Microcoleus (Cyanobacteria) form watershed-wide populations without strong gradients in population structure

The relative importance of separation by distance and by environment to population genetic diversity can be conveniently tested in river networks, where these two drivers are often independently distributed over space. To evaluate the importance of dispersal and environmental conditions in shaping microbial population structures, we performed genome-resolved metagenomic analyses of benthic Microcoleus-dominated cyanobacterial mats collected in the Eel and Russian River networks (California, USA). The 64 Microcoleus genomes were clustered into three species that shared >96.5% average nucleotide identity (ANI). Most mats were dominated by one strain, but minor alleles within mats were often shared, even over large spatial distances (>300 km). Within the most common Microcoleus species, the ANI between the dominant strains within mats decreased with increasing spatial separation. However, over shorter spatial distances (tens of kilometres), mats from different subwatersheds had lower ANI than mats from the same subwatershed, suggesting that at shorter spatial distances environmental differences between subwatersheds in factors like canopy cover, conductivity, and mean annual temperature decreases ANI. Since mats in smaller creeks had similar levels of nucleotide diversity (π) as mats in larger downstream subwatersheds, within-mat genetic diversity does not appear to depend on the downstream accumulation of upstream-derived strains. The four-gamete test and sequence length bias suggest recombination occurs between almost all strains within each species, even between populations separated by large distances or living in different habitats. Overall, our results show that, despite some isolation by distance and environmental conditions, sufficient gene-flow occurs among cyanobacterial strains to prevent either driver from producing distinctive population structures across the watershed.     

Genomic analysis of oceanic cyanobacterial myoviruses compared with T4‐like myoviruses from diverse hosts and environments

T4‐like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4‐like genomes, including 10 from non‐cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non‐cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl‐CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage‐encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2‐oxoglutarate. Patterns among non‐core genes that may drive niche diversification revealed that phosphorus‐related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome‐wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross‐infection. Finally, genome‐wide comparisons of both diverse and closely related, co‐isolated genomes provide a locus‐to‐locus variability metric that will prove valuable for interpreting metagenomic data sets.     

Identification of a Marine Cyanophage in a Protist Single‐cell Metagenome Assembly

Analysis of microbial biodiversity is hampered by a lack of reference genomes from most bacteria, viruses, and algae. This necessitates either the cultivation of a restricted number of species for standard sequencing projects or the analysis of highly complex environmental DNA metagenome data. Single‐cell genomics (SCG) offers a solution to this problem by constraining the studied DNA sample to an individual cell and its associated symbionts, prey, and pathogens. We used SCG to study marine heterotrophic amoebae related to Paulinella ovalis (A. Wulff) P.W. Johnson, P.E. Hargraves & J.M. Sieburth (Rhizaria). The genus Paulinella is best known for its photosynthetic members such as P. chromatophora Lauterborn that is the only case of plastid primary endosymbiosis known outside of algae and plants. Here, we studied the phagotrophic sister taxa of P. chromatophora that are related to P. ovalis and found one SCG assembly to contain α‐cyanobacterial DNA. These cyanobacterial contigs are presumably derived from prey. We also uncovered an associated cyanophage lineage (provisionally named phage PoL_MC2). Phylogenomic analysis of the fragmented genome assembly suggested a minimum genome size of 200 Kbp for phage PoL_MC2 that encodes 179 proteins and is most closely related to Synechococcus phage S‐SM2. For this phage, gene network analysis demonstrates a highly modular genome structure typical of other cyanophages. Our work demonstrates that SCG is a powerful approach for discovering algal and protist biodiversity and for elucidating biotic interactions in natural samples.     

Characterization of CRISPR-Cas systems in the Ralstonia solanacearum species complex

Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of an array of short DNA repeat sequences separated by unique spacer sequences that are flanked by associated (Cas) genes. CRISPR-Cas systems are found in the genomes of several microbes and can act as an adaptive immune mechanism against invading foreign nucleic acids, such as phage genomes. Here, we studied the CRISPR-Cas systems in plant-pathogenic bacteria of the Ralstonia solanacearum species complex (RSSC). A CRISPR-Cas system was found in 31% of RSSC genomes present in public databases. Specifically, CRISPR-Cas types I-E and II-C were found, with I-E being the most common. The presence of the same CRISPR-Cas types in distinct Ralstonia phylotypes and species suggests the acquisition of the system by a common ancestor before Ralstonia species segregation. In addition, a Cas1 phylogeny (I-E type) showed a perfect geographical segregation of phylotypes, supporting an ancient acquisition. Ralstoniasolanacearum strains CFBP2957 and K60^T were challenged with a virulent phage, and the CRISPR arrays of bacteriophage-insensitive mutants (BIMs) were analysed. No new spacer acquisition was detected in the analysed BIMs. The functionality of the CRISPR-Cas interference step was also tested in R. solanacearum CFBP2957 using a spacer-protospacer adjacent motif (PAM) delivery system, and no resistance was observed against phage phiAP1. Our results show that the CRISPR-Cas system in R. solanacearum CFBP2957 is not its primary antiviral strategy.     

Biochemical and structural characterization of the cyanophage-encoded phosphate-binding protein: implications for enhanced phosphate uptake of infected cyanobacteria

To acquire phosphorus, cyanobacteria use the typical bacterial ABC-type phosphate transporter, which is composed of a periplasmic high-affinity phosphate-binding protein PstS and a channel formed by two transmembrane proteins PstC and PstA. A putative pstS gene was identified in the genomes of cyanophages that infect the unicellular marine cyanobacteria Prochlorococcus and Synechococcus. However, it has not been determined whether the cyanophage PstS protein is functional during infection to enhance the phosphate uptake rate of host cells. Here we showed that the cyanophage P-SSM2 PstS protein was abundant in the infected Prochlorococcus NATL2A cells and the host phosphate uptake rate was enhanced after infection. This is consistent with our biochemical and structural analyses showing that the phage PstS protein is indeed a high-affinity phosphate-binding protein. We further modelled the complex structure of phage PstS with host PstCA and revealed three putative interfaces that may facilitate the formation of a chimeric ABC transporter. Our results provide insights into the molecular mechanism by which cyanophages enhance the phosphate uptake rate of cyanobacteria. Phosphate acquisition by infected bacteria can increase the phosphorus contents of released cellular debris and virus particles, which together constitute a significant proportion of the marine dissolved organic phosphorus pool.     

Isolation and characterization of a novel Lambda-like phage infecting the bloom-forming cyanobacteria Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a central bloom-forming cyanobacteria. However, despite its ecological significance, little is known of its interactions with the phages that infect it. Currently, only a single sequenced genome of a Cylindrospermopsis-infecting phage is publicly available. Here we describe the isolation and characterization of Cr-LKS3, a second phage infecting Cylindrospermopsis. Cr-LKS3 is a siphovirus with a higher genome similarity to prophages within heterotrophic bacteria genomes than to any other cyanophage/cyano-prophage, suggesting that it represents a novel cyanophage group. The function, order and orientation of the 72 genes in the Cr-LKS3 genome are highly similar to those of Escherichia virus Lambda (hereafter Lambda), despite the very low sequence similarity between these phages, showing high evolutionary convergence despite the substantial difference in host characteristics. Similarly to Lambda, the genome of Cr-LKS3 contains various genes that are known to be central to lysogeny, suggesting it can enter a lysogenic cycle. Cr-LKS3 has a unique ability to infect a host with a dramatically different GC content, without carrying any tRNA genes to compensate for this difference. This ability, together with its potential lysogenic lifestyle shed light on the complex interactions between C. raciborskii and its phages.     

Properties of the new D3-like Pseudomonas aeruginosa bacteriophage phiPMG1: Genome structure and prospects for the use in phage therapy

Results of studying the novel virulent phage phiPMG1 active on Pseudomonas aeruginosa are presented. It is shown that phiPMG1 exhibits significant homology and the similarity in the overall structure with the genome of a temperate phage converts D3. Phage phiPMG1 differs from D3 in that it fails to stably lysogenize bacteria and can grow on strains carrying plasmids that cause growth inhibition of phage D3 and some other phages. This significantly diminishes the probability of horizontal gene transfer with phage phiPMG1 and suggests the possible employment of this phage in phage therapy. A comparison of phages phiPMG1 and D3 structures of genomes in demonstrated not only high homology of 65 genes, but also the presence of 16 genes in the phiPMG1 genome that were not included in the in NCBI database. Apparently, the evolution of genomes in phages of this species is mostly associated with migrations into other species of bacteria, and recombinations with phages of other species (for example, F116). A detailed analysis of structure of one region genomes, which significant nonhomology for the three D3-like phages (D3, phiPMG1 and PAJU2), revealed that the phiPMG1 genome possible closest to a hypothetical genome of ancestral phage of this species.     

新型淡水微囊藻噬藻体vB＿MweS-yong2的分离与基因组分析

【目的】蓝藻(cyanobacteria)水华频繁暴发,引起水质恶化,使水生生物大量死亡,给水产养殖业造成巨大的经济损失;其代谢产物藻毒素具有肝毒性、神经毒性、生殖毒性、遗传毒性和肿瘤促进作用,并可在水生生物中富集,造成饮用水安全风险和水产品食用安全风险。噬藻体(cyanophages)是一类特异性侵染蓝藻的病毒,参与调控蓝藻的种群密度和丰度,被认为是极具潜力的蓝藻水华生物防控工具。以往的研究报道多集中于海水噬藻体,有关淡水噬藻体的报道寥寥无几,迄今尚无惠氏微囊藻(Microcystis wesenbergii)噬藻体的研究报道。本研究的目的在于分离、鉴定惠氏微囊藻噬藻体。【方法】以惠氏微囊藻FACHB-1112为指示宿主,采用双层平板法从淡水中分离出噬藻体vB＿MweS-yong2,对其进行全基因组测序、基因功能注释和系统进化分析。【结果】vB＿MweS-yong2的基因组长44 530 bp,G+C含量为71.6%,有61个开放阅读框(ORF)、1个tRNA基因。成对序列比较(pairwise sequence comparison,PASC)表明,vB＿MweS-yong2与所有...     

Molecular and morphological criteria for revision of the genus Microcoleus (Oscillatoriales,Cyanobacteria)

Ninety‐two strains of Microcoleus vaginatus (=nomenclatural‐type species of the genus Microcoleus Desmazières ex Gomont) and Phormidium autumnale Trevisan ex Gomont from a wide diversity of regions and biotopes were examined using a combination of morphological and molecular methods. Phylogenies based on the 16S rDNA and 16S‐23S ITS (partial) demonstrated that the 92 strains, together with a number of strains in GenBank, were members of a highly supported monophyletic clade of strains (Bayesian posterior probability = 1.0) distant from the species‐cluster containing the generitype of Phormidium. Similarity of the 16S rRNA gene exceeded 95.5% among all members of the Microcoleus clade, but was less than 95% between any Microcoleus strains and species outside of the clade (e.g., Phormidium sensu stricto). These findings, which are in agreement with earlier studies on these taxa, necessitate the revision of Microcoleus to include P. autumnale. Furthermore, the cluster of Phormidium species in the P. autumnale group (known as Group VII) must be moved into Microcoleus as well, and these nomenclatural transfers are included in this study. The main diacritical characters defining Microcoleus are related to the cytomorphology of trichomes, including: narrowed trichome ends, calyptra, cells shorter than wide up to more or less isodiametric, and facultative presence of sheaths. The majority of species are 4–10 μm in diameter. The possession of multiple trichomes in a common sheath is present facultatively in many but not all species.     

Rise and fall of toxic benthic freshwater cyanobacteria (Anabaena spp.) in the Eel river: Buoyancy and dispersal

Benthic cyanobacteria in rivers produce cyanotoxins and affect aquatic food webs, but knowledge of their ecology lags behind planktonic cyanobacteria. The buoyancy of benthic Anabaena spp. mats was studied to understand implications for Anabaena dispersal in the Eel River, California. Field experiments were used to investigate the effects of oxygen bubble production and dissolution on the buoyancy of Anabaena dominated benthic mats in response to light exposure. Samples of Anabaena dominated mats were harvested from the South Fork Eel River and placed in settling columns to measure floating and sinking velocities, or deployed into in situ ambient and low light treatments to measure the effect of light on flotation. Floating and sinking occurred within minutes and were driven by oxygen bubbles produced during photosynthesis, rather than intracellular changes in carbohydrates or gas vesicles. Light experiment results showed that in a natural ambient light regime, mats remained floating for at least 4 days, while in low light mats begin to sink in <24 h. Floating Anabaena samples were collected from five sites in the watershed and found to contain the cyanotoxins anatoxin-a and microcystin, with higher concentrations of anatoxin-a (median 560, max 30,693 ng/g DW) than microcystin (median 30, max 37 ng/g DW). The ability of Anabaena mats to maintain their buoyancy will markedly increase their downstream dispersal distances. Increased buoyancy also allows toxin-containing mats to collect along shorelines, increasing threats to human and animal public health.     

DNA tandem repeats contribute to the genetic diversity of Brevibacterium aurantiacum phages

This report presents the characterization of the first virulent phages infecting Brevibacterium aurantiacum, a bacterial species used during the manufacture of surface-ripened cheeses. These phages were also responsible for flavour and colour defects in surface-ripened cheeses. Sixteen phages (out of 62 isolates) were selected for genome sequencing and comparative analyses. These cos-type phages with a long non-contractile tail currently belong to the Siphoviridae family (Caudovirales order). Their genome sizes vary from 35,637 to 36,825 bp and, similar to their host, have a high GC content (~61%). Genes encoding for an immunity repressor, an excisionase and a truncated integrase were found, suggesting that these virulent phages may be derived from a prophage. Their genomic organization is highly conserved, with most of the diversity coming from the presence of long (198 bp) DNA tandem repeats (TRs) within an open reading frame coding for a protein of unknown function. We categorized these phages into seven genomic groups according to their number of TR, which ranged from two to eight. Moreover, we showed that TRs are widespread in phage genomes, found in more than 85% of the genomes available in public databases.     

诺卡氏菌烈性噬菌体vB_Ncarnea_KYD1的分离纯化与基因组分析

【背景】诺卡氏菌是一种广泛分布的好氧放线菌,可在人体内引起局部或播散性感染,尤其是在免疫功能低下的个体中。诺卡氏菌感染在临床上较难鉴定,而且不断有新型诺卡氏菌种被发现。不同类型、不同地域的诺卡氏菌具有流行差异和抗生素敏感性差异,阻碍了适当治疗方式的选择。利用病灶处的宿主菌分离得到噬菌体来控制诺卡氏菌感染的这种方法在近年来受到了各界的关注。【目的】尝试从环境中分离出能够用于临床治疗的针对诺卡氏菌的烈性噬菌体,并研究其基因组学特征。【方法】利用双层平板法分离得到目标噬菌体,观察其噬菌斑形态,并对噬菌体进行分离纯化,在透射电镜下鉴定其特征。提取噬菌体DNA进行全基因组测序与注释,并与数据库内已知噬菌体基因组进行比较,同时构建系统进化树以进行遗传进化分析。【结果】本文以肉色诺卡氏菌为宿主,从环境样本中分离出一株烈性噬菌体vB_Ncarnea_KYD1,在双层平板上可形成直径<2 mm的透亮均匀的噬菌斑。基因组分析表明,vB_Ncarnea_KYD1DNA为环状,大小为66 621 bp,共发现102个蛋白质编码区(coding sequence,CDS)及一个tRNA-Ser编码序列。透射电镜观察与系统进化树综合分析可以确定,vB_Ncarnea_KYD1为长尾噬菌体科的一个新属。其在进化过程中经历了复杂的基因重组过程。暂未发现毒力因子相关基因与抗性基因,具备实用价值。【结论】从环境水体中分离出一株烈性肉色诺卡氏菌噬菌体vB_Ncarnea_KYD1,通过电镜观察与基因组分析可知,此株噬菌体为长尾噬菌体,基因组中暂未发现不利于临床应用的相关基因,是一株相对安全的烈性诺卡氏菌噬菌体。研究结果丰富了国内噬菌体资源库,并为后续诺卡氏菌感染疾病的治疗提供支持。     

COLEOFASCICULUS GEN. NOV. (CYANOBACTERIA): MORPHOLOGICAL AND MOLECULAR CRITERIA FOR REVISION OF THE GENUS MICROCOLEUS GOMONT1

Species currently classified within the cyanobacterial genus Microcoleus were determined to fall into two distinct clades in a 16S rDNA phylogeny, one containing taxa within the Oscillatoriaceae, the other containing taxa within the Phormidiaceae. The two lineages were confirmed in an analysis of the 16S–23S internal transcribed spacer (ITS) region sequences and secondary structures. The type species for Microcoleus is M. vaginatus Gomont, and this taxon belongs in the Oscillatoriaceae. Consequently, Microcoleus taxa in the Phormidiaceae must be placed in separate genera, and we propose the new genus Coleofasciculus to contain marine taxa currently placed in Microcoleus. The type species for Coleofasciculus is the well‐studied and widespread marine mat‐forming species Microcoleus chthonoplastes (Mert.) Zanardini ex Gomont. Other characters separating the two families include type of cell division and thylakoid structure.     

Newly isolated Nodularia phage influences cyanobacterial community dynamics

Cyanophages, that is, viruses infecting cyanobacteria, are a key component driving cyanobacterial community dynamics both ecologically and evolutionarily. In addition to reducing biomass and influencing the genetic diversity of their host populations, they can also have a wider community‐level impact due to the release of nutrients by phage‐induced cell lysis. In this study, we isolated and characterized a new cyanophage, a siphophage designated as vB_NpeS‐2AV2, capable of infecting the filamentous nitrogen fixing cyanobacterium Nodularia sp. AV2 with a lytic cycle between 12 and 18 hours. The role of the phage in the ecology of its host Nodularia and competitor Synechococcus was investigated in a set of microcosm experiments. Initially, phage‐induced cell lysis decreased the number of Nodularia cells in the cultures. However, around 18%–27% of the population was resistant against the phage infection. Nitrogen was released from the Nodularia cells as a consequence of phage activity, resulting in a seven‐fold increase in Synechococcus cell density. In conclusion, the presence of the cyanophage vB_NpeS‐2AV2 altered the ecological dynamics in the cyanobacterial community and induced evolutionary changes in the Nodularia population, causing the evolution from a population dominated by susceptible cells to a population dominated by resistant ones.     

Prevalence and evolution of core photosystem II genes in marine cyanobacterial viruses and their hosts

Cyanophages (cyanobacterial viruses) are important agents of horizontal gene transfer among marine cyanobacteria, the numerically dominant photosynthetic organisms in the oceans. Some cyanophage genomes carry and express host-like photosynthesis genes, presumably to augment the host photosynthetic machinery during infection. To study the prevalence and evolutionary dynamics of this phenomenon, 33 cultured cyanophages of known family and host range and viral DNA from field samples were screened for the presence of two core photosystem reaction center genes, psbA and psbD. Combining this expanded dataset with published data for nine other cyanophages, we found that 88% of the phage genomes contain psbA, and 50% contain both psbA and psbD. The psbA gene was found in all myoviruses and Prochlorococcus podoviruses, but could not be amplified from Prochlorococcus siphoviruses or Synechococcus podoviruses. Nearly all of the phages that encoded both psbA and psbD had broad host ranges. We speculate that the presence or absence of psbA in a phage genome may be determined by the length of the latent period of infection. Whether it also carries psbD may reflect constraints on coupling of viral- and host-encoded PsbA–PsbD in the photosynthetic reaction center across divergent hosts. Phylogenetic clustering patterns of these genes from cultured phages suggest that whole genes have been transferred from host to phage in a discrete number of events over the course of evolution (four for psbA, and two for psbD), followed by horizontal and vertical transfer between cyanophages. Clustering patterns of psbA and psbD from Synechococcus cells were inconsistent with other molecular phylogenetic markers, suggesting genetic exchanges involving Synechococcus lineages. Signatures of intragenic recombination, detected within the cyanophage gene pool as well as between hosts and phages in both directions, support this hypothesis. The analysis of cyanophage psbA and psbD genes from field populations revealed significant sequence diversity, much of which is represented in our cultured isolates. Collectively, these findings show that photosynthesis genes are common in cyanophages and that significant genetic exchanges occur from host to phage, phage to host, and within the phage gene pool. This generates genetic diversity among the phage, which serves as a reservoir for their hosts, and in turn influences photosystem evolution.     

Bacteriophage exclusion,a new defense system

The ability to withstand viral predation is critical for survival of most microbes. Accordingly, a plethora of phage resistance systems has been identified in bacterial genomes (Labrie et al, 2010 ), including restriction‐modification systems (R‐M) (Tock & Dryden, 2005 ), abortive infection (Abi) (Chopin et al, 2005 ), Argonaute‐based interference (Swarts et al, 2014 ), as well as clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) adaptive immune system (CRISPR‐Cas) (Barrangou & Marraffini, 2014 ; Van der Oost et al, 2014 ). Predictably, the dark matter of bacterial genomes contains a wealth of genetic gold. A study published in this issue of The EMBO Journal by Goldfarb et al ( 2015 ) unveils bacteriophage exclusion (BREX) as a novel, widespread bacteriophage resistance system that provides innate immunity against virulent and temperate phage in bacteria.     

Stimulation of bacterial cytokinesis by bacteriophage predation

Bacteriophage (phage) are ubiquitous in the water column and in the sediments of most natural water. Because of their colloidal nature, they can either aggregate into clumps large enough to settle into the sediment or departing upon the physiochemical conditions or disassociate and reenter the water column. About 80% of the bacterial strains isolated from New River sediment have a virulent phage that can be isolated with them.Liquid cultures of a strain of Pseudomonas aeruginosa isolated from the New River along with its phage were set up. One was infected with the virulent phage and another kept as a control. Daily counts were made of bacterial numbers. After 10 days the control culture was infected and counted for 3 more days.Both cultures divided exponentially at first. The infected culture continued to divide at about half the initial rate. The uninfected culture nearly ceased division, but when phage were added it quickly began to divide.The virulent phage infection clearly stimulated host division. The effect was to establish itself as an endemic infection which did not outpace its host's division rate. Further, the enhanced division rate may act to increase the host's share of available nutrients and benefit its competitive position in the system.     

Argentinean Lactococcus lactis bacteriophages: genetic characterization and adsorption studies

Aims: Characterization of four virulent Lactococcus lactis phages (CHD, QF9, QF12 and QP4) isolated from whey samples obtained from Argentinean cheese plants. Methods and Results: Phages were characterized by means of electron microscopy, host range and DNA studies. The influence of Ca²⁺, physiological cell state, pH and temperature on cell adsorption was also investigated. The double‐stranded DNA genomes of these lactococcal phages showed distinctive restriction patterns. Using a multiplex PCR, phage QP4 was classified as a member of the P335 polythetic species while the three others belong to the 936 group. Ca²⁺ was not needed for phage adsorption but indispensable to complete cell lysis by phage QF9. The lactococci phages adsorbed normally between pH 5 and pH 8, and from 0°C to 40°C, with the exception of phage QF12 which had an adsorption rate significantly lower at pH 8 and 0°C. Conclusions: Lactococcal phages from Argentina belong to the same predominant groups of phages found in other countries and they have the same general characteristics. Significance and Impact of the Study: This work is the first study to characterize Argentinean L. lactis bacteriophages.     

Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes

Background

Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage.

Methodology/Principal Findings

To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling.

Conclusions/Significance

These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.