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1.  Ecological Genomics of Marine Picocyanobacteria†  
Summary: Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus numerically dominate the picophytoplankton of the world ocean, making a key contribution to global primary production. Prochlorococcus was isolated around 20 years ago and is probably the most abundant photosynthetic organism on Earth. The genus comprises specific ecotypes which are phylogenetically distinct and differ markedly in their photophysiology, allowing growth over a broad range of light and nutrient conditions within the 45°N to 40°S latitudinal belt that they occupy. Synechococcus and Prochlorococcus are closely related, together forming a discrete picophytoplankton clade, but are distinguishable by their possession of dissimilar light-harvesting apparatuses and differences in cell size and elemental composition. Synechococcus strains have a ubiquitous oceanic distribution compared to that of Prochlorococcus strains and are characterized by phylogenetically discrete lineages with a wide range of pigmentation. In this review, we put our current knowledge of marine picocyanobacterial genomics into an environmental context and present previously unpublished genomic information arising from extensive genomic comparisons in order to provide insights into the adaptations of these marine microbes to their environment and how they are reflected at the genomic level.
doi:10.1128/MMBR.00035-08
PMCID: PMC2698417  PMID: 19487728
2.  Global distribution patterns of distinct clades of the photosynthetic picoeukaryote Ostreococcus 
The ISME Journal  2011;5(7):1095-1107.
Ostreococcus is a marine picophytoeukaryote for which culture studies indicate there are ‘high-light' and ‘low-light' adapted ecotypes. Representatives of these ecotypes fall within two to three 18S ribosomal DNA (rDNA) clades for the former and one for the latter. However, clade distributions and relationships to this form of niche partitioning are unknown in nature. We developed two quantitative PCR primer-probe sets and enumerated the proposed ecotypes in the Pacific Ocean as well as the subtropical and tropical North Atlantic. Statistical differences in factors such as salinity, temperature and NO3 indicated the ecophysiological parameters behind clade distributions are more complex than irradiance alone. Clade OII, containing the putatively low-light adapted strains, was detected at warm oligotrophic sites. In contrast, Clade OI, containing high-light adapted strains, was present in cooler mesotrophic and coastal waters. Maximal OI abundance (19 555±37 18S rDNA copies per ml) was detected in mesotrophic waters at 40 m depth, approaching the nutricline. OII was often more abundant at the deep chlorophyll maximum, when nutrient concentrations were significantly higher than at the surface (stratified euphotic zone waters). However, in mixed euphotic-zone water columns, relatively high numbers (for example, 891±107 18S rDNA copies per ml, Sargasso Sea, springtime) were detected at the surface. Both Clades OI and OII were found at multiple euphotic zone depths, but co-occurrence at the same geographical location appeared rare and was detected only in continental slope waters. In situ growth rate estimates using these primer-probes and better comprehension of physiology will enhance ecological understanding of Ostreococcus Clades OII and OI which appear to be oceanic and coastal clades, respectively.
doi:10.1038/ismej.2010.209
PMCID: PMC3146286  PMID: 21289652
picoeukaryotes; Ostreococcus; quantitative PCR; mamiellales; prasinophytes; niche differentiation
3.  Modeling Selective Pressures on Phytoplankton in the Global Ocean 
PLoS ONE  2010;5(3):e9569.
Our view of marine microbes is transforming, as culture-independent methods facilitate rapid characterization of microbial diversity. It is difficult to assimilate this information into our understanding of marine microbe ecology and evolution, because their distributions, traits, and genomes are shaped by forces that are complex and dynamic. Here we incorporate diverse forces—physical, biogeochemical, ecological, and mutational—into a global ocean model to study selective pressures on a simple trait in a widely distributed lineage of picophytoplankton: the nitrogen use abilities of Synechococcus and Prochlorococcus cyanobacteria. Some Prochlorococcus ecotypes have lost the ability to use nitrate, whereas their close relatives, marine Synechococcus, typically retain it. We impose mutations for the loss of nitrogen use abilities in modeled picophytoplankton, and ask: in which parts of the ocean are mutants most disadvantaged by losing the ability to use nitrate, and in which parts are they least disadvantaged? Our model predicts that this selective disadvantage is smallest for picophytoplankton that live in tropical regions where Prochlorococcus are abundant in the real ocean. Conversely, the selective disadvantage of losing the ability to use nitrate is larger for modeled picophytoplankton that live at higher latitudes, where Synechococcus are abundant. In regions where we expect Prochlorococcus and Synechococcus populations to cycle seasonally in the real ocean, we find that model ecotypes with seasonal population dynamics similar to Prochlorococcus are less disadvantaged by losing the ability to use nitrate than model ecotypes with seasonal population dynamics similar to Synechococcus. The model predictions for the selective advantage associated with nitrate use are broadly consistent with the distribution of this ability among marine picocyanobacteria, and at finer scales, can provide insights into interactions between temporally varying ocean processes and selective pressures that may be difficult or impossible to study by other means. More generally, and perhaps more importantly, this study introduces an approach for testing hypotheses about the processes that underlie genetic variation among marine microbes, embedded in the dynamic physical, chemical, and biological forces that generate and shape this diversity.
doi:10.1371/journal.pone.0009569
PMCID: PMC2835739  PMID: 20224766
4.  The diversity of cyanomyovirus populations along a North–South Atlantic Ocean transect 
The ISME Journal  2011;5(11):1713-1721.
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.
doi:10.1038/ismej.2011.54
PMCID: PMC3197166  PMID: 21633395
cyanophage; virus; Prochlorococcus; marine; g20; genetic diversity
5.  Niche-Partitioning of Prochlorococcus Populations in a Stratified Water Column in the Eastern North Atlantic Ocean† 
The in situ community structure of Prochlorococcus populations in the eastern North Atlantic Ocean was examined by analysis of Prochlorococcus 16S rDNA sequences with three independent approaches: cloning and sequencing, hybridization to specific oligonucleotide probes, and denaturing gradient gel electrophoresis (DGGE). The hybridization of high-light (HL) and low-light (LL) Prochlorococcus genotype-specific probes to two depth profiles of PCR-amplified 16S rDNA sequences revealed that in these two stratified water columns, an obvious niche-partitioning of Prochlorococcus genotypes occurred. In each water column a shift from the HL to the LL genotype was observed, a transition correlating with the depth of the surface mixed layer (SML). Only the HL genotype was found in the SML in each water column, whereas the LL genotype was distributed below the SML. The range of in situ irradiance to which each genotype was subjected within these distinct niches was consistent with growth irradiance studies of cultured HL- and LL-adapted Prochlorococcus strains. DGGE analysis and the sequencing of Prochlorococcus 16S rDNA clones were in full agreement with the genotype-specific oligonucleotide probe hybridization data. These observations of a partitioning of Prochlorococcus genotypes in a stratified water column provide a genetic basis for the dim and bright Prochlorococcus populations observed in flow cytometric signatures in several oceanic provinces.
PMCID: PMC91382  PMID: 10347047
6.  Ultraviolet stress delays chromosome replication in light/dark synchronized cells of the marine cyanobacterium Prochlorococcus marinus PCC9511 
BMC Microbiology  2010;10:204.
Background
The marine cyanobacterium Prochlorococcus is very abundant in warm, nutrient-poor oceanic areas. The upper mixed layer of oceans is populated by high light-adapted Prochlorococcus ecotypes, which despite their tiny genome (~1.7 Mb) seem to have developed efficient strategies to cope with stressful levels of photosynthetically active and ultraviolet (UV) radiation. At a molecular level, little is known yet about how such minimalist microorganisms manage to sustain high growth rates and avoid potentially detrimental, UV-induced mutations to their DNA. To address this question, we studied the cell cycle dynamics of P. marinus PCC9511 cells grown under high fluxes of visible light in the presence or absence of UV radiation. Near natural light-dark cycles of both light sources were obtained using a custom-designed illumination system (cyclostat). Expression patterns of key DNA synthesis and repair, cell division, and clock genes were analyzed in order to decipher molecular mechanisms of adaptation to UV radiation.
Results
The cell cycle of P. marinus PCC9511 was strongly synchronized by the day-night cycle. The most conspicuous response of cells to UV radiation was a delay in chromosome replication, with a peak of DNA synthesis shifted about 2 h into the dark period. This delay was seemingly linked to a strong downregulation of genes governing DNA replication (dnaA) and cell division (ftsZ, sepF), whereas most genes involved in DNA repair (such as recA, phrA, uvrA, ruvC, umuC) were already activated under high visible light and their expression levels were only slightly affected by additional UV exposure.
Conclusions
Prochlorococcus cells modified the timing of the S phase in response to UV exposure, therefore reducing the risk that mutations would occur during this particularly sensitive stage of the cell cycle. We identified several possible explanations for the observed timeshift. Among these, the sharp decrease in transcript levels of the dnaA gene, encoding the DNA replication initiator protein, is sufficient by itself to explain this response, since DNA synthesis starts only when the cellular concentration of DnaA reaches a critical threshold. However, the observed response likely results from a more complex combination of UV-altered biological processes.
doi:10.1186/1471-2180-10-204
PMCID: PMC2921402  PMID: 20670397
7.  Global gene expression of Prochlorococcus ecotypes in response to changes in nitrogen availability 
Nitrogen (N) often limits biological productivity in the oceanic gyres where Prochlorococcus is the most abundant photosynthetic organism. The Prochlorococcus community is composed of strains, such as MED4 and MIT9313, that have different N utilization capabilities and that belong to ecotypes with different depth distributions. An interstrain comparison of how Prochlorococcus responds to changes in ambient nitrogen is thus central to understanding its ecology. We quantified changes in MED4 and MIT9313 global mRNA expression, chlorophyll fluorescence, and photosystem II photochemical efficiency (Fv/Fm) along a time series of increasing N starvation. In addition, the global expression of both strains growing in ammonium-replete medium was compared to expression during growth on alternative N sources. There were interstrain similarities in N regulation such as the activation of a putative NtcA regulon during N stress. There were also important differences between the strains such as in the expression patterns of carbon metabolism genes, suggesting that the two strains integrate N and C metabolism in fundamentally different ways.
doi:10.1038/msb4100087
PMCID: PMC1682016  PMID: 17016519
cyanobacteria; interstrain; nitrogen; Prochlorococcus; transcription
8.  Out of the Pacific and Back Again: Insights into the Matrilineal History of Pacific Killer Whale Ecotypes 
PLoS ONE  2011;6(9):e24980.
Killer whales (Orcinus orca) are the most widely distributed marine mammals and have radiated to occupy a range of ecological niches. Disparate sympatric types are found in the North Atlantic, Antarctic and North Pacific oceans, however, little is known about the underlying mechanisms driving divergence. Previous phylogeographic analysis using complete mitogenomes yielded a bifurcating tree of clades corresponding to described ecotypes. However, there was low support at two nodes at which two Pacific and two Atlantic clades diverged. Here we apply further phylogenetic and coalescent analyses to partitioned mitochondrial genome sequences to better resolve the pattern of past radiations in this species. Our phylogenetic reconstructions indicate that in the North Pacific, sympatry between the maternal lineages that make up each ecotype arises from secondary contact. Both the phylogenetic reconstructions and a clinal decrease in diversity suggest a North Pacific to North Atlantic founding event, and the later return of killer whales to the North Pacific. Therefore, ecological divergence could have occurred during the allopatric phase through drift or selection and/or may have either commenced or have been consolidated upon secondary contact due to resource competition. The estimated timing of bidirectional migration between the North Pacific and North Atlantic coincided with the previous inter-glacial when the leakage of fauna from the Indo-Pacific into the Atlantic via the Agulhas current was particularly vigorous.
doi:10.1371/journal.pone.0024980
PMCID: PMC3176785  PMID: 21949818
9.  Aerobic Anoxygenic Phototrophic Bacteria in the Mid-Atlantic Bight and the North Pacific Gyre 
The abundance of aerobic anoxygenic phototrophic (AAP) bacteria, cyanobacteria, and heterotrophs was examined in the Mid-Atlantic Bight and the central North Pacific Gyre using infrared fluorescence microscopy coupled with image analysis and flow cytometry. AAP bacteria comprised 5% to 16% of total prokaryotes in the Atlantic Ocean but only 5% or less in the Pacific Ocean. In the Atlantic, AAP bacterial abundance was as much as 2-fold higher than that of Prochlorococcus spp. and 10-fold higher than that of Synechococcus spp. In contrast, Prochlorococcus spp. outnumbered AAP bacteria 5- to 50-fold in the Pacific. In both oceans, subsurface abundance maxima occurred within the photic zone, and AAP bacteria were least abundant below the 1% light depth. The abundance of AAP bacteria rivaled some groups of strictly heterotrophic bacteria and was often higher than the abundance of known AAP bacterial genera (Erythrobacter and Roseobacter spp.). Concentrations of bacteriochlorophyll a (BChl a) were low (∼1%) compared to those of chlorophyll a in the North Atlantic. Although the BChl a content of AAP bacteria per cell was typically 20- to 250-fold lower than the divinyl-chlorophyll a content of Prochlorococcus, the pigment content of AAP bacteria approached that of Prochlorococcus in shelf break water. Our results suggest that AAP bacteria can be quite abundant in some oceanic regimes and that their distribution in the water column is consistent with phototrophy.
doi:10.1128/AEM.72.1.557-564.2006
PMCID: PMC1352302  PMID: 16391092
10.  Photoheterotrophic Microbes in the Arctic Ocean in Summer and Winter▿  
Applied and Environmental Microbiology  2009;75(15):4958-4966.
Photoheterotrophic microbes, which are capable of utilizing dissolved organic materials and harvesting light energy, include coccoid cyanobacteria (Synechococcus and Prochlorococcus), aerobic anoxygenic phototrophic (AAP) bacteria, and proteorhodopsin (PR)-containing bacteria. Our knowledge of photoheterotrophic microbes is largely incomplete, especially for high-latitude waters such as the Arctic Ocean, where photoheterotrophs may have special ecological relationships and distinct biogeochemical impacts due to extremes in day length and seasonal ice cover. These microbes were examined by epifluorescence microscopy, flow cytometry, and quantitative PCR (QPCR) assays for PR and a gene diagnostic of AAP bacteria (pufM). The abundance of AAP bacteria and PR-containing bacteria decreased from summer to winter, in parallel with a threefold decrease in the total prokaryotic community. In contrast, the abundance of Synechococcus organisms did not decrease in winter, suggesting that their growth was supported by organic substrates. Results from QPCR assays revealed no substantial shifts in the community structure of AAP bacteria and PR-containing bacteria. However, Arctic PR genes were different from those found at lower latitudes, and surprisingly, they were not similar to those in Antarctic coastal waters. Photoheterotrophic microbes appear to compete successfully with strict heterotrophs during winter darkness below the ice, but AAP bacteria and PR-containing bacteria do not behave as superior competitors during the summer.
doi:10.1128/AEM.00117-09
PMCID: PMC2725502  PMID: 19502441
11.  Novel lineages of Prochlorococcus and Synechococcus in the global oceans 
The ISME Journal  2011;6(2):285-297.
Picocyanobacteria represented by Prochlorococcus and Synechococcus have an important role in oceanic carbon fixation and nutrient cycling. In this study, we compared the community composition of picocyanobacteria from diverse marine ecosystems ranging from estuary to open oceans, tropical to polar oceans and surface to deep water, based on the sequences of 16S-23S rRNA internal transcribed spacer (ITS). A total of 1339 ITS sequences recovered from 20 samples unveiled diverse and several previously unknown clades of Prochlorococcus and Synechococcus. Six high-light (HL)-adapted Prochlorococcus clades were identified, among which clade HLVI had not been described previously. Prochlorococcus clades HLIII, HLIV and HLV, detected in the Equatorial Pacific samples, could be related to the HNLC clades recently found in the high-nutrient, low-chlorophyll (HNLC), iron-depleted tropical oceans. At least four novel Synechococcus clades (out of six clades in total) in subcluster 5.3 were found in subtropical open oceans and the South China Sea. A niche partitioning with depth was observed in the Synechococcus subcluster 5.3. Members of Synechococcus subcluster 5.2 were dominant in the high-latitude waters (northern Bering Sea and Chukchi Sea), suggesting a possible cold-adaptation of some marine Synechococcus in this subcluster. A distinct shift of the picocyanobacterial community was observed from the Bering Sea to the Chukchi Sea, which reflected the change of water temperature. Our study demonstrates that oceanic systems contain a large pool of diverse picocyanobacteria, and further suggest that new genotypes or ecotypes of picocyanobacteria will continue to emerge, as microbial consortia are explored with advanced sequencing technology.
doi:10.1038/ismej.2011.106
PMCID: PMC3260499  PMID: 21955990
cyanobacteria; Prochlorococcus; Synechococcus; diversity; global ocean; 16S-23S rRNA ITS
12.  Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis 
The ISME Journal  2012;7(1):184-198.
Prochlorococcus is the numerically dominant photosynthetic organism throughout much of the world's oceans, yet little is known about the ecology and genetic diversity of populations inhabiting tropical waters. To help close this gap, we examined natural Prochlorococcus communities in the tropical Pacific Ocean using a single-cell whole-genome amplification and sequencing. Analysis of the gene content of just 10 single cells from these waters added 394 new genes to the Prochlorococcus pan-genome—that is, genes never before seen in a Prochlorococcus cell. Analysis of marker genes, including the ribosomal internal transcribed sequence, from dozens of individual cells revealed several representatives from two uncultivated clades of Prochlorococcus previously identified as HNLC1 and HNLC2. While the HNLC clades can dominate Prochlorococcus communities under certain conditions, their overall geographic distribution was highly restricted compared with other clades of Prochlorococcus. In the Atlantic and Pacific oceans, these clades were only found in warm waters with low Fe and high inorganic P levels. Genomic analysis suggests that at least one of these clades thrives in low Fe environments by scavenging organic-bound Fe, a process previously unknown in Prochlorococcus. Furthermore, the capacity to utilize organic-bound Fe appears to have been acquired horizontally and may be exchanged among other clades of Prochlorococcus. Finally, one of the single Prochlorococcus cells sequenced contained a partial genome of what appears to be a prophage integrated into the genome.
doi:10.1038/ismej.2012.89
PMCID: PMC3526172  PMID: 22895163
HNLC; Prochlorococcus; siderophore
13.  Archaeal amoA gene diversity points to distinct biogeography of ammonia-oxidizing Crenarchaeota in the ocean 
Environmental Microbiology  2013;15(5):1647-1658.
Mesophilic ammonia-oxidizing Archaea (AOA) are abundant in a diverse range of marine environments, including the deep ocean, as revealed by the quantification of the archaeal amoA gene encoding the alpha-subunit of the ammonia monooxygenase. Using two different amoA primer sets, two distinct ecotypes of marine Crenarchaeota Group I (MCGI) were detected in the waters of the tropical Atlantic and the coastal Arctic. The HAC-AOA ecotype (high ammonia concentration AOA) was ≍ 8000 times and 15 times more abundant in the coastal Arctic and the top 300 m layer of the open equatorial Atlantic, respectively, than the LAC-AOA (low ammonia concentration AOA) ecotype. In contrast, the LAC-AOA ecotype dominated the lower meso- and bathypelagic waters of the tropical Atlantic (≍ 50 times more abundant than the HAC-AOA) where ammonia concentrations are well below the detection limit using conventional spectrophotometric or fluorometric methods. Cluster analysis of the sequences from the clone libraries obtained by the two amoA primer sets revealed two phylogenetically distinct clusters. Taken together, our results suggest the presence of two ecotypes of archaeal ammonia oxidizers corresponding to the medium (1.24 µM on average in the coastal Arctic) and low ammonia concentration (< 0.01 µM) in the shallow and the deep waters respectively.
doi:10.1111/j.1462-2920.2012.02801.x
PMCID: PMC3712475  PMID: 22690844
14.  Patterns and Implications of Gene Gain and Loss in the Evolution of Prochlorococcus 
PLoS Genetics  2007;3(12):e231.
Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the “leaves of the tree,” between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild.
Author Summary
Prochlorococcus—the most abundant photosynthetic microbe living in the vast, nutrient-poor areas of the ocean—is a major contributor to the global carbon cycle. Prochlorococcus is composed of closely related, physiologically distinct lineages whose differences enable the group as a whole to proliferate over a broad range of environmental conditions. We compare the genomes of 12 strains of Prochlorococcus representing its major lineages in order to identify genetic differences affecting the ecology of different lineages and their evolutionary origin. First, we identify the core genome: the 1,273 genes shared among all strains. This core set of genes encodes the essentials of a functional cell, enabling it to make living matter out of sunlight and carbon dioxide. We then create a genomic tree that maps the gain and loss of non-core genes in individual strains, showing that a striking number of genes are gained or lost even among the most closely related strains. We find that lost and gained genes commonly cluster in highly variable regions called genomic islands. The level of diversity among the non-core genes, and the number of new genes added with each new genome sequenced, suggest far more diversity to be discovered.
doi:10.1371/journal.pgen.0030231
PMCID: PMC2151091  PMID: 18159947
15.  Patterns and Implications of Gene Gain and Loss in the Evolution of Prochlorococcus 
PLoS Genetics  2007;3(12):e231.
Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the “leaves of the tree,” between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild.
Author Summary
Prochlorococcus—the most abundant photosynthetic microbe living in the vast, nutrient-poor areas of the ocean—is a major contributor to the global carbon cycle. Prochlorococcus is composed of closely related, physiologically distinct lineages whose differences enable the group as a whole to proliferate over a broad range of environmental conditions. We compare the genomes of 12 strains of Prochlorococcus representing its major lineages in order to identify genetic differences affecting the ecology of different lineages and their evolutionary origin. First, we identify the core genome: the 1,273 genes shared among all strains. This core set of genes encodes the essentials of a functional cell, enabling it to make living matter out of sunlight and carbon dioxide. We then create a genomic tree that maps the gain and loss of non-core genes in individual strains, showing that a striking number of genes are gained or lost even among the most closely related strains. We find that lost and gained genes commonly cluster in highly variable regions called genomic islands. The level of diversity among the non-core genes, and the number of new genes added with each new genome sequenced, suggest far more diversity to be discovered.
doi:10.1371/journal.pgen.0030231
PMCID: PMC2151091  PMID: 18159947
16.  Dependence of the Cyanobacterium Prochlorococcus on Hydrogen Peroxide Scavenging Microbes for Growth at the Ocean's Surface 
PLoS ONE  2011;6(2):e16805.
The phytoplankton community in the oligotrophic open ocean is numerically dominated by the cyanobacterium Prochlorococcus, accounting for approximately half of all photosynthesis. In the illuminated euphotic zone where Prochlorococcus grows, reactive oxygen species are continuously generated via photochemical reactions with dissolved organic matter. However, Prochlorococcus genomes lack catalase and additional protective mechanisms common in other aerobes, and this genus is highly susceptible to oxidative damage from hydrogen peroxide (HOOH). In this study we showed that the extant microbial community plays a vital, previously unrecognized role in cross-protecting Prochlorococcus from oxidative damage in the surface mixed layer of the oligotrophic ocean. Microbes are the primary HOOH sink in marine systems, and in the absence of the microbial community, surface waters in the Atlantic and Pacific Ocean accumulated HOOH to concentrations that were lethal for Prochlorococcus cultures. In laboratory experiments with the marine heterotroph Alteromonas sp., serving as a proxy for the natural community of HOOH-degrading microbes, bacterial depletion of HOOH from the extracellular milieu prevented oxidative damage to the cell envelope and photosystems of co-cultured Prochlorococcus, and facilitated the growth of Prochlorococcus at ecologically-relevant cell concentrations. Curiously, the more recently evolved lineages of Prochlorococcus that exploit the surface mixed layer niche were also the most sensitive to HOOH. The genomic streamlining of these evolved lineages during adaptation to the high-light exposed upper euphotic zone thus appears to be coincident with an acquired dependency on the extant HOOH-consuming community. These results underscore the importance of (indirect) biotic interactions in establishing niche boundaries, and highlight the impacts that community-level responses to stress may have in the ecological and evolutionary outcomes for co-existing species.
doi:10.1371/journal.pone.0016805
PMCID: PMC3033426  PMID: 21304826
17.  Diversity and Distribution of Marine Synechococcus: Multiple Gene Phylogenies for Consensus Classification and Development of qPCR Assays for Sensitive Measurement of Clades in the Ocean 
Marine Synechococcus is a globally significant genus of cyanobacteria that is comprised of multiple genetic lineages or clades. These clades are thought to represent ecologically distinct units, or ecotypes. Because multiple clades often co-occur together in the oceans, Synechococcus are ideal microbes to explore how closely related bacterial taxa within the same functional guild of organisms co-exist and partition marine habitats. Here we sequenced multiple gene loci from cultured strains to confirm the congruency of clade classifications between the 16S–23S rDNA internally transcribed spacer (ITS), 16S rDNA, narB, ntcA, and rpoC1 loci commonly used in Synechococcus diversity studies. We designed quantitative PCR (qPCR) assays that target the ITS for 10 Synechococcus clades, including four clades, XV, XVI, CRD1, and CRD2, not covered by previous assays employing other loci. Our new qPCR assays are very sensitive and specific, detecting down to tens of cells per ml. Application of these qPCR assays to field samples from the northwest Atlantic showed clear shifts in Synechococcus community composition across a coastal to open-ocean transect. Consistent with previous studies, clades I and IV dominated cold, coastal Synechococcus communities. Clades II and X were abundant at the two warmer, off-shore stations, and at all stations multiple Synechococcus clades co-occurred. qPCR assays developed here provide valuable tools to further explore the dynamics of microbial community structure and the mechanisms of co-existence.
doi:10.3389/fmicb.2012.00213
PMCID: PMC3377940  PMID: 22723796
microbial ecology; cyanobacteria; Synechococcus; microbial diversity; quantitative PCR; multiple gene locus phylogeny; biogeography; ecotype
18.  The P-SSP7 Cyanophage Has a Linear Genome with Direct Terminal Repeats 
PLoS ONE  2012;7(5):e36710.
P-SSP7 is a T7-like phage that infects the cyanobacterium Prochlorococcus MED4. MED4 is a member of the high-light-adapted Prochlorococcus ecotypes that are abundant in the surface oceans and contribute significantly to primary production. P-SSP7 has become a model system for the investigation of T7-like phages that infect Prochlorococcus. It was classified as T7-like based on genome content and organization. However, because its genome assembled as a circular molecule, it was thought to be circularly permuted and to lack the direct terminal repeats found in other T7-like phages. Here we sequenced the ends of the P-SSP7 genome and found that the genome map is linear and contains a 206 bp repeat at both genome ends. Furthermore, we found that a 728 bp region of the genome originally placed downstream of the last ORF is actually located upstream of the first ORF on the genome map. These findings suggest that P-SSP7 is likely to use the direct terminal repeats for genome replication and packaging in a similar manner to other T7-like phages. Moreover, these results highlight the importance of experimentally verifying the ends of phage genomes, and will facilitate the use of P-SSP7 as a model for the correct assembly and end determination of the many T7-like phages isolated from the marine environment that are currently being sequenced.
doi:10.1371/journal.pone.0036710
PMCID: PMC3350473  PMID: 22606283
19.  Cyanobacterial community structure as seen from RNA polymerase gene sequence analysis. 
PCR was used to amplify DNA-dependent RNA polymerase gene sequences specifically from the cyanobacterial population in a seawater sample from the Sargasso Sea. Sequencing and analysis of the cloned fragments suggest that the population in the sample consisted of two distinct clusters of Prochlorococcus-like cyanobacteria and four clusters of Synechococcus-like cyanobacteria. The diversity within these clusters was significantly different, however. Clones within each Synechococcus-like cluster were 99 to 100% identical, while each Prochlorococcus-like cluster was only 91% identical at the nucleotide level. One Prochlorococcus-like cluster was significantly more closely related to a Mediterranean Sea (surface) Prochlorococcus isolate than to the other cluster, showing the highly divergent nature of this group even in one sample. The approach described here can be used as a general method for examining cyanobacterial diversity, while an oligotrophic ocean ecosystem such as the Sargasso Sea may be an ideal model for examining diversity in relation to environmental parameters.
PMCID: PMC201791  PMID: 7944363
20.  Evidence of a human-mediated invasion of the tropical western Atlantic by the 'world's most common brittlestar'. 
Approximately three million years ago the Isthmus of Panama formed an impenetrable land barrier between the tropical eastern Pacific Ocean and the tropical western Atlantic Ocean. Since this time, isolated geminate species have evolved from once contiguous populations, either side of the barrier. One such organism whose distribution is divided by the Isthmus is the tropical brittlestar Ophiactis savignyi, once suggested to be the most common brittlestar in the world. Rather than showing a genetic pattern consistent with a history of isolation, we show that this species has recently dispersed between the Pacific Ocean and the western Atlantic Ocean. This conclusion is based upon a phylogenetic analysis using sequences of the COI mitochondrial DNA gene from these populations. Identical haplotypes between oceans, and a genetic signature of population expansion, provide compelling evidence that the western Atlantic contains at least one cluster of haplotypes recently derived from the Indo-Pacific. Inadvertent human-aided translocation of individuals, presumably in ballast water or fouling communities, is strongly implicated as a mechanism for dispersal between oceans. We believe that cryptic marine invasions are likely to be common and our awareness of them will rapidly increase as systematic and phylogeographic knowledge of marine taxa grow.
doi:10.1098/rspb.2002.1977
PMCID: PMC1690993  PMID: 12028758
21.  Streamlined Regulation and Gene Loss as Adaptive Mechanisms in Prochlorococcus for Optimized Nitrogen Utilization in Oligotrophic Environments 
Prochlorococcus is one of the dominant cyanobacteria and a key primary producer in oligotrophic intertropical oceans. Here we present an overview of the pathways of nitrogen assimilation in Prochlorococcus, which have been significantly modified in these microorganisms for adaptation to the natural limitations of their habitats, leading to the appearance of different ecotypes lacking key enzymes, such as nitrate reductase, nitrite reductase, or urease, and to the simplification of the metabolic regulation systems. The only nitrogen source utilizable by all studied isolates is ammonia, which is incorporated into glutamate by glutamine synthetase. However, this enzyme shows unusual regulatory features, although its structural and kinetic features are unchanged. Similarly, urease activities remain fairly constant under different conditions. The signal transduction protein PII is apparently not phosphorylated in Prochlorococcus, despite its conserved amino acid sequence. The genes amt1 and ntcA (coding for an ammonium transporter and a global nitrogen regulator, respectively) show noncorrelated expression in Prochlorococcus under nitrogen stress; furthermore, high rates of organic nitrogen uptake have been observed. All of these unusual features could provide a physiological basis for the predominance of Prochlorococcus over Synechococcus in oligotrophic oceans.
doi:10.1128/MMBR.68.4.630-638.2004
PMCID: PMC539009  PMID: 15590777
22.  The transcriptome landscape of Prochlorococcus MED4 and the factors for stabilizing the core genome 
BMC Microbiology  2014;14:11.
Background
Gene gain and loss frequently occurs in the cyanobacterium Prochlorococcus, a phototroph that numerically dominates tropical and subtropical open oceans. However, little is known about the stabilization of its core genome, which contains approximately 1250 genes, in the context of genome streamlining. Using Prochlorococcus MED4 as a model organism, we investigated the constraints on core genome stabilization using transcriptome profiling.
Results
RNA-Seq technique was used to obtain the transcriptome map of Prochlorococcus MED4, including operons, untranslated regions, non-coding RNAs, and novel genes. Genome-wide expression profiles revealed that three factors contribute to core genome stabilization. First, a negative correlation between gene expression levels and protein evolutionary rates was observed. Highly expressed genes were overrepresented in the core genome but not in the flexible genome. Gene necessity was determined as a second powerful constraint on genome evolution through functional enrichment analysis. Third, quick mRNA turnover may increase corresponding proteins’ fidelity among genes that were abundantly expressed. Together, these factors influence core genome stabilization during MED4 genome evolution.
Conclusions
Gene expression, gene necessity, and mRNA turnover contribute to core genome maintenance during cyanobacterium Prochlorococcus genus evolution.
doi:10.1186/1471-2180-14-11
PMCID: PMC3898218  PMID: 24438106
Core genome; Gene expression; Molecular evolution; Prochlorococcus; RNA-Seq; Transcriptome
23.  Glucose Uptake and Its Effect on Gene Expression in Prochlorococcus 
PLoS ONE  2008;3(10):e3416.
The marine cyanobacteria Prochlorococcus have been considered photoautotrophic microorganisms, although the utilization of exogenous sugars has never been specifically addressed in them. We studied glucose uptake in different high irradiance- and low irradiance-adapted Prochlorococcus strains, as well as the effect of glucose addition on the expression of several glucose-related genes. Glucose uptake was measured by adding radiolabelled glucose to Prochlorococcus cultures, followed by flow cytometry coupled with cell sorting in order to separate Prochlorococcus cells from bacterial contaminants. Sorted cells were recovered by filtration and their radioactivity measured. The expression, after glucose addition, of several genes (involved in glucose metabolism, and in nitrogen assimilation and its regulation) was determined in the low irradiance-adapted Prochlorococcus SS120 strain by semi-quantitative real time RT-PCR, using the rnpB gene as internal control. Our results demonstrate for the first time that the Prochlorococcus strains studied in this work take up glucose at significant rates even at concentrations close to those found in the oceans, and also exclude the possibility of this uptake being carried out by eventual bacterial contaminants, since only Prochlorococcus cells were used for radioactivity measurements. Besides, we show that the expression of a number of genes involved in glucose utilization (namely zwf, gnd and dld, encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and lactate dehydrogenase, respectively) is strongly increased upon glucose addition to cultures of the SS120 strain. This fact, taken together with the magnitude of the glucose uptake, clearly indicates the physiological importance of the phenomenon. Given the significant contribution of Prochlorococcus to the global primary production, these findings have strong implications for the understanding of the phytoplankton role in the carbon cycle in nature. Besides, the ability of assimilating carbon molecules could provide additional hints to comprehend the ecological success of Prochlorococcus.
doi:10.1371/journal.pone.0003416
PMCID: PMC2565063  PMID: 18941506
24.  Accelerated evolution associated with genome reduction in a free-living prokaryote 
Genome Biology  2005;6(2):R14.
Prochlorococcus sp. are marine bacteria with very small genomes. The mechanisms by which these reduced genomes have evolved appears, however, to be distinct from those that have led to small genome size in intracellular bacteria.
Background
Three complete genomes of Prochlorococcus species, the smallest and most abundant photosynthetic organism in the ocean, have recently been published. Comparative genome analyses reveal that genome shrinkage has occurred within this genus, associated with a sharp reduction in G+C content. As all examples of genome reduction characterized so far have been restricted to endosymbionts or pathogens, with a host-dependent lifestyle, the observed genome reduction in Prochlorococcus is the first documented example of such a process in a free-living organism.
Results
Our results clearly indicate that genome reduction has been accompanied by an increased rate of protein evolution in P. marinus SS120 that is even more pronounced in P. marinus MED4. This acceleration has affected every functional category of protein-coding genes. In contrast, the 16S rRNA gene seems to have evolved clock-like in this genus. We observed that MED4 and SS120 have lost several DNA-repair genes, the absence of which could be related to the mutational bias and the acceleration of amino-acid substitution.
Conclusions
We have examined the evolutionary mechanisms involved in this process, which are different from those known from host-dependent organisms. Indeed, most substitutions that have occurred in Prochlorococcus have to be selectively neutral, as the large size of populations imposes low genetic drift and strong purifying selection. We assume that the major driving force behind genome reduction within the Prochlorococcus radiation has been a selective process favoring the adaptation of this organism to its environment. A scenario is proposed for genome evolution in this genus.
doi:10.1186/gb-2005-6-2-r14
PMCID: PMC551534  PMID: 15693943
25.  Fine-Scale Distribution Patterns of Synechococcus Ecological Diversity in Microbial Mats of Mushroom Spring, Yellowstone National Park▿† 
Applied and Environmental Microbiology  2011;77(21):7689-7697.
Past analyses of sequence diversity in high-resolution protein-encoding genes have identified putative ecological species of unicellular cyanobacteria in the genus Synechococcus, which are specialized to 60°C but not 65°C in Mushroom Spring microbial mats. Because these studies were limited to only two habitats, we studied the distribution of Synechococcus sequence variants at 1°C intervals along the effluent flow channel and at 80-μm vertical-depth intervals throughout the upper photic layer of the microbial mat. Diversity at the psaA locus, which encodes a photosynthetic reaction center protein (PsaA), was sampled by PCR amplification, cloning, and sequencing methods at 60, 63, and 65°C sites. The evolutionary simulation programs Ecotype Simulation and AdaptML were used to identify putative ecologically distinct populations (ecotypes). Ecotype Simulation predicted a higher number of putative ecotypes in cases where habitat variation was limited, while AdaptML predicted a higher number of ecologically distinct phylogenetic clades in cases where habitat variation was high. Denaturing gradient gel electrophoresis was used to track the distribution of dominant sequence variants of ecotype populations relative to temperature variation and to O2, pH, and spectral irradiance variation, as measured using microsensors. Different distributions along effluent channel flow and vertical gradients, where temperature, light, and O2 concentrations are known to vary, confirmed the ecological distinctness of putative ecotypes.
doi:10.1128/AEM.05927-11
PMCID: PMC3209189  PMID: 21890675

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