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.     

Patterns and implications of gene gain and loss in the evolution of Prochlorococcus

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.     

Transcriptional profiling of Actinobacillus pleuropneumoniae during the acute phase of a natural infection in pigs

Background

The marine cyanobacterium Prochlorococcus marinus, having multiple ecotypes of distinct genotypic/phenotypic traits and being the first documented example of genome shrinkage in free-living organisms, offers an ideal system for studying niche-driven molecular micro-diversity in closely related microbes. The present study, through an extensive comparative analysis of various genomic/proteomic features of 6 high light (HL) and 6 low light (LL) adapted strains, makes an attempt to identify molecular determinants associated with their vertical niche partitioning.

Results

Pronounced strand-specific asymmetry in synonymous codon usage is observed exclusively in LL strains. Distinct dinucleotide abundance profiles are exhibited by 2 LL strains with larger genomes and G+C-content ≈ 50% (group LLa), 4 LL strains having reduced genomes and G+C-content ≈ 35-37% (group LLb), and 6 HL strains. Taking into account the emergence of LLa, LLb and HL strains (based on 16S rRNA phylogeny), a gradual increase in average aromaticity, pI values and beta- & coil-forming propensities and a decrease in mean hydrophobicity, instability indices and helix-forming propensities of core proteins are observed. Greater variations in orthologous gene repertoire are found between LLa and LLb strains, while higher number of positively selected genes exist between LL and HL strains.

Conclusion

Strains of different Prochlorococcus groups are characterized by distinct compositional, physicochemical and structural traits that are not mere remnants of a continuous genetic drift, but are potential outcomes of a grand scheme of niche-oriented stepwise diversification, that might have driven them chronologically towards greater stability/fidelity and invoked upon them a special ability to inhabit diverse oceanic environments.     

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.     

Genomic potential for nitrogen assimilation in uncultivated members of Prochlorococcus from an anoxic marine zone

Cyanobacteria of the genus Prochlorococcus are the most abundant photosynthetic marine organisms and key factors in the global carbon cycle. The understanding of their distribution and ecological importance in oligotrophic tropical and subtropical waters, and their differentiation into distinct ecotypes, is based on genetic and physiological information from several isolates. Currently, all available Prochlorococcus genomes show their incapacity for nitrate utilization. However, environmental sequence data suggest that some uncultivated lineages may have acquired this capacity. Here we report that uncultivated low-light-adapted Prochlorococcus from the nutrient-rich, low-light, anoxic marine zone (AMZ) of the eastern tropical South Pacific have the genetic potential for nitrate uptake and assimilation. All genes involved in this trait were found syntenic with those present in marine Synechococcus. Genomic and phylogenetic analyses also suggest that these genes have not been aquired recently, but perhaps were retained from a common ancestor, highlighting the basal characteristics of the AMZ lineages within Prochlorococcus.Cyanobacteria of the genus Prochlorococcus are the most abundant photosynthetic microorganisms inhabiting the oceans, key factors in the carbon cycle and a model organism in environmental microbiology (Partensky and Garczarek, 2010). They can be broadly classified into high-light and low-light (LL)-adapted ecotypes (Rocap et al., 2002). These ecotypes exhibit distinct distributions both vertically in the water column and geographically across oligotrophic tropical and subtropical waters (Bouman et al., 2006; Johnson et al., 2006; Zwirglmaier et al., 2008).In past years, the genomes of over a dozen isolates of Prochlorocococus have been fully sequenced (for example, Kettler et al., 2007) and over a hundred single-cell-amplified partial genomes have been described (Malmstrom et al., 2013; Kashtan et al., 2014). All of them have revealed that they cannot use nitrate as a nitrogen source. However, new uncultivated lineages of Prochlorocococus have been identified in the environment using culture-independent techniques based on the sequencing of the 16S rRNA gene and related genomic regions (Lavin et al., 2010; West et al., 2011; Mühling, 2012; Malmstrom et al., 2013). On the other hand, nitrate assimilation rates were reported for uncultivated deep populations of Prochlorococcus in the Western Atlantic Ocean (Casey et al., 2007). In adition, genes necessary for nitrate assimilation associated to Prochlorococcus were identified in the global ocean sampling metagenomic database (Martiny et al., 2009) and in metagenomes of flow-cytometry-sorted Prochlorococcus populations (Batmalle et al., 2014).Important uncultivated Prochlorococcus lineages include those thriving in anoxic marine zones (AMZs), where oxygen concentrations fall below the detection limit of modern sensors, light is scarce, but inorganic nutrients are plentiful (Goericke et al., 2000; Ulloa et al., 2012). Phylogenetic analysis using the 16S–23S rRNA internal transcribed spacer region revealed that the AMZ-associated Prochlorococcus assemblages are mainly composed of two novel LL ecotypes (termed LL-V and LL-VI), which correspond to basal groups linking Prochlorococcus with marine Synechococcus (Lavin et al., 2010), the other dominant marine picocyanobacterium. However, no genomic or physiological information exists for these AMZ lineages.Here we report results from a metagenomic analysis carried out on environmental genomic sequences retrieved from a sample collected at 60 m depth within the AMZ of the eastern tropical South Pacific (Supplementary Figure S1), where dissolved oxygen was undetectable and inorganic nutrients were abundant (Supplementary Figure S2a; Thamdrup et al., 2012). The microbial community was enriched in Prochlorococcus, shown to comprise ~10% of cell abundance, versus ~0.7% of Synechococcus, assessed by flow cytometry (Supplementary Figure S2b). Blast analysis of the taxonomic affiliation of sequences matching the rpoC region 1, a taxonomic marker for cyanobacteria based on a single-copy gene (Palenik, 1994), showed an rpoC gene relative abundance of 86% for Prochlorococcus and 14% for Synechococcus (Supplementary Table S1), supporting the flow cytometry results. Moreover, of the 15% protein-coding sequences assigned to cyanobacteria, 10% binned with Prochlorococcus and 5% with Synechococcus (Supplementary Figure S3). Of those assigned to Prochlorococcus, 90% were related to the LL ecotypes MIT9313 and MIT9303, the closest reported relatives to the AMZ lineages with genomes fully sequenced (Lavin et al., 2010). General statistics of this AMZ metagenome are shown in Supplementary Tables S2 and S3.Analysis of de novo-assembled contigs revealed the presence of several large contigs that binned with Prochlorocococus. In particular, a single contig was found to encode genes related to urea and nitrate uptake and assimilation (contig 51148, GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"KM282015","term_id":"729772065","term_text":"KM282015"}}KM282015; 10 300 bp; Figure 1), in synteny with those in Synechococcus WH8102. The genes in the urease gene cluster (ureABCD) presented high identity to those described for Prochlorococcus MIT9313 and MIT9303 (Rocap et al., 2003; Supplementary Figure S4). Notably, the nitrate/nitrite transporter napA and assimilatory nitrate reductase narB were also found within the same contig (Figure 1a), as well as the genes moeA and mobA (Supplementary Figure S5) involved in the biosynthesis of the Mo-cofactor and necessary for the narB function (Flores et al., 2005). None of these genes have been found in any of the genomes of Prochlorococcus sequenced and described so far. However, homologues that presumably come from uncultivated relatives of Prochlorococcus have been found in the global ocean sampling database (Martiny et al., 2009) and in metagenomes of uncultured, sorted Prochlorococcus populations (Batmalle et al., 2014).Open in a separate windowFigure 1Genomic characteristics of the nitrogen assimilation operon found in contig 51148. (a) Schematic representation of syntenies among contig 51148, Prochlorococcus MIT9313 and MIT9303 genomes, and Synechococcus WH7803 and WH8102 genomes centered on nitrate and urea assimilation genes. Identities (%) among sequences are shown in gray. (b) GC content. (c) Contig coverage. (d) Proximity matrix (Euclidean distance) of the difference in codon usage pattern for the genomes of Prochlorococcus (Pro) and Synechococcus (Syn), and of contig 51148. The shortest distance (dark blue) indicates the highest proximity. (e) Spearman rank-order correlation between tetranucleotide frequency of contig 51148 and those of genomes of marine Prochlorococcus (Pro) and marine Synechococcus (Syn). The highest correlation is shown in dark green.The GC content of contig 51148 was ~51.1% (Figure 1b) and similar to that of LL Prochlorococcus and some marine Synechococcus (Kettler et al., 2007). Likewise, the narB gene had a GC content of 52%, which is less than the ~60% of those in the marine Synechococcus strains WH8102 and WH7803 (to which it presented the highest nucleotide identity), but significantly higher than the ~40% GC of the global ocean sampling high-light Prochlorococcus narB (Supplementary Figure S6). Analysis of codon usage patterns (Yu et al., 2012) and tetranucleotide frequencies (see Supplementary Material and Methods) showed that the cyanobacterial portion of the metagenome and contig 51148 exhibit the highest similarity with LL Prochlorococcus MIT9303 (Figures 1d and e). Additionally, nucleotide identities and phylogenetic analysis confirmed that the urease genes of contig 51148 were associated more closely with Prochlorococcus than Synechococcus (Supplementary Table S4 and Supplementary Figure S4).The homogeneous GC content of contig 51148, the differences in codon usage bias with Synechococcus and phylogenetic analyses of AMZ narB and napA (Figures 2a and b) all suggest that the genetic potential for nitrate uptake and assimilation was not obtained recently by horizontal gene transfer, but instead potentially were retained from a common ancestor with Synechococcus. Mapping the presence/absence of the different nitrate utilization genes onto the cyanobacteria 16S rRNA phylogenetic tree is consistent with this hypothesis (Supplementary Figure S7).Open in a separate windowFigure 2Phylogenetic trees for nitrate assimilation and uptake genes. Maximum-likelihood phylogenetic trees of (a) narB- and (b) napA-predicted amino acid sequences found in contig 51148. Evolutionary history was inferred using neighbour joining (NJ), maximum parsimony (MP) and maximum likelihood (ML). Bootstrap support values for 100 replications are shown at the nodes (NJ/MP/ML).In summary, our results indicate that AMZ Prochlorococcus lineages have the genetic potential for urea and nitrate assimilation, likely an adaptation to the unique nutrient-rich environment where they thrive. Additional genomic characteristics that could explain their high abundance in the oxygen-deficient and very-LL waters of AMZs remain to be assessed.     

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.     

Association between the availability of environmental resources and the atomic composition of organismal proteomes: evidence from Prochlorococcus strains living at different depths

The cyanobacteria Prochlorococcus is a cyanbacterial genus, with some strains adapted to sea surface environments, which are poor in nutrients and have high-light intensity, and some strains adapted to deep sea conditions, which have relatively higher concentrations of nitrogen and phosphorus and lower light intensity. Here, we report pairwise comparisons between strains isolated from different depths of the same sea, which reveal a close association between atomic composition of the proteome and the availability nitrogen and phosphorus in the environment. The atomic composition of proteomes differs significantly among Prochlorococcus strains with different supplies of nitrogen in vivo; these different supplies result from different capacities for nitrogen assimilation. We repeated our whole-proteome analysis with the core proteomes of Prochlorococcus and obtained similar results. Our findings indicate that the elemental composition of proteomes is shaped by the availability of resources in the environment.     

Culture Isolation and Culture-Independent Clone Libraries Reveal New Marine Synechococcus Ecotypes with Distinctive Light and N Physiologies

Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.     

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.     

A Tribute to Disorder in the Genome of the Bloom-Forming Freshwater Cyanobacterium Microcystis aeruginosa

Microcystis aeruginosa is one of the most common bloom-forming cyanobacteria in freshwater ecosystems worldwide. This species produces numerous secondary metabolites, including microcystins, which are harmful to human health. We sequenced the genomes of ten strains of M. aeruginosa in order to explore the genomic basis of their ability to occupy varied environments and proliferate. Our findings show that M. aeruginosa genomes are characterized by having a large open pangenome, and that each genome contains similar proportions of core and flexible genes. By comparing the GC content of each gene to the mean value of the whole genome, we estimated that in each genome, around 11% of the genes seem to result from recent horizontal gene transfer events. Moreover, several large gene clusters resulting from HGT (up to 19 kb) have been found, illustrating the ability of this species to integrate such large DNA molecules. It appeared also that all M. aeruginosa displays a large genomic plasticity, which is characterized by a high proportion of repeat sequences and by low synteny values between the strains. Finally, we identified 13 secondary metabolite gene clusters, including three new putative clusters. When comparing the genomes of Microcystis and Prochlorococcus, one of the dominant picocyanobacteria living in marine ecosystems, our findings show that they are characterized by having almost opposite evolutionary strategies, both of which have led to ecological success in their respective environments.     

Establishing the phylogeny of Prochlorococcus with a new alignment‐free method

Prochlorococcus marinus, one of the most abundant marine cyanobacteria in the global ocean, is classified into low‐light (LL) and high‐light (HL) adapted ecotypes. These two adapted ecotypes differ in their ecophysiological characteristics, especially whether adapted for growth at high‐light or low‐light intensities. However, some evolutionary relationships of Prochlorococcus phylogeny remain to be resolved, such as whether the strains SS120 and MIT9211 form a monophyletic group. We use the Natural Vector (NV) method to represent the sequence in order to identify the phylogeny of the Prochlorococcus. The natural vector method is alignment free without any model assumptions. This study added the covariances of amino acids in protein sequence to the natural vector method. Based on these new natural vectors, we can compute the Hausdorff distance between the two clades which represents the dissimilarity. This method enables us to systematically analyze both the dataset of ribosomal proteomes and the dataset of 16s‐23s rRNA sequences in order to reconstruct the phylogeny of Prochlorococcus. Furthermore, we apply classification to inspect the relationship of SS120 and MIT9211. From the reconstructed phylogenetic trees and classification results, we may conclude that the SS120 does not cluster with MIT9211. This study demonstrates a new method for performing phylogenetic analysis. The results confirm that these two strains do not form a monophyletic clade in the phylogeny of Prochlorococcus.     

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.     

Genetic Manipulation of Prochlorococcus Strain MIT9313: Green Fluorescent Protein Expression from an RSF1010 Plasmid and Tn5 Transposition

Prochlorococcus is the smallest oxygenic phototroph yet described. It numerically dominates the phytoplankton community in the mid-latitude oceanic gyres, where it has an important role in the global carbon cycle. The complete genomes of several Prochlorococcus strains have been sequenced, revealing that nearly half of the genes in each genome are of unknown function. Genetic methods, such as reporter gene assays and tagged mutagenesis, are critical to unveiling the functions of these genes. Here, we describe conditions for the transfer of plasmid DNA into Prochlorococcus strain MIT9313 by interspecific conjugation with Escherichia coli. Following conjugation, E. coli bacteria were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus strain MIT9313. When this plasmid was modified to contain green fluorescent protein, we detected its expression in Prochlorococcus by Western blotting and cellular fluorescence. Further, we applied these conjugation methods to show that a mini-Tn5 transposon will transpose in vivo in Prochlorococcus. These genetic advances provide a basis for future genetic studies with Prochlorococcus, a microbe of ecological importance in the world's oceans.     

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     

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 P_II 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.