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1.  Ecological Genomics of Marine Picocyanobacteria†  
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we put our current knowledge of marine picocyanobacterial genomics into an environmental context and present previously unpublished genomic information arising from extensive genomic comparisons in order to provide insights into the adaptations of these marine microbes to their environment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.
doi:10.1128/MMBR.00035-08
PMCID: PMC2698417  PMID: 19487728
2.  Ultraviolet stress delays chromosome replication in light/dark synchronized cells of the marine cyanobacterium Prochlorococcus marinus PCC9511 
BMC Microbiology  2010;10:204.
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.
doi:10.1186/1471-2180-10-204
PMCID: PMC2921402  PMID: 20670397
3.  Transcriptome response of high- and low-light-adapted Prochlorococcus strains to changing iron availability 
The ISME Journal  2011;5(10):1580-1594.
Prochlorococcus contributes significantly to ocean primary productivity. The link between primary productivity and iron in specific ocean regions is well established and iron limitation of Prochlorococcus cell division rates in these regions has been shown. However, the extent of ecotypic variation in iron metabolism among Prochlorococcus and the molecular basis for differences is not understood. Here, we examine the growth and transcriptional response of Prochlorococcus strains, MED4 and MIT9313, to changing iron concentrations. During steady state, MIT9313 sustains growth at an order-of-magnitude lower iron concentration than MED4. To explore this difference, we measured the whole-genome transcriptional response of each strain to abrupt iron starvation and rescue. Only four of the 1159 orthologs of MED4 and MIT9313 were differentially expressed in response to iron in both strains. However, in each strain, the expression of over a hundred additional genes changed, many of which are in labile genomic regions, suggesting a role for lateral gene transfer in establishing diversity of iron metabolism among Prochlorococcus. Furthermore, we found that MED4 lacks three genes near the iron-deficiency-induced gene (idiA) that are present and induced by iron stress in MIT9313. These genes are interesting targets for studying the adaptation of natural Prochlorococcus assemblages to local iron conditions as they show more diversity than other genomic regions in environmental metagenomic databases.
doi:10.1038/ismej.2011.49
PMCID: PMC3176520  PMID: 21562599
cyanobacteria; iron; transcriptome
4.  Global gene expression of Prochlorococcus ecotypes in response to changes in nitrogen availability 
Nitrogen (N) often limits biological productivity in the oceanic gyres where Prochlorococcus is the most abundant photosynthetic organism. The Prochlorococcus community is composed of strains, such as MED4 and MIT9313, that have different N utilization capabilities and that belong to ecotypes with different depth distributions. An interstrain comparison of how Prochlorococcus responds to changes in ambient nitrogen is thus central to understanding its ecology. We quantified changes in MED4 and MIT9313 global mRNA expression, chlorophyll fluorescence, and photosystem II photochemical efficiency (Fv/Fm) along a time series of increasing N starvation. In addition, the global expression of both strains growing in ammonium-replete medium was compared to expression during growth on alternative N sources. There were interstrain similarities in N regulation such as the activation of a putative NtcA regulon during N stress. There were also important differences between the strains such as in the expression patterns of carbon metabolism genes, suggesting that the two strains integrate N and C metabolism in fundamentally different ways.
doi:10.1038/msb4100087
PMCID: PMC1682016  PMID: 17016519
cyanobacteria; interstrain; nitrogen; Prochlorococcus; transcription
5.  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 [35S]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.
doi:10.1128/AEM.69.2.1299-1304.2003
PMCID: PMC143617  PMID: 12571062
6.  Analysis of the 3′ ends of tRNA as the cause of insertion sites of foreign DNA in Prochlorococcus *  
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.
doi:10.1631/jzus.B0900417
PMCID: PMC2932881  PMID: 20803775
Genomic islands; Prochlorococcus; Transfer RNA (tRNA); Palindromic structure; Codon usage
7.  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 PII 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.
doi:10.1128/MMBR.68.4.630-638.2004
PMCID: PMC539009  PMID: 15590777
8.  Cyanobacteria of the Genus Prochlorothrix† 
Green cyanobacteria differ from the blue–green cyanobacteria by the possession of a chlorophyll-containing light-harvesting antenna. Three genera of the green cyanobacteria namely Acaryochloris, Prochlorococcus, and Prochloron are unicellular and inhabit marine environments. Prochlorococcus marinus attracts most attention due to its prominent role in marine primary productivity. The fourth genus Prochlorothrix is represented by the filamentous freshwater strains. Unlike the other green cyanobacteria, Prochlorothrix strains are remarkably rare: to date, living isolates have been limited to two European locations. Taking into account fluctuating blooms, morphological resemblance to Planktothrix and Pseudanabaena, and unsuccessful attempts to obtain enrichments of Prochlorothrix, the most successful strategy to search for this cyanobacterium involves PCR with environmental DNA and Prochlorothrix-specific primers. This approach has revealed a broader distribution of Prochlorothrix. Marker genes have been found in at least two additional locations. Despite of the growing evidence for naturally occurring Prochlorothrix, there are only a few cultured strains with one of them (PCC 9006) being claimed to be axenic. In multixenic cultures, Prochlorothrix is accompanied by heterotrophic bacteria indicating a consortium-type association. The genus Prochlorothrix includes two species: P. hollandica and P. scandica based on distinctions in genomic DNA, cell size, temperature optimum, and fatty acid composition of membrane lipids. In this short review the properties of cyanobacteria of the genus Prochlorothrix are described. In addition, the evolutionary scenario for green cyanobacteria is suggested taking into account their possible role in the origin of simple chloroplast.
doi:10.3389/fmicb.2012.00173
PMCID: PMC3390582  PMID: 22783229
cyanobacteria; Prochlorothrix; Prochlorophytes
9.  Intertwined Evolutionary Histories of Marine Synechococcus and Prochlorococcus marinus 
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.
doi:10.1093/gbe/evp032
PMCID: PMC2817427  PMID: 20333202
marine cyanobacteria; horizontal gene transfer; introgression; quartet decomposition; supertree; genome evolution
10.  Glucose Uptake and Its Effect on Gene Expression in Prochlorococcus 
PLoS ONE  2008;3(10):e3416.
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.
doi:10.1371/journal.pone.0003416
PMCID: PMC2565063  PMID: 18941506
11.  Dependence of the Cyanobacterium Prochlorococcus on Hydrogen Peroxide Scavenging Microbes for Growth at the Ocean's Surface 
PLoS ONE  2011;6(2):e16805.
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.
doi:10.1371/journal.pone.0016805
PMCID: PMC3033426  PMID: 21304826
12.  Cyanobacterial community structure as seen from RNA polymerase gene sequence analysis. 
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.
PMCID: PMC201791  PMID: 7944363
13.  A novel clade of Prochlorococcus found in high nutrient low chlorophyll waters in the South and Equatorial Pacific Ocean 
The ISME journal  2010;5(6):933-944.
A novel high-light (HL)-adapted Prochlorococcus clade was discovered in high nutrient and low chlorophyll (HNLC) waters in the South Pacific Ocean by phylogenetic analyses of 16S ribosomal RNA (rRNA) and 16S–23S internal transcribed spacer (ITS) sequences. This clade, named HNLC fell within the HL-adapted Prochlorococcus clade with sequences above 99% similarity to one another, and was divided into two subclades, HNLC1 and HNLC2. The distribution of the whole HNLC clade in a northwest to southeast transect in the South Pacific (HNLC-to-gyre) and two 8°N to 8°S transects in the Equatorial Pacific was determined by quantitative PCR using specific primers targeting ITS regions. HNLC was the dominant HL Prochlorococcus clade (2–9% of bacterial 16S rRNA genes) at the three westernmost stations in the South Pacific but decreased to less than 0.1% at the other stations being replaced by the eMIT9312 ecotype in the hyperoligotrophic gyre. The highest contributions of HNLC Prochlorococcus in both Equatorial Pacific transects along the latitudinal lines of 170°W and 155°W were observed at the southernmost stations, reaching 16 and 6% of bacterial 16S rRNA genes, respectively, whereas eMIT9312 dominated near the Equator. Spearman Rank Order correlation analysis indicated that although both the HNLC clade and eMIT9312 were correlated with temperature, they showed different correlations with regard to nutrients. HNLC only showed significant correlations to ammonium uptake and regeneration rates, whereas eMIT9312 was negatively correlated with inorganic nutrients.
doi:10.1038/ismej.2010.186
PMCID: PMC3131852  PMID: 21124492
16S rRNA; Equatorial Pacific; HNLC; ITS; Prochlorococcus; qPCR
14.  ProPortal: a resource for integrated systems biology of Prochlorococcus and its phage 
Nucleic Acids Research  2011;40(D1):D632-D640.
ProPortal (http://proportal.mit.edu/) is a database containing genomic, metagenomic, transcriptomic and field data for the marine cyanobacterium Prochlorococcus. Our goal is to provide a source of cross-referenced data across multiple scales of biological organization—from the genome to the ecosystem—embracing the full diversity of ecotypic variation within this microbial taxon, its sister group, Synechococcus and phage that infect them. The site currently contains the genomes of 13 Prochlorococcus strains, 11 Synechococcus strains and 28 cyanophage strains that infect one or both groups. Cyanobacterial and cyanophage genes are clustered into orthologous groups that can be accessed by keyword search or through a genome browser. Users can also identify orthologous gene clusters shared by cyanobacterial and cyanophage genomes. Gene expression data for Prochlorococcus ecotypes MED4 and MIT9313 allow users to identify genes that are up or downregulated in response to environmental stressors. In addition, the transcriptome in synchronized cells grown on a 24-h light–dark cycle reveals the choreography of gene expression in cells in a ‘natural’ state. Metagenomic sequences from the Global Ocean Survey from Prochlorococcus, Synechococcus and phage genomes are archived so users can examine the differences between populations from diverse habitats. Finally, an example of cyanobacterial population data from the field is included.
doi:10.1093/nar/gkr1022
PMCID: PMC3245167  PMID: 22102570
15.  The P-SSP7 Cyanophage Has a Linear Genome with Direct Terminal Repeats 
PLoS ONE  2012;7(5):e36710.
P-SSP7 is a T7-like phage that infects the cyanobacterium Prochlorococcus MED4. MED4 is a member of the high-light-adapted Prochlorococcus ecotypes that are abundant in the surface oceans and contribute significantly to primary production. P-SSP7 has become a model system for the investigation of T7-like phages that infect Prochlorococcus. It was classified as T7-like based on genome content and organization. However, because its genome assembled as a circular molecule, it was thought to be circularly permuted and to lack the direct terminal repeats found in other T7-like phages. Here we sequenced the ends of the P-SSP7 genome and found that the genome map is linear and contains a 206 bp repeat at both genome ends. Furthermore, we found that a 728 bp region of the genome originally placed downstream of the last ORF is actually located upstream of the first ORF on the genome map. These findings suggest that P-SSP7 is likely to use the direct terminal repeats for genome replication and packaging in a similar manner to other T7-like phages. Moreover, these results highlight the importance of experimentally verifying the ends of phage genomes, and will facilitate the use of P-SSP7 as a model for the correct assembly and end determination of the many T7-like phages isolated from the marine environment that are currently being sequenced.
doi:10.1371/journal.pone.0036710
PMCID: PMC3350473  PMID: 22606283
16.  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.
doi:10.1128/AEM.72.1.723-732.2006
PMCID: PMC1352191  PMID: 16391112
17.  Resolution of Prochlorococcus and Synechococcus Ecotypes by Using 16S-23S Ribosomal DNA Internal Transcribed Spacer Sequences 
Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.
doi:10.1128/AEM.68.3.1180-1191.2002
PMCID: PMC123739  PMID: 11872466
18.  Light Variability Illuminates Niche-Partitioning among Marine Picocyanobacteria 
PLoS ONE  2007;2(12):e1341.
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.
doi:10.1371/journal.pone.0001341
PMCID: PMC2129112  PMID: 18092006
19.  Patterns of ecological specialization among microbial populations in the Red Sea and diverse oligotrophic marine environments 
Ecology and Evolution  2013;3(6):1780-1797.
Large swaths of the nutrient-poor surface ocean are dominated numerically by cyanobacteria (Prochlorococcus), cyanobacterial viruses (cyanophage), and alphaproteobacteria (SAR11). How these groups thrive in the diverse physicochemical environments of different oceanic regions remains poorly understood. Comparative metagenomics can reveal adaptive responses linked to ecosystem-specific selective pressures. The Red Sea is well-suited for studying adaptation of pelagic-microbes, with salinities, temperatures, and light levels at the extreme end for the surface ocean, and low nutrient concentrations, yet no metagenomic studies have been done there. The Red Sea (high salinity, high light, low N and P) compares favorably with the Mediterranean Sea (high salinity, low P), Sargasso Sea (low P), and North Pacific Subtropical Gyre (high light, low N). We quantified the relative abundance of genetic functions among Prochlorococcus, cyanophage, and SAR11 from these four regions. Gene frequencies indicate selection for phosphorus acquisition (Mediterranean/Sargasso), DNA repair and high-light responses (Red Sea/Pacific Prochlorococcus), and osmolyte C1 oxidation (Red Sea/Mediterranean SAR11). The unexpected connection between salinity-dependent osmolyte production and SAR11 C1 metabolism represents a potentially major coevolutionary adaptation and biogeochemical flux. Among Prochlorococcus and cyanophage, genes enriched in specific environments had ecotype distributions similar to nonenriched genes, suggesting that inter-ecotype gene transfer is not a major source of environment-specific adaptation. Clustering of metagenomes using gene frequencies shows similarities in populations (Red Sea with Pacific, Mediterranean with Sargasso) that belie their geographic distances. Taken together, the genetic functions enriched in specific environments indicate competitive strategies for maintaining carrying capacity in the face of physical stressors and low nutrient availability.
doi:10.1002/ece3.593
PMCID: PMC3686209  PMID: 23789085
Cyanophage; metagenomics; osmolyte; Pelagibacter; population genomics; Prochlorococcus; SAR11
20.  Distinct, ecotype-specific genome and proteome signatures in the marine cyanobacteria Prochlorococcus 
BMC Genomics  2010;11:103.
Background
The marine cyanobacterium Prochlorococcus marinus, having multiple ecotypes of distinct genotypic/phenotypic traits and being the first documented example of genome shrinkage in free-living organisms, offers an ideal system for studying niche-driven molecular micro-diversity in closely related microbes. The present study, through an extensive comparative analysis of various genomic/proteomic features of 6 high light (HL) and 6 low light (LL) adapted strains, makes an attempt to identify molecular determinants associated with their vertical niche partitioning.
Results
Pronounced strand-specific asymmetry in synonymous codon usage is observed exclusively in LL strains. Distinct dinucleotide abundance profiles are exhibited by 2 LL strains with larger genomes and G+C-content ≈ 50% (group LLa), 4 LL strains having reduced genomes and G+C-content ≈ 35-37% (group LLb), and 6 HL strains. Taking into account the emergence of LLa, LLb and HL strains (based on 16S rRNA phylogeny), a gradual increase in average aromaticity, pI values and beta- & coil-forming propensities and a decrease in mean hydrophobicity, instability indices and helix-forming propensities of core proteins are observed. Greater variations in orthologous gene repertoire are found between LLa and LLb strains, while higher number of positively selected genes exist between LL and HL strains.
Conclusion
Strains of different Prochlorococcus groups are characterized by distinct compositional, physicochemical and structural traits that are not mere remnants of a continuous genetic drift, but are potential outcomes of a grand scheme of niche-oriented stepwise diversification, that might have driven them chronologically towards greater stability/fidelity and invoked upon them a special ability to inhabit diverse oceanic environments.
doi:10.1186/1471-2164-11-103
PMCID: PMC2836286  PMID: 20146791
21.  A motif-based search in bacterial genomes identifies the ortholog of the small RNA Yfr1 in all lineages of cyanobacteria 
BMC Genomics  2007;8:375.
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.
doi:10.1186/1471-2164-8-375
PMCID: PMC2190773  PMID: 17941988
22.  Strong Genome-Wide Selection Early in the Evolution of Prochlorococcus Resulted in a Reduced Genome through the Loss of a Large Number of Small Effect Genes 
PLoS ONE  2014;9(3):e88837.
The smallest genomes of any photosynthetic organisms are found in a group of free-living marine cyanobacteria, Prochlorococcus. To determine the underlying evolutionary mechanisms, we developed a new method to reconstruct the steps leading to the Prochlorococcus genome reduction using 12 Prochlorococcus and 6 marine Synechococcus genomes. Our results reveal that small genome sizes within Prochlorococcus were largely determined shortly after the split of Prochlorococcus and Synechococcus (an early big shrink) and thus for the first time decouple the genome reduction from Prochlorococcus diversification. A maximum likelihood approach was then used to estimate changes of nucleotide substitution rate and selection strength along Prochlorococcus evolution in a phylogenetic framework. Strong genome wide purifying selection was associated with the loss of many genes in the early evolutionary stage. The deleted genes were distributed around the genome, participated in many different functional categories and in general had been under relaxed selection pressure. We propose that shortly after Prochlorococcus diverged from its common ancestor with marine Synechococcus, its population size increased quickly thus increasing efficacy of selection. Due to limited nutrients and a relatively constant environment, selection favored a streamlined genome for maximum economy. Strong genome wide selection subsequently caused the loss of genes with small functional effect including the loss of some DNA repair genes. In summary, genome reduction in Prochlorococcus resulted in genome features that are similar to symbiotic bacteria and pathogens, however, the small genome sizes resulted from an increase in genome wide selection rather than a consequence of a reduced ecological niche or relaxed selection due to genetic drift.
doi:10.1371/journal.pone.0088837
PMCID: PMC3940434  PMID: 24594762
23.  Genetic Manipulation of Prochlorococcus Strain MIT9313: Green Fluorescent Protein Expression from an RSF1010 Plasmid and Tn5 Transposition▿  
Applied and Environmental Microbiology  2006;72(12):7607-7613.
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.
doi:10.1128/AEM.02034-06
PMCID: PMC1694220  PMID: 17041154
24.  Novel lineages of Prochlorococcus and Synechococcus in the global oceans 
The ISME Journal  2011;6(2):285-297.
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.
doi:10.1038/ismej.2011.106
PMCID: PMC3260499  PMID: 21955990
cyanobacteria; Prochlorococcus; Synechococcus; diversity; global ocean; 16S-23S rRNA ITS
25.  Characterization of Cyanate Metabolism in Marine Synechococcus and Prochlorococcus spp. ▿  
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.
doi:10.1128/AEM.01272-10
PMCID: PMC3019706  PMID: 21057026

Results 1-25 (507403)