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.     

The photosynthetic apparatus of Prochlorococcus: Insights through comparative genomics

Within the vast oceanic gyres, a significant fraction of the total chlorophyll belongs to the light-harvesting antenna systems of a single genus, Prochlorococcus. This organism, discovered only about 10 years ago, is an extremely small, Chl b-containing cyanobacterium that sometimes constitutes up to 50% of the photosynthetic biomass in the oceans. Various Prochlorococcus strains are known to have significantly different conditions for optimal growth and survival. Strains which dominate the surface waters, for example, have an irradiance optimum for photosynthesis of 200 μmol photons m⁻² s⁻¹, whereas those that dominate the deeper waters photosynthesize optimally at 30–50 μmol photons m⁻² s⁻¹. These high and low light adapted ‘ecotypes’ are very closely related — less than 3% divergent in their 16S rRNA sequences — inviting speculation as to what features of their photosynthetic mechanisms might account for the differences in photosynthetic performance. Here, we compare information obtained from the complete genome sequences of two Prochlorococcus strains, with special emphasis on genes for the photosynthetic apparatus. These two strains, Prochlorococcus MED4 and MIT 9313, are representatives of high- and low-light adapted ecotypes, characterized by their low or high Chl b/a ratio, respectively. Both genomes appear to be significantly smaller (1700 and 2400 kbp) than those of other cyanobacteria, and the low-light-adapted strain has significantly more genes than its high light counterpart. In keeping with their comparative light-dependent physiologies, MED4 has many more genes encoding putative high-light-inducible proteins (HLIP) and photolyases to repair UV-induced DNA damage, whereas MIT 9313 possesses more genes associated with the photosynthetic apparatus. These include two pcb genes encoding Chl-binding proteins and a second copy of the gene psbA, encoding the Photosystem II reaction center protein D1. In addition, MIT 9313 contains a gene cluster to produce chromophorylated phycoerythrin. The latter represents an intermediate form between the phycobiliproteins of non-Chl b containing cyanobacteria and an extremely modified β phycoerythrin as the sole derivative of phycobiliproteins still present in MED4. Intriguing features found in both Prochlorococcus strains include a gene cluster for Rubisco and carboxysomal proteins that is likely of non-cyanobacterial origin and two genes for a putative and β lycopene cyclase, respectively, explaining how Prochlorococcus may synthesize the α branch of carotenoids that are common in green organisms but not in other cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

NICHE ADAPTATION OF PROCHLOROCOCCUS ECOTYPES: INSIGHTS THROUGH COMPARATIVE GENOMICS

Hess, W. R.¹*, Rocap, G.², Steglich, C.¹, Post, A.², Ting, C. S.³ & Chisholm, S. W.^{2,3 1}Humboldt-University Berlin, Germany; Institute of Biology, Chausseestr. 117, D-10115 Berlin, Germany; ²Massachusetts Institute of Technology, Department of Civil and Environmental Engineering and ³Department of Biology, Massachusetts Institute of Technology, 15 Vassar Street, 48-425 MIT, Cambridge MA 02139, USA Prochlorococcus is an extremely small, chlorophyll b-containing oceanic cyanobacterium. Specific ecotypes of Prochlorococcus have adapted to different ecological conditions with regard to factors as light or the available nutrients. Such differentially adapted ecotypes are less than 3% divergent in their 16S rRNA sequences, which invites speculation as to how their specific gene content has diverged to reflect the particular niche of each strain. Complete genome sequences have been determined of two Prochlorococcus strains, MED4 and MIT9313. These two strains are representatives of high and low light-adapted ecotypes. Intriguing similarities between both genomes include their small size and compact organization (MED4: 1.7 Mbp and MIT9313: 2.3 Mbp), a gene cluster for RubisCo and carboxysomal proteins that is of obviously non-cyanobacterial origin, or genes for two different lycopene cyclases explaining how Prochlorococcus synthesizes alpha-carotene, a carotenoid that is not common to cyanobacteria. Several genes and operons which in cyanobacteria are involved in light harvesting, nitrate utilization or in the generation of circadian rhythms have been reduced to a different degree in the two compared genomes. MED4 has many more genes encoding high light inducible proteins and photolyases. In contrast, MIT9313 possesses more genes to build up more complex light harvesting structures, including a gene cluster to produce chromophorylated phycoerythrin. The latter represents an intermediate between the phycobiliproteins of non-chlorophyll b containing cyanobacteria and a degenerated phycoerythrin present in MED4. Screening of natural samples from the Red Sea suggests that a highly similar phycoerythrin form is wide-spread among high light-adapted ecotypes.     

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.     

Comparison of photosynthetic performances of marine picocyanobacteria with different configurations of the oxygen-evolving complex

The extrinsic PsbU and PsbV proteins are known to play a critical role in stabilizing the Mn₄CaO₅ cluster of the PSII oxygen-evolving complex (OEC). However, most isolates of the marine cyanobacterium Prochlorococcus naturally miss these proteins, even though they have kept the main OEC protein, PsbO. A structural homology model of the PSII of such a natural deletion mutant strain (P. marinus MED4) did not reveal any obvious compensation mechanism for this lack. To assess the physiological consequences of this unusual OEC, we compared oxygen evolution between Prochlorococcus strains missing psbU and psbV (PCC 9511 and SS120) and two marine strains possessing these genes (Prochlorococcus sp. MIT9313 and Synechococcus sp. WH7803). While the low light-adapted strain SS120 exhibited the lowest maximal O₂ evolution rates (P_max per divinyl-chlorophyll a, per cell or per photosystem II) of all four strains, the high light-adapted strain PCC 9511 displayed even higher P^Chl_max and P^PSII_max at high irradiance than Synechococcus sp. WH7803. Furthermore, thermoluminescence glow curves did not show any alteration in the B-band shape or peak position that could be related to the lack of these extrinsic proteins. This suggests an efficient functional adaptation of the OEC in these natural deletion mutants, in which PsbO alone is seemingly sufficient to ensure proper oxygen evolution. Our study also showed that Prochlorococcus strains exhibit negative net O₂ evolution rates at the low irradiances encountered in minimum oxygen zones, possibly explaining the very low O₂ concentrations measured in these environments, where Prochlorococcus is the dominant oxyphototroph.     

Cryo-electron tomography reveals the comparative three-dimensional architecture of Prochlorococcus, a globally important marine cyanobacterium

In an age of comparative microbial genomics, knowledge of the near-native architecture of microorganisms is essential for achieving an integrative understanding of physiology and function. We characterized and compared the three-dimensional architecture of the ecologically important cyanobacterium Prochlorococcus in a near-native state using cryo-electron tomography and found that closely related strains have diverged substantially in cellular organization and structure. By visualizing native, hydrated structures within cells, we discovered that the MED4 strain, which possesses one of the smallest genomes (1.66 Mbp) of any known photosynthetic organism, has evolved a comparatively streamlined cellular architecture. This strain possesses a smaller cell volume, an attenuated cell wall, and less extensive intracytoplasmic (photosynthetic) membrane system compared to the more deeply branched MIT9313 strain. Comparative genomic analyses indicate that differences have evolved in key structural genes, including those encoding enzymes involved in cell wall peptidoglycan biosynthesis. Although both strains possess carboxysomes that are polygonal and cluster in the central cytoplasm, the carboxysomes of MED4 are smaller. A streamlined cellular structure could be advantageous to microorganisms thriving in the low-nutrient conditions characteristic of large regions of the open ocean and thus have consequences for ecological niche differentiation. Through cryo-electron tomography we visualized, for the first time, the three-dimensional structure of the extensive network of photosynthetic lamellae within Prochlorococcus and the potential pathways for intracellular and intermembrane movement of molecules. Comparative information on the near-native structure of microorganisms is an important and necessary component of exploring microbial diversity and understanding its consequences for function and ecology.     

Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast

Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard techniques. Our recent successes in cloning and genetically engineering the AT-rich, 1.1 Mb Mycoplasma mycoides genome in yeast encouraged us to explore similar methods with Prochlorococcus. Prochlorococcus MED4 has an AT-rich genome, with a GC content of 30.8%, similar to that of Saccharomyces cerevisiae (38%), and contains abundant yeast replication origin consensus sites (ACS) evenly distributed around its 1.66 Mb genome. Unlike Mycoplasma cells, which use the UGA codon for tryptophane, Prochlorococcus uses the standard genetic code. Despite this, we observed no toxic effects of several partial and 15 whole Prochlorococcus MED4 genome clones in S. cerevisiae. Sequencing of a Prochlorococcus genome purified from yeast identified 14 single base pair missense mutations, one frameshift, one single base substitution to a stop codon and one dinucleotide transversion compared to the donor genomic DNA. We thus provide evidence of transformation, replication and maintenance of this 1.66 Mb intact bacterial genome in S. cerevisiae.     

Accelerated evolution associated with genome reduction in a free-living prokaryote

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.     

Contrasting mechanisms of proteomic nitrogen thrift in Prochlorococcus

Organisms limited by carbon, nitrogen or sulphur can reduce protein production costs by transitions to less costly amino acids, or by reducing protein expression. These alternative mechanisms of nutrient thrift might respond differently to selection, but this possibility remains untested. We hypothesized that relatively invariant sequence composition responds to long-term variation in nutrient concentrations, whereas dynamic expression profiles vary with nutrient predictability. Prolonged nutrient scarcity favours proteome-wide nutrient reduction. Under stable, nonfluctuating nutrient availability, reduction of nutrient content typically occurs in proteins upregulated when nutrient availability is low, e.g. assimilation and catabolism. We suggest that fluctuating nutrient availability favours mechanisms involving short-term downregulation of nutrient-rich proteins. We analysed protein nitrogen content in six high-light, low-nutrient adapted (HL) vs. six low-light, high-nutrient adapted (LL) Prochlorococcus (marine cyanobacteria) strains, alongside expression data under experimental nitrogen and phosphorus limitation in two strains, MED4 (HL) vs. MIT9313 (LL). HL strains contained less nitrogen, but DNA GC content confounded this relationship. While anabolic and catabolic proteins had normal nitrogen content, most strains showed reduced nitrogen in typical nitrogen stress response proteins. In the experimental data set, though, proteins upregulated under nitrogen limitation were nitrogen-poor only in MIT9313, not MED4. MIT9313 responded similarly to nitrogen and phosphorus limitation, with slow, sustained downregulation of nitrogen-rich ribosomal proteins. In contrast, under nitrogen but not phosphorus limitation, MED4 rapidly downregulated ribosomal proteins. MED4's specific, rapid nitrogen response suggests adaptation to fluctuating conditions, supporting previous work. Thus, we identify contrasting proteomic nitrogen thrift mechanisms within Prochlorococcus consistent with different nutrient regimes.     

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.     

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.     

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.