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

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.

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?

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.

1. Habitat
2. Life-cycle
3. Cell fusion between different vegetative hyphae
4. Presence of conjugative plasmids
5. Nutrition and nutrient capture
   
6. Chromosomal structure
7. QS
8. Membrane physiology
9. Stress - UV exposure, dehydration
10. Aerial hyphae
11. Spore survival

Good luck.

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

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.

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:

1. 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.
2. 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.
3. 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

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.

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.

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.