Communicating with electricity | Lab Rat, Scientific American Blog Network

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.

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.

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

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.

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

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.

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

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

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!

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.