2006 Mar;50(3):306-13.
Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates.
Akçelik O, Tükel C, Ozcengiz G, Akçelik M.
Department of Biotechnology, Middle East Technical University, Ankara, Turkey.

In this study, bacteriocins from two Lactococcus lactis subsp. lactis isolates from raw milk samples in Turkey designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after alpha-amylase treatment suggested that they form aggregates with carbohydrates.
Molecular masses of the purified bacteriocins were determined by SDS-PAGE. PCR amplification was carried out with specific primers for the detection of their structural genes. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Examination of plasmid contents of the isolates and the results of plasmid curing and conjugation experiments showed that in L. lactis subsp. lactis OC1 strain the 39.7-kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity, whereas the 16.0-kb plasmid is responsible for lacticin 481 production and lactose fermentation in L. lactis subsp. lactis OC2 strain.
PMID 16523441 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 1997 Apr;63(4):1434-40.
Application and evaluation of the phage resistance- and bacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures.
Coakley M, Fitzgerald G, Ros RP.
National Dairy Products Research Centre, Fermoy, County Cork, Ireland.

The conjugative 63-kb lactococcal plasmid pMRC01 encodes bacteriophage resistance and production of and immunity to a novel broad-spectrum bacteriocin, designated lacticin 3147 (M.P. Ryan, M.C. Rea, C. Hill, and R.P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). The phage resistance is an abortive infection mechanism which targets the phage-lytic cycle at a point after phage DNA replication. By using the genetic determinants for bacteriocin immunity encoded on the plasmid as a selectable marker, pMRC01 was transferred into a variety of lactococcal starter cultures to improve their phage resistance properties.
Selection of resulting transconjugants was performed directly on solid media containing the bacteriocin. Since the starters exhibited no spontaneous resistance to the bacteriocin as a selective agent, this allowed the assessment of the transfer of the naturally occurring plasmid into a range of dairy starter cultures. Results demonstrate that efficient transfer of the plasmid was dependent on the particular recipient strain chosen, and while high-frequency transfer (10(-3) per donor) of the entire plasmid to some strains was observed, the plasmid could not be conjugated into a number of starters. In this study, transconjugants for a number of lactococcal starter cultures which are phage resistant and bacteriocin producing have been generated. This
bacteriocin-producing phenotype allows for control of nonstarter flora in food fermentations, and the phage resistance property protects the starter cultures in industry. The 63-kb plasmid was also successfully transferred into Lactococcus lactis MG1614 cells via electroporation.
PMID 9097441 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2001 Jun;67(6):2853-8.
Exploitation of plasmid pMRC01 to direct transfer of mobilizable plasmids into commercial lactococcal starter strains.
Hickey RM, Twomey DP, Ross RP, Hill C.
Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork.

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 x 10(-4)
transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01.
All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriT of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.
PMID 11375207 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2001 Feb;67(2):929-37.
Naturally occurring lactococcal plasmid pAH90 links bacteriophage resistance and mobility functions to a food-grade selectable marker.
O' Sullivan D, Ross RP, Twomey DP, Fitzgerald GF, Hill C, Coffey A.
Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Ireland.

The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P.
Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol.
36:866-876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.
PMID 11157264 [PubMed - indexed for MEDLINE]

J Dairy Sci. 2001 Jul;84(7):1610-20.
DNA sequence analysis of three Lactococcus lactis plasmids encoding phage resistance mechanisms.
Boucher I, Emond E, Parrot M, Moineau S.
Department of Biochemistry and Microbiology, Faculté des Sciences et de Génie,
Faculté de Médecine Dentaire, Université Laval, Quebec, Canada.

The three Lactococcus lactis plasmids pSRQ700, pSRQ800, and pSRQ900 encode the previously described anti-phage resistance mechanisms LlaDCHI, AbiK, and AbiQ, respectively. Since these plasmids are likely to be introduced into industrial Lactococcus lactis strains used to manufacture commercial fermented dairy products, their complete DNA sequences were determined and analyzed. The plasmids pSRQ700 (7784 bp), pSRQ800 (7858 bp), and pSRQ900 (10,836 bp) showed a similar genetic organization including a common lactococcal theta-type replicon. A second replication module showing features of the pMV158 family of rolling circle replicons was also found on pSRQ700. The theta replication regions of the three
plasmids were associated with two additional coding regions, one of which encodes for HsdS, the specificity subunit of the type I restriction/modification system.
When introduced into L. lactis IL1403, the HsdS of pSRQ800 and pSRQ900 conferred a weak resistance against phage P008 (936 species). These results indicated that both HsdS subunits can complement the chromosomally encoded type I restriction/modification system in IL1403. The genes involved in the phage resistance systems LlaDCHI, AbiK, and AbiQ were found in close proximity to and downstream of the replication modules. In pSRQ800 and pSRQ900, transfer origins and putative tyrosine recombinases were found upstream of the theta replicons.
Genes encoding recombination proteins were also found on pSRQ700. Finally, open reading frames associated with bacteriocin production were found on pSRQ900, but no anti-lactococcal activity was detected. Based on our current knowledge, these three plasmids are safe and suitable for food-grade applications.
PMID 11467810 [PubMed - indexed for MEDLINE]

Microbiology. 1994 Jun;140 ( Pt 6):1291-300.
The majority of lactococcal plasmids carry a highly related replicon.
Seegers JF, Bron S, Franke CM, Venema G, Kiewiet R.
Department of Genetics, Centre of Biological Sciences, Haren, The Netherlands.

DNA sequence analysis and Southern hybridizations, together with complementation experiments, were used to study relationships between lactococcal plasmid replicons. pWVO2, pWVO4 and pWVO5, which co-exist in Lactococcus lactis subsp. cremoris Wg2, and pIL7 (isolated from another strain) all contained a functional replication region which appeared to be very similar to that of some known lactococcal plasmids. They contain a gene encoding a highly conserved RepB protein (60-80% amino acid identity between pWVO2, pWVO4 and pWVO5), which is essential for replication. When supplied in trans, repB of pWVO2 complemented a repB deficiency of pWVO5. Upstream of the repB gene, all these plasmids contain a strongly conserved region including a 22 bp sequence tandemly repeated three-and-a-half times, and an A/T-rich region. The similarity with pWVO2, which is known to replicate via a theta mechanism, suggests that all plasmids of this family are capable of theta replication. Southern hybridizations revealed that many lactococcal strains contain plasmids of this family.
PMID 8081493 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2001 Apr;67(4):1700-9.
Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis.
Emond E, Lavallée R, Drolet G, Moineau S, LaPointe G.
Centre de recherche STELA, Département des sciences des aliments et de nutrition,
Université Laval, Québec, Canada G1K 7P4. eemond@chr-hansen-us.com

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing
erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile
of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.
PMID 11282624 [PubMed - indexed for MEDLINE]

Lead for drug discovery based on fundamental bacterial genetics.

2007-07-24T19:22:00.000+10:00

New Way to Target and Kill Antibiotic-Resistant Bacteria Found

Item Courtesy Physorg.com
First Published: July 9 2007, 17:20 EST

Antibiotic resistance propagates in bacteria by moving DNA strands containing the resistance genes to neighboring cells. An enzyme called relaxase is essential for this process. Bisphosphonates, already approved to treat bone loss, have now been shown to potently disrupt the relaxase function. Some bisphosphonates prevent the transfer of antibiotic resistance genes and selectively kill bacterial cells that harbor resistance. Credit: Scott Lujan, University of North Carolina at Chapel Hill

The team discovered a key weakness in the enzyme that helps “fertile” bacteria swap genes for drug resistance. Drugs called bisphosphonates, widely prescribed for bone loss, block this enzyme and prevent bacteria from spreading antibiotic resistance genes, the research shows. Interfering with the enzyme has the added effect of annihilating antibiotic-resistant bacteria in laboratory cultures. Animal studies of the drugs are now underway.

“Our discoveries may lead to the ability to selectively kill antibiotic-resistant bacteria in patients, and to halt the spread of resistance in clinical settings,” said Matt Redinbo, Ph.D., senior study author and professor of chemistry, biochemistry and biophysics at UNC-Chapel Hill.

The study appears online the week of July 9, 2007, in the Proceedings of the National Academy of Sciences. Funding was provided by the National Institutes of Health.

The study provides a new weapon in the battle against antibiotic-resistant bacteria, which represent a serious public health problem. In the last decade, almost every type of bacteria has become more resistant to antibiotic treatment. These bugs cause deadly infections that are difficult to treat and expensive to cure.

Every time someone takes an antibiotic, the drug kills the weakest bacteria in the bloodstream. Any bug that has a protective mutation against the antibiotic survives. These drug-resistant microbes quickly accumulate useful mutations and share them with other bacteria through conjugation – the microbe equivalent of mating.

Conjugation starts when two bacteria smoosh their membranes together. After each opens a hole in their membrane, one squirts a single strand of DNA to the other. Then the two go on their merry way, one with new genes for traits such as drug resistance. Many highly-drug resistant bacteria rely on an enzyme, called DNA relaxase, to obtain and pass on their resistance genes. A mutation that provides antibiotic resistance can sweep through a colony as quickly as the latest YouTube hit.

The researchers analyzed relaxase because it plays a crucial role in conjugation. The enzyme starts and stops the movement of DNA between bacteria. “Relaxase is the gatekeeper, and it is also the Achilles’ heel of the resistance process,” Redinbo said.

Led by graduate student Scott Lujan, the team suspected they could block relaxase by searching for vulnerability in a three-dimensional picture of the relaxase protein. Lujan, a biochemistry graduate student in the School of Medicine, confirmed the hunch using x-ray crystallography, which creates nanoscale structural images of the enzyme.

The researchers predicted that the enzyme’s weak link is the spot where it handles DNA. Relaxase must juggle two phosphate-rich DNA strands at the same time. The team suspected a chemical decoy – a phosphate ion – could plug this dual DNA binding site. Redinbo, who has a background in cancer and other disease-related research, realized that bisphosphonates were the right-size decoy.

There are several bisphosphonates on the market; two proved effective. The drugs, called clodronate and etidronate, steal the DNA binding site, preventing relaxase from handling DNA. This wreaks havoc inside E. coli bacteria that are preparing to transfer their genes, the researchers found. Exactly how bisphosphonates destroy each bacterium is still unknown, Redinbo said, but the drugs are potent, wiping out any E. coli carrying relaxase. “That it killed bacteria was a surprise,” he said. By targeting these bacteria, the drugs act like birth control and prevent antibiotic resistance from spreading.

see also National Review of Medicine

(Note J. Chan is considering this topic for her bioproduct proposal)

Velcome to my site: Time to start verkink hard every day

2007-07-20T09:11:00.000+10:00

Just a gentle hint to say to get good grades, hard effort and thorough engagement with unfamiliar problems is essential.

But please have fun too.

(image thank to Daniel Yuen.)

Energy and gold among the SliMEs

2007-06-16T16:22:00.000+10:00

Clean fuel research deal agreed by BP and Synthetic Genomics
15.jun.07
Biofuel Review
Synthetic Genomics Inc., a privately-held company dedicated to commercializing synthetic genomic processes and naturally occurring processes for alternative energy solutions, has announced a significant, long-term research and development deal with BP.
The deal between BP and Synthetic Genomics is centered on developing biological conversion processes for subsurface hydrocarbons that could lead to cleaner energy production and improved recovery rates. As part of the agreement, BP has also made an equity investment in Synthetic Genomics.
Microbes are key components in sustaining and maintaining life on Earth, and genomics is leading to an enhanced understanding of these organisms. In the first phase of the BP/Synthetic Genomics program, the research will focus on gaining a better understanding of microbial communities in various hydrocarbon formations such as oil, natural gas, coal and shale. Synthetic Genomics, which was founded by genome pioneer J Craig Venter, Ph.D., will use its expertise in environmental DNA sequencing and microbial cell culturing to produce the first comprehensive genomic study of microbial populations living in these environments. Once the basic science research phases are complete, BP and Synthetic Genomics will seek to jointly commercialize the technologies developed.
“We believe that one of the most promising solutions to producing cleaner fuels will be found through genomic-driven advances,” said Dr. Venter. “Through our research collaboration with BP, we will achieve a new and better understanding of the subsurface hydrocarbon bioconversion process which we are confident will yield substantial cleaner energy sources.”
The overall goal of Synthetic Genomics is to discover and/or design new genomes that will code for new types of cells with desired properties for bioenergy or specific chemical production. In this project, Synthetic Genomics scientists hope to better understand hydrocarbon metabolism by sequencing the genomes and culturing the cells of the naturally occurring microbes that thrive in subsurface hydrocarbons.
Tony Meggs, Group Vice President of Technology at BP, adds: “This collaboration is an exciting development that could lead to an unprecedented understanding of the microbial activity in the subsurface; and eventually to the development of more environmentally-friendly and efficient energy production and recovery techniques.”

Welcome to Microbe Pundit , all students of biomolecular engineering!

2007-03-22T11:16:00.000+11:00

Study questions on the fueling reactions of biosynthesis

Question A
*What is the justification for combining energy and reducing power into a single entity-driving force?

Question B
*ATP generation by transmembrane ion gradients involves two steps: establishing the gradient, and using its energy to produce ATP. What mechanisms have microbes evolved for these two steps?

Respond in the comments and get feedback from The Pundit.

Q and A on BACs and Metagenomics. Why large inserts?

2006-10-31T12:51:00.000+11:00

Question from student (edited):

I am reviewing the Soil Metagenome paper that we reviewed in class (Rondon et al., 2000, Appl. Environ. Microbiol. 66: 2541-2547), and have a written note claiming that there are two important reasons to clone large DNA fragments into the BAC vectors. I can only think of one reason, which is that many soil microbes produce important bioactive compounds, and genes required for production along with regulatory genes are often clustered in one continuous segment on the chromosome called an operon. A large fragment of DNA inserted into BAC plasmid would increase the likelihood that the entire operon included. Is this correct, and is there another reason why having large DNA fragments in the BAC plasmids is important?

Answer:

Yes, you are correct, and yes, there is another reason. Large inserts mean that each clone (Escherichia coli clone line) is likely to contain genes encoding for multiple activities that may be of interest. This means that fewer clones need to be maintained than if the inserts were smaller, i.e., the same amount of metagenomic information is maintained in fewer clones (or more metagenomic information can be maintain in any given number of clone lines). Using screening assays for different activities, the same amount of metagenomic material can therefore be interrogated for the presence of specific activities while manipulating fewer cultures.

A question of cycles of oxidation in mineral leaching.

2006-10-31T11:05:00.000+11:00

Question from student (edited):

Regarding acid mine drainage, does the oxidation of sulfides to H2SO4 result in the release of the metal ions which get leached out (eg, the release of Fe2+ from FeS2)? Does the H2SO4 that is produced cause the leaching out of other metals from their ores because of its acidic nature? Can the microorganisms also oxidise the S2- in the FeS2 in addition to oxidising the Fe2+ that is produced from this reaction?

Answer (from Pundit's Environmental Expert Jan Pundit):

Initially, the H2SO4 formed by microorganisms helps to solubilise metals released from the ores. They oxidise the S2- (in the metal sulfides) to produce the H2SO4, which helps to stabilise Fe2+ (which oxidises in air under neutral or alkaline conditions), and keeps Fe3+ in solution, which would otherwise react with water to produce ferric oxy-hydroxides (rust) at neutral and alkaline pH. Once the system is acidic, and there is a high concentration of Fe3+, then the propagation cycle becomes important. There is direct chemical oxidation of the sulfides in the ores, by Fe3+, to produce more H2SO4, and releasing more (reduced) metal ions, and the Fe3+ is chemically reduced to Fe2+. The bacteria are now important for the biological reoxidation of Fe2+ to Fe3+, which is required to keep the chemical oxidation going.

Analogue resistant mutants and the discovery of metabolic regulation are inextricably linked in history.

2006-10-31T09:40:00.000+11:00

The History of Using Metabolic Analogs to Reveal Regulation of Metabolism.

Metabolic analogues are compounds that structurally resemble natural metabolites.

A name for a metabolic analogue that interferes with cell functions is antimetabolite.

Antimetabolities will often inhibit cell growth.

Sulfa drugs were perhaps the first antimetabolites.

In 1958, Ed Adelberg discovered that Escherichia coli MUTANTS, that had been selected as being resistant to growth inhibition by toxic metabolic analogues, secreted excess quantities of certain metabolites into their growth medium that the wild-type parental strains DID NOT secrete.

For instance, E. coli mutants selected for resistance to p-fluorophenylalanine secreted tyrosine and only tyrosine into the medium.

p-Fluorophenylalanine is a tyrosine analogue.

The compounds secreted by E. coli mutants differed when different types of antimetabolite were used to select mutants.

Ethioninione resistant mutants secreted methionine. (The structure of ethionine is Ethyl replacing the Methyl in methioninine.)

The patterns of secretion suggested to Adelberg that resistance was based on COMPETITION between metabolite and analogue.

Interestingly, metabolic analogues were provided to Ed Edelberg by Arthur Pardee, one of the discovers of feed-back inhibitable biosynthetic enzymes (allosteric enzymes).

In 1960 Pardee reported 3-Methylaspartic acid as a potent antimetabolite of aspartic acid in pyrimidine biosynthesis, showing these ideas are not confined to amino acid pathways.

Selection of bacterial mutants which excrete antagonists of antimetabolites.
J Bacteriol. 1958 Sep;76(3):326.Click here to read Links
ADELBERG EA
Now marvellously available online via Pubmed
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=13575393

Famously, Pardee and Yates had discovered feedback-inhibition in 1956, in a key paper revealing the existence of biochemical regulation.

Dick Yates went on to work in applied microbiology in Wilmington DE, and the Pundit is honoured to have met this gracious American scientist.

J Biol Chem. 1956 Aug;221(2):757-70.
Control of pyrimidine biosynthesis in Escherichia coli by a feed-back mechanism.
PARDEE AB, YATES RA.
PMID: 13357469 [PubMed - OLDMEDLINE for Pre1966]
http://www.jbc.org/cgi/reprint/221/2/757

Related Links
Pyrimidine biosynthesis in Escherichia coli. [J Biol Chem. 1956]
PMID:13357468
Studies on the biosynthesis of bacterial and viral pyrimidines. IV.
Utilization of pyrimidine bases and nucleosides by bacterial mutants. [J Biol
Chem. 1957] PMID:13475346
3-Methylaspartic acid as a potent antimetabolite of aspartic acid in
pyrimidine biosynthesis. [J Biol Chem. 1960] PMID:13786646

Discovering novel antifungal agents with antimitotic activity from diverse soil and water sources.

2006-10-30T20:47:00.000+11:00

C W S Fen, Melbourne

Abstract

In response to the side effects and increasing amount of resistance met by antifungal drugs targeting the ergosterol pathway in fungi, we propose to bring antimitotic antifungal agents to a reformation, enlisting the use of high throughput screening to discover novel microtubule-inhibiting compounds produced by fungi from a diverse range of soil and water sources. We intend to screen large amounts of secondary metabolites produced by these fungi against 10 specifically chosen common infection-causing fungi species. After narrowing down the candidate compounds to a few lead compounds, these will be tested for fungal-specific antimitotic activity in vitro with fungal microtubule dimers and in vivo against the 10 fungi species utilizing GFP-labelling technology. Toxicity to human cells will be assessed as well before clinical trials can be proposed. Further experiments are necessary to determine optimum dosage and method of delivery of the drug, and further research into potential anticancer drug applications may yield additional commercial benefit from investment in this initiative.

Introduction
Imagine if a harmless scab that you thought would just go away became the cause of your hospitalisation. Or the joy and relief of being discharged after an operation only to find yourself back in bed being treated for a post-surgical fungal infection. Fungal infections have a significant impact on the quality of life of affected individuals despite little attention dedicated to it when compared to other life-threatening diseases. However, most predominantly, it is patients who have undergone surgery, are immunocompromised, have undergone chemotherapy or excessive antibiotic doses who are severely affected and whose life may well be in danger if the infection is not addressed immediately (White et al., 1998). In view of this, biotechnological innovations and advances over the past few years can be put to good use in fuelling the initiative of discovering new or improved anti-fungal compounds which targets fungi specifically while sparing human cells.

In light of this, we propose to bring fungal-specific antimitotic drugs to a reformation, achievable by exploiting the fungal cells’ dependence on microtubule function to proliferate, and thus prevent the progression of infection. From a marketability standpoint, the diverse range of pharmaceuticals that target the ergosterol pathway in fungi are saturating the market, resulting in resistance among different drugs targeting this same biochemical pathway (White et al., 1998). Therefore the guaranteed effectiveness of this new drug minus the side effects of its competitors (which are the main source of concern), coupled with increased efficacy, is highly promising in guaranteeing a high return in investment. Accordingly, we intend to perform high-throughput screening of soil and water samples from a diverse range of sources to screen for fungi that have the ability to produce such a compound. Following that, we will run carefully designed assays to engineer additional broad-spectrum activity of the said compound. Miracles are more likely to happen if we make them and we intend on doing just that.

PROPOSAL:

The Scientific Basis

Microtubules are essential to all eukaryotic cells; in essence, fungal cell shape, transport, motility and most importantly for this proposal, cell division, rely wholly on the proper function of this critical organelle. The fundamental property of microtubules that we look to exploit in a biotechnological sense is its non-equilibrium behaviour, better known as microtubule dynamic instability, which determines the shortening or lengthening of microtubules to facilitate them in performing the functions listed (Nogales 2001). With this in mind, we analysed fungal cell characteristics to determine a logical strategy of attack.

Many secondary metabolites produced by fungi have potent pharmacological uses through exploiting its initial intended function. An obvious example of this is Penicillin produced by the mould Penicillium notatum a commonly used antibiotic for treating bacterial infections and diseases. However, this same species of mould from which penicillin is derived also produces another compound, Griseofulvin, synthesised by a close relative, Penicillium griseofulvum, which prevents microtubule formation in fungal cells during mitosis. Although griseofulvin requires a longer duration of action and exhibits less efficacy than other commonly used antifungals, nevertheless, it boasts minimal side effects and a currently still unexplored biochemical mechanism as to why it targets fungal cells specifically, partially sparing human cells and therefore resulting in milder side effects (Jimenez et al, 1990). Logically assuming that it is the specific action or higher affinity of the drug for fungi microtubules that results in the milder side effects, we propose to further expand on the merit of this particular target protein of the drug and search for novel compounds which utilises a similar mechanism of attack to halt the growth of fungi, making it a potent fungistatic agent. Subsequently (not covered by this proposal), chemical modification or further subjection of the fungi species of interest to selective chemicals or mutagenic agents can be run to develop a compound that will bind irreversibly to the microtubules and thus effectively kill off the target fungal cells (Refer to Figure 1 and Figure 2 below).

Figure 1: Two possible models of microtubule nucleation. (a) End-on model of nucleation; (b) Lateral interaction of γ-Tubulin protofilament with αβ-Tubulin dimers. (Modified from Nogales, 2001). Irreversible binding of the antifungal compound (in red – whether to the protofilament as whole or to individual dimers) can prevent prolongation of αβ-Tubulin protofilament or interaction between filaments, disrupting microtubule instability and therefore mitosis.

Figure 2: Ribbon diagram of molecular structure of α- and β-tubulin dimer of microtubules. A compound that can bind irreversibly to a point on this most basic unit of microtubule structure at e.g. the taxol binding site (circled in red) can trap these tubulin dimers and cause stabilization of microtubules, preventing mitosis (Revised from Nogales, 2001).

The Source

We plan to run high throughput screening of a range of soil and water samples obtained from diverse geographical locations around the world to isolate as many species of fungal cells as possible, with a more bias focus on mycoparasites, as toxic compounds produced by fungi against other fungi will be more specific and therefore more effective. Then, we will narrow down the candidates of these fungal species to those that may harbour antimitotic activities.

By diluting the soil sample in water and partially diluting the water samples, multiple cultures (100 odd petri dishes) of fungi are obtained using growth media that will stimulate fungal growth, e.g. YES media (Torres et al., 1987). After this primary culture, each fungal colony will be extracted and cultured at diluted concentrations in zone-based bioassays overlaid with a layer of one of 10 designated common infection-causing fungi, i.e. the Tinea spp., Candida spp., Aspergillus sp., Cryptococcus spp., Fusarium spp., etc. (Mays et al, 2006). It was shown by Panda et al., 2005, after a few weeks, any fungal colony whose surrounding cells are trapped in interphase and highly mononuclear exhibits antimitotic activity. These colonies are isolated and the compound produced by each colony is extracted and purified by chemical means. These compounds are then subjected to high throughput screening to test for antimitotic activity.

High Throughput Screening

The screen we have in mind is designed to be a fungal-specific antimitotic screen. Exploiting the success of genomic sequence technology and the completion of function determination of thousands of genes, we will search for genes encoding microtubule function in the 10 species of common infection-causing fungi using the available genome libraries online. By ensuring that the 10 chosen species of fungi have been studied intensively enough to for us to genetically manipulate the microtubule and its associated proteins’ genes, hypersensitive mutants susceptible to antimitotic drugs will then be generated and these mutant fungi will be used to screen for compounds that can inhibit their growth among the potential candidate compounds produced by the initial fungal culture. Testing will be done at increasing concentrations of antimitotic/antifungal compounds. Following positive results, the wild type strain for each of the 10 common disease causing fungi is then exposed to these 10 compounds as a control to identify any of the compounds can arrest the cell cycle of the parent strain (Lila et al, 2003). In this way, the antimitotic drug-sensitive fungi will increase the likelihood of discovering a drug that has antimitotic potential which we can improve the properties of through further screening.

Thus, narrowing down the search to one lead compound, we will proceed to test this in vitro with microtubules obtained from fungal cells. By tagging the fungal tubulin dimer proteins with GFP tags, we will then monitor the action of the compound on the polymerization of individual tubulin entities with increasing lead compound concentration.

From in vitro studies, we will extrapolate success of the compound in inhibiting microtubule function to in vivo testing by a reporter system where cloning technology is used to introduce fluorescence into microtubule dimers by incorporating the GFP gene into the tubulin gene (cf. protein) of each of the 10 fungal species which is then transcribed into tubulin dimers that will fluoresce, thus enabling us to monitor the growth or shrinking of microtubules (mitotic spindle) during mitosis (Implication of concept by Kumagai et al., 2003 and Alberts et al., 2002). Further proof can be obtained by running flow cytometry analysis of fungal cells treated with cytotoxic concentrations of the lead compound – if there is no increase in nuclear DNA content, then there is a lack of tubulin-related activity in vitro (Lila et al, 2003).

These same set of in vitro and in vivo experiments are repeated with human tubulin and in human cells from various tissues across the body respectively to test for toxicity of the compound (Priestly and Brown, 1978). Thus, if luck it working on our side, we may be able to determine a distinct fungal specificity of the drug and determine the exact concentration of the compound which is the threshold of toxicity for human cells. Complicated but necessary further experimentation should be done in whole organism model systems such as rats, mice and other mammals to mimic possible side effects in humans before proposing the extrapolation of these results to humans in clinical trials.

Potential Problems

Microtubules, despite being in different organisms, are highly conserved organelles, especially between organisms of the same domain (Eukaryota). We are taking a significant risk in gambling with nature on the possibility that antifungal compounds produced by fungi themselves against their fungal counterparts might specifically inhibit these competitors for survival antimitotically. At the very least however, we can design these novel compounds specifically for a distinct group of fungal species (e.g. specifically one of superficial, subcutaneous, systemic or opportunistic infection-causing groups of fungi) if not for broad-spectrum activity against most of them.

As in any microbial biotechnology process, transition from a small-scale experiment to culturing of these fungi of interest in the large scale will present us with many more problems to come. However, given the appropriate equipment, competent technicians and proficient biotechnologists, this should be a challenge we can overcome.

Further Research

As part and parcel of an antifungal treatment, side effects are inevitable as human cells are very similar structurally to fungal cells, as mentioned earlier. Thus, drugs that target fungal cells will affect our cells to a certain extent as well. Therefore, although the side effects will be specifically tailored to be minimal, further chemical modification or manipulation (if possible) to obtain compounds that have near-negligible side effects will be needed following the isolation of the fungal microtubule inhibitor protein-producing fungi..

In addition to that, also on a pharmacological basis, we need to take into account the stability of the compounds (determining optimal pH), scaling up of production and the efficiency of cell growth of the cell of interest in culture. Other than that, solubility of antifungal compounds is a major concern in the pharmaceutical industry as most antifungal compounds are contrary to that requirement. Added to that oral bioavailability, potential side effects and possible allergic reactions have to be accounted for to guarantee its economic value.

Potential Future Benefits (Integration of Science and Business)

One of the key valuable gains from the production of this initial microtubule formation inhibitor compound is a better understanding of the mechanism of microtubule inhibition and prevention of mitosis. From investigating so many fungi species with antimitotic activity, we can gain an insight into the many potential ways the microtubule can be inhibited and by manipulating this knowledge as well as other biotechnological screening and assay methods to slightly modify the compound, we could extrapolate the research to involve discovery of similar compounds from fungi which will specifically inhibit microtubule formation of tumour cells and thus secure a spin-off opportunity to produce a potential anti-cancer drug. Much research efforts and funds have been poured into a similar initiative involving Griseofulvin (Panda et al., 2005). Thus, this presents a further bonus for investing in this research.

Conclusion

In conclusion, this initiative of discovering novel antimitotic antifungal drugs presents many commercial, scientific and medical benefits to every player: investors, consumers and the biotechnologists. Besides solving a demanding medical problem and gaining much knowledge about fungi and its infection-causing mechanisms, there is much to gain from investment in the long run, with spin-off opportunities that are becoming a reality in the anticancer drug industry. “Fortune favours the prepared mind - Louis Pasteur.” This statement could not hold truer in this fascinating hybrid between business and science.

References
1. Lila, T., Renau, T.E., Wilson, L., Philips, J., Natsoulis, G., Cope, M.J., Watkins, W.J. and Buysse, J. 2003. Molecular Basis for Fungal Selectivity of Novel Antimitotic Compounds. Antimicrobial Agents and Chemotherapy. 47: 2273-2282.

2. Nogales, E. 2001. Structural Insights into Microtubule Function. Annual Review of Biophysics and Biomolecular Structure. 30:397-420.

3. Kumagai, F., Nagata, T., Yahara, N., Moriyama, Y., Horio, T., Naoi, K., Hashimoto, T., Murata, T. and Hasezawa, S. 2003. Gamma-tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells. European Journal of Cell Biology. 82:43-51.

4. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. 2002. Molecular Biology of The Cell, 4th edition. Garland Science, New York, USA.

5. White, T.C., Marr, K.A. and Bowden, R.A. 1998. Clinical, Cellular, and Molecular Factors That Contribute to Antifungal Drug Resistance. Clinical Microbiology Review. 11: 382-402.

6. Torres, M., Canela, R., Riba, M. and Sanchis, V. 1987. Production of patulin and griseofulvin by a strain of Penicillium griseofulvum in three different media. Mycopathologia. 99: 85-89.

7. Mays, S.R., Bogle, M.A. and Bodey, G.P. 2006. Cutaneous Fungal Infections in the Oncology Patient: Recognition and Management. American Journal of Clinical Dermatology. 7: 31-43.

8. Priestly, G.C. and Brown, J.C. 1978. Effects of griseofulvin on the morphology, growth and metabolism of fibroblasts in culture. British Journal of Dermatology. 99: 245.

Step up: Bacterial Lipase to Substitute Pancreatic Lipase For Enzyme Therapy

2006-10-30T20:05:00.000+11:00

D. G. Sani, Melbourne

Summary

Microbial lipases have been more commonly used than lipases derived from plants and animals due to higher stability, rapid production, higher yields and more convenient manipulations of microorganisms. For example, lipase from Mucor miehei is used in cheese production, Candida antartica in surfactants, Serratia marcescens lipase in drugs (diltiazem). Medical area is another promising area where lipase can be used as digestive aids. To date, only pancreatic enzyme (lipase) therapy is used to treat fat malabsorption in Cystic Fibrosis and pancreatitis patients. However, pancreatic lipase is susceptible to low pH (acidic gastric environment) and protease which can render the lipase inactive. Therefore, it is proposed that an alternative bacterial lipase might have the ability to retain its activity under acidic condition and also be protease tolerant. Screenings for this novel bacterial lipase would be done from bacterial populations that are known to survive in the gastric environment. Moreover, different assays would be done to observe the lipase production, activity, and specificity under certain conditions.

A. INTRODUCTION

Bacterial lipases have been recognized since nearly 100 years ago in lipase-producing bacteria such as Bacillus pyocyaneus (today is named Pseudomonas aeruginosa), Staphylococcus pyogenes (S. aureus), B. fluorescens (P. fluorescens) [1]. For a lipase to be defined as a true lipase, it must exhibit interfacial activation where a presence of triglyceride should rapidly increase its activity; it should also contain a loop allowing substrate entry to the active site. However, these two criteria are unsuitable for some exceptions where the lipases have the loop but do not exhibit interfacial activation. Hence, a simpler definition of lipases is carboxylesterases that catalyse the hydrolysis and synthesis of long-chain acylglycerols [1]. The prospect for industrial enzymes (technical, food, and animal feed enzyme) is very promising with an estimated increase to $2.4 billion in 2009. Technical enzyme such as in detergents, pulp production, and medical treatment is predicted to have the highest share at 52% [2].

Global Enzyme Markets Based on Application Sectors, 2002-2009
($ Millions)

Source: BCC research [2]

Application of lipase in industrial enzyme production has covered a wide area: flavour development in food technology; as oil removal in detergents; textile making; personal care products e.g. cosmetics; digestive aids for medical treatment [3]. As digestive aids, pancreatic lipase has been widely used in the enzyme therapy to treat lipid malabsorption e.g. in cystic fibrosis and pancreatitis patients. However, there are some barriers with the pancreatic enzyme therapy that prevent its maximal efficacy [3]. Hence, the need of finding an alternative lipase from microorganisms has been raised. Microbial enzymes have several advantages than animals or plants enzymes due to the stability, high yields and rapid production from microbes, and variety in catalytic activity [3]. This paper will firstly describe the background and target market of enzyme therapy for lipase, followed by proposal of methods to isolate lipase-producing bacteria and assays to obtain the acid and protease tolerant lipase.

B. BACKGROUND AND TARGET MARKET
Cystic Fibrosis (CF) is an autosomal genetic disorder affecting mostly Caucasian populations with approximately one in every 3500 newborns is born with CF each year in the United States alone [4]. Being a multi-system disease, CF affects many organs: lungs, pancreas, liver, bones (osteoporosis) [5]. The altered gene in CF disrupts the function of salt transports in organs leading to an excess production of thick, sticky mucus that blocks the ducts in these organs mainly the lungs and the pancreas. Patients with affected pancreas tend to have digestion and growth problems due to pancreatic insufficiency where there is a lack of pancreatic enzyme produced by the body [4,5]. This condition leads to lipid malabsorption because the body cannot break down the essential fatty acid (EFA) molecules coming from external nutrients [6]. Poor nutritional status due to EFA deficiency is correlated with weight and height retardation in children with CF. Moreover, a research by CDC in the US has shown a correlation between nutritional status and the lung function showing increase in body weight in CF patients (measured by BMI) is accompanied by increase in the lung function [4].

Many treatments have been developed to treat the malnutrition in CF patients and improve their life expectancy. To date, pancreatic enzyme (lipase) therapy from porcine origin has been the most widely used since 1930s with about 90% of the patients taking pancreatic enzyme supplements in 2004 [4, 7]. However, the efficiency of this treatment has not reached maximal yet. The pancreatic lipase is found to be denatured by gastric acids before reaching the small intestine where the FA hydrolysis and absorption mainly occur. Although coating the enzyme with an acid resistant (enteric) coating appears to improve the delivery of the enzyme into the small intestine, the low intestinal pH in CF patients after meal (pH less than 4) would still irreversibly denature the pancreatic lipase [7].

Another barrier for the existing pancreatic enzyme therapy is that pancreatic lipase is also denatured by proteases especially chymotrypsin in the intestinal lumen [7]. Hence, in search of ways to improve the enzyme therapy, the aim is to find an alternative lipase that would be acid resistant, protease resistant and would still possess the pancreatic lipase activity.

C. SCIENTIFIC BACKGROUND FOR BACTERIA ISOLATION
In order to find a lipase which shares features with pancreatic lipase, one must know the characteristics of pancreatic lipase. It is known that pancreatic lipase hydrolyses long-chain triacylglyceride (TG) into FAs to be readily absorbed in the small intestine at the optimal pH of 8 [6]. When the pH falls below six the pancreatic lipase is rendered inactive and if the pH less than 4.5, it is irreversibly inactivated; the later is the case in CF patients causing denaturation of the pancreatic lipase in the small intestine during the enzyme treatment [6, 7]. Hence, the alternative lipase would have to be able to survive under acidic environment (pH less than 4). Moreover, knowledge of the active site structures of pancreatic lipase can be a valuable information in finding a new lipase. The active site residues of pancreatic lipase are serine (Ser 152), aspartate (Asp 176), and histidine (His 263) [8]. These are found to be conserved in bacterial lipases where the active site has a nucleophilic residue (Ser, Cys or Asp), a catalytic triad residue (Asp or Glu), and a histidine [1].

Acid resistant lipase could most probably be obtained from bacteria living in acidic environment. A study shows that Salmonella, a gastrointestinal pathogen, produces proteins: RpoS, PhoP, and Fur that help them survive under the harsh gastric environment. RpoS and Fur are responsible for survival against organic (weak) acid stress, while PhoP and RpoS act against inorganic acid (low pH) stress [9]. Therefore, bacteria that produce RpoS, PhoP and Fur would presumably be able to survive under low pH in the gastric environment. A database search from the genbank of NCBI returns 33 bacterial strains that have RpoS, PhoP, and Fur proteins in their genome. Most of these strains are from Salmonella, Pseudomonas, E. coli, Yersinia, Shewanella, and Shigella [10].

Out of these bacterial populations, previous studies have shown that some Pseudomonas strains produce true lipase while both E. coli and Salmonella typhimurium produce esterases [1]. Bacteria surviving from the gastric environment would most probably be present in faeces. Hence, novel lipase-producing bacteria can be screened from faecal specimens and looking specifically for Yersinia, and Shigella as no lipases have been identified from these genera before. These specific strains can be isolated from faecal specimens using selective media. For example, MacConkey agar (MAC) is used to isolate lactose-fermenting, gram negative enteric bacteria while xylose lysine desoxycholate (XLD) agar is specific for isolation of Salmonella and Shigella [11]. Yersinia can be isolated on DYS medium containing peptone, ox-bile, arginine, lysine, arabinose, casein where Yersinia will ferment arabinose and appear as bright red colonies [12].

Another consideration in searching for a new lipase is the specificity of the lipase. Since the aim is to screen for a bacterial lipase that mimics pancreatic lipase, the bacterial lipase should be able to hydrolyse long-chain TGs- the substrate for pancreatic lipase [6]. Although the term ‘long-chain’ TG is not strictly defined, in general, glycerolesters with an acyl chain length more than ten carbon atoms can be considered as lipase substrates e.g. trioleoylglycerol. On the other hand, esterase substrates would normally have an acyl chain length less than 10 carbon atoms e.g. tributyrylglycerol. Most lipases, however, are capable of hydrolysing esterase substrates in addition of the lipase substrates [1]. Hence, screening for the alternative lipase would differentiate between lipase and esterase specificities.

D. ASSAYS
Assays that would be used for screening of an alternative bacterial lipase are assays for lipase production, lipase purification, activity and specificity, pH effects on activity (low pH), and tolerance on protease (chymotrypsin).

Identification of lipase-producing bacteria from the formerly isolated strains can be done by a plate assay where the agar medium contains triolein as the lipase substrate. Lipase production on triolein plates is indicated by orange-red fluorescence of the colonies at 350 nm UV lamp [1]. The lipase-producing colonies can then be purified by inoculating a single colony onto agar plates containing essential compounds for growth. Then the strains would be grown in minimal media containing some essential nutrients for growth and enhance extracellular lipase production. It has been studied that exolipase production in Serratia marcescens can be enhanced by addition of certain polysaccharides as shown in the table below [13].

Table from Winkler, U., Stuckmann, M. (1979). Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia
marcescens. J Bacteriol 138: 663-670. Not displayed.

Table Source: Winkler [13].

It can be seen that laminarin exhibits the highest exolipase inducing ability followed by glycogen and pectin B, then hyaluronate. Moreover, the exoprotease production should be kept to minimum so that it would not degrade other essential proteins. Amongst these exolipase enhancers, the most suitable polysaccharide would be pectin B since it has high inducing ability with less exoprotease activity produced compared to laminarin and glycogen. Furthermore, due to interfacial activation of most lipases, addition of long-chain triglyceride such as olive oil might enhance the activity. Hence, pectin B and olive oil can be included in the medium as exolipase enhancers.

There are different assays to test lipase activity in the supernatants namely: the photometric assay and titration method [1]. The photometric assay is done using p-nitrophenylpalmitate (pNPP) as the substrate which is mixed with the supernatant [13]. Lipase activity is then monitored by detecting the hydrolysis of various p-nitrophenolesters of FAs (greater than 10 carbon atoms) into p-nitrophenol at 410 nm. Different p-nitrophenolesters would be separately included in the reaction mixture to test for lipase specificity [14]. The titration method measures lipase activity by recording the amount of NaOH used to maintain pH 8 as the fatty acid is liberated after addition of lipase solution into the supernatants [15].

Assay to screen for acid tolerant lipase can be performed by setting up a reaction mixture mimicking the gastric environment where the pH can be as low as 2.5 to 4. The synthetic gastric fluid has been widely used by mixing pepsin, lysozyme, and bile. Although the synthetic gastric fluid is more convenient to prepare, it has been demonstrated that certain bacteria are more sensitive in an ex vivo porcine gastric fluid than in the synthetic gastric fluid at a same pH of 2.5. This suggests that there are some components which are present only in the porcine stomach and can interfere with bacterial survival [9]. Hence, a more reliable assay for acid tolerant lipase can be done using porcine gastric fluid collected from live pigs instead of the synthetic gastric fluid. The pH of porcine gastric fluid can be adjusted from 3.47 to 4.15 by mixing supernatants of several stomach contents with pH ranging from 1.42 to 4.44 [9]. This mixture (synthetic or porcine gastric fluid) can then be added into the bacterial supernatants and then assayed for lipase activity (photometric assay). The acid resistant lipase would retain its ability to hydrolyse p-nitrophenylesters after treatment with the gastric fluid.

Protease tolerant lipase can be assayed by adding a specific protease into the bacterial supernatants prior to the lipase activity assay. For this experiment, chymotrypsin would be used since it is found to denature pancreatic lipase in the small intestine [7]. If successful, the bacterial lipase that retains its activity would be the protease tolerant lipase. After confirming the lipase which possesses the specific characteristics, purification of the lipase can be done by chromatographic methods where the lipase is bound to beads in a column and then eluted with corresponding buffer. For instance, extracellular lipase of Pseudomonas was purified by anion-exchange chromatography and HIC [15]. The main advantage is that purified enzymes have high activity and can be re-used in other reactions.

CONCLUSION
Technical enzymes application in various biotechnology areas has shared the highest part in the global enzymes market since 2002 [2]. One of the major areas is in medical application where pancreatic lipase therapy is used as digestive aids mostly in CF patients. However, the efficacy has not reached maximal due to degradation of the lipase under acidic condition in the stomach and small intestine of these patients [7]. Hence, searching an alternative acid-tolerant lipase from bacterial origin to substitute pancreatic lipase as digestive aids would have high market returns in the future. If successful, the novel bacterial lipase would be acid and protease tolerant where it can retain its activity at pH less than 4. Moreover, purification would increase its activity implying for higher end products yields from fewer lipase mass and hence, effective production cost.

REFERENCES

1. Jaeger, K. E., Dijkstra, B. W., Reetz, M. T. (1999). Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological application of lipases. Annu Rev Microbiol 53: 315-51.

2. BCC report on global enzyme market. http://www.bccresearch.com/bio/BIO030D.asp. Retrieved 5/10/2006.

3. Hasan, F., et. al. (2006). Industrial applications of microbial lipases. http://www.aseanbiotechnology.info/Abstract/21019024.pdf

4. Cystic Fibrosis Foundation. (2004). Patient registry annual data report 2004.

5. Gotz, M. H. (2002). Cystic Fibrosis literature review annual report 2002.

6. Peretti, N., et. al. (2005). Mechanisms of lipid malabsorption in Cystic Fibrosis: the impact of essential fatty acids deficiency. Nutrition & Metabolism 2:11.

7. Durie, P., et. al. (1998). Uses and abuses of enzyme therapy in cystic fibrosis. J R Soc Med 91: 2-13.

8. Olivecrona, G. et al. (1994). “Medical aspects of triglyceride lipases.” In P. Woolley & S.B Petersen (Eds.), Lipases: their structure, biochemistry and application pp.316-318.

9. Bearson, S. M. D., et. al. (2006). Identification of Salmonella enterica serovar Typhimurium genes important for survival in the swine gastric environment. Applied and Environmental Microbiology 72: 2829-36.

10. NCBI web site: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Genome&cmd=search&term=RpoS,+Fur,+PhoP+containing+bacteria Retrieved 29/9/2006.

11. CDC: Ch4: Identification and isolation of Shigella. http://www.cdc.gov/ncidod/dbmd/diseaseinfo/cholera/ch4.pdf#search='isolation%20of%20shigella'. Retrieved 4/10/2006.

12. Agbonlahor, D. E., et. al. (1982). Differential and selective medium for isolation of Yersinia enterocolitica from stools. J Clin Microbio 15: 599-602.

13. Winkler, U., Stuckmann, M. (1979). Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia
marcescens. J Bacteriol 138: 663-670.

14. Kanwar, S. S., et. al. (2006). Purification and properties of a novel extracellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate. Protein Expression & Purification 46: 421- 428.

15. Kordel, M., et. al. (1991). Extracellular lipase of Pseudomonas sp. strain ATCC 21808: Purification, characterization, crystallization, and preliminary X-Ray diffraction data. J Bacteriol 173: 4836-41.

Another Question for the crowd to kick around.

2006-10-12T14:17:00.000+10:00

Update 30th October
Thanks for putting in questions from the list given in the study Question list not posted here.

THIS IS THE PLACE WHERE I ANSWER THEM, and will happily answer any more that are placed in the comments.

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Compare and contrast fructose syrup and steroid manufacturing processes as used in industry.

Both compare and contrast please.

Questions 2 and 3 for discussion.

2006-10-12T14:16:00.000+10:00

Explain why corticosteroids are relatively cheap drugs, and why fructose syrup is a cheap sweetener.

Question for discussion 1

2006-10-12T14:14:00.000+10:00

Explain the differences in use of alpha-amylase and glucoamylase in the sugar syrup industry.

Screening for Novel Anti-cancer Bioactive Compounds Produced by Bacteria Found in Marine Sponges.

2006-10-09T14:00:00.001+10:00

A Proposal for Novel Compound Discovery

H. A. Ward, Melbourne

Abstract

The chemical and biological diversity of the marine environment is immeasurable and is therefore an extraordinary resource for the discovery of novel anti-cancer compounds. Recent technological and methodological advances in structure elucidation, biological assays and organic synthesis have enabled the isolation and clinical evaluation of various novel anti-cancer agents from marine microorganisms. This report provides insight into the processes involved in the isolation and identification of novel marine microorganisms, the screening for novel bioactive anti-cancer compounds produced by the microorganisms, as well as the evaluation and characterization of their structure and bioactivity.

Introduction
With an estimated 7.6 million deaths reported in 2005, cancer is the second leading cause of death amongst the world’s population . Each year, several billion dollars is invested in cancer research in an attempt to understand the disease processes involved and to try to discover possible therapies to combat this debilitating disease. The potential market for immune drugs is huge and is a rapidly growing area in the biopharmaceutical arena. It is expected that there will be a US $15 billion worldwide market opportunity for therapeutic immune drugs by the year 2010 .

During the past 5 decades of research in anti-cancer drug discovery, about 100 products have been provided for clinical treatment of malignancy (Wagner-Dobler et al., 2002). Significant progress has been made in the chemotherapeutic management of hematologic malignancies, however, more than 50% of patients with tissue tumours either fail to respond or will die from the disease (Wagner-Dobler et al., 2002). Hence, the discovery of novel anti-cancer therapeutic agents remains critically important.

The tremendous biochemical diversity of marine microorganisms and their biotechnological potential, as extraordinary resources for the discovery of new anti-cancer compounds, is becoming more and more recognized by both microbiologists as well as the pharmaceutical industry (Schweder et al., 2005). Of these microorganism species, the microorganisms living in marine sponges have attracted significant attention as potential sources of bioactive compounds (Garcia Camacho et al., 2006). Because of their phenomenal potency, even very small quantities of these compounds can be of significant value in a commercial sense (Simmons et al., 2005).

Culture based techniques are inadequate for studying bacterial diversity from marine samples as many bacteria cannot be cultured using artificial laboratory conditions (Webster et al., 2001) and thus, is not accessible for detailed taxonomical and physiological characterizations. The tools of molecular biology and the phylogenetic framework, which is now available as 16S rDNA sequence alignments, allow a complementary strategy to be pursued. This is based on phylogenetic screening of marine isolates and an in-depth investigation of the biological activity and chemical diversity of selected phylogenetic groups in order to identify a new hotspot for the production of bioactive compounds (Schweder et al., 2005). Selected isolates can then be cultivated on a large scale and under a variety of cultivation conditions.
This report aims to provide an insight into the steps which are involved in screening for these novel anti-cancer bioactive compounds which are produced by marine bacteria.

Proposal
Searching for novel bioactive compounds is a multi-step procedure which begins with the selection of a suitable source to be investigated- in this case, it is a sample of sponge tissue which will contain numerous bacteria, producing bioactive compounds (Wagner-Dobler et al., 2002). The sponge tissue is collected by scuba-diving in the region where the marine sponges are located. The phylogenetic affiliation of the sponge-associated bacteria is assessed using16S rDNA analysis which is mainly based on the selective amplification of 16S rRNA gene sequences, of taxon-specific lengths, by the application of primers of conserved regions of the 16S rDNA in combination with PCR and gel electrophoresis (Schweder et al., 2005). Due to its highly conserved and variable sequence regions, the 16S rDNA sequence is used as a phylogenetic marker (Schweder et al., 2005). This method of direct isolation of DNA from the environment and cloning and sequencing of 16S rRNA genes enables identification and taxonomical affiliation of bacterial species in different natural marine habitats without the cultivation of the microbial cells.

Production of secondary metabolites is usually a strain-specific trait. Thus, typing of the isolated bacteria with a high resolution is necessary to assess the genetic diversity of the strains within a given phylogenetic group (Wagner-Dobler et al., 2002). This high resolution is obtained by using genomic fingerprint methods- in this case a RAPD (Random Amplified Polymorphic DNA) technique with arbitrary primers is used (Wagner-Dobler et al., 2002).

Additionally, phylogenetic data on microbial community composition in sponges can indicate possible nutritional requirements and physiological niches of many microbes based on information already available for known phylogenetic relatives (Webster et al., 2001). This may assist in the experimental manipulation of culture conditions to provide the correct growth environment for targeted bacteria (Webster et al., 2001).

One of the limitations associated with the construction of 16S rDNA clone libraries from total environmental DNA is that it requires the use of PCR, which precludes quantitative estimates of abundance for each organism (Webster et al., 2001). This can be overcome to some degree by the use of fluorescence in situ hybridization (FISH) probing. FISH with rRNA specific probes allows phylogenetic identification of bacteria in mixed assemblages and enables the cells to be visualized and semi-quantified (Webster et al., 2001).

The crude extracts are investigated to evaluate anti-bacterial, anti-fungal, phytotoxic or cytotoxic activity. Individual bacterial colonies are obtained by serially diluting the sample and spread-plating appropriate dilutions on agar plates containing a variety of marine media (Wagner-Dobler et al., 2002). Brine shrimp toxicity has a strong correlation with cytotoxicity and is therefore a good indicator for potential anti-cancer activity (Wagner-Dobler et al., 2002). Secondary metabolite production can only be assigned to the bacteria when synthesis has been demonstrated in cultures isolated from the host species (Webster et al., 2001).

The plates are then incubated at room temperature for up to 4 weeks. The growth of eukaryotes and protozoan grazing is prevented by the addition of the antibiotic cycloheximide (Wagner-Dobler et al., 2002). The isolated bacteria are then compared to the total community structure of the sample determined by the small subunit rDNA approach. Novel bacterial status is assigned to isolates after comparison of colony morphotype and microscopic appearance of gram-stained preparations with previously obtained isolates (Webster et al., 2001).

The strongly positive hits obtained using the serial dilution and agar diffusion tests are then screened further using tumour cell-line based screening (‘in-vitro’ testing) (Wagner-Dobler et al., 2002). In the current National Cancer Institute (NCI) anti-cancer screen, each extract is tested against 60 human tumour cell lines derived from several cancer types (Wagner-Dobler et al., 2002). The most active extracts are then selected for further testing for the following criteria: (i.) potency, (ii.) cell-type specificity, (iii.) unique structure and (iv.) unique mechanism of action (Wagner-Dobler et al., 2002). To obtain information on the mechanism of action, the most active extracts are subjected to cell-cycle analysis. Continuously dividing tumour cells go from one mitosis (M) to the next, passing through the G1-, S- and G2-phases (Wagner-Dobler et al., 2002). Potential anti-cancer compounds will alter the cell-cycle in a specific manner, as is shown below in Figure 1. Hence, cell-cycle analysis can be used as a first indicator to identify the mechanism of action of the new compounds produced by the bacteria (Wagner-Dobler et al., 2002).

Figure 1. Cell-cycle analysis showing the M-, G1-, S- and G2-phases
(Details not posted) Source: Advances in Biochemical Engineering/Biotechnology, Vol. 74

The results from this screening enable the selection of strains whose bioactive compounds can now be isolated and their structure determined. Since even modern methods for structure determination and evaluation require at least 10mg of every compound, a scale-up fermentation is necessary (Wagner-Dobler et al., 2002). Depending on the strain of bacteria, the fermentation conditions can be optimized to achieve maximum yield of metabolite and to increase the genetic stability of the bacteria.

Typically, small-scale 100ml shake flask experiments are initially used, after which the cultivation experiments can then be transferred to glass or stainless steel bio-reactors for cultivation on a large-scale (Wagner-Dobler et al., 2002). The bioreactor design, aeration and agitation are chosen accordingly, depending on the individual strain requirement. Depending on the strain, the cultivation time is generally in the range 24-72 hours. For downstream processing, cells and supernatant are separated by centrifugation and extracted separately using organic solvents such as chloroform/methanol or ethyl acetate (Wagner-Dobler et al., 2002). These experiments provide the material for structural elucidation. In addition, they provide significant quantities of the bioactive compounds in order to carry out the in-vivo anti-tumour tests as well as the pre-clinical pharmacokinetic and toxicological studies before proceeding to the Phase I-III clinical trials.

The isolation and structural determination of natural products is a time consuming and expensive process, hence, it is very important to recognize and exclude known compounds at the earliest possible stage by a process which is called dereplication (Wagner-Dobler et al., 2002) . For this, easily accessible properties of metabolites are compared with literature data. There are databases, such as the Dictionary of Natural Products, where substructures, NMR or UV data and a variety of other molecular description can be searched using computers. Also widely used is the comparison of UV or MS data and HPLC retention times with appropriate reference data collections (Wagner-Dobler et al., 2002).

The most common methods for structural elucidation is mass spectrometry, using electro spray ionization techniques or MALDI-TOF, and NMR spectrometry which are often combined with chromatographic methods (Schweder et al., 2005). The hyphenated techniques HPLC-NMR and HPLC-MS, which need only microgram amounts of compound and have a high resolution, are shown to be powerful tools in combination with databases (Wagner-Dobler et al., 2002).

The in-vivo anti-tumour tests are carried out by testing the bioactive compounds in mice bearing the tumour cell line which was shown to be most sensitive in the in-vitro screen (Wagner-Dobler et al., 2002). Compounds that show significant tumour growth inhibition are then selected for further in-vivo evaluation against more advanced-stage tumours.
Next, the pre-clinical pharmacokinetic studies (absorption, bioavailability, distribution and excretion) and pre-clinical toxicology studies are carried out. The vast majority of anti-cancer drugs are cytotoxic compounds which have significant side-effects and a very small therapeutic index. The objective of these studies is to find a safe initial dose for Phase I clinical studies and to define the qualitative and quantitative organ toxicities (Wagner-Dobler et al., 2002).

Once this data has been established, the potential anti-cancer drug needs to be extensively reviewed by a range of regulatory authorities and committees to determine whether the drug is safe for carrying out studies on humans. Once it has been approved, it is possible to start the Phase I-III clinical trials. If the drug proves to be safe and effective in the clinical studies and once full regulatory approval is granted then the drug can then be scaled up in order to be sold in the market3. It is critical to protect the intellectual property surrounding the drug through the acquisition of various patents that will prevent others from making, using or selling what is described in the patent.

The productivity of the past decade in terms of the discovery of new clinical anti-cancer leads from diverse marine bacteria should translate into a number of new treatments for cancer in the decades to come. Exploitation of the potential of these marine microorganisms as producers of bioactive metabolites, with a wide range of potential pharmacological activities, is only just beginning. With the recent advances in both molecular biology and marine biotechnology, it is indeed very promising to see that the marine microbial environment is likely to continue to be prolific source of novel natural bioactive compounds for many years to come. In the future, further innovations in media development (chemical engineering), bioreactor design (bioprocess engineering) and transgenic production (molecular engineering), coupled with efficient downstream processing and product recovery (Pomponi, 1999), will continue to further enhance both the discovery and bulk production of these novel marine bioactive compounds.

References

Garcia Camacho, F., Chileh, T., Ceron Garcia, M.C., Sanchez Miron, A., Belarbi, E.H., Chisti, Y. & Molina Grima, E. (2005). A bioreaction-diffusion model for growth of marine sponge explants in bioreactors. Applied Microbiology and Biotechnology (details missing)

National Cancer Institute: www.cancer.gov

Pomponi, S.A. (1999). The Potential for the Marine Biotechnology Industry. Trends and Future Challenges for U.S. National Ocean and Coastal Policy: Workshop Materials, pp.101-104. Retrieved from website:
http://oceanservice.noaa.gov/websites/retiredsites/natdia_pdf/17pomponi.pdf

Schweder, T., Lindequist, U. & Lalk, M. (2005). Screening for New Metabolites from Marine Microorganisms. Advances in Biochemical Engineering/Biotechnology, Vol. 96, pp.1-48

Simmons, T.L., Andrianasolo, E., McPhail,K., Flatt, P. & Gerwick, W.H. (2005). Marine natural products as anticancer drugs. Molecular Cancer Therapeutics 2005, Vol.4, No.2, pp.333-342

Virax : www.virax.com.au

Wagner-Dobler, I., Beil, W., Lang, S., Meiners, M. & Laatsch, H. (2002). Integrated Approach to Explore the Potential of Marine Microorganisms for the Production of Bioactive Metabolites. Advances in Biochemical Engineering/Biotechnology, Vol. 74, pp.208-238

Webster, N.S., Wilson, K.Y., Blackall, L.L., Hill, R.T. (2001). Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile. Applied and Environmental Microbiology, Vol.67, No.1, pp.434-444

World Health Organisation: Ten Statistical Highlights in Global Public Health Retrieved from: http://www.who.int/whosis/whostat2006_10highlights.pdf

All you need to now about scale-up and scale down, overflow metabolism and glucose feeding when using Escherichia coli as a process tool.

2006-09-05T07:47:00.000+10:00

Figure 1. Experimental set-up for scale-down simulation studies. (a) Large-scale STR. Va is the addition zone where the most extreme concentration gradients are known to exist and Vb is the bulk region that can be considered to be well mixed. (b) Scale-down simulation equipment. Here Va is represented by a 0.544-L PFR and Vb is represented by a 4-L STR.

Bioprocess Engineering Considerations
The salient feature of the fed-batch fermentation process is the continuous feed of a growth-limiting substrate, usually the carbon source, characterized by an ever-increasing level of energy limitation and an ever-decreasing specific growth rate. This feeding protocol avoids problems associated with catabolic regulation, oxygen limitation, and heat generation that can occur during unlimited batch processes (Minihane and Brown, 1986). Importantly, the build-up of toxic concentrations of metabolic byproducts via so-called “overflow” metabolic routes can also be avoided. Overflow metabolism has been reported for Saccharomyces sp. (George et al., 1993) and Escherichia coli.

For E. coli, an accumulation of an inhibitory concentration of acetic acid occurs via the redirection of acetyl-coenzyme A (CoA) from the Krebs cycle, during fast aerobic growth when a rapidly metabolizable carbon source, such as glucose, is available in excess (Andersson, 1996). In batch fermentation, overflow metabolism can be avoided by the use of a slowly metabolizable carbon source such as glycerol (Elsworth et al., 1968), but the preferred method used by industry (BioGaia AB, Sweden and Pharmacia, Sweden) for increasing the volumetric productivity of such bacterial products as amino acids (Forberg and Haggstrom, 1987) and high-value recombinant proteins (Riesenberg and Schulz, 1991) is the fed-batch strategy (see Lee [1996] for a comprehensive review).

However, problems still arise in the scale-up of such processes in that the production scale often does not give the same performance as the bench scale. There are many possible reasons for this difference and, in this study, flow cytometry is used to highlight a difference that has not been shown previously.

Scale-up is usually the final step in a research and development program leading to the large-scale synthesis of microbial products by fermentation (Einsele, 1978). It is known that chemical gradients exist in large-scale bioreactors (Xu, 1999) where additions of a concentrated, often viscous, substrate at a single point indicate that mixing times are high (greater than ~ 50 seconds even at the 20-m3 scale [Vrabel et al., 1999]). In laboratory-scale bioreactors, where much development work is done, mixing times are low (less than ~ 5 seconds) and such gradients essentially do not exist (Nienow, 1998). The composition of the cells’ microenvironment is a product of both fluid dynamics and the cells’ physiological response to it. Cells circulating around a large-scale bioreactor will undergo rapidly changing microenvironments.

Knowledge of how a cell reacts to such changes is essential if mathematical models are to be derived that can accurately predict process times and product yields on scale-up.

Large-scale studies are difficult and expensive to carry out. In addition, when the results differ from those of bench scale, they are often difficult to interpret. Therefore, equipment and techniques that allow large-scale studies to be simulated at the small scale have become important research tools (see Larsson [1990] for a comprehensive review).

For simulating the phenomenon of poor mixing, one technique used is to divide the large-scale reactor into two compartments (George et al., 1993). The conditions established in each compartment depend on the type of poor mixing situation at the large scale that is to be simulated. For fed-batch fermentations, these two compartments can represent an addition zone where the most severe nutrient concentration gradients exist and the bulk region where the system can be considered to be essentially well mixed. The latter is therefore at a much lower concentration with respect to the nutrient feed. The relative size of the addition zone in the simulation should be of the order of the size of the region in which higher concentrations of substrate exist at the large scale.

The size of this region may be estimated by intuition, flow visualization (again on the small scale), or computational fluid dynamics. Typically, in the small-scale simulation, the addition zone is represented by a plug-flow reactor (PFR), and the bulk region by a stirred-tank reactor (STR) (Larsson, 1990). The volumetric ratio between these two reactors is equal to the estimated ratio of the addition zone to the bulk region in the large-scale reactor (Fig. 1).

Studies Related to the Scale-Up of High-Cell-Density E. coli Fed-Batch Fermentations Using Multiparameter Flow Cytometry: Effect of a Changing
Microenvironment with Respect to Glucose and Dissolved Oxygen Concentration
Christopher J. Hewitt, Gerhard Nebe-Von Caron, Britta Axelsson, Caroline M. McFarlane, Alvin W. Nienow
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 70, NO. 4, NOVEMBER 20, 2000, page 381

Questions to answer:

A. What is overflow metabolism, and what problems does it cause in attempts to grow E. coli to high cell densities in a manufacturing reactor?
B. What is a STR?
C. What is a PFR?
D. What is an addition zone?

Twisted News item for students of bacterial cells: How transport proteins work.

2006-09-02T09:33:00.000+10:00

Schematic of Staphylococcus membrane transport protein in the ATP binding conformation.

A structure an ABC family transport protein that exports dyes and drugs from Staphylococcus aureus has just been reported in the journal Nature. The key discovery is that the two protein dimers in the molecule are intimately twisted around each other. The structure provides many important clues on how the transport mechanism is driven by ATP hyrdrolysis.

Structure of a bacterial multidrug ABC transporter

Roger J. P. Dawson and Kaspar P. Locher
Abstract

Multidrug transporters of the ABC family facilitate the export of diverse cytotoxic drugs across cell membranes. This is clinically relevant, as tumour cells may become resistant to agents used in chemotherapy. To understand the molecular basis of this process, we have determined the 3.0 Å crystal structure of a bacterial ABC transporter (Sav1866) from Staphylococcus aureus. The homodimeric protein consists of 12 transmembrane helices in an arrangement that is consistent with cross-linking studies and electron microscopic imaging of the human multidrug resistance protein MDR1, but critically different from that reported for the bacterial lipid flippase MsbA. The observed, outward-facing conformation reflects the ATP-bound state, with the two nucleotide-binding domains in close contact and the two transmembrane domains forming a central cavity—presumably the drug translocation pathway—that is shielded from the inner leaflet of the lipid bilayer and from the cytoplasm, but exposed to the outer leaflet and the extracellular space.

Nature advance online publication 30 August 2006 | doi:10.1038/nature05155; Received 9 May 2006; Accepted 11 August 2006; Published online 30 August 2006

Entry of dyes can be sensitive to the state of the membrane

2006-08-29T21:43:00.000+10:00

Schematic diagram of a living (bacterial) cell with its polarised (charged) membrane. The cytoplamic membrane is represented in red. PI is a cationic dye (propidium iodide) that binds to DNA, and it is not lipid soluble. BOX is lipophilic oxanol dye, negatively ionised, and about 500 MW. Both PI and BOX are fluorescent.

Question: What are the factors affecting staining of the cell by these dyes?

Assays for compounds that make existing antibiotics effective against antibiotic resistant bacteria.

2006-07-28T16:50:00.000+10:00

The figure above shows structures of the beta-lactam semi-synthetic antibiotic amoxycillin (top) and the different compound clavulanate (lower). These two compounds are used together as a powerful synergistic antibacterial combination therapy in current medical treatments. Although amoxycillin is a semi-synthetic penicillin, clavulanate is not even an antibiotic, but an inhibitor of the bacterial enzyme beta-lactamase, which is enzyme which makes many bacteria resistant to penicillins and the related cephalosporins. Clavulanate is thus augments or extends the effectiveness of beta-lactam antibiotics, and it is produced naturally by a soil Streptomycete. Note that amoxycillin has a side chain (with a phenol group) and the beta-lactam bicyclic ring system which includes N and S atoms. (Image from Sandoz.Com.)

The useful beta lactamase inhibitor clavulanate was discovered around 1970 using a clever but simple assay strategy.

It required a simple way of measuring inhibition of beta lactamase enzyme activity.

Thus the biological activity being sought was inhibition of enzyme catalysis.

For this another beta-lactam antibiotic, Nitrocefin, was most useful. This antibiotic is pretty useless in treating infection as it may cause cancer and many bacteria are resistant to it. But it is superb for detecting chemicals that inhibit beta-lactamases, such as clavulanate.

This is because it changes from yellow to red colour when exposed to beta-lactamase.

The diagram below illustrated how on overlay of soft agar containing a freshly made mixture of Nitrocefin plus beta-lactamase enzyme can reveal beta-lactamase inhibitors diffusing from a bacterial colony. the diagram shows schematically the cross section of petric dish culture on which an unknown type of soil bacteria have developed as a colony.

The most practically useful part of this strategy is that it can be cheaply and conveniently adapted to screen hundreds of thousands of novel bacterial isolates. It does this without demanding much expensive labour.

It is thus a cheap high through-put assay or screen.

This approach enabled a drug company to find soil microbes that produce then unknown biological activities of beta-lactamase inhibition in the 1970s. These activities included clavulanate. Late work by chemists led to other antibiotic extenders such as sulbactam and tazobactam being synthesised.

Further reading:
Link on Nitrocefin colour changes

More ambitious microtitre dish assays based on this idea.

Using microbes to mine copper.

2006-07-27T22:47:00.000+10:00

The Escondida copper mine in Chile- the worlds largest copper mine. Photo BHP Billiton Group from here.

Thomas Barton has recently written a great book - The Australian Miracle: An Innovative Nation Revisited (Picador 2006) - which has many interesting things to say about commercial and technological innovation in Australia.

It has an interesting discussion of the use of bacteria to extract copper in the following passage (pages 223-4):

For many years, people in Australia have talked about the need to shift from an economy based on the production of physical goods to an economy based on the production of knowledge. This idea is so commonplace that it is difficult to imagine anyone interested in Australian technological and economic competitiveness not taking it up.

What does it mean, though? When most people hear this recommendation, they usually imagine it means that Australia needs to shift out of mining, agriculture and other traditional sources of wealth, and into design, computer programming, biotechnology and the arts.

In actuality, though, what the shift might imply is quite the opposite.

One of Australia's biggest biotechnology companies is now BHP Billiton, the mining giant. Little known to the outside world, the company has a biotech laboratory with around eighty employees whose main job is to develop the use of bacteria for minerals leaching. The group specialises in identifying bugs in hot springs and other extreme environments, and then breeds them up so that they become efficient at breaking down intractable ores. Furthermore, unlike a good number of other biotech companies, BHP Billiton is actually putting its products to work.

As part of a one-billion-dollar exercise at Escondida in Chile - the world's largest copper mine - the company is currently bioleaching a heap of ore that stretches across nine square kilometres. Biomining, for the first time in human history, is a reality.

How to find microbes that make Gold.

2006-07-26T11:50:00.000+10:00

The production of microbial products is a huge global industry with many billions of dollars of products sold world wide that are originally derived from microbes. There is much scope to add new bioproducts to that list.

It requires innovative thinking, a good bioproduct discovery strategy, and alertness to opportunities that you might come across while you are reading about biology, science, and business.

Remember, during your searches for bioproduct candidates, the success of French microbiologist Louis Pasteur, who is famous for observing that fortune favours the prepared mind.

Some general approaches and ideas for discovery of novel bioactive compounds from microbes are:

Tap into microbial biodiversity
Search for novel organisms
Be alert to microbial culture and enrichment concepts. These include using indicator agar plates, for example containing enzyme substrates that change colour during a reaction.
Search in novel or extreme microbial environments.
Find and grow previously unculturable organisms.
Find compounds through gene cloning (metagenomics) from organisms that cannot be grown. see eg How to Find New Antibiotics, Jo Handelsman, The Scientist Volume 19 | Issue 19 | Page 20 | Oct. 10, 2005
Exploit suitable assays or screens to find novel biological activities. eg Coloured compounds produced by microbial colonies are easier to find than colourless compounds. Similarly substrates that produce colour (chromogenic compounds) or fluorescence (fluorogenic compounds) by a chemical reaction are extremly useful if a relevant or practically useful way of exploiting a reaction can be devised.
It's possible to find a novel microbe to make or provide a useful biological activity previously unknown in microbes using a well chosen assay.
Exploit specific or sensitive assay approaches.
Possibly identify a novel "drug" target to help develop an assay.
Use a high through-put assay or screening approach ( eg screens of millions of colonies on plates, screens in microtitre dishes).
Screen pools of different compounds or microbes in the one assay to find which pool has the posive microbe.
Use of chemical analysis techniques such as Mass spectroscopy, in innovative ways to allow high through-put detection on novel molecules
Encapsulate individual cells or samples in polmeric microscopic beads to facilitate screening

Pundit is going to add useful hyper links to this list to expand this information.

But why not help the Pundit and put your questions and comments about them below so we can discover good ideas by dialog.

For example, answer this question I'm putting forward to start you thinking about useful approaches:

How are sensitive and specific assays helpful to the bioproduct discovery process, and can you think of particular opportunities they open up?

There is plenty of helpful general reading that can help you prepare for the task of exploiting microbial novelty:

Pages 26- 32 of The textbook Microbial Biotechnology by A.N. Glazer and H. Nikado, Freeman, 1995 are a good short introduction to some previously discovered microbial bioproducts such as plastics and drugs.
Chapter 15. of Microbe, M. Schaechter et al ASM Press 2006 is very good background reading about bacterial diversity.
Several previous posts at this site deal with compounds, such as siderophores, that are produced by soil streptomycete bacteria (these posts are readily accessible via the index hyper-link in the side-bar on the right.)

Process improvement in the antibiotic industry.

2006-07-05T13:35:00.000+10:00

Year

Process improvement also occurs in the antibiotic industry. This example shows how microbiologists improve the process levels (concentrations) in production of an antibiotic over time. The slide is from an old talk by Arnold Demain of MIT.

The point it that many different experiments, such as genetic selections for improved microbe strains, and changes to growth media based on physiology experiments, allow process costs to be driven down over time.

Can you suggest examples of different biotechnology process improvements from previous published scientific work and document and explain how they were achieved?

There are so many different products to chose from, and so many different ways of reducing costs of production, so let me have your ideas in the comments. Let's work them up together for a series of Microbe Pundit Posts to share around.

For example how have ethanol costs been reduced?

Technological Learning Curves: Renewable Energy Redux.

2006-07-05T13:19:00.000+10:00

Renewable Energy Redux. Data showing learning curve demonstrating reduction in cost for Brazilian ethanol (blue) undercuts price of petroleum based liquid fuel (red) by 2005. From a talk by S. Coelho April 2006.

Welcome back to students of the Microbe. This semester, The Pundit is going to concentrate on Microbial Biotechnology.

He's especially emphasising technological innovation. It's this innovation that has delivered most economic growth this last century, and that's what solves problems in this world - prosperity and resources that better management other problems in societies feasible.

The message is clear: intellectual assets and imagination saves lives, rain-forest, and pristine wilderness.

To start the innovation ball rolling, The Pundit is posting some examples of how innovation over time allows biotechnology products to be made more cheaply and efficiently.

This is the slow burn of incremental improvement year after year, only a few percent per year, but eliminating much poverty over decades.

A super example is ethanol fermentation used in Brazil to make liquid fuels. That is, sugar cane based biofuel.

This started out being relatively energy, land, and cost inefficient in the early 1980s, and bioethanol fuel nearly collapsed as an industry in Brazil when oil prices fell because it was very costly. It dragged subsidies from other areas of agriculture where investment was sorely needed. Not so today.

In this case many technological innovations have allowed ethanol from sugar cane to become energy efficient (with fuel energy output being 3-4 times the fuel energy input EOIR) .

Brazilian ethanol is now cost competitive with petroleum liquid fuels without any subsidy. These innovations, including biotechnology-based method changes in fermentation and strain improvement, are ongoing, and there is ample room for further efficiency improvement and more innovation.

A detailed economic documentation of this case is given by Goldemberg et al 2004.

Biomass and Bioenergy 26 (2004) 301 – 304
Short communication
Ethanol learning curve—the Brazilian experience
Jose Goldemberg, Suani Teixeir Coelhob, Plinio Mario Nastaric, Oswaldo Lucond

Abstract

Economic competitiveness is a very frequent argument against renewable energy (RE). This paper demonstrates, through the Brazilian experience with ethanol, that economies of scale and technological advances lead to increased competitiveness of this renewable alternative, reducing the gap with conventional fossil fuels.

Where the wild-things are, and where avermectin comes from.

2006-05-22T23:20:00.000+10:00

The previous post is actually very demanding reading if you read the whole thing. Dr Pundit recognises that biochemistry is an acquired taste, and not always a fatal attraction. And with the molecular biology and genomics of secondary metabolites in streptomyces, Even more so .

Geek territory?

Many of us would prefer to just read the paper's abstract.

Thus for those who want a kindler gentler commentary on the metabolic capabilities of Streptomyces, Fam has kindly provided the following. Thanks Fam.

An overview of secondary metabolites of
Streptomyces avermitilis

Fam W. N

Abstract

Streptomyces avermitilis is the producer of Avermectin, an anthelmintic macrolide which was isolated by Omura et al. Having the largest bacterial genome sequence, S.avermitilis has an interesting ability to produce a variety of secondary metabolites. Genome analysis has led to identification of total of 30 gene clusters involved in secondary metabolite biosynthesis in S.avermitilis, and more than half of them are located near at its chromosome ends. In this review, structure, general characteristics and practical applications of Avermectin will be addressed as well as analysis of these gene clusters in S.avermitilis which give insight into complex diversity of secondary metabolites and their physiological roles.

1. Introduction
Streptomyces is a genus of gram-positive Actinomycete bacteria. These filamentous bacteria are found mainly in soil and marine habitats (Lamb et al., 2003). The unique characteristic of streptomycete bacteria is that they are able to exhibit a complicated life cycle involving formation of a lawn of aerial hyphae on the colony surface that stands up into the air and differentiates into chains of spore (Kelemen and Buttner, 1998). This process is highly controled and requires specialized coordination of metabolism. Because of diversity of their secondary metabolic pathway, streptomycetes are being extensively studied for their biologically active products which are widely used in human and veterinary medicine, as antibiotics, antiparasitic agents, as well as other pharmaceutical activities (Ikeda et al., 2003).

Streptomyces avermilitis is one soil bacterium in this genus. Streptomyces avermilitis was first isolated by Omura et al. of the Kitasato Institute from the soil sample which collected in Ito city, Shizuoka Prefecture, Japan (Burg, 1979). It has a large genome (9.02 Mb) and its chromosome exists as linear which is similar in the cases of other bacteria of the same genus. Taxonomic studies revealed its morphology with sporophores forming spirals as side branches on aerial mycelia (Fig. 1). The spore surface was smooth by observation made under electron microscopy (Burg, 1979). Merck Sharp and Dohme discovered the effective activity of Avermectin in a broth of S.avermilitis by chance. Testing found that it was non-toxic towards the lab mice. It also showed potentially as an anti-helminthic agent, and the activity was ten-fold higher than any synthetic antihelminthic agent (Demain, 1983).

Fig. 1: (Left) Scanning electron micrograph of Streptomyces avermitilis, from http://www.nih.go.jp/saj/atlas/sample.html. (Right) Transmission electron micrograph showing its smooth surface (Burg, 1979) [not shown].

Avermectin is well-known as an excellent anthelminthic agent and it is highly against a broad spectrum of nematode and arthropod parasites. Due to its superior activity, avermectin market exploded during 1980s and reached U.S $1 billion at the end of 1990 (Hwang et al., 2003). Its semisynthetic derivative, Ivermectin is also widely used in veterinary and agricultural fields (Ikeda et al., 1999).

Genome sequence completion has revealed 30 gene cluster in Streptomyces avermitilis that are proposed to be involved in biosynthesis of secondary metabolites (Ikeda et al., 1999). This indicates that there could be a large number of metabolic pathways and hence a variety of potential bioactive compounds that could be as useful as Avermectin awaiting discovery.

2. Avermectin
Avermectins are a complex of macrocyclic lactone derivatives which in contrast to macrolide or polyene antibiotics, lacking significant antibacterial or antifungal activity (Burg, 1999).

2.1 Biosynthesis of Avermectin
Basically, Avermectin biosynthesis can be divided into 3 stages. The first stage is the formation of polyketide-derived initial aglycon. The avermectin polyketide synthase, PKS uses a range of acyl units (Ikeda et al., 1999). The starting acyl group for synthesis of Avermectin derived from valine to form a components and isoleucine to form b components (Lamb et al., 2003). The second stage involves the modification of initial aglycon to generate Avermectin aglycons. This is formed by extension of started unit with addtition of 7 acetate and 5 propionate units and involves a few modification steps which include ketoreduction, methylation and furan ring closure. The final stage involves O-glycosylation at C13 and C4’ to form Avermectins from Avermectin aglycons. This glycosylation step is performed by deoxythymidine diphosphate (dTDP)-oleandrose.

Fig. 2: Chemical structure of Avermectin
(figure from http://www.plantaanalytica.com/avermectins.htm)

2.2 Mode of action of Avermectin
Avermectin and its chemically derived compound, Ivermectin can affect a variety of ligand- and voltage-gated chloride channels (Bloomquist, 1993). It has high affinity to bind glutamate-gated chloride ion channels which is found in peripheral nervous system of invertebrates, establishing its selective activity against human parasites. Binding of Avermectin in this ion channel block electrical activity in vertebrate and invertebrate nerve and muscle cells by increasing the permeability of cell membrane to chloride ions. A consequence of this is the hyperpolarization followed by paralysis and death of the parasite.(Santoro et al., 2003) In normal condition, Avermectin and Ivermectin does not cross mammalian blood-brain barrer and human are spared from serious central nervous system effects that brought by this drug.

2.3 Practical application of Avermectin and Ivermectin
Initially, Avermectin was used as insecticide to protect the crops. It was later used to treat infection in livestock and domestic animals caused by nematodes and arthropods (Santoro et al., 2003). Most importantly, Avermectin has been made as promising drug in human onchocerciasis. It has conferred some protection to a huge population in sub-Saharan Africa in which the disease called river blindness was prevalent at a certain point of time (Hopwood, 2003).

It has become evident that Ivermectin is effective in treating other filariases such as loiasis, bancroftian filariasis and other intestinal nematodes. Inoculation of this drug successfully inhibits the maturation of larvae at certain developmental stages of filarial species. This could be used to protect the domestic animals from diseases such as heartworm disease (Campbell et al., 1983). Some nematodes can enter hypobiosis. However, this does not deter the action of Ivermectin unlike most of other anthelmintic agents. Thus, it is considered strong weapon in the field of parastic dermatology, where it can act against its target at any stage of development (Pascal et al., 2003).

Ivermectin also emerges as an oral antiscabietic to treat scabies. It is shown to be as safe and effective as topical antiscabietics. Recent report revealed that all groups of population tested show good response to Ivermectin in the treatment scabies. These populations include immunocompromised, immunocompetent as well as in other high-risk populations such as those with Down’s syndrome (Santoro et al., 2003).

3. Review of potential of Streptomyces avermitilis as producer of a wide variety of secondary metabolites .
Soil contains highly diversity of bacterial communities. It is a complex environment in which bacteria will encounter chemical, physical as well as biological stress. To combat the stress and compete with other microorganisms, Streptomyces avermilitis which is non-motile possess a large number of genes encoding nutritional enzymes, transport proteins, cell regulators and other useful secondary metabolites (Challis and Hopwood, 2003).

Genome analysis revealed Streptomyces avermitilis has at least 30 kind of secondary metabolite gene clusters in chromosome (Ikeda et al., 2003). Twenty five clusters that have studied earlier involved in biosynthesis of compounds such as carotenoid, melanin, polyketide, siderophore and peptides (Ikeda et al., 1999). Recent studies identified 5 more gene clusters which involved in biosynthesis of terpene and polyketide compounds (Ikeda et al., 2003). The total length of these gene clusters was estimated to be 594 kb, which indicate that about 6.6% of S.avermilitis genome is occupied by genes involved in biosynthesis of secondary metabolites. Most of these gene clusters were observed to be located on the ends of the chromosome and contained many transposable elements. These transposases might be responsible for transferring some secondary metabolite genes into S.avermitilis via horizontal transfer and contribute to high production of secondary metabolites (Omura et al., 2001). Genetic studies suggest that its genome might have undergone evolution by acquisition of novel gene functions in order to survive in extremely variable soil environment with rapid changes in its physical conditions as well as to face tough competitors in obtaining available nutrients. (Ikeda et al., 2003).

Gene clusters analysis discovered that S.avermitilis has the ability to produce two anti-fungal compounds which are oligomycin and polyene macrolide. These two compounds are believed to potentially act synergistically against fungal competitor in its natural environment. (Challis and Hopwood, 2003). In addition, genes encode for two extracellular enzymes with glucanase and chitinase activities are found highly expressed in S.avermitilis. They also play important role against fungi. (Wu et al., 2005).

Cytochrome P450, CYP genes encode a family of heme-thiolate-containing enzymes. The CYP genes are normally located in macrolide antibiotic biosynthetic gene clusters. There are 33 cytochromes P450 (CYPs) found in newly completed S.avermitilis genome (Chater, 1989). It is predicted that 11 of this CYPs might be involved in biosynthesis of secondary metabolism and remaining CYPs might play a role in protecting S.avermitilis against toxic compound in soil environment (Lamb et al., 2003).

Siderophores are involved in transportation of iron in bacteria. Recent genome analysis also found a gene cluster in S.avermitilis which is presumably involved in biosynthesis of desferrioxamine derivatives (Ikeda et al., 2003). This can be used to treat acute iron poisoning especially in small children and proven to be an effective treatment for Ataxia-telangiectasia which is a genetic neurological disorder (Olivieri,1990).

The emergence of antibiotic resistant and new infectious diseases has brought a great demand in discovering novel antibiotics. Genome mining of S.avermitilis provides valuable information of this strain in biosynthesis of wide variety of secreted molecules/secondary metabolites which could be as useful as Avermectin and contributing to novel drug discovery. In addition, various bioinformatics resources such as BLAST (http://www.ncbi.nlm.nih.gov/blast/), ScanProsite ((http://ca.expasy.org/prosite/), enable us to do genome comparative analysis of streptomyces strains to gain insight the evolution of streptomyces family and conserved useful genes that contribute to novel antibiotics production which might advantageous not only to streptomyces itself but as well as in biomedical, pharmaceutical and biotechnological industry.

Acknowledgements
I thank Dr. M. Pundit for his valuable advice and guidance on different aspects of doing this proposal.

References
1. Bloomquist, J. R. 1993. Toxicology, mode of action, and target site-mediated resistance to insecticides acting on chloride channels. Mini Review, Comp. Biochem. Physiol 106: 301-314.

2. Burg, R.W. 1979. Avermectins, new family of potent antihelminthic agents: producing organism and fermentation. Antimicrob. Agents Chemother. 15:361-367.

3. Campbell, W.C., M.H. Fisher, E.O. Staphley, G. Albers-Schonberg, and T.A. Jacob. 1983. Ivermectin: A potent new antiparasitic agent. Sci. 221:823-828.

4. Challis, G. L., and D. A. Hopwood. 2003. Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc. Natl. Acad. Sci. USA 100(Suppl. 2):14555-14561.

5. Chater, K.F. 1989. Multilevel regulation of Streptomyces differentiation. Trends Genet. 5:372–377.

6. Demain, A.L. 1983. New applications of microbial products. Sci. 219(4585):709-714.

7. Hopwood, D.A. 2003. The Streptomyces genome- be prepared! Nature Biotech. 21:505-506.

8. Hwang, Y.S., Kim, E.S., Biro S. and Choi C.Y. 2003. Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various Streptomyces avermitilis Strains. Appl. Envir. Microbiol. 69(2): 1263 - 1269.

9. Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Omura S. 2003. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol. 21;526-531.

10. Ikeda, H., T. Nonomiya, M. Usami, T. Ohta, and S. Omura. 1999. Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis. Proc. Natl. Acad. Sci. USA 96:9509-9514

11. Kelemen, G.H., and Buttner, M.J. 1998. Initiation of aerial mycelium formation in Streptomyces. Curr. Opin. Microbiol. 1: 656–662.

12. Lamb, David C, Ikeda, Haruo, Nelson, David R, Ishikawa, Jun, Skaug, Tove, Jackson, Colin, Omura, Satoshi, Waterman, Michael R, Kelly, Steven L. 2003. Cytochrome p450 complement (CYPome) of the avermectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coelicolor A3(2). Biochem Biophys Res Commun, 307(3):610-9.

13. Lamb, David C., Ikeda, Haruo, Nelson, David R., Ishikawa, Jun, Skaug, Tove, Jackson, Colin, Omura, Satoshi, Waterman, Michael R., Kelly, Steven L. 2003. Cytochrome p450 complement (CYPome) of the avermectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coelicolor A3(2). Biochem Biophys Res Commu., 307(3):610-9.

14. Omura, S., Ikeda, H., Ishikawa, J., Hanamoto, A., Takahashi, C., Shinose, M., Takahashi, Y., Horikawa, H., Nakazawa, H., Osonoe, T., Kikuchi, H., Shiba, T., Sakaki, Y., and Hattori, M. 2001. Genome sequence of an industrial microorganism Streptomyces avermilitis: Deducing the ability of producing secondary metabolites. Proc. Natl. Acad. Sci. U. S. A. 98:12215–12220.

15. Pascal, D.G., Olivier C., and Caumes E. J. 2003. Ivermectin in dermatology. J. of Drugs in Derma. 2:327-313.

16. Santoro, A. F., M. A. Rezac, and J. B. Lee. 2003. Current Trend in Ivermectin Usage for Scabies. J. of Drugs in Derma. 2:397-401.

17. Wu, G., Culley, D. E. and Zhang, W. 2005. Predicted highly expressed genes in the genomes of Streptomyces coelicolor and Streptomyces avermitilis and the implications for their metabolism. Microbio. 15:2175–2187.

18. Olivieri NF, Koren G, Hermann C, Bentur Y, Chung D, Klein J, St Louis P, Freedman MH, McClelland RA, Templeton, DM. 1990. Comparison of oral iron chelator L1 and desferrioxamine in iron-loaded patients. Lancet 336: 8726

Synergistic Substances and Several Siderophores Synthesised by Sessile Soil Saphrophytes.

2006-05-22T20:53:00.000+10:00

Here is an link to a published essay about synergy of antibiotics, as promised by Dr Pundit.

Students may wish to stop at the abstract, as the full essay was written for professional scientists who are investigating streptomycetes in research. The two key words though, to remember, are synergy and contingency. The discussion in the essay about how ability to make several siderophores deals with contingencies faced by streptomyces in the soil is really relevant to classroom discussions.

But before moving on to that, when Pundit read through this fine essay, it reminded him of important points about streptomyces life-cycle that were not explicitly emphasised in the classes, but should be added to your notes.

Note Bene

Aerial hyphae derive their nutrients from dead and lysing vegetative hyphae.
In other words, vegetative hyphae sacrifice themselves for their spores.
This involves substantial reorganisation of cell activities during the transition from vegetative to aerial hyphal growth.

Before reading on, it would be sensible to be clear in your mind what "contingency" means. Helpfully, Challis and Hopwood provide a definition:

Note that our use of "contingency" in this article relates to multiple metabolites acting on the same biological target to provide an organism with a contingency plan to combat unforeseeable biological competition. Moxon and coworkers have used contingency to describe hypermutable loci coding for variable surface proteins in Haemophilus influenzae and Nesseria meningitidis. The two uses of the word should not be confused

Roughly speaking then, contingency means "just in case".

If you read further, as a bonus you also discover the Fatal Attraction hypothesis of soil ecology. But to see the nitty-gritty of Fatal Attraction you have to read the whole thing.

Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species.
Challis GL, Hopwood DA.

In this article we briefly review theories about the ecological roles of microbial secondary metabolites and discuss the prevalence of multiple secondary metabolite production by strains of Streptomyces, highlighting results from analysis of the recently sequenced Streptomyces coelicolor and Streptomyces avermitilis genomes.

We address this question: Why is multiple secondary metabolite production in Streptomyces species so commonplace? We argue that synergy or contingency in the action of individual metabolites against biological competitors may, in some cases, be a powerful driving force for the evolution of multiple secondary metabolite production. This argument is illustrated with examples of the coproduction of synergistically acting antibiotics and contingently acting siderophores: two well-known classes of secondary metabolite. We focus, in particular, on the coproduction of beta-lactam antibiotics and beta-lactamase inhibitors, the coproduction of type A and type B streptogramins, and the coregulated production and independent uptake of structurally distinct siderophores by species of Streptomyces.

Possible mechanisms for the evolution of multiple synergistic and contingent metabolite production in Streptomyces species are discussed. It is concluded that the production by Streptomyces species of two or more secondary metabolites that act synergistically or contingently against biological competitors may be far more common than has previously been recognized, and that synergy and contingency may be common driving forces for the evolution of multiple secondary metabolite production by these sessile saprophytes.

Proc Natl Acad Sci U S A. 2003 Nov 25;100 Suppl 2:14555-61. Epub 2003 Sep 11.

Why do Streptomycetes have ability to produce so many specialised molecules?

2006-05-22T14:32:00.000+10:00

Various streptomycete soil bacteria have ability to produce a range of molecules called secondary metabolites.

See if you can relate a specialised molecule, or streptomycete general metabolic versatility, to one of the following aspects of streptomycete biology.

Habitat
Life-cycle
Cell fusion between different vegetative hyphae
Presence of conjugative plasmids
Nutrition and nutrient capture
Chromosomal structure
QS
Membrane physiology
Stress - UV exposure, dehydration
Aerial hyphae
Spore survival

Good luck.

Hopanoids- not well known, but important molecules for the microbial cognoscenti

2006-05-22T14:25:00.000+10:00

A typical hopanoid molecule (Wikipedia).

Hopanoids have a chemical structure somewhat like cholesterol. They are produced by many different bacteria , have an interesting history and perform a range of functions in membranes.

Berry 1993

Hopanoids occur in cell membranes in a wide range of microorganisms, where these lipids contribute to membrane stability and alter phase transition properties. Hopanoids are particularly important for microbial survival in extreme thermal environments and have been identified as a major component of oil shales.

Archean molecular fossils and the early rise of eukaryotes.
Brocks JJ, Logan GA, Buick R, Summons RE.
Science. 1999 Aug 13;285(5430):1033-6.
Comment on: Science. 1999 Aug 13;285(5430):1025-6.
School of Geosciences, University of Sydney, Sydney, NSW 2006,

Australia

.
jochen.brocks@agso.gov.au

Molecular fossils of biological lipids are preserved in 2700-million-year-old shales from the Pilbara Craton, Australia. Sequential extraction of adjacent samples shows that these hydrocarbon biomarkers are indigenous and syngenetic ton the Archean shales, greatly extending the known geological range of such molecules. The presence of abundant 2alpha-methylhopanes, which are characteristic of cyanobacteria, indicates that oxygenic photosynthesis evolved well before the atmosphere became oxidizing. The presence of steranes, particularly cholestane and its 28- to 30-carbon analogs, provides persuasive evidence for the existence of eukaryotes 500 million to 1 billion years before the extant fossil record indicates that the lineage arose.

Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2).
Poralla K, Muth G, Hartner T.
FEMS Microbiol Lett. 2000 Aug 1;189(1):93-5.
Microbiological Institute, Microbiology/Biotechnology, University of Tubingen,
Auf der Morgenstelle 28, D-72076, Tubingen, Germany. poralla@uni-tuebingen.de

Streptomyces coelicolor A3(2) contains a cluster of putative isoprenoid and hopanoid biosynthetic genes. The strain does not produce the pentacyclic hopanoids in liquid culture but produces them on solid medium when sporulating.

Mutants defective in the formation of aerial mycelium and spores (bld), with the exception of bldB, do not synthesize hopanoids, whereas mutants, which form aerial mycelium but no spores (whi), do. The membrane condensing hopanoids possibly may alleviate stress in aerial mycelium by diminishing water permeability across the membrane.

Hopanoid Lipids Compose the Frankia Vesicle Envelope, Presumptive Barrier of Oxygen Diffusion to Nitrogenase
AM Berry, OT Harriott, RA Moreau, SF Osman, DR Benson and AD Jones
Proceedings of the National Academy of Sciences, Vol 90, 6091-6094

Biological nitrogen fixation in aerobic organisms requires a mechanism for excluding oxygen from the site of nitrogenase activity. Oxygen exclusion in Frankia spp., members of an actinomycetal genus that forms nitrogen-fixing root-nodule symbioses in a wide range of woody Angiosperms, is accomplished within specialized structures termed vesicles, where nitrogen fixation is localized. The lipidic vesicle envelope is apparently a functional analogue of the cyanobacterial heterocyst envelope, forming an external gas-diffusion barrier around the nitrogen-fixing cells. We report here that purified vesicle envelopes consist primarily of two hopanoid lipids, rather than of glycolipids, as is the case in cyanobacteria. One envelope hopanoid, bacteriohopanetetrol phenylacetate monoester, is vesicle-specific. The Frankia vesicle envelope thus represents a layer specific to the locus of nitrogen fixation that is biosynthetically uniquely derived.

Signals - Red, Amber, and Green.

2006-05-19T14:43:00.000+10:00

A practice long assessment question.

Take about 30 minutes to give an answer, devoting about equal time to each part.

Specialised signaling compounds are very important in enabling bacterial cells to adapt and survive in different environments, and to coordinate cell responses.

Such bacterially made chemicals include compounds that can be involved in either inter-cellular communication outside the cell, or intra-cellular communication within the cell.

Questions:

Give examples of two structurally distinct intercellular (ie extra-cellular) signaling chemicals used by bacteria, and explain what you know about their functional roles and mechanisms of action.
Give examples of two structurally distinct intracellular signaling chemicals used by bacteria, and explain what you know about their functional roles and mechanisms of action.
Explain how a signaling pathway can activate expression of a set of unlinked genes, and give one specific example of a set of bacterial genes whose activity is triggered by an extra-cellular signal.

Quorum Sensing in the pathogenicity of Pseudomonas aeruginosa

2006-05-18T20:36:00.000+10:00

M. I-S Low

Abstract

Pseudomonas aeruginosa is a bacterium responsible for a wide range of infections. Quorum sensing (QS) is used in P. aeruginosa as a means of cell-to-cell communication and is particularly important in its pathogenicity. The QS system is composed of two AHL systems: las and rhl; and a non-AHL system: PQS. The involvement of these systems in the virulence of this pathogen is discussed in this report. It has been observed that quorum sensing play an important part in the virulence of this pathogen. Therefore, it is important that novel antimicrobials should be able to target this system to reduce virulence in animal model infections.

Introduction
Pseudomonas aeruginosa is a Gram negative, motile, anaerobic rod that belongs to the genus pseudomonads. It is known for its versatility for survival in a range of ecological niches. This bacterium inhabits soil, water and vegetation. It is an opportunistic pathogen in humans, causing a variety of diseases such as urinary and gastrointestinal tract infections, and respiratory system infections. In immunocompromised patients infected with HIV or cystic fibrosis (CF), this pathogen is found to cause a mortality rate of about 50% (Todar, 2004). The bacteria can be isolated from the skin, throat and stool. It is spread by fomites and contaminated water (Arevalo-Ferro et al, 2003). P. aeruginosa is known for its resistance to many antibiotics because of the presence of the lipopolysaccharide, which prevents host immune cells from recognising it. In addition, the ability to form biofilms by the bacteria makes the cells more resistant to antibiotics as higher concentrations are needed to disrupt biofilm formation (Todar, 2004). P. aeruginosa has a large genome, consisting of over 6 million base pairs and over 5000 ORFs, encoding cellular genes (Stover et al., 2000). The versatility of the organism is most probably attributed to its large genome size and complexity.

Quorum sensing (QS) is a mechanism whereby bacteria communicate with one another, relying on bacterial population density. Gram negative bacteria, such as P. aeruginosa rely on N-acyl-homoserine lactones (AHL) as signal molecules in QS systems (Rasmussen et al., 2006). The paradigm of quorum sensing states that AHLs are constitutively produced at low cell densities. The AHLs will then accumulate in the environment, in proportion to the increase in bacterial population. At a certain threshold concentration of AHL, the molecules will be able to bind to its respective receptor and a series of target gene regulations will be activated (Juhas et al., 2005). This system of regulation ensures that bacteria are able to form organised communities and to exchange information with other members to coordinate cellular activities. Among the processes regulated by quorum sensing are the synthesis of secondary metabolites, enzymes and virulence factors which allow bacteria to colonize various ecological niches (as reviewed by Juhas et al. 2005).

In P. aeruginosa, the quorum sensing systems are made up of the two AHL systems: las and rhl systems; and one non-AHL system: 2-heptyl-3-hydroxy-4-quinolone (PQS) (Pesci et al, 1999). The QS systems in P. aeruginosa regulate about 6-10% of the bacterial genome, indicating that this system play an important role in the survival of the bacteria (Arevalo-Ferro et al., 2003, Todar, 2004). QS in P. aeruginosa is important in pathogenicity as it ensures the bacteria do not express pathogenic traits until population has reached its critical density to be able to overwhelm host defences and establish an infection (Arevalo-Ferro et al., 2003). Among the virulence factors regulated by QS are proteases, exotoxin A, rhamnolipids, pyocyanin and sideophores (Wagner et al., 2003). In this report, the QS system is discussed in terms of its importance in the virulence of the organism as it causes serious implications in immunocompromised patients.

Mode of synthesis
The las system in P. aeruginosa quorum sensing comprise of LasI and LasR, where LasI synthesises the signal molecule N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12- HSL) and LasR acts as a transcriptional regulator.

The second quorum sensing system, the rhl system, is comprised of RhlI and RhlR, where RhlI synthesises the production of signal molecule N-butanoyl-L-homoserine lactone (C4-HSL) and RhlR is the transcriptional regulator (Erikson et al., 2002, Rasmussen et al., 2006). LasR and RhlR are able to induce transcription of their own genes, creating a positive feedback loop that increases AHL production and dissemination (Schuster et al., 2006).

The PQS signalling system links the las and rhl systems. LasI and RhlI have been shown to control the synthesis of PQS which in turn, controls the expression of RhlI and RhlR. PQS gene transcription is positively regulated by LasR, and at the same time, it is negatively regulated by rhl. Therefore, the ratio of 3-oxo-C12-HSL:C4-HSL is important (as reviewed by Juhas et al., 2005). The las and rhl systems do not act independently; they are arranged in a hierarchical fashion where 3-oxo-C12-HSL positively regulates the rhl system (Erikson et al., 2002). Figure 1 represents a simplified depiction of the las and rhl system.

Figure 1 Quorum sensing in P. aeruginosa. The las act in a hierarchical fashion, whereby it controls the rhl. The LasR/3-oxo-C12-HSL complex activates the transcription of rhlR. Additionally, 3-oxo-C12-HSL can also block activation of RhlR by C4-HSL. Both quorum signaling systems regulate the expression of various genes (lasB: LasB elastase, lasA: LasA elastase, toxA: exotoxin A, aprA: alkaline protease, xcpP and xcpR: genes of the xcp secretory pathway, rhlAB: rhamnosyltransferase and rpoS: stationary phase sigma factor (Van Delden et al., 1998).

Structure
The structures of the AHL molecules and the PQS molecule are shown in Figure 2.

Fig. 2. Structures of PQS and AHLs exploited for cell-to-cell communication by P. aeruginosa (Juhas et al., 2005).

Biological Roles
The las system is mainly involved in the regulation of various virulence factors, while the rhl system regulates a broad spectrum of P. aeruginosa genes (Juhas et al., 2005). In CF patients, lasR transcripts have been detected in the sputum samples, and this accumulation correlated with lasA, lasB, toxA, arpA, lasI and rhlR mRNA transcripts, which are virulence factors (Pesci et al, 1997, Rumbaugh et al, 1999), indicating a functional link on the regulation of these genes. RhlR has been shown to bind to a specific upstream sequence of the rhlAB gene independently of the presence or absence of C4-HSL. When C4-HSL is present, transcription of rhlAB is activated, while transcription is repressed in the absence of C4-HSL (as reviewed by Juhas et al., 2005). C4-HSL has also been shown to regulate virulence genes such as lasB, rpoS, rhlA and rhlI (Rumbaugh et al., 1999). The PQS signalling system is able to exert antimicrobial activity and is shown to promote biofilm formation (as reviewed by Juhas et al., 2005).

Quorum sensing also play a part in host-pathogen interactions by modulating the host immune system. The AHL mainly responsible in immunomodulation of the immune system is 3-oxo-C12-HSL (Wagner et al., 2006). It is able to induce inflammation in infected hosts, which produces further damage. When 3-oxo-C12-HSL is produced, immune cells, such as lymphocytes, macrophages and antibodies are activated. The long chain AHL of 3-oxo-C12-HSL suppresses IL12 and TNFα secretion, skewing the T cell response to a Th2 type response (Telford et al., 1998). 3-oxo-C12-HSL has also been shown to induce Cox2 [an inflammation associated enzyme in macrophages]. Cox2 induces inflammation, fever and pain, which enables the pathogen to disseminate and cause septicemia. Therefore, this shows that 3-oxo-C12-HSL does not only regulate virulence genes, but it is a virulence factor by itself (Smith et al., 2002). 3-oxo-C12-HSL, which controls PQS production through the regulation of pqsH show that these compounds synergise to reduce T cell proliferation at sub-cytotoxic doses (Pritchard, 2006). PQS is able to act in the T cell signalling pathway by enhancing IL2 production, which in turn inhibits T cell proliferation. This suggests that the pathogen is capable of influencing the immune system to optimise its survival (Pritchard 2006, Smith et al., 2003).

To show that 3-oxo-C12-HSL and C4-HSL are directly accountable for pathogenicity, mutations in the las and rhl systems were made and the effects on pathogenicity were observed. Various experiments have shown that there is a general decrease in virulence in these mutants. In lung infection models, a lasI and rhlI double mutant produced less virulence factors, resulting in a faster immune response, stronger oxidative bursts of blood PMNs and accumulate antibodies faster (Rasmussen et al., 2006). In burn wound infections, lasI and rhlI double mutants showed reduced virulence as the pathogen was less efficient in dissemination compared to its wild type counterpart. This is because virulence factors, such as alkaline protease and elastase which interfere with phagocytosis of neutrophils are absent (Rumbaugh et al., 1999). In acute pulmonary infections (Pearson et al., 2000), burn wound infections (Rumbaugh et al., 1999) and chronic lung infections (Wu et al., 2001), mutants in QS genes have been able to reduce mortality compared to wild type infections. In a study done by Arevalo-Ferro et al. (2003), lasI and rhlI double mutant showed that cellular protein composition was significantly reduced compared to the cell’s transcriptome. This suggests that the LasR and RhlR transcriptional regulators are able to activate or repress the transcription of target genes. However, it should be noted that double mutants are not completely avirulent. Virulence of the bacteria is multi-factorial and quorum sensing is only a system that plays a part in virulence (Smith et al., 2003).

Practical Applications
The understanding of quorum sensing in P aeruginosa is important as it controls one third of the virulence genes. Hence, it can be exploited as a target for antimicrobials. Mutations made in the QS regulatory genes have been shown to reduce virulence and mortality. There are three ways to interfere with the QS system: blocking AHL synthesis, degrading the AHL signal molecule or inhibiting the binding of the signal molecule to the receptor (Juhas et al., 2005, Rasmussen et al., 2006). These methods minimise the selective pressure for resistant bacteria as it does not affect its growth (Juhas et al., 2005).

As precursors such as acyl-ACP and SAM are used in the synthesis of the AHL molecules, analogues of SAM, such as Holo ACP, L/D-S-adenosyl-homocystein, sinefugin and butyryl-SAM can inhibit synthesis of AHL synthetases, such as RhlI (as reviewed by Juhas et al., 2005). Therefore, there will be no accumulation of AHLs despite the increase in bacterial population. The AHL signal molecule could be degraded using chemical, enzymic or metabolic methods (Rasmussen et al., 2006). AiiA enzyme from Bacillus sp can inactivate AHLs by hydrolysing the lactone bond, also known as quorum quenching (as reviewed by Juhas et al., 2005).

Lactonolysis, or ring opening of the AHL lactones could be triggered by high pH as well as high temperatures. These result in lowered amounts of bioactive AHLs (Lee et al., 2002). Activation of the transcription regulators could be blocked using analogues of signalling molecules. Substitutions at the 3-oxo-C6-HSL acyl side chain are able to displace C6-HSL from activating the regulator (as reviewed by Juhas et al. 2005). The HSL ring can also be replaced with an alternative ring structure, such as exchanging the lactone ring for the amino cycloalcohol or amino cycloketone. These were shown to inhibit QS controlled expression of a plasI-gfp fusion and selected virulence factors (Smith et al., 2003).

Antimicrobials produced by other organisms could also block QS signalling. Delisea pulchra produces furanone, which is able to block cell-to-cell communication and is shown to be able to clear P. aeruginosa lung infections in mouse models (as reviewed by Juhas et al., 2005). QS inhibitors, such as para-benzoquinone and 4-nitro-pyridine-N-oxide (4-NPO) can also be used to target RhlR and LasR (Rasmussen et al., 2005). Eukaryotic organisms, such as fungi have also been shown to produce antimicrobial compounds such as patulin and penicillic acid to downregulate 45-60% of QS regulated genes.

QS systems are important in regulating the virulence of P. aeruginosa. Therefore, continued research to produce novel antibiotics that will be able to efficiently target this system would be greatly beneficial to reduce nosocomial infections and reduce mortality in immunocompromised patients.

References

Arevalo-Ferro, C., M. Hentzer, G. Reil, A. Görg, S. Kjelleberg, M. GIvskov, K. Riedel, and L. Eberl. 2003. Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics. Environmental Microbiology. 5: 1350-1369.

Erikson, D. L., R. Endersby, A. Kirkham, K. Stuber, D. D. Vollman, H. R. Rabin, I. Mitchell, and D. G. Storey. 2002. Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cyctic fibrosis. Infection and Immunity. 70: 1783-1790.

Heurlier, K., V. Dénervaud, and D. Haas. 2006. Impact of quorum sensing on fitness of Pseudomonas aeruginosa. International Journal of Medical Microbiology. 296: 93-102.

Juhas, M., L. Eberl, and B. Tümmler. 2005. Quorum sensing: the power of cooperation in the world of Pseudomonas. Environmental Microbiology. 7: 459-471.

Lee, S. J., S.Y. Park, J.J. Lee, D.Y. Yum, B.T. Koo and J.K. Lee. 2002. Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Applied Environmental Microbiology. 68: 3919–3924.

Pearson, J. P., M. Feldman, B. H. Iglewski, and A. Prince. 2000. Pseudomonas aeruginosa cell-to-cell signalling is required for virulence in a model of acute pulmonary infection. Infection and Immunity. 68: 4331-4334.

Pesci E. C., Pearson J. P., Seed P. C., Iglewski B. H. 1997. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. Journal of Bacteriology 179:3127–3132

Pritchard, D. I. 2006. Immune modulation by Pseudomonsa aeruginosa quorum-sensing signal molecules. International Journal of Medical Microbiology. 296: 111-116.

Rasmussen, T. B., and M. Givskov. 2006. Quorum-sensing inhibitors as anti-pathogenic drugs. International Journal of Medical Microbiology. 296: 149-161.
Rasmussen, T. B., T. Bjarnsholt, M.E. Skindersoe, M. Hentzer, P. Kristoffersen, M. Kote, J. Nielsen, L. Eberl and M. Givskov. 2005. Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector. Journal of Bacteriology. 187: 1799–1814.

Rumbaugh, K. P., J. A. Griswold, B. H. Iglewski, and A. N. Hamood. 1999. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections. Infection and Immunity. 67: 5854-5862

Schuster, M., and E. P. Greenberg. 2006. A network of networks: quorum sensing gene regulation in Pseudomonas aeruginosa. International Journal of Medical Microbiology. 296: 73-81.

Smith, R. J., S. G. Harris, R. Phipps, and B. Iglewski. 2002. The Pseudomonas aeruginosa quorum-sensing molecule N-(3-Oxododecanoyl)Homoserine Lactone contributes to virulence and induces inflammation in vivo. Journal of Bacteriology. 184: 1132-1139.

Smith, R. S., and B. H. Iglewski. 2003. P. aeruginosa quorum-sensing systems and virulence. Current Opinion in Microbiology. 6: 56-60.

Stover, C. K., X. Q. Pham, A. L. Erwin, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F.S. L. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K.-S. Wong, Z. Wu, I. T. Paulsenk, J. Reizer, M. H. Saier, R. E. W. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature. 406: 959-964

Telford, G., D. Wheeler, P. Williams, P.T. Tomkins, P. Appleby, H. Sewell, G. Stewart, B.W. Bycroft and D.I. Pritchard, 1998. The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone has immunomodulatory activity. Infection and Immunity. 66: 36–42.

Todar’s Online Textbook of Bacteriology. http://textbookofbacteriology.net/pseudomonas.html

Van Delden, C., and B. H. Iglewski.1998.Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerging Infectious Diseases 4: 551-560

Wagner, V. E., Bushnell, D., Passador, L., Brooks, A. I. & Iglewski, B. H. 2003. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. Journal of Bacteriology. 185: 2080–2095.

Wagner, V. E., J. G. Frelinger, R. K. Barth, and B. H. Iglewski. 2006. Quorum sensing: dynamic response of Pseudomonas aeruginosa to external signals. Trends in Microbiology 14: 55-58

Wu, H., Z. Song, M. Givskov, G. Doring, D. Worlitzsch, K. Mathee, J. Rygaard, and N. Høiby. 2001. Pseudomonas aeruginosa mutations in lasI and rhlI quorum sensing systems result in milder chronic lung infection. Microbiology. 147: 1105-1113.

A signal challenge for the mother cell.

2006-05-18T18:54:00.000+10:00

A signal from the spore cell facilitates the expression of late genes in the mother cell of Bacillus subtilis during sporulation.

S. Vickery.

Summary

During sporulation in Bacillus subtilis, a late regulon of genes under control of sigma factor K must be expressed in the mother cell to implement the final maturation and release of the spore cell. SpoIVB protein is necessary for intercompartmental signalling between the forespore and mother cell, resulting in activation of precursor pro-σK to active σK. This is achieved via a complex pathway through which SpoIVB protein, produced in the spore cell, traverses through the forespore cell membrane into the intermembrane space where it acts on components from the mother cell to mediate pro-σK processing. SpoIVB acts to overcome an inhibitory effect upon SpoIVFB enzyme in the mother cell, which cleaves pro-σK to σK. SpoIVFA protein draws together and tethers SpoIVFB and BofA proteins in the mother cell, allowing BofA to impose an inhibitory effect upon SpoIVFB. The SpoIVB signal from the spore cell cleaves SpoIVFA, thus disengaging SpoIVFB and BofA, enabling SpoIVFB to activate pro-σK.

Endospore formation in Bacillus subtilis is induced by envrironmental pressures such as limited nutrients or a deviation of pH from the permissible range, which pose a threat to the organism. (Prince et al., 2005). Sporulation results in death of the mother cell allowing release of a resistant spore into the environment, which greatly increases the probability of that cell's survival in extreme conditions. Following the detection of an environmental threat, the cell proceeds through a series of morphological changes, collectively called 'sporulation' which results in the production and consequent release of the developed spore cell. Firstly, DNA is replicated and the two chromosomes are directed to opposite poles by an axial filament. Secondly, a septum is formed close to one pole of the cell, resulting in one large and one small cellular compartment known as the mother cell and forespore respectively (Stragier and Losick, 1996). The mother cell then engulfs the forespore, which becomes a free endospore cell within the mother cell (Stragier and Losick, 1996).

In the figure:

An axial filament directs the separation of chromosomes
A septum is formed
The mother cell engulfs the spore cell
The mother cell is lysed and the spore cell is released.

Figure 1. The morphological stages of sporulation. Source: Stragier and Losick, 1996. (Original Figure was unavailable. An alternative from the net at http://www.ibpc.fr/UPR9073/psfr.html was substituted)

During sporulation in Bacillus subtilis, the forespore and the mother cell follow different patterns of gene expression as defined by the presence of unique sigma factors that are confined to either cell (Stragier and Losick, 1996; Zhang et al., 1998). σF and σG are present only in the forespore while σE and σK are present exclusively in the mother cell (Rudner and Losick, 2002). These sigma factors induce cell-specific gene expression and account for the varying morphological and physiological requirements in the two cells. For example, a regulon of late genes under the control of σK, is expressed exclusively in the mother cell and facilitates three key processes; spore coat biosynthesis, final spore maturation and lysis of the mother cell via apoptosis to implement release of the spore cell (Zhang et al., 1998). The activation of these late genes in the mother cell is a particularly well-studied and intriguing example of the complex interactions that occur between the mother cell and forespore throughout sporulation, via intercellular signalling.

The cascade of signalling events required to activate sigma factors, and thus induce gene expression in the two cells, ensures that sporulation is tightly regulated and is maintained in an orderly sequential process. The sigma factors in both the mother cell and the forespore cell exist in an inactive form until the cells receive signals from each other, allowing the sigma factors to be activated, thus inducing the expression of a specific set of genes (Rudner and Losick, 2002; Stragier and Losick, 1996; Wakeley et al., 2000). In the case of pro-σK in the mother cell, it is the forespore-derived protein SpoIVB that acts on components in the mother cell to mediate proteolytic cleavage of pro-σK to active σK (Zhang et al., 1998; Dong and Cutting 2003; 2004; Wakeley et al., 2000).

SpoIVB does not directly process pro-σK to its active state. Rather, it relieves an inhibitory effect that is imposed upon SpoIVFB enzyme in the mother cell which functions to process pro-σK (Zhang et al., 1998; Rudner and Losick, 2002; Wakeley et al., 2000, Dong and Cutting, 2003). SpoIVFB enzyme is maintained in an inactive form by BofA protein in the mother cell until the SpoIVB signal is received from the spore cell (Zhang et al., 1998). This is made possible by the presence of an additional protein in the mother cell (SpoIVFA) which plays a central role in BofA's inhibitory function. Research to date supports a model whereby SpoIVFA facilitates the interaction between BofA and SpoIVFB by serving as a platform to draw these two proteins into proximity to one another (Rudner and Losick, 2002). Only once BofA and SpoIVFB are adjacent can BofA impose its inhibitory effect upon SpoIVFB (Zhang et al., 1998; Rudner and Losick, 2002). A multimeric complex is formed between the three mother cell proteins and embeds in the mother-cell membrane that surrounds the forespore, permeating into the intermembrane space, which is where it interacts with SpoIVB from the forespore (Zhang et al., 1998). The subcellular localisation of the SpoIVFB-SpoIVFA-BofA complex is thought to take place via a conserved domain in SpoIVFA which interacts with peptidoglycan in the intermembrane space between the mother and forespore cells (Zhang et al., 1998).

Fig.2. Proteolytic processing of pro-σK in the mother cell is catalyzed by SpoIVfB which exists in complex with its two regulators, SpoIVFA and BofA, until SpoIVB activates processing by reversing the inhibition imposed on SpoIVFB by BofA. Source: Rudner and Losick, 2002.

SpoIVB enzyme produced in the forespore is able to relieve an inhibitory event in the mother cell by interacting with the pro-σK complex in intermembrane space. SpoIVB crosses the forespore membrane to reside in the intermembrane space (Wakeley et al., 2000). The PDZ domain in SpoIVB recognises the COOH terminus of another SpoIVB molecule in the forespore (Dong and Cutting, 2004). The interaction between the two SpoIVB molecules activates their cleavage into a form that can be released across the spore cell membrane into the intermembrane space. The knowledge that SpoIVB can move across a phospholipid bilayer in Escherichia coli invoked the suggestion that it may also cross a membrane in this system, which was consequently proved by experiment (Wakeley et al., 2000).

Once in the intermembrane space, SpoIVB is able to induce the activation of pro-σK, made possible by the fact that the pro-σK initiating complex is conveniently embedded in the mother cell membrane surrounding the forespore awaiting a signal from the forespore (Dong and Cutting 2004). SpoIVB undergoes a second cleavage event; autoproteolysis, to produce short protein fragments which interact with the pro-σK initiation complex inducing pro-σK processing (Wakeley et al., 2000). This cleavage event is a critical aspect of this protein's ability to signal pro-σK processing, as demonstrated through examination of spoIVB gene mutants in which autoproteolysis was blocked resulting in failed activation of pro-σK processing (Wakeley et al., 2000).

The PDZ domain in the active products resulting from the second cleavage event target the pro-σK processing complex embedded in the mother cell membrane surrounding the forespore. SpoIVB binds the COOH terminus of BofA in the pro-σK initiating complex which draws SpoIVB into proximity to SpoIVFA. Using its PDZ domain, SpoIVB recognises an internal motif in SpoIVFA and associates with it, inducing cleavage of SpoIVFA and subsequent dissassemby of the multimeric complex (Stragier and Losick, 1996). SpoIVFA releases BofA and SpoIVFB from its clasp once it is cleaved, so BofA can no longer inhibit the enzymatic action of SpoIVFB. SpoIVFB is then available to process pro-σK to σK. As a result of interaction with SpoIVB, SpoIVFB is released from its inhibited state in the complex and is available to activate proteolytic processing of pro-σK to produce the active σK. This occurs at the mother cell membrane into which inactive pro-σk has also embedded, proximal to the multimeric complex (Rudner and Losick, 2002; Wakeley et al., 2000; Zhang et al., 1998). Presumably the migratory mechanism employed by pro-σk to reside in the mother cell membrane has evolved to maximise the efficiency of its processing as it enables pro-σK to be present when SpoIVFB is activated.

Figure 3. SpoIVB protein undergoes at least 3 cleavage events.

1. The PDZ domain of SpoIVB recognising the COOH terminus of another SpoIVB molecule prompting cleavage which releases the molecules into the intermembrane space.
2. Autoproteolysis of SpoIVB produces an active protein fragment which targets the Pro-sigmaK signalling complex.
3. Further cleavage occurs to inactivate SpoIVB.

A third and final cleavage event inactivates SpoIVB (Dong and Cutting 2003; 2004). This series of cleavage events is heavily reliant on the presence of a PDZ domain in the SpoIVB protein, as defined by experiments in which SpoIVB proteins carrying various PDZ mutations had impaired cleavage ability (Dong and Cutting 2003).

The signal communicated by SpoIVB from the forespore induces a cascade of proteolytic cleavage events in the mother cell that enable pro-σK to be processed and the late genes under σK control to be expressed (Stragier and Losick, 1996). The interaction between SpoIVB and SpoIVFA in the intermembrane space between the spore cell and mother cell is where products from the two cells meet. It is by way of these proteins intersecting that the spatially discrete cells can communicate despite the restrictive presence of two cellular membranes. This is achieved by employment of a complex pathway through which SpoIVB targets SpoIVFA to overcome its inhibitory effect upon SpoIVFB. As this example of signal transduction involves the signalling protein, SpoIVB, acting across a cell membrane, this system offers an important insight into the mechanisms at work in intercompartmental signalling. In particular, that this system of transmembrane signalling essentially relies upon the PDZ domains recognising DNA motifs, and regulated proteolysis. This defines the mechanism of intercompartmental signalling by which SpoIVB induces pro-σk processing.

In order to further expand upon the knowledge of how pro-σK processing pathway operates, it would be useful to understand the interaction between BofA and SpoIVFB proteins in the mother cell in more detail. It is known that BofA is most certainly responsible for inhibiting SpoIVFB, but the current models surrounding an interaction between the two proteins are based on speculation. It is known that SpoIVFA draws these two proteins together, and the current models favour BofA and SpoIVFB directly interacting but the more complicated model has not yet been ruled out, whereby BofA induces a confomational change in SpoIVFA, the altered state of which is responsible for inhibiting SpoIVFB enzyme (Rudner and Losick, 2002).

Also, further investigation of the function of SpoIVB protein from the spore cell would provide greater insight into the process of sporulation in Bacillus subtilis. Observation of cells carrying a spoIVB null mutation imply that this protein is not only active in pro-σK processing. In spoIVB null mutants pro-σK processing is constitutively active, but cells fail to make intact heat-resistant spores, so SpoIVB protein must have a second, as yet undefined role in spore formation (Dong and Cutting, 2004)

References

T.C. Dong and S.M. Cutting (2004) The PDZ domain of the SpoIVB transmembrane signalling protein enables cis-trans interactions involving multiple partners leading to the activation of the pro-σK processing complex in Bacillus subtilis. The Journal of Biological Chemistry 279(42): 43468-43478.

T.C. Dong and S.M. Cutting (2003) SpoIVB-mediated cleavage of SpoIVFA could provide the intercellular signal to activate processing of pro-σK in Bacillus subtilis. Molecular Microbiology 49(5): 1425-1434.

D.Z. Rudner and R. Losick (2002) A sporulation membrane protein tethers the pro-sk processing enzyme to its inhibitor and dictates its subcellular localization. Genes and Development 16: 1007-1018.

P.R. Wakeley, R. Dorazi, N. Thi Hoa, J.R. Bowyer and S.M. Cutting (2000) Proteolysis of SpoIVB is a critical determinant in signalling of pro-σK processing in Bacillus subtilis. Molecular Microbiology 36(6): 1336-1348.

B. Zhang, A. Hofmeister and L. Kroos (1998) The prosequence of pro-σK promotes membrane association and inhibits RNA polymerase core binding. Journal of Bacteriology 180(9): 2434-2441.

P. Stragier and R. Losick (1996) Molecular genetics of sporulation in Bacillus Subtilis. Annual Review of Genetics 30: 297-341.

H. Prince, R. Zhou and L. Kroos (2005) Substrate requirements for regulated intramembrane proteolysis of Bacillus subtilis pro-σK. Journal of Bacteriology 187: 961-971.

Using Atomic Force Microscopy.

2006-05-05T01:00:00.000+10:00

This great stuff is what Grad Students can do on the web:

Studying Bacteria with Atomic Force Microscopy (AFM)
Posted 17 April 2006 by André under André’s Research

AFM has been widely used in the life sciences since the application of optical lever detection by Hansma and co workers in the late ‘80s so it was no surprise that when I searched the literature I found a bunch of papers describing experiments on bacteria using AFM. In case you’re interested in doing something similar here’s an annotated bibliography with most of the papers I found [pdf]. As always, the list is incomplete—especially the section on bacterial adhesion since that’s not my primary interest right now even though it forms the bulk of the literature. Never the less, you’ll get a good survey of the field if you have a look at the papers in the list and dive into the web of citations...continues at link in more detail.

Study Questions relating to mechanisms of nutrient uptake by bacteria.

2006-04-26T13:43:00.000+10:00

Q1. How are proton gradients created?

Q2. What is the role performed by outer-membrane pores (porins) of gram-negative bacteria?

Q3. Give two examples of facilitated diffusion as a substrate uptake mechanism in bacteria?

Q4. How is facilitated diffusion selective?

Q5. The group translocation process accomplishes transport by chemically modifying the solute, which arrives inside the cell as a different molecule.

Give an example of substrate uptake by group translocation in bacteria, and describe the source of energy used for active uptake.

Your responses in comments please.(They can be anonymous!)

Ammonia assimilation is an important part of the biosynthesis stage of growth metabolism.

2006-04-23T12:55:00.000+10:00

After the fueling step of growth metabolism has occurred, ammonia is incorporated into metabolites. This part of biosynthesis is known as nitrogen assimilation.

Study question:

How much of the cell is nitrogen?

Two alternative assimilation routes for nitrogen:

Although some nutritional sources of nitrogen for bacterial cells (e.g. nitrate) are in a more oxidised chemical state than ammonia, these are uniformly converted to ammonia before assimilation, and almost all cell nitrogen is at the same oxidation state as ammonia. Ammonia is fully reduced, fitting in with the concept that early life evolved in an oxygen free environment.

Escherichia coli has two different first steps for incorporating ammonia into building blocks for polymers such as proteins and nucleic acids, each alternative relying on a different enzyme.

One of these alternative uses glutamate as an acceptor of ammonia, ATP as a co-substrate, and the enzyme glutamine synthetase to catalise the reaction:

The other alternative first step for ammonia assimilation uses the metobolite 2-oxoglutarate (also called alpha ketoglurate), to accept ammonia, NADPH and the enzyme GDH.

Study Questions:

What are the products of the two alternative first steps in ammonia assimilation?

How do the two reactions differ in terms of reaction products?

More importantly, what are the different advantages and opportunities offered by the two alternatives to the cell in its survival under varous different environmental challenges?

Reference Sources:

Chapter 7, Biosynthesis, in Microbe, Schaechter 2006

EcoCyc, Encyclopedia of Escherichia coli K-12 Genes and Metabolism

Why does Escherichia coli have two primary pathways for synthesis of glutamate?
Helling RB.
J Bacteriol. 1994 Aug;176(15):4664-8. Erratum J Bacteriol 1997 Jul;179(13):4455.

Biosynthesis Stage of Growth Metabolism.

2006-04-22T14:54:00.000+10:00

Biosynthesis of building blocks for polymers - such as amino acids and nucleotides- follows the Fueling reaction stage of microbial growth metabolism.

Biosynthesis differs from the Fueling stage in that nitrogen (as ammonia) and sometimes sulphur are assimilated into the molecules of metabolism. Nitrogen is not found in the fuelling precursor metabolites.

Generally the end products of biosynthesis are more reduced than the precursor metabolites, and this reduction is achieved by use of NADPH to donate hydrogen. Additionally ATP is also used, so that ATP and NADPH provide the driving force for biosynthesis.

Quoting from Schaechter 2006 Chapter 7:

Biosynthesis, then, more than fueling, reflects the essentially close relation of microbes to plants and animals. While the fueling metabolism of microbes expresses the potential for life forms to scavenge for food and thereby recycle organic material, the biosynthetic metabolism of microbes reminds us of what we have lost in evolving into beings higher in the food chain.

Study Question:
Explain in what way the biosynthesis of amino acids and other nitrogen-containing components of human cells depends ultimately on the biochemical activities of Earth's prokaryotes.

Responses in comments please.

The best way to find information about bacteria is through effective use of both publication and gene databases.

2006-04-11T11:19:00.000+10:00

La Cosa Nostra Genetica

This post is to help people use computer searches effectively to find biological information about cell functions.

When it comes to functional information on singled celled organisms that have a genome sequence completed, a very powerful way forward is to take these steps

Start by using a relevant words searche via PubMed or equivalent service, to locate an appropriate protein sequence in a sequence data base (say at NCBI website),and
Then use the retrieved protein sequence to BLASTP for relevant other genes in any organism of interest.
Search the BLASTP output for clues to relevance of for new ideas.

The reason why this approach is powerful is that it enables natural evolution to be traced in silico. Evolutionary traces make definite connections between unknown functions in one putative gene to known functions in other well studied genes, and are supported by huge and constantly growing genome databases.

To illustrate this, Microbe Pundit will show the trail of results obtained in a student discussion about secretion of biologically active compounds by Streptomyces avermitilis.

Pundit started by assuming Quorum sensing systems would be involved in secretion of important compounds in this organism- the question is how to find the genes involved.

First find a studied secretion system in any Streptomycete. (Evolutionary relatedness neans that all Streptomycete species will share many important genes).

To find this, go to Pubmed, the guide for published medical scientific papers, and type in "QUORUM SENSING STREPTOMYCES" as a search item, press buttons and scan the output for a better more specific lead in the form of a relevant paper.

This is computer detective work in action. It helps if you imagine you are Sherlock Holmes or Mrs Marples.

The trick is the use you own brain to find useful further clues in this computer output.

This approach lead me to the following paper:

1: J Bacteriol. 2005 Jan;187(1):135-42.

Dual transcriptional control of amfTSBA, which regulates the onset of cellular differentiation in Streptomyces griseus.
Ueda K, Takano H, Nishimoto M, Inaba H, Beppu T.

The amf gene cluster encodes a probable secretion system for a peptidic morphogen, AmfS, which induces aerial mycelium formation in Streptomyces griseus. Here we examined the transcriptional control mechanism for the promoter preceding amfT (PamfT) directing the transcription of the amfTSBA operon.
High-resolution S1 analysis mapped a transcriptional start point at 31 nucleotides upstream of the translational start codon of amfT. Low-resolution analysis showed that PamfT is developmentally regulated in the wild type and completely abolished in an amfR mutant. The -35 region of PamfT contained the consensus sequence for the binding of BldD, a pleiotropic negative regulator for morphological and physiological development in Streptomyces coelicolor A3(2).
The cloned bldD locus of S. griseus showed high sequence similarity to the S. coelicolor counterpart. Transcription of bldD occurred constitutively in both the wild type and an A-factor-deficient mutant of S. griseus, which suggests that the regulatory role of BldD is independent of A-factor. The gel retardation
assay revealed that purified BldD and AmfR recombinant proteins specifically bind PamfT. Overproduction of BldD in the wild-type cell conferred a bald phenotype (defective in aerial growth and streptomycin production) and caused marked repression of PamfT activity. An amfT-depleted mutant also showed a bald
phenotype but PamfT activity was not affected. Both the bldD-overproducing wild-type strain and the amfT mutant were unable to induce aerial growth of an amfS mutant in a cross-feeding assay, which indicates that these strains are defective in the production of an active AmfS peptide. The results overall suggests that two independent regulators, AmfR and BldD, control PamfT activity via direct binding to determine the transcriptional level of the amf operon responsible for the production and secretion of AmfS peptide, which induces the erection of aerial hyphae in S. griseus.

The bolded sections are key facts (always skip over details at this stage you are trolling for pearls).

Pundit assumed one of these amf genes must code for a transport (peptide secretion) system. Lets assume it is gene amfB

Next go to a protein sequence database that takes word query searches such as this one at NCBI.

Type in amfB and press the right buttons, use common sense and you will find this entry:

LOCUS NP_828677 595 aa linear BCT 16-FEB-2006
DEFINITION ABC transporter ATP-binding membrane translocator, AmfB [Streptomyces avermitilis MA-4680].
ACCESSION NP_828677
VERSION NP_828677.1 GI:29834043
DBSOURCE UNKNOWN
KEYWORDS .
SOURCE Streptomyces avermitilis MA-4680
ORGANISM Streptomyces avermitilis MA-4680
Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales;
Streptomycineae; Streptomycetaceae; Streptomyces.
REFERENCE 1
AUTHORS Ikeda,H., Ishikawa,J., Hanamoto,A., Shinose,M., Kikuchi,H.,
Shiba,T., Sakaki,Y., Hattori,M. and Omura,S.
TITLE Complete genome sequence and comparative analysis of the industrial
microorganism Streptomyces avermitilis
JOURNAL Nat. Biotechnol. 21 (5), 526-531 (2003)
PUBMED 12692562
REFERENCE 2
AUTHORS Omura,S., Ikeda,H., Ishikawa,J., Hanamoto,A., Takahashi,C.,
Shinose,M., Takahashi,Y., Horikawa,H., Nakazawa,H., Osonoe,T.,
Kikuchi,H., Shiba,T., Sakaki,Y. and Hattori,M.
TITLE Genome sequence of an industrial microorganism Streptomyces
avermitilis: deducing the ability of producing secondary
metabolites
JOURNAL Proc. Natl. Acad. Sci. U.S.A. 98 (21), 12215-12220 (2001)
PUBMED 11572948

SNIP

FEATURES Location/Qualifiers
source 1..595
/organism="Streptomyces avermitilis MA-4680"
/strain="MA-4680; ATCC 31267; NCIMB 12804; NRRL 8165"
/db_xref="ATCC:31267"
/db_xref="taxon:227882"
Protein 1..595
/product="ABC transporter ATP-binding membrane
translocator, AmfB"
/calculated_mol_wt=61203
Region 82..589
/region_name="ABC-type multidrug transport system, ATPase
and permease components [Defense mechanisms]"
/note="MdlB"
/db_xref="CDD:10852"
Region 375..570
/region_name="ABC (ATP-binding cassette) transporter
nucleotide-binding domain"
/note="ABC_ATPase"
/db_xref="CDD:29340"
CDS 1..595
/gene="amfB"
/locus_tag="SAV7501"
/coded_by="complement(NC_003155.3:8939202..8940989)"
/note="AmfB/RamA homolog protein"
/transl_table=11
/db_xref="GeneID:1211743"
ORIGIN
1 mrghsrmktp sghgaapdgt gaadeaaart llrsaarhsr srcvalcltt aaasgaslll
61 paalgraldl lltrpgdaag thwvlwctgl vllialldac htvlagttda ratawlrqrl
121 vghvlavgpr agerfgpgel varlvgnaaq agtapataat llaalagpvg avvalglidp
181 llaavflgga pvltlllraf ardssqcvar yqdvqgriag alaeaiggar tiaaggtadk
241 evarilrplp elsregrrmw rvqgraaaqa vavapllqlg vvavggvllv hhrlsvgell
301 aasryavlat gvgvlvgqls gliraraaar rlgevltepa pvygtrqlpp gegrlelrsv
361 tvrrggrtvl dgvdlvvpag rtvavvgrsg sgksllaala grladpddgh vlldgvplrd
421 ldrtalrrav ghaferpall gdtiedtiaf gipspppdrv rqaaatarad sfvrrlpdgy
481 atpcaeapls ggecqrlgla rafahdsrll vlddalssld tvterhitea llrhtpgssr
541 liiahrvsta aradavvwla agrvravgth aelwrsaayr evfgssgter nggag
//

This means somebody has already annotated a similar putative gene to amfB in S. avermititis. The implied membrane located active transport sytem is of the ABC or ATP binding cassette type.

There are hundreds of known ABC-transporters. Some secrete compounds, other import compounds.

The next question is to ask what are the evolutionarily related transport systems to this one?, particularly one that have been investigated in the lab. (Hypothetical functions predicted by computers are "dime-a-hundred". Experimentally characterised functions are pearls.)

For this you take the protein sequence (bolded above) and go to the BLASTP search tool here.
http://www.ncbi.nlm.nih.gov/BLAST/

Paste in the protein sequence from the entry above and again push buttons.

The output is huge.

This a a small selection of what you get:

gi|29834043|ref|NP_828677.1|  ABC transporter ATP-binding memb...   686    0.0

gi|4928927|gb|AAD33775.1|  putative ATP binding membrane trans...   275    4e-72

gi|21224979|ref|NP_630758.1|  ABC transporter ATP-binding prot...   273    2e-71

gi|432992|gb|AAA21388.1|  potential ATP-binding membrane transpor   273    2e-71

gi|31044106|dbj|BAA33538.2|  membrane translocator [Streptomyces    267    1e-69

gi|50905835|ref|XP_464406.1|  putative multidrug resistance p-...   117    2e-24

gi|34913530|ref|NP_918112.1|  putative multidrug resistance pr...   115    6e-24

gi|85813525|emb|CAF33031.1|  putative ABC-type aminoglycoside ...   114    2e-23

gi|35214709|dbj|BAC92076.1|  HlyB/MsbA family ABC transporter ...   108    5e-22

gi|7023646|dbj|BAA92038.1|  unnamed protein product [Homo sapiens   108    9e-22

gi|27378906|ref|NP_770435.1|  ABC transporter HlyB/MsbA family...   106    3e-21

gi|7688707|gb|AAF67494.1|  NovA [Streptomyces caeruleus]            102    5e-20

gi|26991605|ref|NP_747030.1|  toxin secretion ABC transporter ...   102    5e-20

gi|2633146|emb|CAB12651.1|  yfiC [Bacillus subtilis subsp. sub...   102    5e-20

gi|85813784|emb|CAF31837.1|  putative hygromycin B exporter [S...  99.4    4e-19

Try pressing a few of the hyperlinks above to inspect database entry details.
The hits that are connected with export of antibiotics in Streptomyces are the most interesting finds- a reward for due diligence.

Sherlock Holmes and the Pundit can find these easily in the output.

Computers by themselves are stupid and can't do this well.
Humans are also needed to hop effectively between databases, as shown above.
After a while you will get good at this thing of ours, so lets call it La Cosa Nostra Genetica

Il Signor Pundit
;0)

Replication mechanisms of the bacterial chromosome.

2006-03-13T14:54:00.000+11:00

A timeless experiment in cell biology by an Australian scientist. FIGURE 5-2 from here. Autoradiograph of intact replicating chromosome of E coli. Bacteria were radioactively labeled with tritiated thymidine for approximately two generations and were lysed gently. Bacterial DNA was then examined by autoradiography. Insert shows replicating bacterial chromosome in diagrammatic form. The chromosome is circular, and two forks (X and Y) are present in replicating structure. Bar, 100 µm. From Cairns, J.P.: Cold Spring Harbor Symposia on Quantitative Biology 28:44, 1963

Schaecter et al. Microbe Chapters 8, 10 are very useful reading on this topic.

There is also the Baron free online textbook.

The chromosome of bacteria is a structure whose organisation and means of accurate and reliable duplication and distribution to daughter cells has been shaped by billions of years of natural evolution. As time passes we biological scientists are getting to understand much more of what this intense natural selection has achieved in terms of functional design.

The major points made in the teaching session were:

The structure of a DNA molecule including the backbone and pairing of bases.
How DNA and RNA differ.
How a DNA molecule is replicated and what constraints the structure puts on the replication process
A description of the proteins, including their function, that make up the replication machinery.
How all of the steps in replication are coordinated.

Study questions to reinforce and extend points made in the teaching session:

How are topoisomerases different to nucleases?
What is an oriC, what are its features and what takes place there?
What is a DNA editing activity, how does it work and why is it needed?

Proposal for how the TolC channel passes through murein layer.

2006-03-11T18:06:00.000+11:00

Artistic impression of purple TolC channel protein penetrating pores of peptidoglycan.

Just two days ago a proposed structure for the murein cell wall polymer was first reported.

In this paper there are wonderful colour images showing how the TolC "drainpipe" will just fit though proposed small pores in murein sheets. Another piece of the detailed stuctural reconstruction of bacterial surfaces just fell into place.

Three-dimensional structure of the bacterial cell wall peptidoglycan

The 3D structure of the bacterial peptidoglycan, the major constituent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmuramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacterium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its complexity and the lack of pure samples.

Herein we report the 3D solution structure as determined by NMR of the 2-kDa NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) synthetic fragment of the cell wall. The glycan backbone of this peptidoglycan forms a right-handed helix with a periodicity of three for the NAG-NAM repeat (per turn of the helix). The first two amino acids of the pentapeptide adopt a limited number of conformations. Based on this structure a model for the bacterial cell wall is proposed.

Samy O. Meroueh, Krisztina Z. Bencze, Dusan Hesek, Mijoon Lee, Jed F. Fisher, Timothy L. Stemmler, and Shahriar Mobashery
Three-dimensional structure of the bacterial cell wall peptidoglycan
PNAS published March 9, 2006, 10.1073/pnas.0510182103

Discussion thread on molecules secreted by Streptomyces.

2006-03-11T10:42:00.000+11:00

Image from here.

How to start this assignment?

Discuss the biological importance of specialised molecules secreted by Streptomyces avermitilis. Discuss their structure, mode of synthesis, possible biological roles and practical applications.

(This topic might interest people who are not specially interested in medicine, but who like practical topics like biochemical engineering, biotechnology, human welfare advances to benefit Africa, or even animal husbandry. Avermectins are used to treat animal and human parasite infections.)

The first step in tackling the assignment topic is to read what David Hopwood had to say in 2003:

The Streptomyces genome—be prepared!
David A. Hopwood

Selected quotes from Hopwood 2003:
The completion of the sequence of a second Streptomyces chromosome further establishes these soil-dwelling bacteria as nature's most prolific producers of potentially useful pharmaceuticals.

Streptomyces avermitilis became famous for producing the anti-parasitic agent avermectin, which is used to rid livestock of worm and insect infestations and to protect large numbers of people from river blindness in sub-Saharan Africa. Knowledge of the sequence of its genome, reported in this issue by Ikeda et al.1, should help in the development of higher yielding strains. However, the significance of the work goes far beyond such an objective.

In the context of biotechnology, the most interesting finding in the two Streptomyces genomes is the abundance of genes that would encode enzymes for secondary metabolism. Before the genome was sequenced, three antibiotics and a spore pigment were known to be encoded in the S. coelicolor genome, but the sequence revealed two dozen clusters, for pigments, complex lipids, signaling molecules, and iron-scavenging siderophores. In a preliminary report on the S. avermitilis genome before completion [Ref]7, 25 such clusters were described, and this number has increased to 30 in the complete sequence. Even more remarkable, nearly all the clusters probably encode different compounds in the two species, indicating the large number of pathways that await discovery in the Actinomycetes as a whole. In both species, a majority of the clusters lie in or near the arm regions, suggesting that their products are conditionally adaptive. Indeed, there is increasing evidence that the full capacity for secondary metabolite production in soil microorganisms is not expressed under typical conditions used for antibiotic screening in the laboratory but that particular compounds are made only in response to specific physical, chemical, or biological stresses (see Fig. 1).

A recent report by Zazopoulos et al.8 provides striking support for this view. These authors identified a cassette of five genes responsible for biosynthesis of the DNA-damaging warhead of the enediyne class of antibiotics, and detected it in 15% of a random collection of Streptomycetes. None at first made the predicted compounds, but all could be persuaded to do so under special fermentation conditions.

News and Views
Nature Biotechnology 21, 505 - 506 (2003)

Clearly there is a lot of choice as far as what Streptomycete molecules you can write about, because the message from David Hopwood's article is that Steptomycetes produce mumerous different compounds, and potentially produce compounds that have not yet even been detected directly. Thus there is good scope for discussing secreted molecules other than avermectins.

As far as their biological functions, there is a lot to be said too. Streptomycete "secondary metabolites" as they are called, are not just antibiotics.

One can even ask:

In what chemical languages do streptomycete cells communicate with one another to cooperate when they kill worms for food in the soil?

As a small (undersized for my age) boy I played rugby. (Rugby Union of course). With the right tackling technique and complete absence of fear, I could bring down the big boys by taking out the ankles of a runner. That's why I admire tiny Streptomyces avermitilis. It can take out the big guys like flatworms.

(Side note: rugby injuries put me in bed for about one month at age 11 years and I never played football again after that. However I have retained since then an obsessive fear of doctor's needles, which were used at that time to inject me intamuscularly with penicillin to combat the Clostridium bacteria that infected the injury, and whose neurotoxins made me delerious and accentuated my fear of needles.)

Discussion thread on compound secretion by Pseudomonas.

2006-03-10T14:06:00.000+11:00

Image from here.

This discussion thread is to assist people who want to write an assignment essay about molecules secreted by Pseudomonas aeruginosa, a gram negative bacterium commonly found in natural waters, that can also be a highly antibiotic resistant human pathogen.

The same general comments made in the previous thread about how to start researching the topic of cell division apply also here. Use NCBI-PubMed, and put your comments, questions, and discoveries in the comments please.

This current posting is designed to explain a different way of quickly finding the scientific status of a topic. I suggest here to try exploiting the Introduction section of a relevant and very recent research paper.

These Introduction sections are actually short up to date reviews by very competant scientists. They may be concise, but they are packed with the benefits of special expertise, and can be much more up to date than the most recently available mini-review, or recent longer review article.

The tricky challenge is to quickly locate a suitable recent paper.

You need to start somewhere close to the topic and leverage off that starting point.

Microbe Pundit has already suggested that multi-drug efflux pumps of Pseudomonas are involved in secretion of many different compounds. Why not start at this factoid?

He's mentioned already that Greenberg's journalistic style article Pumping up the versatility in a Nature journal is a good place to start discovering what's been learned about Pseudomonas.

But when reading Greenberg's article, you will find there is surprizingly little detail about molecule secretion, and it's a few years out of date.

But it does have the words :

RND pump multidrug efflux

mentioned so plug them into an NCBI-Pub Med search (Google NCBI first if you're lost for a URL).

With this literature search short-cut you come quickly (if you use a bit of intelligence) to:

Nehme D, Li XZ, Elliot R, Poole K. Related Articles, Links
Free in PMC Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa: identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB.
J Bacteriol. 2004 May;186(10):2973-83.

The bolded words show why Pundit thought this was gold.

Now get to the full text version of the J Bacteriol paper via your friendly university library or better, from FREE IN PMC over the internet anywhere with a terminal. Remember to have your USB thumb drive with you at the computer. (An USB MP3 player will also suffice, you just have to delete all those Kylie Minogue MP3s.)

With this you get to:

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobials (21). The resistance has historically been attributed to the presence in this organism of an outer membrane (OM) of low permeability (55), but it is increasingly clear that resistance owes much to the operation of broadly specific, so-called multidrug efflux systems (58-60, 64) that work synergistically with limited OM permeability (18, 40, 60). Several multidrug efflux systems in P. aeruginosa have been described to date (61), although the major system contributing to intrinsic multidrug resistance is encoded by the mexAB-oprM operon (38). Hyperexpression of this system also occurs in so-called nalB (27, 28, 75, 88)- and nalC (75, 88)-type multidrug-resistant mutants. MexAB-OprM accommodates a broad range of structurally diverse antimicrobials, including dyes, detergents, inhibitors of fatty acid biosynthesis, organic solvents, disinfectants, and clinically relevant antibiotics (10, 34, 37, 39, 41, 42, 48, 70, 74, 74, 76), and is implicated in the export of homoserine lactones involved in quorum sensing (17, 57) and, possibly, virulence factors (22).

Evans, K., L. Passador, R. Srikumar, E. Tsang, J. Nezezon, and K. Poole. 1998. Influence of the MexAB-OprM multidrug efflux system on quorum-sensing in Pseudomonas aeruginosa. J. Bacteriol. 180:5443-5447.

Pearson, J. P., C. Van Delden, and B. H. Iglewski. 1999. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J. Bacteriol. 181:1203-1210.

Hirakata, Y., R. Srikumar, K. Poole, N. Gotoh, T. Suematsu, S. Kohno, S. Kamihira, R. E. Hancock, and D. P. Speert. 2002. Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa. J Exp. Med. 196:109-118.

The MexAB-OprM efflux system, like the other tripartite Mex efflux systems in P. aeruginosa, consists of an inner membrane (IM) drug-proton antiporter of the resistance-nodulation-cell division (RND) family (MexB), an OM channel-forming component (OprM; also called OM factor [OMF]), and a periplasmic membrane fusion protein (MFP) (MexA) (58, 86). Crystal structures have not yet been reported for any of the efflux components of P. aeruginosa, although structures are available for the homologous OM (TolC [35]) and RND (AcrB [52]) components of the Mex-like AcrAB-TolC multidrug efflux system of Escherichia coli. The TolC channel is a trimer and spans both the OM (as a ß-barrel) and periplasm (as a {alpha}-helical barrel) (35). Measuring 140 Å in length, the channel is open at the distal (extracellular) end and tapers almost to a close at the proximal (periplasmic) end, which likely interacts with the RND component, AcrB (35). Modeling studies suggest that OprM adopts a similar structure (43, 80). The AcrB RND component also exists as a trimer, composed of a 50-Å-thick transmembrane region and a 70-Å headpiece that protrudes into the periplasm (52). This headpiece has a funnel-like opening at the top that is connected to a central cavity at the bottom, which, in turn, opens to the periplasm via three vestibules that likely play a role in substrate recognition by and/or access to the pump (52). Indeed, several studies highlight the role played by the periplasmic portion of the RND transporters of E. coli and P. aeruginosa in substrate (i.e., drug) recognition (14, 15, 45, 53, 79, 83). A high-resolution crystal structure for an MFP efflux component is not yet available, although preliminary studies indicate that, e.g., AcrA is also likely trimeric (85) and that monomer AcrA is a highly asymmetric, elongated molecule of sufficient length to span the periplasm (4, 84).

Koronakis, V., A. Sharff, E. Koronakis, B. Luisi, and C. Hughes. 2000. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405:914-919.

Bold indicates the words that provide leads to interesting aspects of secretion by Pseudomonas.

A mini-review essay topic could well focus entirely on homoserine lactone or maybe protein virulence factor secretion by Pseudomonas, and could involve careful reading, and re-reading, of 2 to 3 relevant review papers and 4 to 6 good research papers on the topic. That's enough work for an assigment.

PS Remember to ITALICISE all those species binomials in your final assignment. Cite references professionally too.

And finally, trust your luck .

In an effort to brighten up this post with a colourful image (posted at the top), I did a google image search for bacterial multi-drug efflux pumps and found this.

Discussion thread on Cell Division in Bacillus and Caulobacter.

2006-03-10T12:18:00.000+11:00

Discussion thread on cell division in Bacillus subtilis and Caulobacter crescentus
This is designed to help with an assignment.

Put your questions and findings in the comments please.

Cell division is one of the most rapidly developing and exciting areas of bacteriology today .

How can a novice find out about it and do literature research on the topic for a short review of what is currently known?

The first professional step is to find a high quality recent scientific mini-review in the literature.

First go the NCBI-PubMed literature search engine here.

(If in doubt google NCBI and press the PubMed button.)

Type in something like this,

cell division Caulobacter review

and press enter (or return).

A literature search output will appear on the screen. Be aware there are various options for display and textdownloading. The full summary option is most useful. Buying a cheapish USB thumb-drive to help harvest this information will be a good investment too.

You get this high up on the output list

Trends in Cell Biology
Volume 15, Issue 7 , July 2005, Pages 343-345

doi:10.1016/j.tcb.2005.05.002 Research Focus

Bacterial DNA segregation by the actin-like MreB protein

Thomas Kruse and Kenn Gerdes E-mail The Corresponding Author

Department of Biochemistry and Molecular Biology, Campusvej 55, DK-5230 Odense M, University of Southern Denmark, Denmark

Available online 25 May 2005.

Faithful chromosome segregation is vital to all organisms. Eukaryotic cells use the tubulin-based cytoskeleton to segregate their chromosomes during mitosis. A handful of papers have provided convincing evidence that, in bacteria, this task is accomplished by the actin homolog MreB. In particular, a recent study by Gitai et al. demonstrates that MreB specifically binds to and segregates the replication origin of the bacterial chromosome.

Before we go on, note that the PubMed outut for this article has a Related papers hyperlink button. Pressing it will virtually complete your assignment research for you.

Now go to a good library and search for the full copy of the journal article. An electronic version is best, as you can cut and paste your own notes onto your brand new thumdrive just as I do here.

In the mini-review full text article you find all sorts of goodies about Bacillus Caulobacter and MreB

(Note MreB has been mentioned in the lectures.)

Microbe Pundit found this :

Could MreB form a bacterial mitotic-like machine?

It is now clear that bacteria contain true homologs of both tubulin (FtsZ) and actin (MreB and ParM). Could cytoskeletal elements contribute to DNA segregation in bacteria? Indeed, the DNA segregation machinery encoded by the E. coli plasmid R1 specifies a simple prokaryotic analog of the eukaryotic spindle apparatus. The plasmid-encoded ParM protein, an actin homolog, forms F-actin-like filaments that are responsible for the active movement of plasmid copies to opposite cell poles [Ref] 5 and 6. The chromosome of rod-shaped bacteria encodes another actin homolog called MreB. The structure of monomeric MreB is very similar to yeast actin (Figure 1), thus placing the evolutionary root of actin in the prokaryotic domain [7].

[...]

In addition to its role in cell shape determination, results from several research groups have linked MreB to a function in chromosome segregation. Expression of mutant forms of MreB with impaired ATPase activity in otherwise wild-type E. coli produced rod-shaped cells with unevenly distributed and abnormal MreB filament morphologies, leaving parts of the cells with no detectable MreB signal. Under these circumstances, severe chromosome segregation defects were observed [10]. Consistently, depletion of MreB in both B. subtilis and C. crescentus leads to a rapid defect in chromosome segregation, where replication origins fail to localize in a regular bipolar fashion 17 and 18. Together with the observation that MreB forms dynamic filaments that move away from the mid-cell towards opposite cell poles in B. subtilis, these result indicate that MreB could be part of a mitotic machinery involved in chromosome segregation 4 and 13.

[...]
Inhibitor confirms role for MreB in chromosome segregation

Recent convincing evidence for a direct role of MreB in chromosome segregation in Caulobacter crescentus was presented by the Shapiro laboratory [19]. They used a small molecule, S-(3,4-dichlorobenzyl)isothiourea (A22), that was originally identified in a screen for compounds that induce anucleate E. coli cells and a change in cell morphology from the normal rod-shape to a spherical form [20]. When administered to Caulobacter cells, A22 mimics the effects of MreB depletion and causes a rapid and reversible disintegration of the MreB cables.

[...]

9 L.J. Jones et al., Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis, Cell 104 (2001), pp. 913–922.

11 R.M. Figge et al., MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus, Mol. Microbiol. 51 (2004), pp. 1321–1332.

I have bolded portions of the text which are pure gold nuggets for an assignment.
If I had time I would mention them in a lecture.

Note how two major concepts developed the classroom, MreB "cables" and chromosome movement/partition are connected to one-another by this review. This field is surely moving fast.

Note also the quality of writing and the currentness of the information in the mini-review. It is a fine example of an assigment that I would award an 110% grade (or more).

Using flow cytometry to study the bacterial cell cycle.

2006-03-06T17:28:00.000+11:00

Diagram of flow cytometry experimental set up from here (which has extensive notes on the theory and practice of flow cytometry).

Flow cytometry is a tool for making light scattering and fluorescence emission measurements on individual cells. The equipment can collect data from numerous individual cells passing through the detector and present the collected data graphically. Usually light scattering is measured as an indicator of cell size and fluorescence staining as a measure of some component, such as stained DNA or amount of bound antigen specific antibody that is detectable by fluorescence because it has been coupled chemically to a dye.

Chapter 9 of Schaechter presents data of this type in Figure 9.3.

Fig. 9.3 describes a bacterial culture (probably E. coli) that has been treated with antibiotic rifampin, and then allowed 90 minutes to complete DNA replication before being analysed by flow cytometry.

Study Questions:

What is the target of action of rifampin?

What is the basic model for chromosome replication in these cells?

How can cells have 4 chromosomes?

How do you think DNA content per cell was measured?

Making a Cell Part 2

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Adeninine related nucleotides including ATP, and NAD(P), shown here, are important coupling compounds in metabolism

One of the important roles of Fueling reactions is to generate chemical energy for cellular activities.

This energy is in the form of either ATP or PMF.

Energy sources ATP and PMF are intra convertible by cells.

This energy can be used for either Growth related activities or Growth independent activities.

Another major fueling reaction is production of reducing power (carried by NAD(P) as Hydrogen atoms attached the nicotinamide ring) which is also consumed in growth-related processes.

Study questions:

What are examples of non-growth related processes that consume energy?

Quantitatively speaking:

What are the main growth related processes that consume ATP?

What are the main growth related processes that consume reducing power?

Making a cell.

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The broad processes of bacterial metabolism (fueling, biosynthesis, polymerisation, assembly, and cell division) are interrelated by the fact that each requires as a starting material the products of the preceding phase.

Study Questions:
For each phase list the starting materials and products.
(No more than one phase answered per student, to spread out the activity.)

Could any phase be dispensible? If so under what circumstances?

Operation and assembly of the bacterial flagellum.

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Image from here.

Chapter three describes the three main parts of a flagellum.

Basal body, hook and filament.

The filament is hollow and made of flagellin, and rigid.The flagellin subunits assemble spontaneously to give the filament.

The hook is at the base of the filament and appears to act like a "universal joint" when the flagella is rotating.

The basal body is imbedded in the cell membranes, and include some 15 different proteins.

It acts as an electric rotating motor driven by PMF.

The flagellum is assembled in a stepwise fashion. First the basal body is inserted into the membrane, next the hook, finally the filament.

Units of flagellin reach the tip through its hollow centre.

Regulatory Loops.
In E. coli synthesis of flagellin subunits is coupled to their assembly at the tip by a special inhibitor of flagellin synthesis. When the basal body is inserted into the membrane it accomplishes secretion of this inhibitor into the growth medium which thus allows filament subunit synthesis to start.

Proteins needed to allow the flagella to rotate are added late in organelle assembly.

Study Questions:

What is PMF and where does it come from?

How were the stages of flagellum assembly first identified?

Protection of the cell membrane

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Protection of the cell membrane in Gram positive and Gram negative bacteria is a important topic that is discussed very well in Chapter 2 of Schaechter 2006.

How do these two different types of bacterial outer cell layers (G+ versus G-) exclude toxic hydrophobic compounds?

How do hydrophilic compounds less than 700 molecular mass enter Gram negative cells?

The Bacterial Cell Interior.

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Image from this site.

Chapter 3 of Microbe explains how the cell interior of bacteria is a very busy, crowded, and viscous place. A major component taking up space in this region is the nucleoid, and there is much more that can be said about it than there is space for in the chapter.

The DNA of the nucleoid is heavily folded and super-coiled. The DNA of Escherichia coli, if it were uncoiled, would be about 1000 times the length of the cell. Bacteria are thus very compact with genetic information storage.

An interesting point made by Schaechter in Chapter 3 that transcription of DNA into RNA only occurs at the nucleoid-cytosol interface, so organisation of the nucleoid's coiled domains is functionally important. Diagrams of this are presented in teaching sessions.

Recently, many dramatic advances have been made in scientific understanding of bacterial cytoplasmic filament proteins such as MreB and FtsZ , and these are already mentioned in chapter 2 of Schaechter.

Study Question:

How were MreB and FtsZ proteins first identified, and where do their names come from? Where are they located in the cell?

Prokaryotic cell envelopes and appendages.

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(EM thin section image from here.)

Chapter 2 of Microbe deals with the outer layers of bacterial cells. It explains how these are complex in structure.
Important points are
The composition, content and function of the cell membrane.
Protection of the cell membrane in Gram positive and Gram negative bacteria.
The operation and assembly of the flagellum

Other links with images
Cells alive

Study question:

How are bacterial flagella and pili (=fimbrae) different from one-another in terms of structure, mode of assembly, and function?

The World of Microbes . Question 2.

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In what ways have Bacteria and Archaea influenced the evolution of eukaryotes in the past?

How do they influence it in the present?

Discuss in the comments panel.

Microbe. The World of Microbes.

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Epulopiscium fishelsoni

As a start to this discussion forum, it's good to go to the website devoted to book Microbe that inspired this weblog. This website is here.

We are starting discussions with chapter 1 of this book- The World of Microbes.

Let's get started by posting a study question for discussion in the comments thread.

Question
Bacteria range in volume over a million-fold. Discuss some of the consequences of being larger or smaller than average?

Update of question and posting with more information to assist students. 4th March 2007
Here is some extra information that can assist students understand how size has implications for available membrane surface area to service the metabolic requirement of a unit cell volume.

To understand the point of the question about size, students need to think about have the ration of surface are:cell volume changes with increase in size. SA/Vol Ratio~diameter squared/diameter cubed~inversely proportional to diameter, with similar cell shapes.

To see some consequencences raised by the biology of large bacteria you need to read about observations that have been made on Epulopiscium bacteria, usually called epulos. The paper by K D Clements and S Bullivant (1991) An unusual symbiont from the gut of surgeonfishes may be the largest known prokaryote. J Bacteriol. 1991 September; 173(17): 5359–5362 provides some good interesting insights.

Figure 1 of this paper shows how large epulos are.
J Bacteriol. 1991 September; 173(17): 5359–5362. Figure 1. Light micrograph of an Epulo. The letter C indicates a smaller, eukaryotic ciliate.

J Bacteriol. 1991 September; 173(17): 5359–5362. Figure 3. Electron micrograph of a thin section of an Epulopiscium bacterium showing concolutions to cytoplasmic membrane. Such convolutions would increase the membrane area to cell volume ratio of these large bacteria

Thus epulos have a peripheral layer of highly convoluted cytoplasmic membrane - which has been interpreted as a mechanism increase in membrane area to compensate for some surface area to volume related challenges that they face because of their large size.

Extra reading for the high achieving student:
In the links at the Microbe Chapter 1 webpage, this is worth studying several times in the course.