MicrobiologyBytes

How proteins and nucleic acids assemble, often spontaneously, into structurally well-defined three-dimensional objects is an intriguing question. The limited size of the phage genome and the multicomponent composition of bacteriophages make them well suited for assembly investigations. Genetic manipulation of phages has made it easy to observe the effects of gene inactivation on protein-protein association, providing information on the sequence of assembly processes. Over the past fifty years, mutational, biochemical and biophysical analyses, X-ray crystallography, NMR, cryo-electron microscopy (cryo-EM), thin sectioning and single molecule methods have been used to study bacteriophages. This review describes what has been achieved and contemplates what still needs to be accomplished, focusing mostly on dsDNA tailed phages.

Bacteriophage Assembly. Viruses 2011, 3(3), 172-203; doi:10.3390/v3030172
Bacteriophages have been a model system to study assembly processes for over half a century. Formation of infectious phage particles involves specific protein-protein and protein-nucleic acid interactions, as well as large conformational changes of assembly precursors. The sequence and molecular mechanisms of phage assembly have been elucidated by a variety of methods. Differences and similarities of assembly processes in several different groups of bacteriophages are discussed in this review. The general principles of phage assembly are applicable to many macromolecular complexes.

It is well established that the genomes of many dsDNA tailed bacteriophages have mosaic relationships, where the mosaicism is defined as patchy sequence similarity when two related phage genomes are compared. The regions with different extents of similarity have been interpreted to be genome sections with different evolutionary histories that have been horizontally exchanged among phages. Such exchangeable genome segments have been called “modules”, and these modules are hypothesized to be minimal autonomously functional units, such as groups of genes that must function together or single proteins or even protein domains that function independently. Phage genome mosaicism can range from quantitative differences in the extent of sequence similarity between homologous regions to parallel non-homologous genome sections that encode completely different proteins.

A quantitative understanding of the extant diversity, exchange rates and precise boundaries of these genetic “modules” has remained elusive. Recent advances in the ease of DNA sequence determination have resulted in a rapid increase in the number of phage genome sequences available, and these provide an opportunity for much more detailed and robust analyses of phage genome mosaicism. This paper focuses on the virion assembly genes of fifty-seven different of phages that are “closely” related to Salmonella enterica phage P22. Analysis of such a large number of unambiguously orthologous gene sets is powerful, and analysis of phage P22 is particularly informative because of the extensive body of experimental work on phage P22 virion structure and the specific roles and interactions of its morphogenetic proteins.

Evolution of mosaically related tailed bacteriophage genomes seen through the lens of phage P22 virion assembly. Virology. 2011 411(2): 393-415
The mosaic composition of the genomes of dsDNA tailed bacteriophages (Caudovirales) is well known. Observations of this mosaicism have generally come from comparisons of small numbers of often rather distantly related phages, and little is known about the frequency or detailed nature of the processes that generate this kind of diversity. Here we review and examine the mosaicism within fifty-seven clusters of virion assembly genes from bacteriophage P22 and its “close” relatives. We compare these orthologous gene clusters, discuss their surprising diversity and document horizontal exchange of genetic information between subgroups of the P22-like phages as well as between these phages and other phage types. We also point out apparent restrictions in the locations of mosaic sequence boundaries in this gene cluster. The relatively large sample size and the fact that phage P22 virion structure and assembly are exceptionally well understood make the conclusions especially informative and convincing.

Bacteriophages are the most numerous biological entities in the biosphere, with an estimated 10³¹ particles. The global population is highly dynamic with an estimated 10²³ phage infections per second, and has likely been evolving for perhaps two to four billion years. Not surprisingly, this has given rise to a genetically highly diverse population. Most bacteriophages do not extend their host range beyond a single bacterial genus, and host specificity likely offers a substantial impediment to the free exchange of genetic material between phages of different bacterial hosts. Consequently, it is unusual to find extensive nucleotide sequence similarity among phages of different hosts; such phages often share few if any genes identifiable through amino acid sequence comparisons.

Remarkably, phages capable of infecting a single bacterial species can also be highly diverse, as are for example the genetically distinct DNA phages of Escherichia coli, such as φX174, M13, lambda, T1, T4, T5, and T7. This is further exemplified with the mycobacteriophages – viruses infecting mycobacterial hosts – of which sixty-two genomes of phages known to infect Mycobacterium smegmatis mc2155 have been sequenced. All of these are dsDNA tailed phages, restricted to two morphotypes, the Siphoviridae and the Myoviridae.

This paper describes the isolation and characterization of new mycobacteriophages by student microbiologists, a valuble contribution to our understanding of these organisms.

Expanding the Diversity of Mycobacteriophages: Insights into Genome Architecture and Evolution. (2011) PLoS ONE 6(1): e16329. doi:10.1371/journal.pone.0016329
Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Bacteria constantly encounter numerous enemies in microbial communities. For example, ubiquitous bacteriophages, i.e. parasitic viruses that replicate within bacterial cells, can effectively constrain bacterial survival in nature. A wide array of laboratory experiments has shown that bacteriophages can drive rapid bacterial evolution by imposing strong selection for phage-resistant bacteria. Furthermore, phage-resistance has been shown to correlate negatively with some other bacterial life-history traits, such as growth efficiency and motility, which both are important traits for bacterial pathogenicity. Motility, for example, helps pathogens colonise suitable niches within the host, while growth efficiency can determine how fast bacteria can exploit their hosts. If phage-resistance leads to trade-off with bacterial virulence factors phages could potentially select for lowered bacterial pathogenicity in environmental reservoirs.

This study investigates how parasitic phages and thermal environment affect the evolution of bacterial pathogenicity traits in vitro, and how these changes correlate with bacterial virulence in vivo.

High Temperature and Bacteriophages Can Indirectly Select for Bacterial Pathogenicity in Environmental Reservoirs. (2011) PLoS ONE 6(3): e17651. doi:10.1371/journal.pone.0017651
The coincidental evolution hypothesis predicts that traits connected to bacterial pathogenicity could be indirectly selected outside the host as a correlated response to abiotic environmental conditions or different biotic species interactions. To investigate this, an opportunistic bacterial pathogen, Serratia marcescens, was cultured in the absence and presence of the lytic bacteriophage PPV (Podoviridae) at 25°C and 37°C for four weeks (N = 5). At the end, we measured changes in bacterial phage-resistance and potential virulence traits, and determined the pathogenicity of all bacterial selection lines in the Parasemia plantaginis insect model in vivo. Selection at 37°C increased bacterial motility and pathogenicity but only in the absence of phages. Exposure to phages increased the phage-resistance of bacteria, and this was costly in terms of decreased maximum population size in the absence of phages. However, this small-magnitude growth cost was not greater with bacteria that had evolved in high temperature regime, and no trade-off was found between phage-resistance and growth rate. As a result, phages constrained the evolution of a temperature-mediated increase in bacterial pathogenicity presumably by preferably infecting the highly motile and virulent bacteria. In more general perspective, our results suggest that the traits connected to bacterial pathogenicity could be indirectly selected as a correlated response by abiotic and biotic factors in environmental reservoirs.

Bacteriophages represent one of the most abundant biological entities in nature and have long been recognized for their potential use as therapeutic agents. In recent years overprescription of antibiotics and the concomitant development of antibiotic-resistant ‘super-bugs’ have highlighted the need for alternative strategies to combat infectious diseases. Consequently, a lot of phage research in the past two decades was aimed at assessing whether phage can be used to eliminate undesirable bacteria. Traceability is a requirement in modern food production, incorporating every step in the production process, commonly known as the ‘farm to fork’ concept (European Commission White paper on Food Safety, January 2000). Phages are omnipresent and are accidentally, yet regularly, consumed through ingestion of water and food. For this reason they are presumed to be safe as undesirable effects have not been reported. This, together with their specificity, makes them excellent tools for food safety purposes.

The ‘farm to fork’ concept identifies quality assurance steps at which bacterial contamination may occur, and which also represent critical points where phage treatments may be applied. The most frequently encountered food pathogens belong to one of the four dominant genera, Salmonella, enterotoxigenic Escherichia coli, Campylobacter and Listeria, along with less common infections by Clostridium spp., Staphylococcus aureus, Streptococcus suis and Cronobacter sakazakii. Phages targeting strains of each of these species have been identified and this review discusses the pros and cons of the use of phages as biocontrol, biosanitation and detection agents.

Bacteriophages as biocontrol agents of food pathogens. Curr Opin Biotechnol. Nov 4 2010
Bacteriophages have long been recognized for their potential as biotherapeutic agents. The recent approval for the use of phages of Listeria monocytogenes for food safety purposes has increased the impetus of phage research to uncover phage-mediated applications with activity against other food pathogens. Areas of emerging and growing significance, such as predictive modelling and genomics, have shown their potential and impact on the development of new technologies to combat food pathogens. This review will highlight recent advances in the research of phages that target food pathogens and that promote their use in biosanitation, while it will also discuss its limitations.

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There are estimated to be on the order of 10¹⁰ phage per liter of sea water and roughly 10²⁴ phage infections per second. Despite the frequency of phage infection and its ecological importance, relatively little is known about the molecular mechanics of the infection process. Questions such as what provides the driving force for genome exit from the capsid, what signals the conduit to open to allow exit and how the nucleic acid enters the host cell during infection remain unanswered. However, advances in electron microscopy and image analysis are allowing us to capture a glimpse of this remarkable process. The T7-like podovirus P-SSP7 infects Prochlorococcus marinus, the most abundant photosynthetic microorganism. A recent cryo-electron microscopy study provides insight into the molecular details of the P-SSP7 infection process, and given the similarity between P-SSP7 and other podoviruses is likely to provide a paradigm for understanding the process of phage infection.

Mind the Gap: How Some Viruses Infect Their Hosts. (2010) Viruses 2(11): 2536-2540; doi:10.3390/v2112536
Cryo-electron microscopy (Cryo-EM) and cryo-electron tomography (Cryo-ET) provide structural insights into complex biological processes. The podoviridae are dsDNA containing phage with short, non-contractile tails which nevertheless translocate their DNA into the cytoplasm of their host cells. Liu et al. [1] used a combination of cryo-EM and cryo-ET to study the structural changes accompanying infection of P. marinus by the phage P-SSP7 and thereby provide unique molecular insight into the process by which the DNA transits from phage to host during infection.

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• Modelling bacteriophage
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• Bacteriophage resistance

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

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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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