Genomic and proteomic characterization of two novel siphovirus infecting the sedentary facultative epibiont cyanobacterium Acaryochloris marina.

Acaryochloris marina is a symbiotic species of cyanobacteria that is capable of utilizing far-red light. We report the characterization of the phages A-HIS1 and A-HIS2, capable of infecting Acaryochloris. Morphological characterization of these phages places them in the family Siphoviridae. However, molecular characterization reveals that they do not show genetic similarity with any known siphoviruses. While the phages do show synteny between each other, the nucleotide identity between the phages is low at 45-67%, suggesting they diverged from each other some time ago. The greatest number of genes shared with another phage (a myovirus infecting marine Synechococcus) was four. Unlike most other cyanophages and in common with the Siphoviridae infecting Synechococcus, no photosynthesis-related genes were found in the genome. CRISPR (clustered regularly interspaced short palindromic repeats) spacers from the host Acaryochloris had partial matches to sequences found within the phages, which is the first time CRISPRs have been reported in a cyanobacterial/cyanophage system. The phages also encode a homologue of the proteobacterial RNase T. The potential function of RNase T in the mark-up or digestion of crRNA hints at a novel mechanism for evading the host CRISPR system.

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

Detailed analysis of the microdiversity of Prochlorococcus populations along a north-south Atlantic ocean transect.

In order to understand how environmental factors shape the diversity of Prochlorococcus in the Atlantic Ocean, we have elucidated the microdiversity along a north-south transect. The polymerase chain reaction-restriction fragment length polymorphism analysis of the genetic diversity of rpoC1 gene fragments of Prochlorococcus at 12 sampling sites revealed a latitudinal pattern in Prochlorococcus RFLP-type diversity in the samples collected from two depths. At the depth to which 14% of surface irradiance penetrated, HLII clones dominated the stations closest to the equator. The percentage of HLI clones increased with distance from the equator and LL clones were found only at the most northern and southern stations. In contrast, deeper (1% light depth) water samples did not show any overall trend in Prochlorococcus diversity or clade dominance. Multivariate statistical analyses indicated that Prochlorococcus diversity was linked to water temperature (partially an effect of latitude) and depth (which was linked to light penetration and turbidity). Phylogenetic analysis of the sequences obtained from the 423 different environmental RFLP-types detected in this study indicated that the HLII and HLI populations were composed of a wide range of genetically different clones, while the LL Prochlorococcus clade was less diverse, although half of the samples screened in this study derived from the 1% light depth.

T4 genes in the marine ecosystem: studies of the T4-like cyanophages and their role in marine ecology.

From genomic sequencing it has become apparent that the marine cyanomyoviruses capable of infecting strains of unicellular cyanobacteria assigned to the genera Synechococcus and Prochlorococcus are not only morphologically similar to T4, but are also genetically related, typically sharing some 40-48 genes. The large majority of these common genes are the same in all marine cyanomyoviruses so far characterized. Given the fundamental physiological differences between marine unicellular cyanobacteria and heterotrophic hosts of T4-like phages it is not surprising that the study of cyanomyoviruses has revealed novel and fascinating facets of the phage-host relationship. One of the most interesting features of the marine cyanomyoviruses is their possession of a number of genes that are clearly of host origin such as those involved in photosynthesis, like the psbA gene that encodes a core component of the photosystem II reaction centre. Other host-derived genes encode enzymes involved in carbon metabolism, phosphate acquisition and ppGpp metabolism. The impact of these host-derived genes on phage fitness has still largely to be assessed and represents one of the most important topics in the study of this group of T4-like phages in the laboratory. However, these phages are also of considerable environmental significance by virtue of their impact on key contributors to oceanic primary production and the true extent and nature of this impact has still to be accurately assessed.

Comparative genomics of marine cyanomyoviruses reveals the widespread occurrence of Synechococcus host genes localized to a hyperplastic region: implications for mechanisms of cyanophage evolution.

The vast majority of cyanophages isolated to date are cyanomyoviruses, a group related to bacteriophage T4. Comparative genome analysis of five cyanomyoviruses, including a newly sequenced cyanophage S-RSM4, revealed a 'core genome' of 64 genes, the majority of which are also found in other T4-like phages. Subsequent comparative genomic hybridization analysis using a pilot microarray showed that a number of 'host' genes are widespread in cyanomyovirus isolates. Furthermore, a hyperplastic region was identified between genes g15-g18, within a highly conserved structural gene module, which contained a variable number of inserted genes that lacked conservation in gene order. Several of these inserted genes were host-like and included ptoX, gnd, zwf and petE encoding plastoquinol terminal oxidase, 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase and plastocyanin respectively. Phylogenetic analyses suggest that these genes were acquired independently of each other, even though they have become localized within the same genomic region. This hyperplastic region contains no detectable sequence features that might be mechanistically involved with the acquisition of host-like genes, but does appear to be a site specifically associated with the acquisition process and may represent a novel facet of the evolution of marine cyanomyoviruses.

Differential grazing of two heterotrophic nanoflagellates on marine Synechococcus strains.

Grazing of heterotrophic nanoflagellates on marine picophytoplankton presents a major mortality factor for this important group of primary producers. However, little is known of the selectivity of the grazing process, often merely being thought of as a general feature of cell size and motility. In this study, we tested grazing of two heterotrophic nanoflagellates, Paraphysomonas imperforata and Pteridomonas danica, on strains of marine Synechococcus. Both nanoflagellates proved to be selective in their grazing, with Paraphysomonas being able to grow on 5, and Pteridomonas on 11, of 37 Synechococcus strains tested. Additionally, a number of strains (11 for Paraphysomonas, 9 for Pteridomonas) were shown to be ingested, but not digested (and thus did not support growth of the grazer). Both the range of prey strains that supported growth as well as those that were ingested but not digested was very similar for the two grazers, suggesting a common property of these prey strains that lent them susceptible to grazing. Subsequent experiments on selected Synechococcus strains showed a pronounced difference in grazing susceptibility between wild-type Synechococcus sp. WH7803 and a spontaneous phage-resistant mutant derivative, WH7803PHR, suggesting that cell surface properties of the Synechococcus prey are an important attribute influencing grazing vulnerability.

The potential of phages to prevent MRSA infections.

This short review attempts to examine whether there is a potential for the use of phages capable of infecting Staphylococcus aureus to eradicate or reduce nasal colonisation, thereby reducing the overall infection burden in patient populations identified as being at risk from MRSA infections. There is clear evidence that nasal decolonisation may be of benefit to certain patient groups and also that phages can effectively combat experimentally induced S. aureus infections in animals. However, this is not in itself enough to validate the use of phages for decolonisation and, given the appearance of strains resistant to currently used topical antibiotics, there is a need for clinical trials of this prophylactic use of phages.

Exploring the prokaryotic virosphere.

The world of prokaryotic viruses, including the "traditional" bacteriophages and the viruses of Archaea, is currently in a period of renaissance, brought about largely by our new capabilities in (meta)genomics and by the isolation of diverse novel virus-host systems. In this review, we highlight some of the directions where we believe research on the prokaryotic virosphere will lead us in the near future.

Infection by the 'photosynthetic' phage S-PM2 induces increased synthesis of phycoerythrin in Synechococcus sp. WH7803.

Phycoerythrin-containing Synechococcus strains are unicellular cyanobacteria that are of great ecological importance in the marine environment. These organisms are known to be susceptible to infection by cyanophages (viruses that infect cyanobacteria). The infection cycle takes several hours and during this time the cyanophages may potentially modify the cyanobacterial light-harvesting apparatus. This study based on a model system consisting of Synechococcus sp. WH7803 and cyanophage S-PM2 revealed a progressive increase in the content of phycoerythrin per cell and per phycobilisome postinfection using absorption and emission spectrophotometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. An increased cellular content of chlorophyll a was also revealed using absorption spectrophotometry. The transcript levels of the phycoerythrin-coding operons, mpeBA and cpeBA, were found to increase after phage infection using quantitative real-time PCR. This phage-induced increase in light-harvesting capacity could potentially increase the photosynthetic activity of the host to satisfy the phage's energy demand for reproduction.

Photosynthetic genes in viral populations with a large genomic size range from Norwegian coastal waters.

This study reports the diversity of uncultured environmental viruses harbouring photosynthetic genes (psbA and psbD) in samples from cold seawater (latitude above 60 degrees ). The viral community in coastal Norwegian waters was separated according to genome size using pulse field gel electrophoresis. Viral populations within a wide genome size range (31-380 kb) were investigated for the presence of the psbA and psbD genes using PCR, combined with cloning and sequencing. The results show the presence of photosynthetic genes in viral populations from all size ranges. Thus, valuable information could be obtained about the size class to which viral particles that encode photosynthesis genes belong. The wide genomic size range detected implies that a different cyanophage profile has been observed than has been reported previously. Thus, the method of phage gene detection applied here may represent a truer picture of phage diversity in general or that there is a larger range of size profile for viruses with psbA and psbD in higher latitudes than for the better-studied lower latitudes. Alternatively, a picture of diversity based on a different set of biases than that from either isolation-based research or from conventional metagenomic approaches may be observed.

The third age of phage.

The third age of phage has begun with the recognition that phages may be key to the great planetary biogeochemical cycles and represent the greatest potential genetic resource in the biosphere.

Pigment composition and adaptation in free-living and symbiotic strains of Acaryochloris marina.

Acaryochloris marina strains have been isolated from several varied locations and habitats worldwide demonstrating a diverse and dynamic ecology. In this study, the whole cell photophysiologies of strain MBIC11017, originally isolated from a colonial ascidian, and the free-living epilithic strain CCMEE5410 are analyzed by absorbance and fluorescence spectroscopy, laser scanning confocal microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent protein analysis. We demonstrate pigment adaptation in MBIC11017 and CCMEE5410 under different light regimes. We show that the higher the incident growth light intensity for both strains, the greater the decrease in their chlorophyll d content. However, the strain MBIC11017 loses its phycobiliproteins relative to its chlorophyll d content when grown at light intensities of 40 microE m(-2) s(-1) without shaking and 100 microE m(-2) s(-1) with shaking. We also conclude that phycobiliproteins are absent in the free-living strain CCMEE5410.

Phages of the marine cyanobacterial picophytoplankton.

Cyanobacteria of the genera Synechococcus and Prochlorococcus dominate the prokaryotic component of the picophytoplankton in the oceans. It is still less than 10 years since the discovery of phages that infect marine Synechococcus and the beginning of the characterisation of these phages and assessment of their ecological impact. Estimations of the contribution of phages to Synechococcus mortality are highly variable, but there is clear evidence that phages exert a significant selection pressure on Synechococcus community structure. In turn, there are strong selection pressures on the phage community, in terms of both abundance and composition. This review focuses on the factors affecting the diversity of cyanophages in the marine environment, cyanophage interactions with their hosts, and the selective pressures in the marine environment that affect cyanophage evolutionary biology.

The occurrence of rapidly reversible non-photochemical quenching of chlorophyll a fluorescence in cyanobacteria.

Cyanobacteria have previously been considered to differ fundamentally from plants and algae in their regulation of light harvesting. We show here that in fact the ecologically important marine prochlorophyte, Prochlorococcus, is capable of forming rapidly reversible non-photochemical quenching of chlorophyll a fluorescence (NPQf or qE) as are freshwater cyanobacteria when they employ the iron stress induced chlorophyll-based antenna, IsiA. For Prochlorococcus, the capacity for NPQf is greater in high light-adapted strains, except during iron starvation which allows for increased quenching in low light-adapted strains. NPQf formation in freshwater cyanobacteria is accompanied by deep Fo quenching which increases with prolonged iron starvation.

Cyanophage infection and photoinhibition in marine cyanobacteria.

Members of two cyanobacterial genera, Synechococcus and Prochlorococcus, are dominant within the prokaryotic component of the picophytoplankton and contribute significantly to global photosynthetic productivity. These organisms are known to be susceptible to infection by bacteriophages (viruses that infect bacteria) and it is believed that phage infection in the oceans has exerted selective pressures on the evolution of both phage and host and continues to influence community structure. Understanding of the processes of host-phage interaction within the marine environment is limited; however, new insights have arisen from sequence analysis of the genome of the bacteriophage S-PM2, which infects Synechococcus strains. The phage was found to encode homologs of the key photosystem II reaction center core polypeptides, D1 and D2. These reaction center polypeptides are known to be rapidly turned over in uninfected cells in a repair cycle that helps to protect oxygenic phototrophs against photoinhibition. This finding suggests that bacteriophages infecting marine cyanobacteria may play an active role in protecting their hosts against photoinhibition, thereby ensuring an energy supply for replication by preventing the deleterious effects on host cell integrity seen during acute photoinhibition.

Comparative genomics defines the core genome of the growing N4-like phage genus and identifies N4-like Roseophage specific genes.

Two bacteriophages, RPP1 and RLP1, infecting members of the marine Roseobacter clade were isolated from seawater. Their linear genomes are 74.7 and 74.6 kb and encode 91 and 92 coding DNA sequences, respectively. Around 30% of these are homologous to genes found in Enterobacter phage N4. Comparative genomics of these two new Roseobacter phages and 23 other sequenced N4-like phages (three infecting members of the Roseobacter lineage and 20 infecting other Gammaproteobacteria) revealed that N4-like phages share a core genome of 14 genes responsible for control of gene expression, replication and virion proteins. Phylogenetic analysis of these genes placed the five N4-like roseophages (RN4) into a distinct subclade. Analysis of the RN4 phage genomes revealed they share a further 19 genes of which nine are found exclusively in RN4 phages and four appear to have been acquired from their bacterial hosts. Proteomic analysis of the RPP1 and RLP1 virions identified a second structural module present in the RN4 phages similar to that found in the Pseudomonas N4-like phage LIT1. Searches of various metagenomic databases, including the GOS database, using CDS sequences from RPP1 suggests these phages are widely distributed in marine environments in particular in the open ocean environment.

The diversity of cyanomyovirus populations along a North-South Atlantic Ocean transect.

Viruses that infect the marine cyanobacterium Prochlorococcus have the potential to impact the growth, productivity, diversity and abundance of their hosts. In this study, changes in the microdiversity of cyanomyoviruses were investigated in 10 environmental samples taken along a North-South Atlantic Ocean transect using a myoviral-specific PCR-sequencing approach. Phylogenetic analyses of 630 viral g20 clones from this study, with 786 published g20 sequences, revealed that myoviral populations in the Atlantic Ocean had higher diversity than previously reported, with several novel putative g20 clades. Some of these clades were detected throughout the Atlantic Ocean. Multivariate statistical analyses did not reveal any significant correlations between myoviral diversity and environmental parameters, although myoviral diversity appeared to be lowest in samples collected from the north and south of the transect where Prochlorococcus diversity was also lowest. The results were correlated to the abundance and diversity of the co-occurring Prochlorococcus and Synechococcus populations, but revealed no significant correlations to either of the two potential host genera. This study provides evidence that cyanophages have extremely high and variable diversity and are distributed over large areas of the Atlantic Ocean.

Light-dependent adsorption of photosynthetic cyanophages to Synechococcus sp. WH7803.

Cyanophages infecting marine Synechococcus strains are abundant in the world's oceans and are of considerable ecological significance by virtue of their hosts' role as prominent primary producers in the marine environment. In nature, cyanobacteria experience diel light-dark (LD) cycles, which may exert significant effects on the phage life cycle. An investigation into the role of light revealed that cyanophage S-PM2 adsorption to Synechococcus sp. WH7803 was a light-dependent process. Phage adsorption assays were carried out under illumination at different wavelengths and also in the presence of photosynthesis inhibitors. Furthermore, phage adsorption was also assayed to LD-entrained cells at different points in the circadian cycle. Cyanophage S-PM2 exhibited a considerably decreased adsorption rate under red light as compared with blue, green, yellow light or daylight. However, photosynthesis per se was not required for adsorption as inhibitors such as dichlorophenyldimethyl urea did not affect the process. Neither was S-PM2 adsorption influenced by the circadian rhythm of the host cells. The presence or absence of the photosynthetic reaction centre gene psbA in cyanophage genomes was not correlated with the light-dependent phage adsorption.

Phage therapy.

There is a renaissance of interest in the antimicrobial potential of phages as more pathogens become multiply antibiotic resistant. Phage therapy is not a new concept, and it is important to ask why it is not part of the current repertoire of western medicine despite the fact that it has been continuously and extensively used in Eastern Europe for almost a century. Answering this question successfully will, largely, determine whether phage therapy can gain the credibility needed to overcome the scientific, financial and regulatory hurdles facing its adoption in mainstream clinical practice. Despite a paucity of such information from human studies, pharmacokinetic data and clinical outcomes from animal studies are currently providing convincing evidence for the safety and efficacy of phage therapy.