Widespread known and novel phosphonate utilization pathways in marine bacteria revealed by functional screening and metagenomic analyses

Phosphonates (Pn), compounds with a direct C–P bond instead of the more common C–O–P ester bond, constitute a significant fraction of marine dissolved organic phosphorus and recent evidence suggests that they may be an alternative source of P for marine microorganisms. To further characterize the microorganisms and pathways involved in Pn utilization, we screened bacterioplankton genomic libraries for their ability to complement an Escherichia coli strain unable to use Pns as a P source. Using this approach we identified a phosphonatase pathway as well as a novel pair of genes that allowed utilization of 2-aminoethylphosphonate (2-AEPn) as the sole P source. These pathways are present in diverse bacteria common in marine plankton including representatives of Proteobacteria , Planctomycetes and Cyanobacteria . Analysis of metagenomic databases for Pn utilization genes revealed that they are widespread and abundant among marine bacteria, suggesting that Pn metabolism is likely to play an important role in P-depleted surface waters, as well as in the more P-rich deep-water column.     

Occurrence of phosphate acquisition genes in Prochlorococcus cells from different ocean regions

The cyanobacterium Prochlorococcus is the numerically dominant phototroph in oligotrophic parts of the oceans. Recently, it was shown that the distribution of phosphate acquisition genes did not match the 16S rRNA phylogeny among isolates from this group but rather appeared related to phosphate availability where the strains had been isolated. To further understand adaptation to phosphate limitation in Prochlorococcus , the distribution of phosphate acquisition genes was investigated in different ocean regions and related to local ortho-phosphate concentration. In regions characterized by less than 0.1 μM phosphate, most Prochlorococcus cells contain genes involved in phosphate uptake, regulation and utilization of organic phosphates. In contrast, most of these genes are absent in regions with more than 0.1 μM phosphate with the exception of genes involved in transport of phosphate ( pho E and pst ABCS) and three genes of unknown function. This pattern of phosphate acquisition genes showed no significant correspondence to the distribution of rRNA phylotypes. In addition, it was demonstrated that several genes in a separate genomic island were commonly present in low-P sites while absent in high-P sites. Overall, this study further demonstrates a linkage between environmental conditions in the ocean and genome content of Prochlorococcus .     

Expression of PTHrP and its cognate receptor in the rheumatoid synovial microcirculation

Genome sequence analyses revealed the occurrence of two paralogous ppa genes potentially encoding distinct Family I inorganic pyrophosphatases (sPPases, EC3.6.1.1) in the marine unicellular cyanobacteria Prochlorococcus marinus strains MED4 and MIT9313 and Synechococcus sp. WH8102. Protein sequence alignment and phylogenetic analysis indicated that the ppa gene proper of cyanobacteria (ppa1) encodes a presumably inactive mutant enzyme whereas the second gene (ppa2) might encode an active sPPase closely related to those of some proteobacteria. Heterologous expression of the two cloned P. marinus MED4 ppa genes in Escherichia coli confirmed this proposal, only the inactive ppa1 product being immunodetected by anti-cyanobacterial sPPase antibodies. A possible scenario of ppa gene inactivation and replacement in the context of the postulated rapid diversification of marine unicellular cyanobacteria, the most abundant photosynthetic prokaryotes in the oceans, is discussed.     

An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton.

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P1) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P1 concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P1 typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.     

Phosphorus scavenging in the unicellular marine diazotroph Crocosphaera watsonii

Through the fixation of atmospheric nitrogen and photosynthesis, marine diazotrophs play a critical role in the global cycling of nitrogen and carbon. Crocosphaera watsonii is a recently described unicellular diazotroph that may significantly contribute to marine nitrogen fixation in tropical environments. One of the many factors that can constrain the growth and nitrogen fixation rates of marine diazotrophs is phosphorus bioavailability. Using genomic and physiological approaches, we examined phosphorus scavenging mechanisms in strains of C. watsonii from both the Atlantic and the Pacific. Observations from the C. watsonii WH8501 genome suggest that this organism has the capacity for high-affinity phosphate transport (e.g., homologs of pstSCAB) in low-phosphate, oligotrophic systems. The pstS gene (high-affinity phosphate binding) is present in strains isolated from both the Atlantic and the Pacific, and its expression was regulated by the exogenous phosphate supply in strain WH8501. Genomic observation also indicated a broad capacity for phosphomonoester hydrolysis (e.g., a putative alkaline phosphatase). In contrast, no clear homologs of genes for phosphonate transport and hydrolysis could be identified. Consistent with these genomic observations, C. watsonii WH8501 is able to grow on phosphomonoesters as a sole source of added phosphorus but not on the phosphonates tested to date. Taken together these data suggest that C. watsonii has a robust capacity for scavenging phosphorus in oligotrophic systems, although this capacity differs from that of other marine cyanobacterial genera, such as Synechococcus, Prochlorococcus, and Trichodesmium.     

Analysis of the hli gene family in marine and freshwater cyanobacteria

Certain cyanobacteria thrive in natural habitats in which light intensities can reach 2000 micromol photon m(-2) s(-1) and nutrient levels are extremely low. Recently, a family of genes designated hli was demonstrated to be important for survival of cyanobacteria during exposure to high light. In this study we have identified members of the hli gene family in seven cyanobacterial genomes, including those of a marine cyanobacterium adapted to high-light growth in surface waters of the open ocean (Prochlorococcus sp. strain Med4), three marine cyanobacteria adapted to growth in moderate- or low-light (Prochlorococcus sp. strain MIT9313, Prochlorococcus marinus SS120, and Synechococcus WH8102), and three freshwater strains (the unicellular Synechocystis sp. strain PCC6803 and the filamentous species Nostoc punctiforme strain ATCC29133 and Anabaena sp. [Nostoc] strain PCC7120). The high-light-adapted Prochlorococcus Med4 has the smallest genome (1.7 Mb), yet it has more than twice as many hli genes as any of the other six cyanobacterial species, some of which appear to have arisen from recent duplication events. Based on cluster analysis, some groups of hli genes appear to be specific to either marine or freshwater cyanobacteria. This information is discussed with respect to the role of hli genes in the acclimation of cyanobacteria to high light, and the possible relationships among members of this diverse gene family.     

Structural characterization of four prochlorosins: a novel class of lantipeptides produced by planktonic marine cyanobacteria

Prochlorosins make up a class of secondary metabolites produced by strains of Prochlorococcus, single-cell, planktonic marine cyanobacteria. These polycyclic peptides contain lanthionine and methyllanthionine residues that result in thioether cross-links. In Prochlorococcus MIT9313, a single enzyme, ProcM, catalyzes the posttranslational modification of 29 linear peptide substrates to generate a library of highly diverse cyclic peptides. To investigate the catalytic promiscuity of ProcM, we chose four prochlorosins previously demonstrated to be produced by the organism for detailed structural characterization. Nuclear magnetic resonance studies allowed unambiguous assignment of the ring topologies, demonstrating a high degree of topological diversity. The stereochemistry of the lanthionine and methyllanthionine residues was determined by gas chromatography and mass spectrometry for seven prochlorosins. All methyllanthionines had the (2S,3S,6R) configuration, and the lanthionines had the (2S,6R) configuration, irrespective of the direction of cyclization, ring size, or ring topology. These findings indicate that most, if not all, of the rings in prochlorosins are formed enzymatically by ProcM lanthionine synthetase and not by a nonenzymatic process as previously suggested.     

Dependence of the cyanobacterium Prochlorococcus on hydrogen peroxide scavenging microbes for growth at the ocean's surface

The phytoplankton community in the oligotrophic open ocean is numerically dominated by the cyanobacterium Prochlorococcus, accounting for approximately half of all photosynthesis. In the illuminated euphotic zone where Prochlorococcus grows, reactive oxygen species are continuously generated via photochemical reactions with dissolved organic matter. However, Prochlorococcus genomes lack catalase and additional protective mechanisms common in other aerobes, and this genus is highly susceptible to oxidative damage from hydrogen peroxide (HOOH). In this study we showed that the extant microbial community plays a vital, previously unrecognized role in cross-protecting Prochlorococcus from oxidative damage in the surface mixed layer of the oligotrophic ocean. Microbes are the primary HOOH sink in marine systems, and in the absence of the microbial community, surface waters in the Atlantic and Pacific Ocean accumulated HOOH to concentrations that were lethal for Prochlorococcus cultures. In laboratory experiments with the marine heterotroph Alteromonas sp., serving as a proxy for the natural community of HOOH-degrading microbes, bacterial depletion of HOOH from the extracellular milieu prevented oxidative damage to the cell envelope and photosystems of co-cultured Prochlorococcus, and facilitated the growth of Prochlorococcus at ecologically-relevant cell concentrations. Curiously, the more recently evolved lineages of Prochlorococcus that exploit the surface mixed layer niche were also the most sensitive to HOOH. The genomic streamlining of these evolved lineages during adaptation to the high-light exposed upper euphotic zone thus appears to be coincident with an acquired dependency on the extant HOOH-consuming community. These results underscore the importance of (indirect) biotic interactions in establishing niche boundaries, and highlight the impacts that community-level responses to stress may have in the ecological and evolutionary outcomes for co-existing species.     

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.     

Glutamine synthetase from the marine cyanobacteria Prochlorococcus spp: characterization,phylogeny and response to nutrient limitation

The regulation of glutamine synthetase (EC 6.3.1.2) from Prochlorococcus was previously shown to exhibit unusual features: it is not upregulated by nitrogen starvation and it is not inactivated by darkness (El Alaoui et al. (2001) Appl Environ Microbiol 67: 2202-2207). These are probably caused by adaptations to oligotrophic environments, as confirmed in this work by the marked decrease in the enzymatic activity when cultures were subjected to iron or phosphorus starvation. In order to further understand the adaptive features of ammonium assimilation in this cyanobacterium, glutamine synthetase was purified from two Prochlorococcus strains: PCC 9511 (high-light adapted) and SS120 (low-light adapted). We obtained approximately 100-fold purified samples of glutamine synthetase electrophoretically homogeneous, with a yield of approximately 30%. The estimated molecular mass of the subunits was roughly the same for both strains: 48.3 kDa. The apparent Km constants for the biosynthetic activity were 0.30 mM for ammonium, 1.29 mM for glutamate and 1.35 mM for ATP; the optimum pH was 8.0. Optimal temperature was surprisingly high (55 degrees C). Phylogenetic analysis of glnA from three Prochlorococcus strains (MED4, MIT9313 and SS120) showed they group closely with marine Synechococcus isolates, in good agreement with other studies based on 16 S RNA sequences. All of our results suggest that the structure and kinetics of glutamine synthetase in Prochlorococcus have not been significantly modified during the evolution within the cyanobacterial radiation, in sharp contrast with its regulatory properties.     

Glucose uptake and its effect on gene expression in prochlorococcus

The marine cyanobacteria Prochlorococcus have been considered photoautotrophic microorganisms, although the utilization of exogenous sugars has never been specifically addressed in them. We studied glucose uptake in different high irradiance- and low irradiance-adapted Prochlorococcus strains, as well as the effect of glucose addition on the expression of several glucose-related genes. Glucose uptake was measured by adding radiolabelled glucose to Prochlorococcus cultures, followed by flow cytometry coupled with cell sorting in order to separate Prochlorococcus cells from bacterial contaminants. Sorted cells were recovered by filtration and their radioactivity measured. The expression, after glucose addition, of several genes (involved in glucose metabolism, and in nitrogen assimilation and its regulation) was determined in the low irradiance-adapted Prochlorococcus SS120 strain by semi-quantitative real time RT-PCR, using the rnpB gene as internal control. Our results demonstrate for the first time that the Prochlorococcus strains studied in this work take up glucose at significant rates even at concentrations close to those found in the oceans, and also exclude the possibility of this uptake being carried out by eventual bacterial contaminants, since only Prochlorococcus cells were used for radioactivity measurements. Besides, we show that the expression of a number of genes involved in glucose utilization (namely zwf, gnd and dld, encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and lactate dehydrogenase, respectively) is strongly increased upon glucose addition to cultures of the SS120 strain. This fact, taken together with the magnitude of the glucose uptake, clearly indicates the physiological importance of the phenomenon. Given the significant contribution of Prochlorococcus to the global primary production, these findings have strong implications for the understanding of the phytoplankton role in the carbon cycle in nature. Besides, the ability of assimilating carbon molecules could provide additional hints to comprehend the ecological success of Prochlorococcus.     

Detection and expression of the phosphonate transporter gene phnD in marine and freshwater picocyanobacteria

We describe a PCR-based assay designed to detect expression of the phosphonate assimilation gene phnD from picocyanobacteria. The phnD gene encodes the phosphonate binding protein of the ABC-type phosphonate transporter, present in many of the picocyanobacterial genome sequences. Detection of phnD expression can indicate a capacity of picoplankton to utilize phosphonates, a refractory form of phosphorus that can represent 25% of the high-molecular-weight dissolved organic phosphorus pool in marine systems. Primer sets were designed to specifically amplify phnD sequences from marine and freshwater Synechococcus spp., Prochlorococcus spp. and environmental samples from the ocean and Laurentian Great Lakes. Quantitative RT-PCR from cultured marine Synechococcus sp. strain WH8102 and freshwater Synechococcus sp. ARC-21 demonstrated induction of phnD expression in P-deficient media, suggesting that phn genes are regulated coordinately with genes under phoRB control. Last, RT-PCR of environmental RNA samples from the Sargasso Sea and Pacific Ocean detected phnD expression from the endemic picocyanobacterial population. Synechococcus spp. phnD expression yielded a depth-dependent pattern following gradients of P bioavailability. By contrast, the Prochlorococcus spp. primers revealed that in all samples tested, phnD expression was constitutive. The method described herein will allow future studies aimed at understanding the utilization of naturally occurring phoshonates in the ocean as well as monitoring the acquisition of synthetic phosphonate herbicides (e.g. glyphosate) by picocyanobacteria in freshwaters.     

High rate of uptake of organic nitrogen compounds by Prochlorococcus cyanobacteria as a key to their dominance in oligotrophic oceanic waters

Direct evidence that marine cyanobacteria take up organic nitrogen compounds in situ at high rates is reported. About 33% of the total bacterioplankton turnover of amino acids, determined with a representative [(35)S]methionine precursor and flow sorting, can be assigned to Prochlorococcus spp. and 3% can be assigned to Synechococcus spp. in the oligotrophic and mesotrophic parts of the Arabian Sea, respectively. This finding may provide a mechanism for Prochlorococcus' competitive dominance over both strictly autotrophic algae and other bacteria in oligotrophic regions sustained by nutrient remineralization via a microbial loop.     

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.     

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.     

Basin-scale distribution patterns of picocyanobacterial lineages in the Atlantic Ocean

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus are major contributors to oceanic primary production. The genera are genetically diverse, comprising several known ecotypes or lineages. However, little is known of the distribution of these lineages over large geographic areas. Here, we analysed the relative abundance of Prochlorococcus ecotypes and Synechococcus lineages at the ocean basin scale along an Atlantic Meridional Transect (AMT) using dot blot hybridization and fluorescence in situ hybridization (FISH) techniques. The transect covered several contrasting oceanic provinces (gyres, upwelling, temperate regions) as well as environmentally 'equivalent' regions in the northern and southern hemisphere (northern and southern gyres and temperate regions). Flow cytometric data revealed a discrete separation in abundance of major picocyanobacterial genera. Prochlorococcus reached highest abundance in oligotrophic regions, while more mesotrophic waters were dominated by Synechococcus. Individual genetic lineages of both Prochlorococcus and Synechococcus showed highly similar distributions in corresponding regions in the northern and southern hemisphere. In addition, Prochlorococcus showed a distinctive depth distribution, with HLI and HLII ecotypes near the surface and co-occurring LL ecotypes further down in the water column. Conversely, Synechococcus generally revealed no obvious depth preference, but did show highly specific distribution at the horizontal scale, with clades I and IV particularly dominating temperate, mesotrophic waters in both the northern and southern hemispheres. The data clearly reveal that specific picocyanobacterial lineages proliferate in similar oceanic provinces separated by large spatial scales. Furthermore, comparison with an earlier AMT dataset suggests that basin scale distribution patterns for Prochlorococcus ecotypes are remarkably reproducible from year to year.     

Attenuation of phosphate starvation responses by phosphite in Arabidopsis.

When inorganic phosphate is limiting, Arabidopsis has the facultative ability to metabolize exogenous nucleic acid substrates, which we utilized previously to identify insensitive phosphate starvation response mutants in a conditional genetic screen. In this study, we examined the effect of the phosphate analog, phosphite (Phi), on molecular and morphological responses to phosphate starvation. Phi significantly inhibited plant growth on phosphate-sufficient (2 mM) and nucleic acid-containing (2 mM phosphorus) media at concentrations higher than 2.5 mM. However, with respect to suppressing typical responses to phosphate limitation, Phi effects were very similar to those of phosphate. Phosphate starvation responses, which we examined and found to be almost identically affected by both anions, included changes in: (a) the root-to-shoot ratio; (b) root hair formation; (c) anthocyanin accumulation; (d) the activities of phosphate starvation-inducible nucleolytic enzymes, including ribonuclease, phosphodiesterase, and acid phosphatase; and (e) steady-state mRNA levels of phosphate starvation-inducible genes. It is important that induction of primary auxin response genes by indole-3-acetic acid in the presence of growth-inhibitory Phi concentrations suggests that Phi selectively inhibits phosphate starvation responses. Thus, the use of Phi may allow further dissection of phosphate signaling by genetic selection for constitutive phosphate starvation response mutants on media containing organophosphates as the only source of phosphorus.