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1.  Diel Expression of Cell Cycle-Related Genes in Synchronized Cultures of Prochlorococcus sp. Strain PCC 9511 
Journal of Bacteriology  2001;183(3):915-920.
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.
doi:10.1128/JB.183.3.915-920.2001
PMCID: PMC94958  PMID: 11208789
2.  Cell Cycle Regulation by Light in Prochlorococcus Strains 
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.
doi:10.1128/AEM.67.2.782-790.2001
PMCID: PMC92648  PMID: 11157244
3.  Prochlorococcus and Synechococcus have Evolved Different Adaptive Mechanisms to Cope with Light and UV Stress 
Prochlorococcus and Synechococcus, which numerically dominate vast oceanic areas, are the two most abundant oxygenic phototrophs on Earth. Although they require solar energy for photosynthesis, excess light and associated high UV radiations can induce high levels of oxidative stress that may have deleterious effects on their growth and productivity. Here, we compared the photophysiologies of the model strains Prochlorococcus marinus PCC 9511 and Synechococcus sp. WH7803 grown under a bell-shaped light/dark cycle of high visible light supplemented or not with UV. Prochlorococcus exhibited a higher sensitivity to photoinactivation than Synechococcus under both conditions, as shown by a larger drop of photosystem II (PSII) quantum yield at noon and different diel patterns of the D1 protein pool. In the presence of UV, the PSII repair rate was significantly depressed at noon in Prochlorococcus compared to Synechococcus. Additionally, Prochlorococcus was more sensitive than Synechococcus to oxidative stress, as shown by the different degrees of PSII photoinactivation after addition of hydrogen peroxide. A transcriptional analysis also revealed dramatic discrepancies between the two organisms in the diel expression patterns of several genes involved notably in the biosynthesis and/or repair of photosystems, light-harvesting complexes, CO2 fixation as well as protection mechanisms against light, UV, and oxidative stress, which likely translate profound differences in their light-controlled regulation. Altogether our results suggest that while Synechococcus has developed efficient ways to cope with light and UV stress, Prochlorococcus cells seemingly survive stressful hours of the day by launching a minimal set of protection mechanisms and by temporarily bringing down several key metabolic processes. This study provides unprecedented insights into understanding the distinct depth distributions and dynamics of these two picocyanobacteria in the field.
doi:10.3389/fmicb.2012.00285
PMCID: PMC3441193  PMID: 23024637
marine cyanobacteria; Synechococcus; Prochlorococcus; light/dark cycle; light stress; UV radiations; oxidative stress; photophysiology
4.  Patterns and Implications of Gene Gain and Loss in the Evolution of Prochlorococcus 
PLoS Genetics  2007;3(12):e231.
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.
doi:10.1371/journal.pgen.0030231
PMCID: PMC2151091  PMID: 18159947
5.  ProPortal: a resource for integrated systems biology of Prochlorococcus and its phage 
Nucleic Acids Research  2011;40(Database issue):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
6.  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
7.  Accelerated evolution associated with genome reduction in a free-living prokaryote 
Genome Biology  2005;6(2):R14.
Prochlorococcus sp. are marine bacteria with very small genomes. The mechanisms by which these reduced genomes have evolved appears, however, to be distinct from those that have led to small genome size in intracellular bacteria.
Background
Three complete genomes of Prochlorococcus species, the smallest and most abundant photosynthetic organism in the ocean, have recently been published. Comparative genome analyses reveal that genome shrinkage has occurred within this genus, associated with a sharp reduction in G+C content. As all examples of genome reduction characterized so far have been restricted to endosymbionts or pathogens, with a host-dependent lifestyle, the observed genome reduction in Prochlorococcus is the first documented example of such a process in a free-living organism.
Results
Our results clearly indicate that genome reduction has been accompanied by an increased rate of protein evolution in P. marinus SS120 that is even more pronounced in P. marinus MED4. This acceleration has affected every functional category of protein-coding genes. In contrast, the 16S rRNA gene seems to have evolved clock-like in this genus. We observed that MED4 and SS120 have lost several DNA-repair genes, the absence of which could be related to the mutational bias and the acceleration of amino-acid substitution.
Conclusions
We have examined the evolutionary mechanisms involved in this process, which are different from those known from host-dependent organisms. Indeed, most substitutions that have occurred in Prochlorococcus have to be selectively neutral, as the large size of populations imposes low genetic drift and strong purifying selection. We assume that the major driving force behind genome reduction within the Prochlorococcus radiation has been a selective process favoring the adaptation of this organism to its environment. A scenario is proposed for genome evolution in this genus.
doi:10.1186/gb-2005-6-2-r14
PMCID: PMC551534  PMID: 15693943
8.  The Challenge of Regulation in a Minimal Photoautotroph: Non-Coding RNAs in Prochlorococcus 
PLoS Genetics  2008;4(8):e1000173.
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.
doi:10.1371/journal.pgen.1000173
PMCID: PMC2518516  PMID: 18769676
9.  Genetic structure of the dnaA region of the cyanobacterium Synechocystis sp. strain PCC6803. 
Journal of Bacteriology  1995;177(15):4245-4251.
We have cloned and sequenced the dnaA region of Synechocystis sp. strain PCC6803, a bacterium with a light-dependent cell cycle. The dnaA gene product, DnaA, is the central factor for replication initiation in bacteria. The deduced amino acid sequence of the protein encoded by the cyanobacterial dnaA gene is 45% identical to DnaA of Bacillus subtilis and fits very well into the homology pattern of the known eubacterial DnaA proteins. The genetic environment of the Synechocystis sp. strain PCC6803 dnaA gene is completely different from the one in other eubacteria. An open reading frame of unknown function, orf134, was detected upstream of dnaA. The purT gene homolog encoding the glycinamide ribonucleotide transformylase T starts about 200 bp away from this open reading frame in the opposite direction. Downstream of the dnaA gene we detected the start of the psbDC operon, which codes for the photosystem II reaction center proteins D2 and CP43 that are involved in the positioning of chlorophyll a.
PMCID: PMC177169  PMID: 7635812
10.  Ultradian Growth in Prochlorococcus spp. 
Species of the widespread marine prokaryote Prochlorococcus exhibited ultradian growth (faster than 1 division per day) both in situ and in culture, even though cell division is strictly phased to the light-dark cycle. Under optimal conditions a second DNA replication and cell division closely followed, but did not overlap with, the first division. The timing of cell cycle events was not affected by light intensity or duration, suggesting control by a light-triggered timer or circadian clock rather than by completion of a light-dependent assimilation phase. This mode of ultradian growth has not been observed previously and poses new questions about the regulation of cellular rhythms in prokaryotes. In addition, it implies that conclusions regarding the lack of nutrient limitation of Prochlorococcus in the open ocean, which were based on the appearance that cells were growing at their maximal rate, need to be reconsidered.
PMCID: PMC106368  PMID: 16349513
11.  Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511 
PLoS ONE  2014;9(7):e103380.
The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.
doi:10.1371/journal.pone.0103380
PMCID: PMC4111581  PMID: 25061751
12.  Choreography of the Transcriptome, Photophysiology, and Cell Cycle of a Minimal Photoautotroph, Prochlorococcus 
PLoS ONE  2009;4(4):e5135.
The marine cyanobacterium Prochlorococcus MED4 has the smallest genome and cell size of all known photosynthetic organisms. Like all phototrophs at temperate latitudes, it experiences predictable daily variation in available light energy which leads to temporal regulation and partitioning of key cellular processes. To better understand the tempo and choreography of this minimal phototroph, we studied the entire transcriptome of the cell over a simulated daily light-dark cycle, and placed it in the context of diagnostic physiological and cell cycle parameters. All cells in the culture progressed through their cell cycles in synchrony, thus ensuring that our measurements reflected the behavior of individual cells. Ninety percent of the annotated genes were expressed, and 80% had cyclic expression over the diel cycle. For most genes, expression peaked near sunrise or sunset, although more subtle phasing of gene expression was also evident. Periodicities of the transcripts of genes involved in physiological processes such as in cell cycle progression, photosynthesis, and phosphorus metabolism tracked the timing of these activities relative to the light-dark cycle. Furthermore, the transitions between photosynthesis during the day and catabolic consumption of energy reserves at night— metabolic processes that share some of the same enzymes — appear to be tightly choreographed at the level of RNA expression. In-depth investigation of these patterns identified potential regulatory proteins involved in balancing these opposing pathways. Finally, while this analysis has not helped resolve how a cell with so little regulatory capacity, and a ‘deficient’ circadian mechanism, aligns its cell cycle and metabolism so tightly to a light-dark cycle, it does provide us with a valuable framework upon which to build when the Prochlorococcus proteome and metabolome become available.
doi:10.1371/journal.pone.0005135
PMCID: PMC2663038  PMID: 19352512
13.  In Vivo Regulation of Glutamine Synthetase Activity in the Marine Chlorophyll b-Containing Cyanobacterium Prochlorococcus sp. Strain PCC 9511 (Oxyphotobacteria)† 
The physiological regulation of glutamine synthetase (GS; EC 6.3.1.2) in the axenic Prochlorococcus sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable for Prochlorococcus growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of Prochlorococcus for coping with a light- and nutrient-limited environment.
doi:10.1128/AEM.67.5.2202-2207.2001
PMCID: PMC92856  PMID: 11319101
14.  Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast 
Nucleic Acids Research  2012;40(20):10375-10383.
Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard techniques. Our recent successes in cloning and genetically engineering the AT-rich, 1.1 Mb Mycoplasma mycoides genome in yeast encouraged us to explore similar methods with Prochlorococcus. Prochlorococcus MED4 has an AT-rich genome, with a GC content of 30.8%, similar to that of Saccharomyces cerevisiae (38%), and contains abundant yeast replication origin consensus sites (ACS) evenly distributed around its 1.66 Mb genome. Unlike Mycoplasma cells, which use the UGA codon for tryptophane, Prochlorococcus uses the standard genetic code. Despite this, we observed no toxic effects of several partial and 15 whole Prochlorococcus MED4 genome clones in S. cerevisiae. Sequencing of a Prochlorococcus genome purified from yeast identified 14 single base pair missense mutations, one frameshift, one single base substitution to a stop codon and one dinucleotide transversion compared to the donor genomic DNA. We thus provide evidence of transformation, replication and maintenance of this 1.66 Mb intact bacterial genome in S. cerevisiae.
doi:10.1093/nar/gks823
PMCID: PMC3488255  PMID: 22941652
15.  Regulation of the Activity of the Dual-Function DnaA Protein in Caulobacter crescentus 
PLoS ONE  2011;6(10):e26028.
DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A)]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA). We found that the expression of the DnaA(R357A) mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A) protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A) could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.
doi:10.1371/journal.pone.0026028
PMCID: PMC3193534  PMID: 22022497
16.  Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants 
Molecular microbiology  2008;71(2):291-304.
The genetic aspects of oriC replication initiation in Mycobacterium tuberculosis are largely unknown. A two-step genetic screen was utilized for isolating M. tuberculosis dnaA cold-sensitive (cos) mutants. First, a resident plasmid expressing functional dnaA integrated at the attB locus in dnaA null background was exchanged with an incoming plasmid bearing a mutagenized dnaA gene. Next, the mutants that were defective for growth at 30°C, a nonpermissive temperature, but resumed growth and DNA synthesis when shifted to 37°C, a permissive temperature, were subsequently selected. Nucleotide sequencing analysis located mutations to different regions of the dnaA gene. Modulation of the growth temperatures led to synchronized DNA synthesis. The dnaA expression under synchronized DNA replication conditions continued to increase during the replication period, but decreased thereafter reflecting autoregulation. The dnaAcos mutants at 30°C were elongated suggesting that they may possibly be blocked during the cell division. The DnaA115 protein is defective in its ability to interact with ATP at 30°C, but not at 37°C. Our results suggest that the optimal cell cycle progression and replication initiation in M. tuberculosis requires that the dnaA promoter remains active during the replication period and that the DnaA protein is able to interact with ATP.
doi:10.1111/j.1365-2958.2008.06523.x
PMCID: PMC2733369  PMID: 19019143
17.  UV hyper-resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase 
Environmental Microbiology  2010;12(7):1978-1988.
Exposure to solar radiation can cause mortality in natural communities of pico-phytoplankton, both at the surface and to a depth of at least 30 m. DNA damage is a significant cause of death, mainly due to cyclobutane pyrimidine dimer formation, which can be lethal if not repaired. While developing a UV mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated a UV-hyper-resistant variant of high light-adapted strain MED4. The hyper-resistant strain was constitutively upregulated for expression of the mutT-phrB operon, encoding nudix hydrolase and photolyase, both of which are involved in repair of DNA damage that can be caused by UV light. Photolyase (PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy from visible light in the process known as photoreactivation. Nudix hydrolase (MutT) hydrolyses 8-oxo-dGTP, an aberrant form of GTP that results from oxidizing conditions, including UV radiation, thus impeding mispairing and mutagenesis by preventing incorporation of the aberrant form into DNA. These processes are error-free, in contrast to error-prone SOS dark repair systems that are widespread in bacteria. The UV-hyper-resistant strain contained only a single mutation: a 1 bp deletion in the intergenic region directly upstream of the mutT-phrB operon. Two subsequent enrichments for MED4 UV-hyper-resistant strains from MED4 wild-type cultures gave rise to strains containing this same 1 bp deletion, affirming its connection to the hyper-resistant phenotype. These results have implications for Prochlorococcus DNA repair mechanisms, genome stability and possibly lysogeny.
doi:10.1111/j.1462-2920.2010.02203.x
PMCID: PMC2955971  PMID: 20345942
18.  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
19.  Relationship between Abundance and Specific Activity of Bacterioplankton in Open Ocean Surface Waters 
Marine microbial communities are complex and dynamic, and their ecology impacts biogeochemical cycles in pelagic ecosystems. Yet, little is known about the relative activities of different microbial populations within genetically diverse communities. We used rRNA as a proxy for activity to quantify the relative specific activities (rRNA/ribosomal DNA [rDNA or rRNA genes]) of the eubacterial populations and to identify locations or clades for which there are uncouplings between specific activity and abundance. After analyzing 1.6 million sequences from 16S rDNA and rRNA (cDNA) libraries from two euphotic depths from a representative site in the Pacific Ocean, we show that although there is an overall positive relationship between the abundances (rDNAs) and activities (rRNAs) among populations of the bacterial community, for some populations these measures are uncoupled. Different ecological strategies are exemplified by the two numerically dominant clades at this site: the cyanobacterium Prochlorococcus is abundant but disproportionately more active, while the heterotrophic SAR11 is abundant but less active. Other rare populations, such as Alteromonas, have high specific activities in spite of their low abundances, suggesting intense population regulation. More detailed analyses using a complementary quantitative PCR (qPCR)-based approach of measuring relative specific activity for Prochlorococcus populations in the Pacific and Atlantic Oceans also show that specific activity, but not abundance, reflects the key drivers of light and nutrients in this system; our results also suggest substantial top-down regulation (e.g., grazing, viruses, or organismal interactions) or transport (e.g., mixing, immigration, or emigration) of these populations. Thus, we show here that abundance and specific activity can be uncoupled in open ocean systems and that describing both is critical to characterizing microbial communities and predicting marine ecosystem functioning and responses to change.
doi:10.1128/AEM.02155-12
PMCID: PMC3536108  PMID: 23087033
20.  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
21.  Coordination of chromosome replication initiation in Escherichia coli: effects of different dnaA alleles. 
Journal of Bacteriology  1988;170(2):852-858.
The synchrony of initiation of chromosomal replication in single cells was determined in ten different dnaA(Ts) mutants. After inhibiting the initiation of replication but allowing initiated rounds of replication to terminate, we measured the number of fully replicated chromosomes per individual cell by flow cytometry. Synchronous initiation at the several independent origins (oriC) in single rapidly growing cells would give 2'' (n = 0,1,2,3,...) chromosomes per cell, whereas asynchronous initiation was indicated by the presence of a different number of chromosomes. Mutations mapping in the central part of the dnaA gene (dnaA5, dnaA46, dnaA601, dnaA602, and dnaA604) lead to a high degree of asynchrony (class I mutants), whereas mutations mapping in either of the distal parts of the gene (dnaA508, dnaA167, dnaA203, and dnaA204) yielded a low degree of asynchrony at the permissive temperature (class 2 mutants). The dnaA205 mutant exhibited an intermediate degree of asynchrony. Mutants dnaA203 and dnaA204 (promoter distal) differed from the other class 2 mutants (dnaA167, dnaA508; promoter proximal) in that asynchrony increased no more than twofold between 25 and 37 degrees C compared with the more-than-fourfold increase in the latter. The high degree of asynchrony in class 1 mutants was independent of temperature and was not due to insufficient functional DnaA protein, because overproduction of DnaA46 protein did not decrease the asynchrony. The data demonstrate that the DnaA protein has functions in addition to acting positively in the initiation process and negatively as its own repressor, namely in coordinating initiations at all oriC sites within a single cell.
PMCID: PMC210732  PMID: 2828328
22.  DnaA and the timing of chromosome replication in Es-cherichia coli as a function of growth rate 
BMC Systems Biology  2011;5:201.
Background
In Escherichia coli, overlapping rounds of DNA replication allow the bacteria to double in faster times than the time required to copy the genome. The precise timing of initiation of DNA replication is determined by a regulatory circuit that depends on the binding of a critical number of ATP-bound DnaA proteins at the origin of replication, resulting in the melting of the DNA and the assembly of the replication complex. The synthesis of DnaA in the cell is controlled by a growth-rate dependent, negatively autoregulated gene found near the origin of replication. Both the regulatory and initiation activity of DnaA depend on its nucleotide bound state and its availability.
Results
In order to investigate the contributions of the different regulatory processes to the timing of initiation of DNA replication at varying growth rates, we formulate a minimal quantitative model of the initiator circuit that includes the key ingredients known to regulate the activity of the DnaA protein. This model describes the average-cell oscillations in DnaA-ATP/DNA during the cell cycle, for varying growth rates. We evaluate the conditions under which this ratio attains the same threshold value at the time of initiation, independently of the growth rate.
Conclusions
We find that a quantitative description of replication initiation by DnaA must rely on the dependency of the basic parameters on growth rate, in order to account for the timing of initiation of DNA replication at different cell doubling times. We isolate two main possible scenarios for this, depending on the roles of DnaA autoregulation and DnaA ATP-hydrolysis regulatory process. One possibility is that the basal rate of regulatory inactivation by ATP hydrolysis must vary with growth rate. Alternatively, some parameters defining promoter activity need to be a function of the growth rate. In either case, the basal rate of gene expression needs to increase with the growth rate, in accordance with the known characteristics of the dnaA promoter. Furthermore, both inactivation and autorepression reduce the amplitude of the cell-cycle oscillations of DnaA-ATP/DNA.
doi:10.1186/1752-0509-5-201
PMCID: PMC3309966  PMID: 22189092
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.  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
25.  Signature proteins for the major clades of Cyanobacteria 
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.
doi:10.1186/1471-2148-10-24
PMCID: PMC2823733  PMID: 20100331

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