Differential global distribution of marine picocyanobacteria gene clusters reveals distinct niche-related adaptive strategies
Different picocyanobacterial communities exhibit distinct gene repertoiresTo analyze the distribution of Prochlorococcus and Synechococcus reads along the Tara Oceans transect, metagenomic reads corresponding to the bacterial size fraction were recruited against 256 picocyanobacterial reference genomes, including SAGs and MAGs representative of uncultured lineages (e.g., Prochlorococcus HLIII-IV, Synechococcus EnvA or EnvB). This yielded a total of 1.07 billion recruited reads, of which 87.7% mapped onto Prochlorococcus genomes and 12.3% onto Synechococcus genomes, which were then functionally assigned by mapping them onto the manually curated Cyanorak v2.1 CLOG database [19]. In order to identify picocyanobacterial genes potentially involved in niche adaptation, we analyzed the distribution across the oceans of flexible (i.e. non-core) genes. Clustering of Tara Oceans stations according to the relative abundance of flexible genes resulted in three well-defined clusters for Prochlorococcus (Fig. 1A), which matched those obtained when stations were clustered according to the relative abundance of Prochlorococcus ESTUs, as assessed using the high-resolution marker gene petB, encoding cytochrome b6 (Fig. 1A; [24]). Only a few discrepancies can be observed between the two trees, including stations TARA-070 that displayed one of the most disparate ESTU compositions and TARA-094, dominated by the rare HLID ESTU (Fig. 1A). Similarly, for Synechococcus, most of the eight assemblages of stations discriminated based on the relative abundance of ESTUs (Fig. 1B) were also retrieved in the clustering based on flexible gene abundance, except for a few intra-assemblage switches between stations, notably those dominated by ESTU IIA (Fig. 1B). Despite these few variations, four major clusters can be clearly delineated in both Synechococcus trees, corresponding to four broadly defined ecological niches, namely (i) cold, nutrient-rich, pelagic or coastal environments (blue and light red in Fig. 1B), (ii) Fe-limited environments (purple and grey), (iii) temperate, P-depleted, Fe-replete areas (yellow) and (iv) warm, N-depleted, Fe-replete regions (dark red). This correspondence between taxonomic and functional information was also confirmed by the high congruence between distance matrices based on ESTU relative abundance and on CLOG relative abundance (p-value 0.01) are marked by a cross. Φsat: index of iron limitation derived from satellite data. PAR30: satellite-derived photosynthetically available radiation at the surface, averaged on 30 days. DCM: depth of the deep chlorophyll maximum.Full size imageIdentification of individual genes potentially involved in niche partitioningTo identify genes relevant for adaptation to a specific set of environmental conditions and enriched in specific ESTU assemblages, we selected the most representative genes from each module (Dataset 5; Figs. 3, S2). Most genes retrieved this way encode proteins of unknown or hypothetical function (85.7% of 7,485 genes). However, among the genes with a functional annotation (Dataset 6), a large fraction seems to have a function related to their realized environmental niche (Figs. 3, S2). For instance, many genes involved in the transport and assimilation of nitrite and nitrate (nirA, nirX, moaA-C, moaE, mobA, moeA, narB, M, nrtP; [6]) as well as cyanate, an organic form of nitrogen (cynA, B, D, S), are enriched in the Prochlorococcus blue module, which is correlated with the HLIIA-D ESTU and to low inorganic N, P, and silica levels and anti-correlated with Fe availability (Fig. 2A–C). This is consistent with previous studies showing that while only a few Prochlorococcus strains in culture possess the nirA gene and even less the narB gene, natural Prochlorococcus populations inhabiting N-poor areas do possess one or both of these genes [40,41,42]. Similarly, numerous genes amongst the most representative of Prochlorococcus brown, red and turquoise modules are related to adaptation of HLIIIA/IVA, HLIA and LLIA ESTUs to Fe-limited, cold P-limited, and cold, mixed waters, respectively (Fig. 3). Comparable results were obtained for Synechococcus, although the niche delineation was less clear than for Prochlorococcus since genes within each module exhibited lower correlations with the module eigenvalue (Fig. S2). These results therefore constitute a proof of concept that this network analysis was able to retrieve niche-related genes from metagenomics data.Fig. 3: Violin plots highlighting the most representative genes of each Prochlorococcus module.For each module, each gene is represented as a dot positioned according to its correlation with the eigengene for each module, the most representative genes being localized on top of each violin plot. Genes mentioned in the text and/or in Dataset 6 have been colored according to the color of the corresponding module, indicated by a colored bar above each module. The text above violin plots indicates the most significant environmental parameter(s) and/or ESTU(s) for each module, as derived from Fig. 2.Full size imageIdentification of eCAGs potentially involved in niche partitioningIn order to better understand the function of niche-related genes, notably of the numerous unknowns, we then integrated global distribution data with gene synteny in reference genomes using a network approach (Datasets 7, 8). This led us to identify clusters of adjacent genes in reference genomes, and thus potentially involved in the same metabolic pathway (Figs. 4, S3, S4; Dataset 6). These clusters were defined within each module and thus encompass genes with similar distribution and abundance in situ. Hereafter, these environmental clusters of adjacent genes will be called “eCAGs”.Fig. 4: Delineation of Prochlorococcus eCAGs, defined as a set of genes that are both adjacent in reference genomes and share a similar in situ distribution.Nodes correspond to individual genes with their gene name (or significant numbers of the CK number, e.g. 1234 for CK_00001234) and are colored according to their WGCNA module. A link between two nodes indicates that these two genes are less than five genes apart in at least one genome. The bottom insert shows the most significant environmental parameter(s) and/or ESTU(s) for each module, as derived from Fig. 2.Full size imageeCAGs related to nitrogen metabolismThe well-known nitrate/nitrite gene cluster involved in uptake and assimilation of inorganic forms of N (see above), which is present in most Synechococcus genomes (Dataset 6), was expectedly not restricted to a particular niche in natural Synechococcus populations, as shown by its quasi-absence from WGCNA modules. In Prochlorococcus, this cluster is separated into two eCAGs enriched in low-N areas (Fig. S5A, B), most genes being included in Pro-eCAG_002, present in only 13 out of 118 Prochlorococcus genomes, while nirA and nirX form an independent eCAG (Pro-eCAG_001) due to their presence in many more genomes. The quasi-core ureA-G/urtB-E genomic region was also found to form a Prochlorococcus eCAG (Pro-eCAG_003) that was impoverished in low-Fe compared to other regions (Fig. S5C, D), in agreement with its presence in only two out of six HLIII/IV genomes. We also uncovered several other Prochlorococcus and Synechococcus eCAGs that seem to be involved in the transport and/or assimilation of more unusual and/or complex forms of nitrogen, which might either be degraded into elementary N molecules or possibly directly used by cells for e.g. the biosynthesis of proteins or DNA. Indeed, we detected in both genera an eCAG (Pro-eCAG_004 and Syn-eCAG_001; Fig. S6A, B; Dataset 6) that encompasses speB2, an ortholog of Synechocystis PCC 6803 sll1077, previously annotated as encoding an agmatinase [29, 43] and which was recently characterized as a guanidinase that degrades guanidine rather than agmatine to urea and ammonium [44]. E. coli produces guanidine under nutrient-poor conditions, suggesting that guanidine metabolism is biologically significant and potentially prevalent in natural environments [44, 45]. Furthermore, the ykkC riboswitch candidate, which was shown to specifically sense guanidine and to control the expression of a variety of genes involved in either guanidine metabolism or nitrate, sulfate, or bicarbonate transport, is located immediately upstream of this eCAG in Synechococcus reference genomes, all genes of this cluster being predicted by RegPrecise 3.0 to be regulated by this riboswitch (Fig. S6C; [45, 46]). The presence of hypA and B homologs within this eCAG furthermore suggests that, in the presence of guanidine, these homologs could be involved in the insertion of Ni2+, or another metal cofactor, in the active site of guanidinase. The next three genes of this eCAG, which encode an ABC transporter similar to the TauABC taurine transporter in E. coli (Fig. S6C), could be involved in guanidine transport in low-N areas. Of note, the presence in most Synechococcus/Cyanobium genomes possessing this eCAG of a gene encoding a putative Rieske Fe-sulfur protein (CK_00002251) downstream of this gene cluster, seems to constitute a specificity compared to the homologous gene cluster in Synechocystis sp. PCC 6803. The presence of this Fe-S protein suggests that Fe is used as a cofactor in this system and might explain why this gene cluster is absent from picocyanobacteria thriving in low-Fe areas, while it is present in a large proportion of the population in most other oceanic areas (Fig. S6A, B).Another example of the use of organic N forms concerns compounds containing a cyano radical (C ≡ N). The cyanate transporter genes (cynABD) were indeed found in a Prochlorococcus eCAG (Pro-eCAG_005, also including the conserved hypothetical gene CK_00055128; Fig. S7A, B). While only a small proportion of the Prochlorococcus community possesses this eCAG in warm, Fe-replete waters, it is absent from other oceanic areas in accordance with its low frequency in Prochlorococcus genomes (present in only two HLI and five HLII genomes). In Synechococcus these genes were not included in a module, and thus are not in an eCAG (Dataset 6; Fig. S7C), but seem widely distributed despite their presence in only a few Synechococcus genomes (mostly in clade III strains; [6, 47, 48]). Interestingly, we also uncovered a 7-gene eCAG (Pro-eCAG_006 and Syn-eCAG_002), encompassing a putative nitrilase gene (nitC), which also suggests that most Synechococcus cells and a more variable fraction of the Prochlorococcus population could use nitriles or cyanides in warm, Fe-replete waters and more particularly in low-N areas such as the Indian Ocean (Fig. 5A, B). The whole operon (nitHBCDEFG; Fig. 5C), called Nit1C, was shown to be upregulated in the presence of cyanide and to trigger an increase in the rate of ammonia accumulation in the heterotrophic bacterium Pseudomonas fluorescens [49], suggesting that like cyanate, cyanide could constitute an alternative nitrogen source in marine picocyanobacteria as well. However, given the potential toxicity of these C ≡ N-containing compounds [50], we cannot exclude that these eCAGs could also be devoted to cell detoxification [45, 47]. Such an example of detoxification has been described for arsenate and chromate that, as analogs of phosphate and sulfate respectively, are toxic to marine phytoplankton and must be actively exported out of the cells [51, 52].Fig. 5: Global distribution map of the eCAG involved in nitrile or cyanide transport and assimilation.A Prochlorococcus Pro-eCAG_006. B Synechococcus Syn-eCAG_002. C The genomic region in Prochlorococcus marinus MIT9301. The size of the circle is proportional to relative abundance of each genus as estimated based on the single-copy core gene petB and this gene was also used to estimate the relative abundance of other genes in the population. Black dots represent Tara Oceans stations for which Prochlorococcus or Synechococcus read abundance was too low to reach the threshold limit.Full size imageWe detected the presence of an eCAG encompassing asnB, pyrB2, and pydC (Pro-eCAG_007, Syn-eCAG_003, Fig. S8), which could contribute to an alternative pyrimidine biosynthesis pathway and thus provide another way for cells to recycle complex nitrogen forms. While this eCAG is found in only one fifth of HLII genomes and in quite specific locations for Prochlorococcus, notably in the Red Sea, it is found in most Synechococcus cells in warm, Fe-replete, N and P-depleted niches, consistent with its phyletic pattern showing its absence only from most clade I, IV, CRD1, and EnvB genomes (Fig. S8; Dataset 6). More generally, most N-uptake and assimilation genes in both genera were specifically absent from Fe-depleted areas, including the nirA/narB eCAG for Prochlorococcus, as mentioned by Kent et al. [36] as well as guanidinase and nitrilase eCAGs. In contrast, picocyanobacterial populations present in low-Fe areas possess, in addition to the core ammonium transporter amt1, a second transporter amt2, also present in cold areas for Synechococcus (Fig. S9). Additionally, Prochlorococcus populations thriving in HNLC areas also possess two amino acid-related eCAGs that are present in most Synechococcus genomes, the first one involved in polar amino acid N-II transport (Pro-eCAG_008; natF-G-H-bgtA; [53]; Fig. S10A, B) and the second one (leuDH-soxA-CK_00001744, Pro-eCAG_009, Fig. S10C, D) that notably encompasses a leucine dehydrogenase, able to produce ammonium from branched-chain amino acids. This highlights the profound difference in N acquisition mechanisms between HNLC regions and Fe-replete, N-deprived areas: the primary nitrogen sources for picocyanobacterial populations dwelling in HNLC areas seem to be ammonium and amino acids, while N acquisition mechanisms are more diverse in N-limited, Fe-replete regions.eCAGs related to phosphorus metabolismAdaptation to P depletion has been well documented in marine picocyanobacteria showing that while in P-replete waters Prochlorococcus and Synechococcus essentially rely on inorganic phosphate acquired by core transporters (PstSABC), strains isolated from low-P regions and natural populations thriving in these areas additionally contain a number of accessory genes related to P metabolism, located in specific genomic islands [6, 14, 30,31,32, 54]. Here, we indeed found in Prochlorococcus an eCAG containing the phoBR operon (Pro-eCAG_010) that encodes a two-component system response regulator, as well as an eCAG including the alkaline phosphatase phoA (Pro-eCAG_011), both present in virtually the whole Prochlorococcus population from the Mediterranean Sea, the Gulf of Mexico and the Western North Atlantic Ocean, which are known to be P-limited [30, 55] (Fig. S11A, B). By comparison, in Synechococcus, we only identified the phoBR eCAG (Syn-eCAG_005, Fig. S11C) that is systematically present in warm waters whatever the limiting nutrient, in agreement with its phyletic pattern in reference genomes showing its specific absence from cold thermotypes (clades I and IV, Dataset 6). Furthermore, although our analysis did not retrieve them within eCAGs due to the variability of gene content and synteny in this genomic region, even within each genus, several other P-related genes were enriched in low-P areas but partially differed between Prochlorococcus and Synechococcus (Figs. 3, S2, S11; Dataset 6). While the genes putatively encoding a chromate transporter (ChrA) and an arsenate efflux pump ArsB were present in both genera in different proportions, a putative transcriptional phosphate regulator related to PtrA (CK_00056804; [56]) was specific to Prochlorococcus. Synechococcus in contrast harbors a large variety of alkaline phosphatases (PhoX, CK_00005263 and CK_00040198) as well as the phosphate transporter SphX (Fig. S11).Phosphonates, i.e. reduced organophosphorus compounds containing C–P bonds that represent up to 25% of the high-molecular-weight dissolved organic P pool in the open ocean, constitute an alternative P form for marine picocyanobacteria [57]. We indeed identified, in addition to the core phosphonate ABC transporter (phnD1-C1-E1), a second previously unreported putative phosphonate transporter phnC2-D2-E2-E3 (Pro-eCAG_012; Fig. 6A). Most of the Prochlorococcus population in strongly P-limited areas of the ocean harbored these genes, while they were absent from other areas, consistent with their presence in only a few Prochlorococcus and no Synechococcus genomes. Furthermore, as previously described [58,59,60], we found a Prochlorococcus eCAG encompassing the phnYZ operon involved in C-P bond cleavage, the putative phosphite dehydrogenase ptxD, and the phosphite and methylphosphonate transporter ptxABC (Pro-eCAG_0013, Dataset 6; Fig. 6B, [60,61,62]). Compared to these previous studies that mainly reported the presence of these genes in Prochlorococcus cells from the North Atlantic Ocean, here we show that they actually occur in a much larger geographic area, including the Mediterranean Sea, the Gulf of Mexico, and the ALOHA station (TARA_132) in the North Pacific, even though they were present in a fairly low fraction of Prochlorococcus cells. These genes occurred in an even larger proportion of the Synechococcus population, although not found in an eCAG for this genus (Fig. S12; Dataset 6). Synechococcus cells from the Mediterranean Sea, a P-limited area dominated by clade III [24], seem to lack phnYZ, in agreement with the phyletic pattern of these genes in reference genomes, showing the absence of this two-gene operon in the sole clade III strain that possesses the ptxABDC gene cluster. In contrast, the presence of the complete gene set (ptxABDC-phnYZ) in the North Atlantic, at the entrance of the Mediterranean Sea, and in several clade II reference genomes rather suggests that it is primarily attributable to this clade. Altogether, our data indicate that part of the natural populations of both Prochlorococcus and Synechococcus would be able to assimilate phosphonate and phosphite as alternative P-sources in low-P areas using the ptxABDC-phnYZ operon. Yet, the fact that no picocyanobacterial genome except P. marinus RS01 (Fig. 6C) possesses both phnC2-D2-E2-E3 and phnYZ, suggests that the phosphonate taken up by the phnC2-D2-E2-E3 transporter could be incorporated into cell surface phosphonoglycoproteins that may act to mitigate cell mortality by grazing and viral lysis, as recently suggested [63].Fig. 6: Global distribution map of eCAGs putatively involved in phosphonate and phosphite transport and assimilation.A Prochlorococcus Pro-eCAG_012 putatively involved in phosphonate transport. B Prochlorococcus Pro-eCAG_013, involved in phosphonate/phosphite uptake and assimilation and phosphonate C-P bond cleavage. C The genomic region encompassing both phnC2-D2-E2-E3 and ptxABDC-phnYZ specific to P. marinus RS01. The size of the circle is proportional to relative abundance of Prochlorococcus as estimated based on the single-copy core gene petB and this gene was also used to estimate the relative abundance of other genes in the population. Black dots represent Tara Oceans stations for which Prochlorococcus read abundance was too low to reach the threshold limit.Full size imageeCAGs related to iron metabolismAs for macronutrients, it has been hypothesized that the survival of marine picocyanobacteria in low-Fe regions was made possible through several strategies, including the loss of genes encoding proteins that contain Fe as a cofactor, the replacement of Fe by another metal cofactor, and the acquisition of genes involved in Fe uptake and storage [14, 15, 36, 39, 64]. Accordingly, several eCAGs encompassing genes encoding proteins interacting with Fe were found in modules anti-correlated to HNLC regions in both genera. These include three subunits of the (photo)respiratory complex succinate dehydrogenase (SdhABC, Pro-eCAG_014, Syn-eCAG_006, Fig. S13; [65]) and Fe-containing proteins encoded in most abovementioned eCAGs involved in N or P metabolism, such as the guanidinase (Fig. S6), the NitC1 (Fig. 5), the pyrB2 (Fig. S8), the phosphonate (Fig. 6, S12), and the urea and inorganic nitrogen eCAGs (Fig. S5). Most Synechococcus cells thriving in Fe-replete areas also possess the sodT/sodX eCAG (Syn-eCAG_007, Fig. S14A, B) involved in nickel transport and maturation of the Ni-superoxide dismutase (SodN), these three genes being in contrast core in Prochlorococcus. Additionally, Synechococcus from Fe-replete areas, notably from the Mediterranean Sea and the Indian Ocean, specifically possess two eCAGs (Syn-eCAG_008 and 009; Fig. S14C, D), involved in the biosynthesis of a polysaccharide capsule that appear to be most similar to the E. coli groups 2 and 3 kps loci [66]. These extracellular structures, known to provide protection against biotic or abiotic stress, were recently shown in Klebsiella to provide a clear fitness advantage in nutrient-poor conditions since they were associated with increased growth rates and population yields [67]. However, while these authors suggested that capsules may play a role in Fe uptake, the significant reduction in the relative abundance of kps genes in low-Fe compared to Fe-replete areas (t-test p-value More
