MicrobiologyBytes

Pathogenicity islands have a major role in spreading virulence genes among bacterial populations. A notable example are the phage-related pathogenicity islands of staphylococci, the SaPIs, which are responsible for the inter- as well as intrageneric spread of toxins – such as TSST-1 (toxic shock syndrome toxin) and other superantigens – through the exploitation of specific staphylococcal helper phages for high-frequency transfer within phage-encoded particles. Toxic shock syndrome is a rare, potentially fatal illness that can be caused by the release of toxins from Staphylococcus. The toxic particles are encoded by discrete genetic units called pathogenicity islands, which reside passively in the host chromosome, under the control of the global repressor Stl, unless activated by a helper phage. This paper shows that a non-essential and specific protein from the helper phage 80α is responsible for de-repression of the pathogenicity island, providing the mechanism for the first step of its mobilization. The proteins involved are ‘moonlighters’, because they have two different and genetically distinct activities. Through a remarkable evolutionary adaptation, various related pathogenicity islands co-opt entirely unrelated phage proteins to aid in their mobilization.

Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands. 2010 Nature. 465(7299): 779-782
Staphylococcal superantigen-carrying pathogenicity islands (SaPIs) are discrete, chromosomally integrated units of ~15 kilobases that are induced by helper phages to excise and replicate. SaPI DNA is then efficiently encapsidated in phage-like infectious particles, leading to extremely high frequencies of intra- as well as intergeneric transfer. In the absence of helper phage lytic growth, the island is maintained in a quiescent prophage-like state by a global repressor, Stl, which controls expression of most of the SaPI genes4. Here we show that SaPI derepression is effected by a specific, non-essential phage protein that binds to Stl, disrupting the Stl–DNA complex and thereby initiating the excision-replication-packaging cycle of the island. Because SaPIs require phage proteins to be packaged, this strategy assures that SaPIs will be transferred once induced. Several different SaPIs are induced by helper phage 80α and, in each case, the SaPI commandeers a different non-essential phage protein for its derepression. The highly specific interactions between different SaPI repressors and helper-phage-encoded antirepressors represent a remarkable evolutionary adaptation involved in pathogenicity island mobilization.

Related:

• Genomic islands in bacterial evolution
• Spread of Pathogenicity Islands in Escherichia coli

Phages are now acknowledged as the most abundant microorganisms on the planet and are also possibly the most diversified. This diversity is mostly driven by their dynamic adaptation when facing selective pressure such as phage resistance mechanisms, which are widespread in bacterial hosts. When infecting bacterial cells, phages face a range of antiviral mechanisms, and they have evolved multiple tactics to avoid, circumvent or subvert these mechanisms in order to thrive in most environments. This review highlights the most important antiviral mechanisms of bacteria as well as the counter-attacks used by phages to evade these systems.

Bacteriophage resistance mechanisms. Nature Reviews Microbiology 8: 317-327 (2010). doi:10.1038/nrmicro2315

Related:

• Coevolution with viruses drives bacterial mutations

Phage lambda is one of the key model organisms on which molecular biology was built. Such a wealth of existing knowledge on this organism makes it an ideal test model for systems biology. In this article in Microbiology Today (pdf) Rosalind Allen explores the usefulness of this bacteriophage to systems biology and vice versa:

Phage lambda, discovered in 1950, infects sensitive strains of E. coli. For a genome of only 48.5kb, packed into a particle of only 50nm diameter, this phage has attracted a lot of attention. Phage lambda became one of the key model organisms on which modern molecular biology was built. Over a period of intense study lasting 40 years, the genes in the phage lambda genome, the proteins encoded by them and the interactions between these genes and proteins were investigated in great detail. The humble phage lambda was the source of discoveries such as repression and activation mechanisms for gene regulation, chaperone proteins, DNA recombination and restriction enzymes, which microbiologists and many others now take for granted. This huge body of knowledge makes it the ideal test case for systems biology modelling.

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• Bugs Against Biofilms
• Bacteriophages for plant disease control
• Bacteriophage lysogeny

Bacteriophage lysis is a precisely scheduled process controlled by proteins of the holin family. Holins are a diverse class of small phage-encoded membrane proteins. The best studied holin is S105, a 105-residue polypeptide with three transmembrane domains (TMDs) encoded by the S gene of phage λ. Throughout the period of late gene expression and particle assembly, S105 accumulates in the cytoplasmic membrane of Escherichia coli without any effect on its integrity. Suddenly, at a programmed time, S105 triggers to form a lesion, or hole, in the membrane; this allows the λ-endolysin, R, to escape from the cytoplasm and attack the cell wall. In phages of Gram-negative hosts, there is a third step to complete the lysis pathway involving a protein or protein complex, the spanin, which connects the cytoplasmic and outer membranes. In λ, the spanin complex consists of the cytoplasmic membrane protein, Rz, and the outer membrane lipoprotein, Rz1. This complex is thought to act by disrupting the outer membrane, possibly by fusion with the inner membrane. The large holes observed can be viewed as supporting the notion that at the time of lethal triggering, the S105 holin exists in such large aggregates, leading to one or a small number of holes rather than many smaller holes distributed throughout the membrane.

Micron-scale holes terminate the phage infection cycle. PNAS USA January 11 2010. doi: 10.1073/pnas.0914030107
Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the phage infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or “holes.” In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.

Related:

• How can lytic viruses be evolutionarily competitive?

E. coli has been in the media a lot recently (Latest News), so MicrobiologyBytes thinks it’s time for:

10 things you should know about E. coli O157:

1. Escherichia coli (E. coli) is a normal inhabitant of the human gut. It’s been with us for millions of years and overall does us a lot of good, e.g. helping with digestion and providing vitamins we can’t make for ourselves.

2. There are many different strains of E. coli, which all look much alike. They are identified by the antigens on the surface of the cell. These include somatic (O antigens) on the surface of the cell, flagellar (H antigen) and capsular (K antigens) associated with polysaccharide capsules on some strains.

3. A few strains of E. coli are pathogenic and cause disease. Enterotoxigenic (ETEC) strains cause diarrhea but are non-invasive and do not leave the intestine. Enteropathogenic (EPEC) strains also cause diarrhea and enter epithelial cells around the intestine. Enteroinvasive (EIEC) strains cause severe diarrhea and high fever. Enterohemorrhagic (EHEC) strains such as E. coli O157:H7 cause bloody diarrhea, hemolytic-uremic syndrome and kidney failure.

4. E. coli O157:H7 infections often case to bloody diarrhea and occasionally acute kidney failure, especially in young children and elderly people.

5. Most infections are associated with eating undercooked, contaminated ground beef (e.g. burgers), drinking unpasteurized milk, swimming in or drinking contaminated water, and eating contaminated salad vegetables. Infection can also be aquired via direct contact with animal faeces, for example on farms.

6. A bit of dirt never did me any harm… E. coli O157:H7 is new. It was first recognized around 25 years ago and is now widespread, possibly due to agricultural practices.

7. Where did it come from? This strain of E. coli contains lysogenic bacteriophages which encode Shiga toxins (these strains are known as STECs: Shiga Toxin Producing Escherichia coli). E. coli O157:H7 has two stx toxins, stx1 and stx2.

8. How does it cause disease? E. coli O157:H7 is an EHEC strain which kills epithelial cells in the gut, resulting in bloody diarrhea. It also invades the urinary tract causing an ascending infection which damages the kidneys. But it gets worse. Broad spectrum fluoroquinolone antibiotics such as ciprofloxacin which are often used to treat infections cause an SOS response in E. coli cells which in turn induces the lytic cycle of the lysogenic toxin-carrying phages. This results in a thousand-fold increase in toxin expression. Treatment with some some beta-lactam antibiotics also increase stx toxin production.

9. Many people recover without antibiotics or other specific treatment in 5–10 days. There is no clinical evidence that antibiotics improve the course of disease, and some may make it much worse (see above). Haemolytic-uremic syndrome is a life-threatening condition usually treated in an intensive care unit. Blood transfusions and kidney dialysis are often required. Even with intensive care, the death rate for haemolytic uremic syndrome is 3%–5%.

10. Wash your hands thoroughly with soap and warm water after contact with animals. Wash raw vegetables such as salads well before eating. Cook meat thoroughly all the way through, especially burgers and sasuages where external contamination of meat is transferred to the inside by mincing.

11. E. coli is a Gram-negative bacterium. IT’S NOT A VIRUS! So next time a journalist talks or writes about “the E. coli virus” – do us all a favour and yell at them!

Related:

• The continuing evolution of E. coli O157:H7
• E. coli O157:H7 – getting to the bottom of the burger bug
• Bacterial toxins

The force of Bacillus anthracis, the ancient scourge that causes anthrax, can sweep through and overpower a two-ton animal in under 72 hours. But when it isn’t busy claiming livestock and humans throughout the world – up to 100,000 annually – it resides ominously in the soil as a spore waiting for its next victim. Researchers now reveal that this deadly bacterium isn’t the only master of its fate. Its survival is directed and shaped by the DNA of bacteria-infecting viruses in what appears to be an evolutionary contract written to benefit both parties. The research revamps the way scientists think about how pathogens exist in the environment in between outbreaks, focusing on the role viruses play during this dormant stage in the life cycle. The implications reach far and wide, from the sequencing of genomes to the recurrent and cyclical nature of disease.

B. anthracis leads a much more complicated life than we had ever known. Small, infecting viruses dramatically alter the survival capabilities of B. anthracis. It is more or less a symbiotic relationship in which the interests of both the bacterium and virus are kept in balance. The secret life of anthrax-causing bacteria emerged from a seemingly innocuous observation made by Louis Pasteur more than 100 years ago. The famous bacteriologist found that earthworms were associated with anthrax-infected animal carcasses in the ground and hypothesized that the earthworm could play an important role in the life cycle of the deadly pest. For the first time, scientists have now confirmed Pasteur’s early hunch. They found that in the gut of the earthworm, B. anthracis infected with bacteriophages live longer than virus-free bacteria. The gut of the earthworm provides the infected bacteria with a safe niche in which to exist.

The researchers further show that in both the gut of the earthworm and the stark confines of a Petri dish, viruses can alter the lifestyle of B. anthracis in two principal ways. One is associated with the ability to build communities, the state in which bacteria prefer to live in the environment; the other affects the bacterium’s ability to produce spores: round, dormant cells with a thick cell wall that enables them to endure harsh environmental conditions that the rod-shaped bacteria cannot. What is more, they found that depending on the conditions of the environment, the virus’s DNA manipulates the bacterium’s genome to toggle between spore production and community building. The relationship appears to result from some sort of evolutionary contract that keeps the interests of bacterium and virus in balance. Since viruses cannot infect and grow in spores, they have an interest in silencing genes that ramp up spore production and in activating genes that help build B. anthracis communities. But when soil conditions threaten the survival of anthrax-causing bacteria, spawning a tougher line of defense to weather the soil’s extreme conditions benefits both parties.

The unveiling of the bacterium’s life cycle opens up completely new strategies to combat anthrax infection. In it was shown that infected anthrax-causing bacteria become more resistant to a natural antibiotic found in the soil. The new studies now go further, showing how these survival capabilities are not just affected by bacteriophages but actually depend on them. Bacteriophages exert their control via molecules known as sigma factors, which delegate proteins to turn specific host genes on or off. Different viruses encode different sigma factors, so the appearance of different traits depends on which virus infects the bacterium. While the DNA of some bacteriophages gets incorporated into the bacterium’s single chromosome, the DNA of others exists as separate circular entities called episomes. These episomes can either stay inside one bacterium or flit in and out, infecting several bacteria in a matter of hours. The finding has implications for the sequencing of genomes. There are more than 1,000 known isolates of anthrax and there is little genetic variation between one isolate and the next. But the phage DNA, which works together with the anthrax genome, has been overlooked. If bacteriophages can govern the fate of bacteria and bacteria affect human health, the transformation of these bacteria may be able to explain the recurrent and cyclical nature of certain diseases. Humans have 10 times more bacteria on them or in them than the number of human cells. And there are 10 times more bacteriophages than there are bacteria.

The Secret Life of the Anthrax Agent Bacillus anthracis: Bacteriophage-Mediated Ecological Adaptations. 2009 PLoS ONE 4(8): e6532. doi:10.1371/journal.pone.0006532
Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.

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• A bacteriophage contributes to the pathogenicity of the plague bacillus
• Bacteriophage lysogeny
• Novel method predicts impact of anthrax release
• Incidence of bacteriophage lysogeny in temperate and extreme soil environments

A recent paper in Trends in Microbiology examined the possibilities for using bacteriophages in controlling biofilms which might result in healthcare-associated infections (Preventing biofilms of clinically relevant organisms using bacteriophage. 2009 Trends in Microbiology 17: 66-72). Biofilms are firmly attached microbial communities in which the organisms produce an extracellular polymeric matrix. Biofilm organisms might cause disease by detachment of individual cells or clumps of cells, by production of endotoxin, or by providing a niche for the development of antibiotic-resistant organisms. Biofilm organisms are usually tolerant to antimicrobial agents and the treatment of indwelling medical device-associated infections with systemic antimicrobial agents is usually ineffective.

Bacteriophages have been used for the treatment of infectious diseases in plants and animals, although few clinical trials with stringent negative controls have been carried out. There is a renewed interest in phage therapy in light of growing concerns with antimicrobial resistance in healthcare institutions worldwide. The use of phages for the treatment of device-associated infections could reduce the use of antibiotics and might limit the spread of resistant organisms.

There is some evidence for the potential of phages in biofilm control. Bacteriophage T4 phage was effective against E. coli biofilms in a glucose-limited chemostat, although the rate of phage synthesis and assembly were directly proportional to the amount of protein synthesis in the host cell. Some phages produce polysaccharide depolymerases that have the potential to degrade the biofilm matrix.

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However, there are several important characteristics of phage that should be considered when evaluating the potential of phages to control clinically relevant biofilms. The specificity of receptors for a single phage strain will determine its host range; some phage have specificity at the strain level, such as strain typing phages, whereas some are more broad-spectrum and can infect multiple strains or related species. This high degree of specificity could be a drawback, especially in the case of polymicrobial biofilms.

Few studies have explored the role of biofilms in the development of phage-resistance. Phage cocktails developed via the isolation of host range mutants and broad spectrum phage could be advantageous. Another important question is how a patient’s immune system will respond to the therapeutic introduction of phage. Phages are antigenic and elicit a response by serum antibodies and the cellular immune system. Repeated exposure to the phage results in increasing antibody titers and studies in animal models have shown that phage is cleared from the bloodstream by the cellular immune system. In a short-lived treatment, the antibody response to phage is weak except in cases were serum antibody titers are present before phage treatment. It is possible, although unproven, that phage might associate with biofilms and thereby be protected from inactivation. It might also be possible to design mutant phages with enhanced ability to resist clearance by the cellular immune system.

Related:

• Biofilms
• Saturday Cimema: Bacteriophage Therapy
• Bacteriophages for plant disease control
• Coevolution with viruses drives bacterial mutations

A surprising example of interspecies competition is the production by certain bacteria of hydrogen peroxide at concentrations that are lethal for others. A case in point is the displacement of Staphylococcus aureus by Streptococcus pneumoniae in the nasopharynx, which is of considerable clinical significance. How it is accomplished, however, has been a great mystery, because H₂O₂ is a very well known disinfectant whose lethality is largely due to the production of hyperoxides through the abiological Fenton reaction. In this report, we have solved the mystery by showing that H₂O₂ at the concentrations typically produced by pneumococci kills lysogenic but not nonlysogenic staphylococci by inducing the SOS response. The SOS response, a stress response to DNA damage, not only invokes DNA repair mechanisms but also induces resident prophages, and the resulting lysis is responsible for H₂O₂ lethality. Because the vast majority of S. aureus strains are lysogenic, the production of H₂O₂ is a very widely effective antistaphylococcal strategy. Pneumococci, however, which are also commonly lysogenic and undergo SOS induction in response to DNA-damaging agents such as mitomycin C, are not SOS-induced on exposure to H₂O₂. This is apparently because they are resistant to the DNA-damaging effects of the Fenton reaction. The production of an SOS-inducing signal to activate prophages in neighboring organisms is thus a rather unique competitive strategy, which we suggest may be in widespread use for bacterial interference. However, this strategy has as a by-product the release of active phage, which can potentially spread mobile genetic elements carrying virulence genes.

Killing niche competitors by remote-control bacteriophage induction. PNAS USA January 13, 2009

Related:

• Pneumococcus: the quorum-sensing killer
• New Insights Could Lead to a Better Pneumococcal Vaccine
• Bacteriophage lysogeny
• Incidence of bacteriophage lysogeny in temperate and extreme soil environments

Huge numbers and variety of bacteriophages exist on our planet. In this article in Microbiology Today, Graham Hatfull describes this massive reservoir of unidentified genetic information:

You may well be under the impression that the largest number of undiscovered species – and the greatest pool of unknown genes – lie within the considerable biodiversity of the tropical rain forests. Not so. A compelling argument can be made that the biggest reservoir of unidentified genetic information is all around us, in the global population of bacteriophages.

Read more

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• MicrobiologyBytes: Bacteriophages
• Bacteriophages for plant disease control
• 5500 Phages examined in the electron microscope
• Bacteriophage infection at the poles