MicrobiologyBytes

Microbial ecologists have devoted considerable effort to understanding the nature of the viruses in seawater, because viruses have key roles in the evolution, ecology and mortality of marine plankton. For at least the past two decades, researchers have assumed that the pool of viruses in the ocean is dominated by bacteriophages with DNA genomes. Perhaps as a consequence, studies of the molecular diversity of marine viruses have most commonly focused on DNA viruses. However, evidence that RNA viruses are important contributors to marine plankton ecology has been steadily accumulating.

A recent paper shows that there are a large number of RNA viruses in surface ocean waters, and concludes that RNA viruses made up between 38 and 63% of the viruses in the sea water. In other words, about half of the viruses in the ocean (or at least, off Hawaii, where such fieldwork is most fun) are RNA viruses, suggesting that our current guess at the total number of viruses on earth, 10³¹, could be a major under estimate.

Are we missing half of the viruses in the ocean? (2013) ISME Journal 7, 672–679 doi: 10.1038/ismej.2012.121
Viruses are abundant in the ocean and a major driving force in plankton ecology and evolution. It has been assumed that most of the viruses in seawater contain DNA and infect bacteria, but RNA-containing viruses in the ocean, which almost exclusively infect eukaryotes, have never been quantified. We compared the total mass of RNA and DNA in the viral fraction harvested from seawater and using data on the mass of nucleic acid per RNA- or DNA-containing virion, estimated the abundances of each. Our data suggest that the abundance of RNA viruses rivaled or exceeded that of DNA viruses in samples of coastal seawater. The dominant RNA viruses in the samples were marine picorna-like viruses, which have small genomes and are at or below the detection limit of common fluorescence-based counting methods. If our results are typical, this means that counts of viruses and the rate measurements that depend on them, such as viral production, are significantly underestimated by current practices. As these RNA viruses infect eukaryotes, our data imply that protists contribute more to marine viral dynamics than one might expect based on their relatively low abundance. This conclusion is a departure from the prevailing view of viruses in the ocean, but is consistent with earlier theoretical predictions.

Pelagibacter ubique is the most successful member of a group of bacteria called SAR11, that jointly constitute about a third of the single-celled organisms in the ocean. But this is not P. ubique’s only claim to fame, for unlike almost every other known cellular creature, it and its relatives have seemed to be untroubled by viruses. But four viruses that parasitise P. ubique have now neen found, and one called HTVC010P was the commonest. It thus displaces its host as the likely winner of the most-common-living-thing prize.

Abundant SAR11 viruses in the ocean. (2013) Nature. doi: 10.1038/nature11921 http://goo.gl/iXVyF

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Phage and their bacterial hosts are the most abundant and genetically diverse group of organisms on the planet. Given their dominance, it is no wonder that many recent studies have found that phage-bacteria interactions strongly influence global biogeochemical cycles, incidence of human diseases, productivity of industrial microbial commodities, and patterns of microbial genome diversity. Unfortunately, given the extreme diversity and complexity of microbial communities, traditional analyses fail to characterize interaction patterns and underlying processes.

Despite increasing recognition that phages play a significant role in shaping microbial ecosystems, fundamental questions remain unanswered. Quantifying who infects whom is essential to understand how infections at the cellular level (such as changes to metabolic rates, gene transfer, and the fate of cells) scale-up to influence ecosystem function in complex environments. This paper reviews systems approaches that combine empirical data with rigorous theoretical analysis to study phage-bacterial interactions as networks rather than as coupled interactions in isolation, and highlights the ways in which a better understanding of phage–bacteria infection networks will aid predictive models of viral effects on microbial communities, from microbiomes to the whole Earth.

Phage-bacteria infection networks. (2012) Trends Microbiol. doi: 10.1016/j.tim.2012.11.003

Molecular piracy is a biological phenomenon in which one replicon (the pirate) uses the structural proteins encoded by another replicon (the helper) to package its own genome and thus allow its propagation and spread. Such piracy is dependent on a complex web of interactions between the helper and the pirate that occur at several levels, from transcriptional control to macromolecular assembly. The best characterized examples of molecular piracy are from the E. coli P2/P4 system and the S. aureus SaPI pathogenicity island/helper system. In both of these cases, the pirate element is mobilized and packaged into phage-like transducing particles assembled from proteins supplied by a helper phage that belongs to the Caudovirales order of viruses (tailed, dsDNA bacteriophages).

This review summarizes and compares the processes that are involved in molecular piracy in these two systems.

Pirates of the Caudovirales. Virology. 03 Nov 2012. doi: 10.1016/j.virol.2012.10.028

Two recent interesting papers from Virology:

Tools from viruses: Bacteriophage successes and beyond. Virology. 11 Oct 2012, pii: S0042-6822(12)00458-8. doi: 10.1016/j.virol.2012.09.017
Viruses are ubiquitous and can infect any of the three existing cellular lineages (Archaea, Bacteria and Eukarya). Despite the persisting negative public perception of these entities, scientists learnt how to domesticate some of them. The study of molecular mechanisms essential to the completion of viral cycles has greatly contributed to deciphering fundamental processes in biology. Nowadays, viruses have entered the biotechnological era and numerous applications have already been developed. Viral-derived tools are used to manipulate genetic information, detect, diagnose, control and cure infectious diseases, or even design new structural assemblies. With the recent advances in the field of metagenomics, an overwhelming amount of information on novel viruses has become available. As current tools have been historically developed from a limited number of viruses, the potential of discoveries from new archaeal, bacterial and eukaryotic viruses may be limited only by our understanding of the multiple facets of viral cycles.

What is needed for phage therapy to become a reality in Western medicine? Virology. 08 Oct 2012,pii: S0042-6822(12)00456-4. doi: 10.1016/j.virol.2012.09.015
The current status of phage therapy approaches is reviewed and possible hurdles to a practical medical application of bacteriophages in Western countries are identified as discussed at a recent EMBO meeting on “Viruses of Microbes” in Brussels. In view of the growing antibiotic resistance crisis, a coordinated effort by the public health sector is needed to evaluate the potential of phage therapy as an adjunct to antibiotics.

Bacteriophages, particularly the tailed bacteriophages, are the most promiscuous organisms known, playing a continual mix and match with their genes. This paper describes a novel phage genome architecture where one phage genome nestles inside the genome of another phage, similar to a “Russian Doll” arrangement.

Novel Bacteriophages Containing a Genome of Another Bacteriophage within Their Genomes. (2012) PLoS ONE 7(7): e40683. doi:10.1371/journal.pone.0040683
A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 3′ protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein-coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit) and B. cereus AH676 (additional regulatory and recombination genes), respectively. The third region represents an almost complete genome (except for the short terminal segments) of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a ‘fast track’ route of virus evolution and horizontal gene transfer. Another phage (BceA1) nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri.

Traditional approaches to phage therapy rely on the ability of viruses to kill their bacterial prey. However, the narrow host range or most bacteriophages and the ability of bacteria to become resistant to infection mean that in practice, using phage to simply replace antibiotics is not feasible. We need smarter approaches, which is where a recent paper comes in. Using phages to engineer sensitivity to antibiotics is a promising approach, but whether this proof-of-principle experiment ever makes it to the clinic is another matter.

Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. (2011)Appl. Environ. Microbiol. 23 Nov 2011 doi: 10.1128/AEM.05741-11
Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that will resensitize pathogens to approved antibiotics therefore holds key advantages. We present a proof-of-principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages encoding the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene’s sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage’s ability to kill pathogens in the infected host, but instead, to deliver genetic constructs into the bacteria, and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phages will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

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This review gives an overview of the current state-of-the-art of bacteriophage therapy and discusses the challenges to be overcome before phage therapy can become a part of Western medicine.

The next generation of bacteriophage therapy Curr Opin Microbiol. (2011)14(5): 524-531
Bacteriophage therapy for bacterial infections is a concept with an extensive but controversial history. There has been a recent resurgence of interest into bacteriophages owing to the increasing incidence of antibiotic resistance and virulent bacterial pathogens. Despite these efforts, bacteriophage therapy remains an underutilized option in Western medicine due to challenges such as regulation, limited host range, bacterial resistance to phages, manufacturing, side effects of bacterial lysis, and delivery. Recent advances in biotechnology, bacterial diagnostics, macromolecule delivery, and synthetic biology may help to overcome these technical hurdles. These research efforts must be coupled with practical and rigorous approaches at academic, commercial, and regulatory levels in order to successfully advance bacteriophage therapy into clinical settings.

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Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. A new paper has examined the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. The authors set out to find out how many protein-protein interactions remain to be found in this phage.

In order to map lambda’s interactions, they cloned 68 out of 73 lambda open reading frames (the “ORFeome”) into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions, including 16 out of 30 previously published interactions (53%). They also also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda.

Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins.

The protein interaction map of bacteriophage lambda. BMC Microbiology 2011, 11:213 doi:10.1186/1471-2180-11-213

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