Modeling Selective Pressures on Phytoplankton in the Global Ocean

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.     

Rapid Diversification of Marine Picophytoplankton with Dissimilar Light-Harvesting Structures Inferred from Sequences of Prochlorococcus and Synechococcus (Cyanobacteria)

Cultured isolates of the unicellular planktonic cyanobacteria Prochlorococcus and marine Synechococcus belong to a single marine picophytoplankton clade. Within this clade, two deeply branching lineages of Prochlorococcus, two lineages of marine A Synechococcus and one lineage of marine B Synechococcus exhibit closely spaced divergence points with low bootstrap support. This pattern is consistent with a near-simultaneous diversification of marine lineages with divinyl chlorophyll b and phycobilisomes as photosynthetic antennae. Inferences from 16S ribosomal RNA sequences including data for 18 marine picophytoplankton clade members were congruent with results of psbB and petB and D sequence analyses focusing on five strains of Prochlorococcus and one strain of marine A Synechococcus. Third codon position and intergenic region nucleotide frequencies vary widely among members of the marine picophytoplankton group, suggesting that substitution biases differ among the lineages. Nonetheless, standard phylogenetic methods and newer algorithms insensitive to such biases did not recover different branching patterns within the group, and failed to cluster Prochlorococcus with chloroplasts or other chlorophyll b-containing prokaryotes. Prochlorococcus isolated from surface waters of stratified, oligotrophic ocean provinces predominate in a lineage exhibiting low G + C nucleotide frequencies at highly variable positions. Received: 18 January 1997 / Accepted: 18 May 1997     

Physiology and evolution of nitrate acquisition in Prochlorococcus

Prochlorococcus is the numerically dominant phototroph in the oligotrophic subtropical ocean and carries out a significant fraction of marine primary productivity. Although field studies have provided evidence for nitrate uptake by Prochlorococcus, little is known about this trait because axenic cultures capable of growth on nitrate have not been available. Additionally, all previously sequenced genomes lacked the genes necessary for nitrate assimilation. Here we introduce three Prochlorococcus strains capable of growth on nitrate and analyze their physiology and genome architecture. We show that the growth of high-light (HL) adapted strains on nitrate is ∼17% slower than their growth on ammonium. By analyzing 41 Prochlorococcus genomes, we find that genes for nitrate assimilation have been gained multiple times during the evolution of this group, and can be found in at least three lineages. In low-light adapted strains, nitrate assimilation genes are located in the same genomic context as in marine Synechococcus. These genes are located elsewhere in HL adapted strains and may often exist as a stable genetic acquisition as suggested by the striking degree of similarity in the order, phylogeny and location of these genes in one HL adapted strain and a consensus assembly of environmental Prochlorococcus metagenome sequences. In another HL adapted strain, nitrate utilization genes may have been independently acquired as indicated by adjacent phage mobility elements; these genes are also duplicated with each copy detected in separate genomic islands. These results provide direct evidence for nitrate utilization by Prochlorococcus and illuminate the complex evolutionary history of this trait.     

Resolution of Prochlorococcus and Synechococcus Ecotypes by Using 16S-23S Ribosomal DNA Internal Transcribed Spacer Sequences

Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.     

Differential Sunlight Sensitivity of Picophytoplankton from Surface Mediterranean Coastal Waters

We tested the sensitivity of coastal picophytoplankton exposed to natural sunlight in short-term experiments. Cell abundance and cell-specific chlorophyll fluorescence were significantly reduced in Prochlorococcus spp. but not in Synechococcus, whereas picoeukaryotes had an intermediate response. These results are the first direct evidence of a differential sensitivity to sunlight of these ubiquitous marine members of unicellular phytoplankton.     

Presence of toxin-antitoxin systems in picocyanobacteria and their ecological implications

Picocyanobacteria (mainly Synechococcus and Prochlorococcus) contribute significantly to ocean’s primary production. Toxin-Antitoxin (TA) systems present in bacteria and archaea are known to regulate cell growth in response to environmental stresses. However, little is known about the presence of TA systems in picocyanobacteria. This study investigated complete genomes of Synechococcus and Prochlorococcus to understand the prevalence of TA systems in picocyanobacteria. Using the TAfinder software, Type II TA systems were predicted in 27 of 33 (81%) Synechococcus strains, but none of 38 Prochlorococcus strains contain TA genes. Synechococcus strains with larger genomes tend to contain more putative type II TA systems. The number of TA pairs varies from 0 to 42 in Synechococcus strains isolated from various environments. A linear correlation between the genome size and the number of putative TA systems in both coastal and freshwater Synechococcus was established. In general, open ocean Synechococcus contain no or few TA systems, while coastal and freshwater Synechococcus contain more TA systems. The type II TA systems inhibit microbial translation via ribonucleases and allow cells to enter the “dormant” stage in adverse environments. Inheritance of TA genes in freshwater and coastal Synechococcus could confer a recoverable persister mechanism important to survive in variable environments.Subject terms: Microbial ecology, Biooceanography     

Ni Uptake and Limitation in Marine Synechococcus Strains

Ni accumulation and utilization were studied in two strains of marine Synechococcus, isolated from both coastal (CC9311; clade I) and open-ocean (WH8102; clade III) environments, for which complete genome sequences are available. Both strains have genes encoding an Ni-containing urease and when grown on urea without Ni become Ni-N colimited. The Ni requirements of these strains also depend upon the genomic complement of genes encoding superoxide dismutase (SOD). WH8102, with a gene encoding only an Ni-SOD, has a novel obligate requirement for Ni, regardless of the N source. Reduced SOD activity in Ni-depleted cultures of WH8102 supports the link of this strain's Ni requirement to Ni-SOD. The genome of CC9311 contains a gene for a Cu/Zn-SOD in addition to a predicted pair of Ni-SODs, yet this strain cannot grow without Ni on NO₃⁻ and can grow only slowly on NH₄⁺ without Ni, implying that the Cu/Zn-SOD cannot completely replace Ni-SOD in marine cyanobacteria. CC9311 does have a greater tolerance for Ni starvation. Both strains increase their Ni uptake capabilities and actively bioconcentrate Ni in response to decreasing extracellular and intracellular Ni. The changes in Ni uptake rates were more pronounced in WH8102 than in CC9311 and for growth on urea or nitrate than for growth on ammonia. These results, combined with an analysis of fully sequenced marine cyanobacterial genomes, suggest that the growth of many marine Synechococcus and all Prochlorococcus strains is dependent upon Ni.     

Chromatic Adaptation in Marine Synechococcus Strains

Characterization of two genetically distinct groups of marine Synechococcus sp. strains shows that one, but not the other, increases its phycourobilin/phycoerythrobilin chromophore ratio when growing in blue light. This ability of at least some marine Synechococcus strains to chromatically adapt may help explain their greater abundance in particular ocean environments than cyanobacteria of the genus Prochlorococcus.     

Ocean time-series reveals recurring seasonal patterns of virioplankton dynamics in the northwestern Sargasso Sea

There are an estimated 10³⁰ virioplankton in the world oceans, the majority of which are phages (viruses that infect bacteria). Marine phages encompass enormous genetic diversity, affect biogeochemical cycling of elements, and partially control aspects of prokaryotic production and diversity. Despite their importance, there is a paucity of data describing virioplankton distributions over time and depth in oceanic systems. A decade of high-resolution time-series data collected from the upper 300 m in the northwestern Sargasso Sea revealed recurring temporal and vertical patterns of virioplankton abundance in unprecedented detail. An annual virioplankton maximum developed between 60 and 100 m during periods of summer stratification and eroded during winter convective mixing. The timing and vertical positioning of this seasonal pattern was related to variability in water column stability and the dynamics of specific picophytoplankton and heterotrophic bacterioplankton lineages. Between 60 and 100 m, virioplankton abundance was negatively correlated to the dominant heterotrophic bacterioplankton lineage SAR11, as well as the less abundant picophytoplankton, Synechococcus. In contrast, virioplankton abundance was positively correlated to the dominant picophytoplankton lineage Prochlorococcus, and the less abundant alpha-proteobacteria, Rhodobacteraceae. Seasonally, virioplankton abundances were highly synchronous with Prochlorococcus distributions and the virioplankton to Prochlorococcus ratio remained remarkably constant during periods of water column stratification. The data suggest that a significant fraction of viruses in the mid-euphotic zone of the subtropical gyres may be cyanophages and patterns in their abundance are largely determined by Prochlorococcus dynamics in response to water column stability. This high-resolution, decadal survey of virioplankton abundance provides insight into the possible controls of virioplankton dynamics in the open ocean.     

Comparative Genomic and Phylogenomic Analyses Reveal a Conserved Core Genome Shared by Estuarine and Oceanic Cyanopodoviruses

Podoviruses are among the major viral groups that infect marine picocyanobacteria Prochlorococcus and Synechococcus. Here, we reported the genome sequences of five Synechococcus podoviruses isolated from the estuarine environment, and performed comparative genomic and phylogenomic analyses based on a total of 20 cyanopodovirus genomes. The genomes of all the known marine cyanopodoviruses are highly syntenic. A pan-genome of 349 clustered orthologous groups was determined, among which 15 were core genes. These core genes make up nearly half of each genome in length, reflecting the high level of genome conservation among this cyanophage type. The whole genome phylogenies based on concatenated core genes and gene content were highly consistent and confirmed the separation of two discrete marine cyanopodovirus clusters MPP-A and MPP-B. The genomes within cluster MPP-B grouped into subclusters mainly corresponding to Prochlorococcus or Synechococcus host types. Auxiliary metabolic genes tend to occur in a specific phylogenetic group of these cyanopodoviruses. All the MPP-B phages analyzed here encode the photosynthesis gene psbA, which are absent in all the MPP-A genomes thus far. Interestingly, all the MPP-B and two MPP-A Synechococcus podoviruses encode the thymidylate synthase gene thyX, while at the same genome locus all the MPP-B Prochlorococcus podoviruses encode the transaldolase gene talC. Both genes are hypothesized to have the potential to facilitate the biosynthesis of deoxynucleotide for phage replication. Inheritance of specific functional genes could be important to the evolution and ecological fitness of certain cyanophage genotypes. Our analyses demonstrate that cyanopodoviruses of estuarine and oceanic origins share a conserved core genome and suggest that accessory genes may be related to environmental adaptation.     

Analysis and Phylogeny of Small Heat Shock Proteins from Marine Viruses and Their Cyanobacteria Host

Small heat shock proteins (sHSPs) are oligomeric stress proteins characterized by an α-crystallin domain (ACD) surrounded by a N-terminal arm and C-terminal extension. Publications on sHSPs have reported that they exist in prokaryotes and eukaryotes but, to our knowledge, not in viruses. Here we show that sHSPs are present in some cyanophages that infect the marine unicellular cyanobacteria, Synechococcus and Prochlorococcus. These phage sHSPs contain a conserved ACD flanked by a relatively conserved N-terminal arm and a short C-terminal extension with or without the conserved C-terminal anchoring module (CAM) L-X-I/V, suggested to be implicated in the oligomerization. In addition, cyanophage sHSPs have the signature pattern, P-P-[YF]-N-[ILV]-[IV]-x(9)-[EQ], in the predicted β2 and β3 strands of the ACD. Phylogenetically, cyanophage sHSPs form a monophyletic clade closer to bacterial class A sHSPs than to cyanobacterial sHSPs. Furthermore, three sHSPs from their cellular host, Synechococcus, are phylogenetically close to plants sHSPs. Implications of evolutionary relationships between the sHSPs of cyanophages, bacterial class A, cyanobacteria, and plants are discussed.     

Codon usage patterns and adaptive evolution of marine unicellular cyanobacteria Synechococcus and Prochlorococcus

Marine unicellular cyanobacteria, represented by Synechococcus and Prochlorococcus, dominate the total phytoplankton biomass and production in oligotrophic ocean. In this study, we employed comparative genomics approaches to extensively investigate synonymous codon usage bias and evolutionary rates in a large number of closely related species of marine unicellular cyanobacteria. Although these two groups of marine cyanobacteria have a close phylogenetic relationship, we find that they are highly divergent not only in codon usage patterns but also in the driving forces behind the diversification. It is revealed that in Prochlorococcus, mutation and genome compositional constraints are the main forces contributing to codon usage bias, whereas in Synechococcus, translational selection. In addition, nucleotide substitution rate analysis indicates that they are not evolving at a constant rate after the divergence and that the average d_N/d_S values of core genes in Synechococcus are significantly higher than those in Prochlorococcus. Our evolutionary genomic analysis provides the first insight into codon usage, evolutionary genetic mechanisms and environmental adaptation of Synechococcus and Prochlorococcus after divergence.     

Cell Cycle Regulation by Light in Prochlorococcus Strains

The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 μmol of quanta · m⁻² s⁻¹, the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the “light on” signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G₁ phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. Shifting Prochlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G₂ phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.     

Phylogenetic Diversity of Sequences of Cyanophage Photosynthetic Gene psbA in Marine and Freshwaters

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.     

Non-coding RNAs in marine Synechococcus and their regulation under environmentally relevant stress conditions

Regulatory small RNAs (sRNAs) have crucial roles in the adaptive responses of bacteria to changes in the environment. Thus far, potential regulatory RNAs have been studied mainly in marine picocyanobacteria in genetically intractable Prochlorococcus, rendering their molecular analysis difficult. Synechococcus sp. WH7803 is a model cyanobacterium, representative of the picocyanobacteria from the mesotrophic areas of the ocean. Similar to the closely related Prochlorococcus it possesses a relatively streamlined genome and a small number of genes, but is genetically tractable. Here, a comparative genome analysis was performed for this and four additional marine Synechococcus to identify the suite of possible sRNAs and other RNA elements. Based on the prediction and on complementary microarray profiling, we have identified several known as well as 32 novel sRNAs. Some sRNAs overlap adjacent coding regions, for instance for the central photosynthetic gene psbA. Several of these novel sRNAs responded specifically to environmentally relevant stress conditions. Among them are six sRNAs changing their accumulation level under cold stress, six responding to high light and two to iron limitation. Target predictions suggested genes encoding components of the light-harvesting apparatus as targets of sRNAs originating from genomic islands and that one of the iron-regulated sRNAs might be a functional homolog of RyhB. These data suggest that marine Synechococcus mount adaptive responses to these different stresses involving regulatory sRNAs.     

Efficient CO2 fixation by surface Prochlorococcus in the Atlantic Ocean

Nearly half of the Earth''s surface is covered by the ocean populated by the most abundant photosynthetic organisms on the planet—Prochlorococcus cyanobacteria. However, in the oligotrophic open ocean, the majority of their cells in the top half of the photic layer have levels of photosynthetic pigmentation barely detectable by flow cytometry, suggesting low efficiency of CO₂ fixation compared with other phytoplankton living in the same waters. To test the latter assumption, CO₂ fixation rates of flow cytometrically sorted ¹⁴C-labelled phytoplankton cells were directly compared in surface waters of the open Atlantic Ocean (30°S to 30°N). CO₂ fixation rates of Prochlorococcus are at least 1.5–2.0 times higher than CO₂ fixation rates of the smallest plastidic protists and Synechococcus cyanobacteria when normalised to photosynthetic pigmentation assessed using cellular red autofluorescence. Therefore, our data indicate that in oligotrophic oceanic surface waters, pigment minimisation allows Prochlorococcus cells to harvest plentiful sunlight more effectively than other phytoplankton.     

Cyanophycin Production in a Phycoerythrin-Containing Marine Synechococcus Strain of Unusual Phylogenetic Affinity

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.