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Background

The marine cyanobacterium Prochlorococcus is very abundant in warm, nutrient-poor oceanic areas. The upper mixed layer of oceans is populated by high light-adapted Prochlorococcus ecotypes, which despite their tiny genome (~1.7 Mb) seem to have developed efficient strategies to cope with stressful levels of photosynthetically active and ultraviolet (UV) radiation. At a molecular level, little is known yet about how such minimalist microorganisms manage to sustain high growth rates and avoid potentially detrimental, UV-induced mutations to their DNA. To address this question, we studied the cell cycle dynamics of P. marinus PCC9511 cells grown under high fluxes of visible light in the presence or absence of UV radiation. Near natural light-dark cycles of both light sources were obtained using a custom-designed illumination system (cyclostat). Expression patterns of key DNA synthesis and repair, cell division, and clock genes were analyzed in order to decipher molecular mechanisms of adaptation to UV radiation.

Results

The cell cycle of P. marinus PCC9511 was strongly synchronized by the day-night cycle. The most conspicuous response of cells to UV radiation was a delay in chromosome replication, with a peak of DNA synthesis shifted about 2 h into the dark period. This delay was seemingly linked to a strong downregulation of genes governing DNA replication (dnaA) and cell division (ftsZ, sepF), whereas most genes involved in DNA repair (such as recA, phrA, uvrA, ruvC, umuC) were already activated under high visible light and their expression levels were only slightly affected by additional UV exposure.

Conclusions

Prochlorococcus cells modified the timing of the S phase in response to UV exposure, therefore reducing the risk that mutations would occur during this particularly sensitive stage of the cell cycle. We identified several possible explanations for the observed timeshift. Among these, the sharp decrease in transcript levels of the dnaA gene, encoding the DNA replication initiator protein, is sufficient by itself to explain this response, since DNA synthesis starts only when the cellular concentration of DnaA reaches a critical threshold. However, the observed response likely results from a more complex combination of UV-altered biological processes.

Summary: Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus numerically dominate the picophytoplankton of the world ocean, making a key contribution to global primary production. Prochlorococcus was isolated around 20 years ago and is probably the most abundant photosynthetic organism on Earth. The genus comprises specific ecotypes which are phylogenetically distinct and differ markedly in their photophysiology, allowing growth over a broad range of light and nutrient conditions within the 45°N to 40°S latitudinal belt that they occupy. Synechococcus and Prochlorococcus are closely related, together forming a discrete picophytoplankton clade, but are distinguishable by their possession of dissimilar light-harvesting apparatuses and differences in cell size and elemental composition. Synechococcus strains have a ubiquitous oceanic distribution compared to that of Prochlorococcus strains and are characterized by phylogenetically discrete lineages with a wide range of pigmentation. In this review, we put our current knowledge of marine picocyanobacterial genomics into an environmental context and present previously unpublished genomic information arising from extensive genomic comparisons in order to provide insights into the adaptations of these marine microbes to their environment and how they are reflected at the genomic level.

Prochlorococcus and Synechococcus picocyanobacteria are dominant contributors to marine primary production over large areas of the ocean. Phytoplankton cells are entrained in the water column and are thus often exposed to rapid changes in irradiance within the upper mixed layer of the ocean. An upward fluctuation in irradiance can result in photosystem II photoinactivation exceeding counteracting repair rates through protein turnover, thereby leading to net photoinhibition of primary productivity, and potentially cell death. Here we show that the effective cross-section for photosystem II photoinactivation is conserved across the picocyanobacteria, but that their photosystem II repair capacity and protein-specific photosystem II light capture are negatively correlated and vary widely across the strains. The differences in repair rate correspond to the light and nutrient conditions that characterize the site of origin of the Prochlorococcus and Synechococcus isolates, and determine the upward fluctuation in irradiance they can tolerate, indicating that photoinhibition due to transient high-light exposure influences their distribution in the ocean.

Picocyanobacteria represented by Prochlorococcus and Synechococcus have an important role in oceanic carbon fixation and nutrient cycling. In this study, we compared the community composition of picocyanobacteria from diverse marine ecosystems ranging from estuary to open oceans, tropical to polar oceans and surface to deep water, based on the sequences of 16S-23S rRNA internal transcribed spacer (ITS). A total of 1339 ITS sequences recovered from 20 samples unveiled diverse and several previously unknown clades of Prochlorococcus and Synechococcus. Six high-light (HL)-adapted Prochlorococcus clades were identified, among which clade HLVI had not been described previously. Prochlorococcus clades HLIII, HLIV and HLV, detected in the Equatorial Pacific samples, could be related to the HNLC clades recently found in the high-nutrient, low-chlorophyll (HNLC), iron-depleted tropical oceans. At least four novel Synechococcus clades (out of six clades in total) in subcluster 5.3 were found in subtropical open oceans and the South China Sea. A niche partitioning with depth was observed in the Synechococcus subcluster 5.3. Members of Synechococcus subcluster 5.2 were dominant in the high-latitude waters (northern Bering Sea and Chukchi Sea), suggesting a possible cold-adaptation of some marine Synechococcus in this subcluster. A distinct shift of the picocyanobacterial community was observed from the Bering Sea to the Chukchi Sea, which reflected the change of water temperature. Our study demonstrates that oceanic systems contain a large pool of diverse picocyanobacteria, and further suggest that new genotypes or ecotypes of picocyanobacteria will continue to emerge, as microbial consortia are explored with advanced sequencing technology.

Photoheterotrophic microbes, which are capable of utilizing dissolved organic materials and harvesting light energy, include coccoid cyanobacteria (Synechococcus and Prochlorococcus), aerobic anoxygenic phototrophic (AAP) bacteria, and proteorhodopsin (PR)-containing bacteria. Our knowledge of photoheterotrophic microbes is largely incomplete, especially for high-latitude waters such as the Arctic Ocean, where photoheterotrophs may have special ecological relationships and distinct biogeochemical impacts due to extremes in day length and seasonal ice cover. These microbes were examined by epifluorescence microscopy, flow cytometry, and quantitative PCR (QPCR) assays for PR and a gene diagnostic of AAP bacteria (pufM). The abundance of AAP bacteria and PR-containing bacteria decreased from summer to winter, in parallel with a threefold decrease in the total prokaryotic community. In contrast, the abundance of Synechococcus organisms did not decrease in winter, suggesting that their growth was supported by organic substrates. Results from QPCR assays revealed no substantial shifts in the community structure of AAP bacteria and PR-containing bacteria. However, Arctic PR genes were different from those found at lower latitudes, and surprisingly, they were not similar to those in Antarctic coastal waters. Photoheterotrophic microbes appear to compete successfully with strict heterotrophs during winter darkness below the ice, but AAP bacteria and PR-containing bacteria do not behave as superior competitors during the summer.

Prochlorococcus is a genus of marine cyanobacteria characterized by small cell and genome size, an evolutionary trend toward low GC content, the possession of chlorophyll b, and the absence of phycobilisomes. Whereas many shared derived characters define Prochlorococcus as a clade, many genome-based analyses recover them as paraphyletic, with some low-light adapted Prochlorococcus spp. grouping with marine Synechococcus. Here, we use 18 Prochlorococcus and marine Synechococcus genomes to analyze gene flow within and between these taxa. We introduce embedded quartet scatter plots as a tool to screen for genes whose phylogeny agrees or conflicts with the plurality phylogenetic signal, with accepted taxonomy and naming, with GC content, and with the ecological adaptation to high and low light intensities. We find that most gene families support high-light adapted Prochlorococcus spp. as a monophyletic clade and low-light adapted Prochlorococcus sp. as a paraphyletic group. But we also detect 16 gene families that were transferred between high-light adapted and low-light adapted Prochlorococcus sp. and 495 gene families, including 19 ribosomal proteins, that do not cluster designated Prochlorococcus and Synechococcus strains in the expected manner. To explain the observed data, we propose that frequent gene transfer between marine Synechococcus spp. and low-light adapted Prochlorococcus spp. has created a “highway of gene sharing” (Beiko RG, Harlow TJ, Ragan MA. 2005. Highways of gene sharing in prokaryotes. Proc Natl Acad Sci USA. 102:14332–14337) that tends to erode genus boundaries without erasing the Prochlorococcus-specific ecological adaptations.

The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 μmol of quanta · m−2 s−1, the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the “light on” signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G1 phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. Shifting Prochlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G2 phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.

The cell cycle of the chlorophyll b-possessing marine cyanobacterium Prochlorococcus is highly synchronized under natural conditions. To understand the underlying molecular mechanisms we cloned and sequenced dnaA and ftsZ, two key cell cycle-associated genes, and studied their expression. An axenic culture of Prochlorococcus sp. strain PCC 9511 was grown in a turbidostat with a 12 h–12 h light-dark cycle for 2 weeks. During the light periods, a dynamic light regimen was used in order to simulate the natural conditions found in the upper layers of the world's oceans. This treatment resulted in strong cell cycle synchronization that was monitored by flow cytometry. The steady-state mRNA levels of dnaA and ftsZ were monitored at 4-h intervals during four consecutive division cycles. Both genes exhibited clear diel expression patterns with mRNA maxima during the replication (S) phase. Western blot experiments indicated that the peak of FtsZ concentration occurred at night, i.e., at the time of cell division. Thus, the transcript accumulation of genes involved in replication and division is coordinated in Prochlorococcus sp. strain PCC 9511 and might be crucial for determining the timing of DNA replication and cell division.

Photoinactivation of photosystem II (PSII), the light-induced loss of ability to evolve oxygen, is an inevitable event during normal photosynthesis, exacerbated by saturating light but counteracted by repair via new protein synthesis. The photoinactivation of PSII is dependent on the dosage of light: in the absence of repair, typically one PSII is photoinactivated per 10(7) photons, although the exact quantum yield of photoinactivation is modulated by a number of factors, and decreases as fewer active PSII targets are available. PSII complexes initially appear to be photoinactivated independently; however, when less than 30% functional PSII complexes remain, they seem to be protected by strongly dissipative PSII reaction centres in several plant species examined so far, a mechanism which we term 'inactive PSII-mediated quenching'. This mechanism appears to require a pH gradient across the photosynthetic membrane for its optimal operation. The residual fraction of functional PSII complexes may, in turn, aid in the recovery of photoinactivated PSII complexes when conditions become less severe. This mechanism may be important for the photosynthetic apparatus in extreme environments such as those experienced by over-wintering evergreen plants, desert plants exposed to drought and full sunlight and shade plants in sustained sunlight.

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.

The abundance of aerobic anoxygenic phototrophic (AAP) bacteria, cyanobacteria, and heterotrophs was examined in the Mid-Atlantic Bight and the central North Pacific Gyre using infrared fluorescence microscopy coupled with image analysis and flow cytometry. AAP bacteria comprised 5% to 16% of total prokaryotes in the Atlantic Ocean but only 5% or less in the Pacific Ocean. In the Atlantic, AAP bacterial abundance was as much as 2-fold higher than that of Prochlorococcus spp. and 10-fold higher than that of Synechococcus spp. In contrast, Prochlorococcus spp. outnumbered AAP bacteria 5- to 50-fold in the Pacific. In both oceans, subsurface abundance maxima occurred within the photic zone, and AAP bacteria were least abundant below the 1% light depth. The abundance of AAP bacteria rivaled some groups of strictly heterotrophic bacteria and was often higher than the abundance of known AAP bacterial genera (Erythrobacter and Roseobacter spp.). Concentrations of bacteriochlorophyll a (BChl a) were low (∼1%) compared to those of chlorophyll a in the North Atlantic. Although the BChl a content of AAP bacteria per cell was typically 20- to 250-fold lower than the divinyl-chlorophyll a content of Prochlorococcus, the pigment content of AAP bacteria approached that of Prochlorococcus in shelf break water. Our results suggest that AAP bacteria can be quite abundant in some oceanic regimes and that their distribution in the water column is consistent with phototrophy.

Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the “leaves of the tree,” between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild.

Author Summary

Prochlorococcus—the most abundant photosynthetic microbe living in the vast, nutrient-poor areas of the ocean—is a major contributor to the global carbon cycle. Prochlorococcus is composed of closely related, physiologically distinct lineages whose differences enable the group as a whole to proliferate over a broad range of environmental conditions. We compare the genomes of 12 strains of Prochlorococcus representing its major lineages in order to identify genetic differences affecting the ecology of different lineages and their evolutionary origin. First, we identify the core genome: the 1,273 genes shared among all strains. This core set of genes encodes the essentials of a functional cell, enabling it to make living matter out of sunlight and carbon dioxide. We then create a genomic tree that maps the gain and loss of non-core genes in individual strains, showing that a striking number of genes are gained or lost even among the most closely related strains. We find that lost and gained genes commonly cluster in highly variable regions called genomic islands. The level of diversity among the non-core genes, and the number of new genes added with each new genome sequenced, suggest far more diversity to be discovered.

Cyanobacteria of the genera Synechococcus and Prochlorococcus are the most abundant photosynthetic organisms on earth, occupying a key position at the base of marine food webs. The cynS gene that encodes cyanase was identified among bacterial, fungal, and plant sequences in public databases, and the gene was particularly prevalent among cyanobacteria, including numerous Prochlorococcus and Synechococcus strains. Phylogenetic analysis of cynS sequences retrieved from the Global Ocean Survey database identified >60% as belonging to unicellular marine cyanobacteria, suggesting an important role for cyanase in their nitrogen metabolism. We demonstrate here that marine cyanobacteria have a functionally active cyanase, the transcriptional regulation of which varies among strains and reflects the genomic context of cynS. In Prochlorococcus sp. strain MED4, cynS was presumably transcribed as part of the cynABDS operon, implying cyanase involvement in cyanate utilization. In Synechococcus sp. strain WH8102, expression was not related to nitrogen stress responses and here cyanase presumably serves in the detoxification of cyanate resulting from intracellular urea and/or carbamoyl phosphate decomposition. Lastly, we report on a cyanase activity encoded by cynH, a novel gene found in marine cyanobacteria only. The presence of dual cyanase genes in the genomes of seven marine Synechococcus strains and their respective roles in nitrogen metabolism remain to be clarified.

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (Fv/Fm). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H2 production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H2 production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD+ ratio revealed that the reductant generated by PFOR contributing to the first phase of H2 production was not in equilibrium with bulk NADH/NAD+ and that the second phase corresponded to the equilibrium NADH-mediated process.

Background

The phylogeny and taxonomy of cyanobacteria is currently poorly understood due to paucity of reliable markers for identification and circumscription of its major clades.

Results

A combination of phylogenomic and protein signature based approaches was used to characterize the major clades of cyanobacteria. Phylogenetic trees were constructed for 44 cyanobacteria based on 44 conserved proteins. In parallel, Blastp searches were carried out on each ORF in the genomes of Synechococcus WH8102, Synechocystis PCC6803, Nostoc PCC7120, Synechococcus JA-3-3Ab, Prochlorococcus MIT9215 and Prochlor. marinus subsp. marinus CCMP1375 to identify proteins that are specific for various main clades of cyanobacteria. These studies have identified 39 proteins that are specific for all (or most) cyanobacteria and large numbers of proteins for other cyanobacterial clades. The identified signature proteins include: (i) 14 proteins for a deep branching clade (Clade A) of Gloebacter violaceus and two diazotrophic Synechococcus strains (JA-3-3Ab and JA2-3-B'a); (ii) 5 proteins that are present in all other cyanobacteria except those from Clade A; (iii) 60 proteins that are specific for a clade (Clade C) consisting of various marine unicellular cyanobacteria (viz. Synechococcus and Prochlorococcus); (iv) 14 and 19 signature proteins that are specific for the Clade C Synechococcus and Prochlorococcus strains, respectively; (v) 67 proteins that are specific for the Low B/A ecotype Prochlorococcus strains, containing lower ratio of chl b/a2 and adapted to growth at high light intensities; (vi) 65 and 8 proteins that are specific for the Nostocales and Chroococcales orders, respectively; and (vii) 22 and 9 proteins that are uniquely shared by various Nostocales and Oscillatoriales orders, or by these two orders and the Chroococcales, respectively. We also describe 3 conserved indels in flavoprotein, heme oxygenase and protochlorophyllide oxidoreductase proteins that are specific for either Clade C cyanobacteria or for various subclades of Prochlorococcus. Many other conserved indels for cyanobacterial clades have been described recently.

Conclusions

These signature proteins and indels provide novel means for circumscription of various cyanobacterial clades in clear molecular terms. Their functional studies should lead to discovery of novel properties that are unique to these groups of cyanobacteria.

PCR was used to amplify DNA-dependent RNA polymerase gene sequences specifically from the cyanobacterial population in a seawater sample from the Sargasso Sea. Sequencing and analysis of the cloned fragments suggest that the population in the sample consisted of two distinct clusters of Prochlorococcus-like cyanobacteria and four clusters of Synechococcus-like cyanobacteria. The diversity within these clusters was significantly different, however. Clones within each Synechococcus-like cluster were 99 to 100% identical, while each Prochlorococcus-like cluster was only 91% identical at the nucleotide level. One Prochlorococcus-like cluster was significantly more closely related to a Mediterranean Sea (surface) Prochlorococcus isolate than to the other cluster, showing the highly divergent nature of this group even in one sample. The approach described here can be used as a general method for examining cyanobacterial diversity, while an oligotrophic ocean ecosystem such as the Sargasso Sea may be an ideal model for examining diversity in relation to environmental parameters.

Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web.

The purpose of this study was to investigate the characteristics of transfer RNA (tRNA) responsible for the association between tRNA genes and genes of apparently foreign origin (genomic islands) in five high-light adapted Prochlorococcus strains. Both bidirectional best BLASTP (basic local alignment search tool for proteins) search and the conservation of gene order against each other were utilized to identify genomic islands, and 7 genomic islands were found to be immediately adjacent to tRNAs in Prochlorococcus marinus AS9601, 11 in P. marinus MIT9515, 8 in P. marinus MED4, 6 in P. marinus MIT9301, and 6 in P. marinus MIT9312. Monte Carlo simulation showed that tRNA genes are hotspots for the integration of genomic islands in Prochlorococcus strains. The tRNA genes associated with genomic islands showed the following characteristics: (1) the association was biased towards a specific subset of all iso-accepting tRNA genes; (2) the codon usages of genes within genomic islands appear to be unrelated to the codons recognized by associated tRNAs; and, (3) the majority of the 3′ ends of associated tRNAs lack CCA ends. These findings contradict previous hypotheses concerning the molecular basis for the frequent use of tRNA as the insertion site for foreign genetic materials. The analysis of a genomic island associated with a tRNA-Asn gene in P. marinus MIT9301 suggests that foreign genetic material is inserted into the host genomes by means of site-specific recombination, with the 3′ end of the tRNA as the target, and during the process, a direct repeat of the 3′ end sequence of a boundary tRNA (namely, a scar from the process of insertion) is formed elsewhere in the genomic island. Through the analysis of the sequences of these targets, it can be concluded that a region characterized by both high GC content and a palindromic structure is the preferred insertion site.

The filamentous cyanobacterium Microcoleus vaginatus, a major primary producer in desert biological sand crusts, is exposed to frequent hydration (by early morning dew) followed by desiccation during potentially damaging excess light conditions. Nevertheless, its photosynthetic machinery is hardly affected by high light, unlike “model” organisms whereby light-induced oxidative stress leads to photoinactivation of the oxygen-evolving photosystem II (PSII). Field experiments showed a dramatic decline in the fluorescence yield with rising light intensity in both drying and artificially maintained wet plots. Laboratory experiments showed that, contrary to “model” organisms, photosynthesis persists in Microcoleus sp. even at light intensities 2–3 times higher than required to saturate oxygen evolution. This is despite an extensive loss (85–90%) of variable fluorescence and thermoluminescence, representing radiative PSII charge recombination that promotes the generation of damaging singlet oxygen. Light induced loss of variable fluorescence is not inhibited by the electron transfer inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), nor the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), thus indicating that reduction of plastoquinone or O2, or lumen acidification essential for non-photochemical quenching (NPQ) are not involved. The rate of QA− re-oxidation in the presence of DCMU is enhanced with time and intensity of illumination. The difference in temperatures required for maximal thermoluminescence emissions from S2/QA− (Q band, 22°C) and S2,3/QB− (B band, 25°C) charge recombinations is considerably smaller in Microcoleus as compared to “model” photosynthetic organisms, thus indicating a significant alteration of the S2/QA− redox potential. We propose that enhancement of non-radiative charge recombination with rising light intensity may reduce harmful radiative recombination events thereby lowering 1O2 generation and oxidative photodamage under excess illumination. This effective photo-protective mechanism was apparently lost during the evolution from the ancestor cyanobacteria to the higher plant chloroplast.

Background

Non-coding RNAs (ncRNA) are regulators of gene expression in all domains of life. They control growth and differentiation, virulence, motility and various stress responses. The identification of ncRNAs can be a tedious process due to the heterogeneous nature of this molecule class and the missing sequence similarity of orthologs, even among closely related species. The small ncRNA Yfr1 has previously been found in the Prochlorococcus/Synechococcus group of marine cyanobacteria.

Results

Here we show that screening available genome sequences based on an RNA motif and followed by experimental analysis works successfully in detecting this RNA in all lineages of cyanobacteria. Yfr1 is an abundant ncRNA between 54 and 69 nt in size that is ubiquitous for cyanobacteria except for two low light-adapted strains of Prochlorococcus, MIT 9211 and SS120, in which it must have been lost secondarily. Yfr1 consists of two predicted stem-loop elements separated by an unpaired sequence of 16–20 nucleotides containing the ultraconserved undecanucleotide 5'-ACUCCUCACAC-3'.

Conclusion

Starting with an ncRNA previously found in a narrow group of cyanobacteria only, we show here the highly specific and sensitive identification of its homologs within all lineages of cyanobacteria, whereas it was not detected within the genome sequences of E. coli and of 7 other eubacteria belonging to the alpha-proteobacteria, chlorobiaceae and spirochaete. The integration of RNA motif prediction into computational pipelines for the detection of ncRNAs in bacteria appears as a promising step to improve the quality of such predictions.

The in situ community structure of Prochlorococcus populations in the eastern North Atlantic Ocean was examined by analysis of Prochlorococcus 16S rDNA sequences with three independent approaches: cloning and sequencing, hybridization to specific oligonucleotide probes, and denaturing gradient gel electrophoresis (DGGE). The hybridization of high-light (HL) and low-light (LL) Prochlorococcus genotype-specific probes to two depth profiles of PCR-amplified 16S rDNA sequences revealed that in these two stratified water columns, an obvious niche-partitioning of Prochlorococcus genotypes occurred. In each water column a shift from the HL to the LL genotype was observed, a transition correlating with the depth of the surface mixed layer (SML). Only the HL genotype was found in the SML in each water column, whereas the LL genotype was distributed below the SML. The range of in situ irradiance to which each genotype was subjected within these distinct niches was consistent with growth irradiance studies of cultured HL- and LL-adapted Prochlorococcus strains. DGGE analysis and the sequencing of Prochlorococcus 16S rDNA clones were in full agreement with the genotype-specific oligonucleotide probe hybridization data. These observations of a partitioning of Prochlorococcus genotypes in a stratified water column provide a genetic basis for the dim and bright Prochlorococcus populations observed in flow cytometric signatures in several oceanic provinces.

Assays of photosynthesis were conducted with a biofilm population of a cyanobacterium, a Synechococcus sp., growing at ∼70°C in a Yellowstone National Park hot spring to test whether cells growing near the upper temperature limit of photosynthetic life are optimally adapted to their mean environmental temperature. Cell suspensions were assayed at 70, 65, and 55°C while being simultaneously exposed to modified solar environments, including reduction of total irradiance and exclusion of UV radiation. Carbon fixation was greatest at 65°C, while 70 and 55°C were always supraoptimal and suboptimal for photosynthesis, respectively. The degree of temperature stress was dependent upon light intensity, and this light-dependent temperature effect may involve both reduced quantum efficiency at subsaturating irradiances and a lower saturating irradiance at both supraoptimal and suboptimal temperatures. The Synechococcus sp. was also more susceptible to UV inhibition of photosynthesis at nonoptimal temperatures. These results suggest that this population is persisting at a nearly lethal temperature and is consequently subject to greater damage by both visible and UV radiation, but it is speculated that these cells may be avoiding competition with other photoautotrophs under these nonoptimal conditions. In separate experiments monitoring diurnal patterns of photosynthesis, cells exhibited peak productivity during the morning, followed by an afternoon decline. No recovery of photosynthesis was observed during the remaining daytime, and carbon fixation was always UV inhibited under conditions of photosynthetically saturating light.

Prochlorococcus, an extremely small cyanobacterium that is very abundant in the world's oceans, has a very streamlined genome. On average, these cells have about 2,000 genes and very few regulatory proteins. The limited capability of regulation is thought to be a result of selection imposed by a relatively stable environment in combination with a very small genome. Furthermore, only ten non-coding RNAs (ncRNAs), which play crucial regulatory roles in all forms of life, have been described in Prochlorococcus. Most strains also lack the RNA chaperone Hfq, raising the question of how important this mode of regulation is for these cells. To explore this question, we examined the transcription of intergenic regions of Prochlorococcus MED4 cells subjected to a number of different stress conditions: changes in light qualities and quantities, phage infection, or phosphorus starvation. Analysis of Affymetrix microarray expression data from intergenic regions revealed 276 novel transcriptional units. Among these were 12 new ncRNAs, 24 antisense RNAs (asRNAs), as well as 113 short mRNAs. Two additional ncRNAs were identified by homology, and all 14 new ncRNAs were independently verified by Northern hybridization and 5′RACE. Unlike its reduced suite of regulatory proteins, the number of ncRNAs relative to genome size in Prochlorococcus is comparable to that found in other bacteria, suggesting that RNA regulators likely play a major role in regulation in this group. Moreover, the ncRNAs are concentrated in previously identified genomic islands, which carry genes of significance to the ecology of this organism, many of which are not of cyanobacterial origin. Expression profiles of some of these ncRNAs suggest involvement in light stress adaptation and/or the response to phage infection consistent with their location in the hypervariable genomic islands.

Author Summary

Prochlorococcus is the most abundant phototroph in the vast, nutrient-poor areas of the ocean. It plays an important role in the ocean carbon cycle, and is a key component of the base of the food web. All cells share a core set of about 1,200 genes, augmented with a variable number of “flexible” genes. Many of the latter are located in genomic islands—hypervariable regions of the genome that encode functions important in differentiating the niches of “ecotypes.” Of major interest is how cells with such a small genome regulate cellular processes, as they lack many of the regulatory proteins commonly found in bacteria. We show here that contrary to the regulatory proteins, ncRNAs are present at levels typical of bacteria, revealing that they might have a disproportional regulatory role in Prochlorococcus—likely an adaptation to the extremely low-nutrient conditions of the open oceans, combined with the constraints of a small genome. Some of the ncRNAs were differentially expressed under stress conditions, and a high number of them were found to be associated with genomic islands, suggesting functional links between these RNAs and the response of Prochlorococcus to particular environmental challenges.

Characterization of two genetically distinct groups of marine Synechococcus sp. strains shows that one, but not the other, increases its phycourobilin/phycoerythrobilin chromophore ratio when growing in blue light. This ability of at least some marine Synechococcus strains to chromatically adapt may help explain their greater abundance in particular ocean environments than cyanobacteria of the genus Prochlorococcus.

A sensor histidine kinase of Synechococcus sp. strain PCC7942, designated nblS, was previously identified and shown to be critical for the acclimation of cells to high-light and nutrient limitation conditions and to influence the expression of a number of light-responsive genes. The nblS orthologue in Synechocystis sp. strain PCC6803 is designated dspA (also called hik33). We have generated a dspA null mutant and analyzed global gene expression in both the mutant and wild-type strains under high- and low-light conditions. The mutant is aberrant for the expression of many genes encoding proteins critical for photosynthesis, phosphate and carbon acquisition, and the amelioration of stress conditions. Furthermore, transcripts from a number of genes normally detected only during exposure of wild-type cells to high-light conditions become partially constitutive in the low-light-grown dspA mutant. Other genes for which transcripts decline upon exposure of wild-type cells to high light are already lower in the mutant during growth in low light. These results suggest that DspA may influence gene expression in both a positive and a negative manner and that the dspA mutant behaves as if it were experiencing stress conditions (e.g., high-light exposure) even when maintained at near-optimal growth conditions for wild-type cells. This is discussed with respect to the importance of DspA for regulating the responses of the cell to environmental cues.