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.     

Prochlorococcus, a marine photosynthetic prokaryote of global significance.

The minute photosynthetic prokaryote Prochlorococcus, which was discovered about 10 years ago, has proven exceptional from several standpoints. Its tiny size (0.5 to 0.7 microm in diameter) makes it the smallest known photosynthetic organism. Its ubiquity within the 40 degrees S to 40 degrees N latitudinal band of oceans and its occurrence at high density from the surface down to depths of 200 m make it presumably the most abundant photosynthetic organism on Earth. Prochlorococcus typically divides once a day in the subsurface layer of oligotrophic areas, where it dominates the photosynthetic biomass. It also possesses a remarkable pigment complement which includes divinyl derivatives of chlorophyll a (Chl a) and Chl b, the so-called Chl a2 and Chl b2, and, in some strains, small amounts of a new type of phycoerythrin. Phylogenetically, Prochlorococcus has also proven fascinating. Recent studies suggest that it evolved from an ancestral cyanobacterium by reducing its cell and genome sizes and by recruiting a protein originally synthesized under conditions of iron depletion to build a reduced antenna system as a replacement for large phycobilisomes. Environmental constraints clearly played a predominant role in Prochlorococcus evolution. Its tiny size is an advantage for its adaptation to nutrient-deprived environments. Furthermore, genetically distinct ecotypes, with different antenna systems and ecophysiological characteristics, are present at depth and in surface waters. This vertical species variation has allowed Prochlorococcus to adapt to the natural light gradient occurring in the upper layer of oceans. The present review critically assesses the basic knowledge acquired about Prochlorococcus both in the ocean and in the laboratory.     

Prochlorococcus, a Marine Photosynthetic Prokaryote of Global Significance

The minute photosynthetic prokaryote Prochlorococcus, which was discovered about 10 years ago, has proven exceptional from several standpoints. Its tiny size (0.5 to 0.7 μm in diameter) makes it the smallest known photosynthetic organism. Its ubiquity within the 40°S to 40°N latitudinal band of oceans and its occurrence at high density from the surface down to depths of 200 m make it presumably the most abundant photosynthetic organism on Earth. Prochlorococcus typically divides once a day in the subsurface layer of oligotrophic areas, where it dominates the photosynthetic biomass. It also possesses a remarkable pigment complement which includes divinyl derivatives of chlorophyll a (Chl a) and Chl b, the so-called Chl a₂ and Chl b₂, and, in some strains, small amounts of a new type of phycoerythrin. Phylogenetically, Prochlorococcus has also proven fascinating. Recent studies suggest that it evolved from an ancestral cyanobacterium by reducing its cell and genome sizes and by recruiting a protein originally synthesized under conditions of iron depletion to build a reduced antenna system as a replacement for large phycobilisomes. Environmental constraints clearly played a predominant role in Prochlorococcus evolution. Its tiny size is an advantage for its adaptation to nutrient-deprived environments. Furthermore, genetically distinct ecotypes, with different antenna systems and ecophysiological characteristics, are present at depth and in surface waters. This vertical species variation has allowed Prochlorococcus to adapt to the natural light gradient occurring in the upper layer of oceans. The present review critically assesses the basic knowledge acquired about Prochlorococcus both in the ocean and in the laboratory.     

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.     

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 .     

Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511

Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed.     

Glutamine synthetase degradation is controlled by oxidative proteolysis in the marine cyanobacterium Prochlorococcus marinus strain PCC 9511

Prochlorococcus is one of the most important primary producers on Earth; its unusual features and ecological importance have made it a model organism, but nutrient assimilation has received little attention. Glutamine synthetase (GS) plays a key role in nitrogen metabolism and its central position justifies the fine regulation of this enzyme. The aim of this work is to demonstrate the involvement of metal-catalyzed oxidation (MCO) in the control of the biological activity and turnover of GS from Prochlorococcus. In order to study the physiological role of MCO, we have first characterized the in vitro biosynthetic inactivation and degradation of GS in the axenic PCC 9511 strain, testing then the effect of several stress conditions, such as the presence of electron transport inhibitors, darkness and aging, on the inactivation and degradation of GS. It is noteworthy that the physiological substrates of GS could protect the enzyme from the oxidative inactivation and ATP partially reverted this inactivation once the enzyme had been oxidized, being this effect higher in the presence of glutamate. We have also found that the GS from aged cultures is degraded to the same smaller size fragments obtained in the in vitro degradation of GS by an oxidative model system (Fe3+/NADH/NADH oxidase/O2). These results suggest the implication of MCO in the age- and oxidative state-dependent degradation of GS from Prochlorococcus.     

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.     

Prochlorococcus ecotype abundances in the North Atlantic Ocean as revealed by an improved quantitative PCR method

The cyanobacterium Prochlorococcus numerically dominates the photosynthetic community in the tropical and subtropical regions of the world's oceans. Six evolutionary lineages of Prochlorococcus have been described, and their distinctive physiologies and genomes indicate that these lineages are "ecotypes" and should have different oceanic distributions. Two methods recently developed to quantify these ecotypes in the field, probe hybridization and quantitative PCR (QPCR), have shown that this is indeed the case. To facilitate a global investigation of these ecotypes, we modified our QPCR protocol to significantly increase its speed, sensitivity, and accessibility and validated the method in the western and eastern North Atlantic Ocean. We showed that all six ecotypes had distinct distributions that varied with depth and location, and, with the exception of the deeper waters at the western North Atlantic site, the total Prochlorococcus counts determined by QPCR matched the total counts measured by flow cytometry. Clone library analyses of the deeper western North Atlantic waters revealed ecotypes that are not represented in the culture collections with which the QPCR primers were designed, explaining this discrepancy. Finally, similar patterns of relative ecotype abundance were obtained in QPCR and probe hybridization analyses of the same field samples, which could allow comparisons between studies.     

Phosphite utilization by the marine picocyanobacterium Prochlorococcus MIT9301

Primary productivity in the ocean's oligotrophic regions is often limited by phosphorus (P) availability. In low phosphate environments, the prevalence of many genes involved in P acquisition is elevated, suggesting that the ability to effectively access diverse P sources is advantageous for organisms inhabiting these regions. Prochlorococcus, the numerically dominant primary producer in the oligotrophic ocean, encodes high-affinity P transporters, P regulatory proteins and enzymes for organic phosphate utilization, but its ability to use reduced P compounds has not been previously demonstrated. Because Prochlorococcus strain MIT9301 encodes genes similar to phnY and phnZ, which constitute a novel marine bacterial 2-aminoethylphosphonate (2-AEPn) utilization pathway, it has been suggested that this organism might use 2-AEPn as an alternative P source. We show here that although MIT9301 was unable to use 2-AEPn as a sole P source under standard culture conditions, it was able to use phosphite. Phosphite utilization by MIT9301 appears to be mediated by an NAD-dependent phosphite dehydrogenase encoded by ptxD. We show that phosphite utilization genes are present in diverse marine microbes and that their abundance is higher in low-P waters. These results strongly suggest that phosphite represents a previously unrecognized component of the marine P cycle.     

Measurement of Prochlorococcus ecotypes using real-time polymerase chain reaction reveals different abundances of genotypes with similar light physiologies

Prochlorococcus is a marine cyanobacterium which is found at high abundances in world's tropical and subtropical oligotrophic oceans. The genus Prochlorococcus can be divided into two major groups based on light physiology. Both of these groups can be further subdivided into genetically distinct lineages, or ecotypes. Real-time polymerase chain reaction (PCR) assays based on sequence differences in the 16S-23S rDNA internal transcribed spacer or the 23S rDNA were developed to examine the distribution of each ecotype in the field. The real-time PCR assays enabled linear quantification of concentrations ranging from 10 to 4 x 10(5) cells ml(-1). These assays were applied to a stratified water column in the Sargasso Sea. The majority of Prochlorococcus cells above 110 m belonged to the one of the low chlorophyll b/a ratio (high-light adapted) ecotypes, while two types of high chlorophyll b/a ratio (low-light adapted) cells dominated below 110 m. The other three types were found at significantly lower numbers or not detected at all. Differences in the abundance of ecotypes within the major light physiology groupings suggest that other factors, such as nutrient utilization and differential mortality, are driving their relative distributions. Real-time PCR assays will enable further exploration of these factors and temporal and geographic variability in ecotype abundance.     

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.     

海洋微型和微微型浮游生物的区域分布与影响因素

微型和微微型浮游生物几乎存在于所有的海洋生态系统中,其群落遍布世界各大洋。在大部分海域,微型浮游生物的优势类群为鞭毛藻;原绿球藻是贫营养海域的优势类群,而聚球藻、微微型真核生物和异养细菌主要在营养盐丰富的海域出现,在热带、亚热带和温带的富营养区域占优势。温度、盐度、光照、营养盐可得性、水体稳定性和摄食压力是影响微型和微微型浮游生物的主要因素,各海域主要调控因子的不同造成了微型和微微型浮游生物类群和丰度分布的差异。本文主要综述海洋微型和微微型浮游生物类群检测方法、区域分布特点及其受环境影响的研究概况,并提出了今后的重点研究内容和发展方向。     

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.     

Community differentiation and population enrichment of Sargasso Sea bacterioplankton in the euphotic zone of a mesoscale mode‐water eddy

Eddies are mesoscale oceanographic features (～ 200 km diameter) that can cause transient blooms of phytoplankton by shifting density isoclines in relation to light and nutrient resources. To better understand how bacterioplankton respond to eddies, we examined depth‐resolved distributions of bacterial populations across an anticyclonic mode‐water eddy in the Sargasso Sea. Previous work on this eddy has documented elevated phytoplankton productivity and diatom abundance within the eddy centre with coincident bacterial productivity and biomass maxima. We illustrate bacterial community shifts within the eddy centre, differentiating populations uplifted along isopycnals from those enriched or depleted at horizons of enhanced bacterial and primary productivity. Phylotypes belonging to the Roseobacter, OCS116 and marine Actinobacteria clades were enriched in the eddy core and were highly correlated with pigment‐based indicators of diatom abundance, supporting developing hypotheses that members of these clades associate with phytoplankton blooms. Typical mesopelagic clades (SAR202, SAR324, SAR406 and SAR11 IIb) were uplifted within the eddy centre, increasing bacterial diversity in the lower euphotic zone. Typical surface oligotrophic clades (SAR116, OM75, Prochlorococcus and SAR11 Ia) were relatively depleted in the eddy centre. The biogeochemical context of a bloom‐inducing eddy provides insight into the ecology of the diverse uncultured bacterioplankton dominating the oligotrophic oceans.     

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.     

Cyanobacterial photosynthesis in the oceans: the origins and significance of divergent light-harvesting strategies

Prochlorococcus and Synechococcus are abundant unicellular cyanobacteria and major participants in global carbon cycles. Although they are closely related and often coexist in the same ocean habitat, they possess very different photosynthetic light-harvesting antennas. Whereas Synechococcus and the majority of cyanobacteria use phycobilisomes, Prochlorococcus has evolved to use a chlorophyll a(2)/b(2) light-harvesting complex. Here, we present a scenario to explain how the Prochlorococcus antenna might have evolved in an ancestral cyanobacterium in iron-limited oceans, resulting in the diversification of the Prochlorococcus and marine Synechococcus lineages from a common phycobilisome-containing ancestor. Differences in the absorption properties and cellular costs between chlorophyll a(2)/b(2) and phycobilisome antennas in extant Prochlorococcus and Synechococcus appear to play a role in differentiating their ecological niches in the ocean environment.