Biosynthesis of open-chain tetrapyrroles in Prochlorococcus marinus

Members of the genus Prochlorococcus belong to the most abundant phytoplankton on earth. In contrast to other cyanobacteria, Prochlorococcus is characterized by divinyl-chlorophyll containing light-harvesting complexes and the lack of phycobilisomes. Despite the lack of phycobilisomes, all sequenced genomes of Prochlorococcus possess genes that putatively encode enzymes involved in the biosynthesis of open-chain tetrapyrrole molecules. Here, biochemical evidence is presented indicating that high-light- and low-light-adapted Prochlorococcus ecotypes possess genes encoding functional enzymes for the biosynthesis of open-chain tetrapyrrole molecules. Experiments on recombinant protein as well as through complementation studies of a cyanobacterial insertion mutant revealed the functionality of the bilin reductases investigated.     

Glucosylglycerate: a secondary compatible solute common to marine cyanobacteria from nitrogen-poor environments

The synthesis and accumulation of compatible solutes represent an essential part of the salt acclimation strategy of microorganisms. Glucosylglycerol is considered to be the typical compatible solute among marine cyanobacteria. However, genes that encode enzymes for the synthesis of glucosylglycerol were not detected in the genome sequences of marine picoplanktonic Prochlorococcus strains. Instead, we noticed the presence of genes that putatively encode for glucosylglycerate (GGA) synthesis among Prochlorococcus and most other closely related marine picocyanobacteria. Recombinant proteins from Prochlorococcus marinus SS120 and Synechococcus sp. PCC 7002 exhibited glucosyl-phosphoglycerate synthase (GpgS) activity, and GpgS is a key enzyme of GGA synthesis. GGA accumulation was found to be salt- as well as nitrogen-regulated in the coastal strain Synechococcus sp. PCC 7002. Moreover, GGA was also detected in all picoplanktonic Prochlorococcus and Synechococcus strains harbouring gpgS genes, especially under N-limiting conditions. These results suggest that marine picocyanobacteria acquired the capacity to synthesize the negatively charged compound GGA during their evolution. Our results establish GGA as the fifth most widespread compatible solute among cyanobacteria. Additionally, GGA appears to replace glutamate as an anion to counter monovalent cations in marine picocyanobacteria from N-poor environments.     

A novel type of lycopene ε-cyclase in the marine cyanobacterium<Emphasis Type="Italic"> Prochlorococcus marinus</Emphasis> MED4

Chlorophyll- b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of alpha- and beta-carotenoids with green algae and higher plants. The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, epsilon- and beta-lycopene cyclase. All cyanobacteria studied so far possess a single beta-cyclase. Here, two different Prochlorococcus sp. MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases. Whereas one is both functionally and in sequence highly similar to the beta-cyclase of Synechococcus sp. strain PCC 7942 and other cyanobacteria, the other showed several intriguing features. It acts as a bifunctional enzyme catalyzing the formation of epsilon- as well as of beta-ionone end groups. Expression of this cyclase in E. coli resulted in the simultaneous accumulation of alpha- beta-, delta-, and epsilon-carotene. Such an activity is in contrast to all lycopene epsilon-cyclases known so far, including those of the higher plants. Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either beta- or epsilon-end groups. These two genes are suggested to be designated as crtL-b and crtL-e. The results indicate that both enzymes might have originated from duplication of a single gene. Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.     

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.     

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.     

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.     

Efficient phage-mediated pigment biosynthesis in oceanic cyanobacteria

Although the oceanic cyanobacterium Prochlorococcus harvests light with a chlorophyll antenna [1-3] rather than with the phycobilisomes that are typical of cyanobacteria, some strains express genes that are remnants of the ancestral Synechococcus phycobilisomes [4]. Similarly, some Prochlorococcus cyanophages, which often harbor photosynthesis-related genes [5], also carry homologs of phycobilisome pigment biosynthesis genes [6, 7]. Here, we investigate four such genes in two cyanophages that both infect abundant Prochlorococcus strains [8]: homologs of heme oxygenase (ho1), 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (pebA), ferredoxin (petF) in the myovirus P-SSM2, and a phycocyanobilin:ferredoxin oxidoreductase (pcyA) homolog in the myovirus P-SSM4. We demonstrate that the phage homologs mimic the respective host activities, with the exception of the divergent phage PebA homolog. In this case, the phage PebA single-handedly catalyzes a reaction for which uninfected host cells require two consecutive enzymes, PebA and PebB. We thus renamed the phage enzyme phycoerythrobilin synthase (PebS). This gene, and other pigment biosynthesis genes encoded by P-SSM2 (petF and ho1), are transcribed during infection, suggesting that they can improve phage fitness. Analyses of global ocean metagenomes show that PcyA and Ho1 occur in both cyanobacteria and their phages, whereas the novel PebS-encoding gene is exclusive to phages.     

Carotenoid biosynthesis in cyanobacteria: structural and evolutionary scenarios based on comparative genomics

Carotenoids are widely distributed pigments in nature and their biosynthetic pathway has been extensively studied in various organisms. The recent access to the overwhelming amount genomic data of cyanobacteria has given birth to a novel approach called comparative genomics. The putative enzymes involved in the carotenoid biosynthesis among the cyanobacteria were determined by similarity-based tools. The reconstruction of biosynthetic pathway was based on the related enzymes. It is interesting to find that nearly all the cyanobacteria share quite similar pathway to synthesize beta-carotene except for Gloeobacter violaceus PCC 7421. The enzymes, crtE-B-P-Qb-L, involved in the upstream pathway are more conserved than the subsequent ones (crtW-R). In addition, many carotenoid synthesis enzymes exhibit diversity in structure and function. Such examples in the families of zeta -carotene desaturase, lycopene cylases and carotene ketolases were described in this article. When we mapped these crt genes to the cyanobacterial genomes, the crt genes showed great structural variation among species. All of them are dispersed on the whole chromosome in contrast to the linear adjacent distribution of the crt gene cluster in other eubacteria. Moreover, in unicellular cyanobacteria, each step of the carotenogenic pathway is usually catalyzed by one gene product, whereas multiple ketolase genes are found in filamentous cyanobacteria. Such increased numbers of crt genes and their correlation to the ecological adaptation were carefully discussed.     

噬藻体光合作用基因研究

感染原绿球藻和聚球藻的噬藻体基因组中普遍存在与psbA、psbD和hli等同源的基因,这些基因编码的蛋白参与光合作用,是光合成反应中心II(photosystem II,PSII)的重要组成成分,在噬藻体感染蓝藻过程中可能发挥着重要的作用。一些假说认为这些基因可能来自于宿主并发生共进化。因此,光合作用基因的功能、起源与演变及基因多样性分布引起了人们的关注。     

Genomic and structural analysis of Syn9, a cyanophage infecting marine Prochlorococcus and Synechococcus

Cyanobacteriophage Syn9 is a large, contractile-tailed bacteriophage infecting the widespread, numerically dominant marine cyanobacteria of the genera Prochlorococcus and Synechococcus . Its 177 300 bp genome sequence encodes 226 putative proteins and six tRNAs. Experimental and computational analyses identified genes likely involved in virion formation, nucleotide synthesis, and DNA replication and repair. Syn9 shows significant mosaicism when compared with related cyanophages S-PM2, P-SSM2 and P-SSM4, although shared genes show strong purifying selection and evidence for large population sizes relative to other phages. Related to coliphage T4 – which shares 19% of Syn9's genes – Syn9 shows evidence for different patterns of DNA replication and uses homologous proteins to assemble capsids with a different overall structure that shares topology with phage SPO1 and herpes virus. Noteworthy bacteria-related sequences in the Syn9 genome potentially encode subunits of the photosynthetic reaction centre, electron transport proteins, three pentose pathway enzymes and two tryptophan halogenases. These genes suggest that Syn9 is well adapted to the physiology of its photosynthetic hosts and may affect the evolution of these sequences within marine cyanobacteria.     

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.     

Genome streamlining results in loss of robustness of the circadian clock in the marine cyanobacterium Prochlorococcus marinus PCC 9511

The core oscillator of the circadian clock in cyanobacteria consists of 3 proteins, KaiA, KaiB, and KaiC. All 3 have previously been shown to be essential for clock function. Accordingly, most cyanobacteria possess at least 1 copy of each kai gene. One exception is the marine genus Prochlorococcus, which we suggest here has suffered a stepwise deletion of the kaiA gene, together with significant genome streamlining. Nevertheless, natural Prochlorococcus populations and laboratory cultures are strongly synchronized by the alternation of day and night, displaying 24-h rhythms in DNA replication, with a temporal succession of G1, S, and G2-like cell cycle phases. Using quantitative real-time PCR, we show here that in Prochlorococcus marinus PCC 9511, the mRNA levels of the clock genes kaiB and kaiC, as well as a few other selected genes including psbA, also displayed marked diel variations when cultures were kept under a light-dark rhythm. However, both cell cycle and psbA gene expression rhythms damped very rapidly under continuous light. In the closely related Synechococcus sp. WH8102, which possesses all 3 kai genes, cell cycle rhythms persisted over several days, in agreement with established cyanobacterial models. These data indicate a correlation between the loss of kaiA and a loss of robustness in the endogenous oscillator of Prochlorococcus and raise questions about how a basic KaiBC system may function and through which mechanism the daily "lights-on" and "lights-off" signal could be mediated.     

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.     

Phycoerythrins of the oxyphotobacterium Prochlorococcus marinus are associated to the thylakoid membrane and are encoded by a single large gene cluster

An intrinsic divinyl-chlorophyll a/b antenna and a particular form of phycobiliprotein, phycoerythrin (PE) III, coexist in the marine oxyphotobacterium Prochlorococcus marinus CCMP 1375. The genomic region including the cpeB/A operon of P. marinus was analysed. It encompasses 10153 nucleotides that encode three structural phycobiliproteins and at least three (possibly five) different polypeptides analogous to cyanobacterial or red algal proteins involved either in the linkage of subunits or the synthesis and attachment of chromophoric groups. This gene cluster is part of the chromosome and is located within a distance of less than 110 kb from a previously characterized region containing the genes aspA-psbA-aroC. Whereas the Prochlorococcus phycobiliproteins are characterized by distinct deletions and amino acid replacements with regard to analogous proteins from other organisms, the gene arrangement resembles the organization of phycobiliprotein genes in some other cyanobacteria, in particular marine Synechococcus strains. The expression of two of the Prochlorococcus polypeptides as recombinant proteins in Escherichia coli allowed the production of individual homologous antisera to the Prochlorococcus and PE subunits. Experiments using these sera show that the Prochlorococcus PEs are specifically associated to the thylakoid membrane and that the protein level does not significantly vary as a function of light irradiance or growth phase.     

Marine cyanophages and light

In contrast to the phages of heterotrophic hosts, light can play a key role in all aspects of the life cycle of phages infecting ecologically important marine unicellular cyanobacteria of the genera Synechococcus and Prochlorococcus. Phage adsorption, replication, modulation of the host cell metabolism, and survival in the environment following lysis, all exhibit light-dependent components. The analysis of cyanophage genomes has revealed the acquisition of key photosynthetic genes during the course of evolution, such as those encoding central components of the light harvesting apparatus. These discoveries are beginning to reveal novel features of the interactions between parasite and host that shape the biology of both.     

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.     

Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean

(35)S-Methionine and (3)H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.