Summary Prochlorococcus, an abundant phototroph in the oceans, are infected by members of three families of viruses: myo-, podo- and siphoviruses. Genomes of myo- and podoviruses isolated on Prochlorococcus contain DNA replication machinery and virion structural genes homologous to those from coliphages T4 and T7 respectively. They also contain a suite of genes of cyanobacterial origin, most notably photosynthesis genes, which are expressed during infection and appear integral to the evolutionary trajectory of both host and phage. Here we present the first genome of a cyanobacterial siphovirus, P-SS2, which was isolated from Atlantic slope waters using a Prochlorococcus host (MIT9313). The P-SS2 genome is larger than, and considerably divergent from, previously sequenced siphoviruses. It appears most closely related to lambdoid siphoviruses, with which it shares 13 functional homologues. The approximately 108 kb P-SS2 genome encodes 131 predicted proteins and notably lacks photosynthesis genes which have consistently been found in other marine cyanophage, but does contain 14 other cyanobacterial homologues. While only six structural proteins were identified from the genome sequence, 35 proteins were detected experimentally; these mapped onto capsid and tail structural modules in the genome. P-SS2 is potentially capable of integration into its host as inferred from bioinformatically identified genetic machinery int, bet, exo and a 53 bp attachment site. The host attachment site appears to be a genomic island that is tied to insertion sequence (IS) activity that could facilitate mobility of a gene involved in the nitrogen-stress response. The homologous region and a secondary IS-element hot-spot in Synechococcus RS9917 are further evidence of IS-mediated genome evolution coincident with a probable relic prophage integration event. This siphovirus genome provides a glimpse into the biology of a deep-photic zone phage as well as the ocean cyanobacterial prophage and IS element 'mobilome'.

BACKGROUND: Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells. METHODOLOGY/PRINCIPAL FINDINGS: We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs. CONCLUSIONS/SIGNIFICANCE: The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.

Microbial life predominates in the ocean, yet little is known about its genomic variability, especially along the depth continuum. We report here genomic analyses of planktonic microbial communities in the North Pacific Subtropical Gyre, from the ocean's surface to near-sea floor depths. Sequence variation in microbial community genes reflected vertical zonation of taxonomic groups, functional gene repertoires, and metabolic potential. The distributional patterns of microbial genes suggested depth-variable community trends in carbon and energy metabolism, attachment and motility, gene mobility, and host-viral interactions. Comparative genomic analyses of stratified microbial communities have the potential to provide significant insight into higher-order community organization and dynamics.

Prochlorococcus ecotypes are a useful system for exploring the origin and function of diversity among closely related microbes. The genetic variability between phenotypically distinct strains that differ by less that 1% in 16S ribosomal RNA sequences occurs mostly in genomic islands. Island genes appear to have been acquired in part by phage-mediated lateral gene transfer, and some are differentially expressed under light and nutrient stress. Furthermore, genome fragments directly recovered from ocean ecosystems indicate that these islands are variable among cooccurring Prochlorococcus cells. Genomic islands in this free-living photoautotroph share features with pathogenicity islands of parasitic bacteria, suggesting a general mechanism for niche differentiation in microbial species.

The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish "core" genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of "cyanobacterial" genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent "signature" cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages-T7 and T4-but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.

Prochlorococcus and Synechococcus are abundant unicellular cyanobacteria and major participants in global carbon cycles. Although they are closely related and often coexist in the same ocean habitat, they possess very different photosynthetic light-harvesting antennas. Whereas Synechococcus and the majority of cyanobacteria use phycobilisomes, Prochlorococcus has evolved to use a chlorophyll a(2)/b(2) light-harvesting complex. Here, we present a scenario to explain how the Prochlorococcus antenna might have evolved in an ancestral cyanobacterium in iron-limited oceans, resulting in the diversification of the Prochlorococcus and marine Synechococcus lineages from a common phycobilisome-containing ancestor. Differences in the absorption properties and cellular costs between chlorophyll a(2)/b(2) and phycobilisome antennas in extant Prochlorococcus and Synechococcus appear to play a role in differentiating their ecological niches in the ocean environment.

Prochlorococcus is the numerically dominant phytoplankter in the oligotrophic oceans, accounting for up to half of the photosynthetic biomass and production in some regions. Here, we describe how the abundance of six known ecotypes, which have small subunit ribosomal RNA sequences that differ by less than 3%, changed along local and basin-wide environmental gradients in the Atlantic Ocean. Temperature was significantly correlated with shifts in ecotype abundance, and laboratory experiments confirmed different temperature optima and tolerance ranges for cultured strains. Light, nutrients, and competitor abundances also appeared to play a role in shaping different distributions.

The cyanobacterium Prochlorococcus is the numerically dominant phototroph in the oligotrophic oceans. This group consists of multiple ecotypes that are physiologically and phylogenetically distinct and occur in different abundances along environmental gradients. Here we examine adaptations to phosphate (P) limitation among ecotypes. First, we used DNA microarrays to identify genes involved in the P-starvation response in two strains belonging to different ecotypes, MED4 (high-light-adapted) and MIT9313 (low-light-adapted). Most of the up-regulated genes under P starvation were unique to one strain. In MIT9313, many ribosomal genes were down-regulated, suggesting a general stress response in this strain. We also observed major differences in regulation. The P-starvation-induced genes comprise two clusters on the chromosome, the first containing the P master regulator phoB and most known P-acquisition genes and the second, absent in MIT9313, containing genes of unknown function. We examined the organization of the phoB gene cluster in 11 Prochlorococcus strains belonging to diverse ecotypes and found high variability in gene content that was not congruent with rRNA phylogeny. We hypothesize that this genome variability is related to differences in P availability in the oceans from which the strains were isolated. Analysis of a metagenomic library from the Sargasso Sea supports this hypothesis; most Prochlorococcus cells in this low-P environment contain the P-acquisition genes seen in MED4, although a number of previously undescribed gene combinations were observed.

The marine unicellular cyanobacterium Prochlorococcus is the smallest-known oxygen-evolving autotroph. It numerically dominates the phytoplankton in the tropical and subtropical oceans, and is responsible for a significant fraction of global photosynthesis. Here we compare the genomes of two Prochlorococcus strains that span the largest evolutionary distance within the Prochlorococcus lineage and that have different minimum, maximum and optimal light intensities for growth. The high-light-adapted ecotype has the smallest genome (1,657,990 base pairs, 1,716 genes) of any known oxygenic phototroph, whereas the genome of its low-light-adapted counterpart is significantly larger, at 2,410,873 base pairs (2,275 genes). The comparative architectures of these two strains reveal dynamic genomes that are constantly changing in response to myriad selection pressures. Although the two strains have 1,350 genes in common, a significant number are not shared, and these have been differentially retained from the common ancestor, or acquired through duplication or lateral transfer. Some of these genes have obvious roles in determining the relative fitness of the ecotypes in response to key environmental variables, and hence in regulating their distribution and abundance in the oceans.

There are numerous examples from the genomes of viruses, mitochondria, and chromosomes that adjacent genes can overlap, sharing at least one nucleotide. Overlaps have been hypothesized to be involved in genome size minimization and as a regulatory mechanism of gene expression. Here we show that overlapping genes are a consistent feature (approximately one-third of all genes) across all microbial genomes sequenced to date, have homologs in more microbes than do non-overlapping genes, and are therefore likely more conserved. In addition, the size, phase (reading frame offset), and distribution, among other characteristics, of overlapping genes are most consistent with the hypothesis that overlaps function in the regulation of gene expression. The upstream sequences and conservation of overlapping orthologs of two model organisms from the genus Prochlorococcus that have significantly different GC-content, and therefore different nucleotide sequences for orthologs, are also consistent with small overlapping sequence regions and programmed shifts in reading frame as a common mechanism in the regulation of microbial gene expression.

Comparative genomics gives us a new window into phage-host interactions and their evolutionary implications. Here we report the presence of genes central to oxygenic photosynthesis in the genomes of three phages from two viral families (Myoviridae and Podoviridae) that infect the marine cyanobacterium Prochlorococcus. The genes that encode the photosystem II core reaction center protein D1 (psbA), and a high-light-inducible protein (HLIP)(hli) are present in all three genomes. Both myoviruses contain additional hli gene types, and one of them encodes the second photosystem II core reaction center protein D2 (psbD), whereas the other encodes the photosynthetic electron transport proteins plastocyanin (petE) and ferredoxin (petF). These uninterrupted, full-length genes are conserved in their amino acid sequence, suggesting that they encode functional proteins that may help maintain photosynthetic activity during infection. Phylogenetic analyses show that phage D1, D2, and HLIP proteins cluster with those from Prochlorococcus, indicating that they are of cyanobacterial origin. Their distribution among several Prochlorococcus clades further suggests that the genes encoding these proteins were transferred from host to phage multiple times. Phage HLIPs cluster with multicopy types found exclusively in Prochlorocococus, suggesting that phage may be mediating the expansion of the hli gene family by transferring these genes back to their hosts after a period of evolution in the phage. These gene transfers are likely to play a role in the fitness landscape of hosts and phages in the surface oceans.

Prochlorococcus is a marine cyanobacterium which is found at high abundances in world's tropical and subtropical oligotrophic oceans. The genus Prochlorococcus can be divided into two major groups based on light physiology. Both of these groups can be further subdivided into genetically distinct lineages, or ecotypes. Real-time polymerase chain reaction (PCR) assays based on sequence differences in the 16S-23S rDNA internal transcribed spacer or the 23S rDNA were developed to examine the distribution of each ecotype in the field. The real-time PCR assays enabled linear quantification of concentrations ranging from 10 to 4 x 10(5) cells ml(-1). These assays were applied to a stratified water column in the Sargasso Sea. The majority of Prochlorococcus cells above 110 m belonged to the one of the low chlorophyll b/a ratio (high-light adapted) ecotypes, while two types of high chlorophyll b/a ratio (low-light adapted) cells dominated below 110 m. The other three types were found at significantly lower numbers or not detected at all. Differences in the abundance of ecotypes within the major light physiology groupings suggest that other factors, such as nutrient utilization and differential mortality, are driving their relative distributions. Real-time PCR assays will enable further exploration of these factors and temporal and geographic variability in ecotype abundance.