The marine cyanobacterium Crocosphaera watsonii WH8501 synthesizes the compatible solute trehalose by a laterally acquired OtsAB fusion protein

Compatible solutes are small organic molecules that are involved in the acclimation to various stresses such as temperature and salinity. Marine or moderate halotolerant cyanobacteria accumulate glucosylglycerol, while cyanobacteria with low salt tolerance (freshwater strains) usually accumulate sucrose or trehalose as the main compatible solutes. The screening of the genome of the marine, unicellular N(2) -fixing cyanobacterium Crocosphaera watsonii WH8501 revealed that instead of genes for glucosylglycerol biosynthesis, a fusion protein for the synthesis of trehalose was found that displayed similarities to trehalose-phosphate-synthase and -phosphatase (OtsAB pathway) from enterobacteria. Accordingly, cells of Crocosphaera showed salt-stimulated expression of the otsAB gene as well as a salt-dependent trehalose accumulation. The biochemical characterization of recombinant full-length OtsAB and truncated OtsB versions revealed that the otsAB gene in Crocosphaera encodes for an active trehalose-phosphate-synthase/phosphatase fusion protein. Genes coding for such proteins were not found in the genomes of other cyanobacteria but were present in many other, non-related marine bacteria, suggesting that otsAB might have been acquired by lateral gene transfer into the Crocosphaera genome.     

Light-dark (12:12) cycle of carbon and nitrogen metabolism in Crocosphaera watsonii WH8501: relation to the cell cycle

This study provides with original data sets on the physiology of the unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501, maintained in continuous culture in conditions of obligate diazotrophy. Cultures were exposed to a 12:12 light-dark regime, representative of what they experience in nature and where growth is expected to be balanced. Nitrogen and carbon metabolism were monitored at high frequency and their dynamics was compared with the cell cycle. Results reveal a daily cycle in the physiological and biochemical parameters, tightly constrained by the timely decoupled processes of N(2) fixation and carbon acquisition. The cell division rate increased concomitantly to carbon accumulation and peaked 6 h into the light. The carbon content reached a maximum at the end of the light phase. N(2) fixation occurred mostly during the dark period and peaked between 9 and 10 h into the night, while DNA synthesis, reflected by DNA fluorescence, increased until the end of the night. Consequently, cells in G1- and S-phases present a marked decrease in their C:N ratio. Nitrogen acquisition through N(2) fixation exceeded 1.3- to 3-fold the nitrogen requirements for growth, suggesting that important amounts of nitrogen are excreted even under conditions supposed to favour balanced, carbon and nitrogen acquisitions.     

Combined effects of CO2 and light on large and small isolates of the unicellular N2-fixing cyanobacterium Crocosphaera watsonii from the western tropical Atlantic Ocean

We examined the combined effects of light and pCO₂ on growth, CO₂-fixation and N₂-fixation rates by strains of the unicellular marine N₂-fixing cyanobacterium Crocosphaera watsonii with small (WH0401) and large (WH0402) cells that were isolated from the western tropical Atlantic Ocean. In low-pCO₂-acclimated cultures (190 ppm) of WH0401, growth, CO₂-fixation and N₂-fixation rates were significantly lower than those in cultures acclimated to higher (present-day ～385 ppm, or future ～750 ppm) pCO₂ treatments. Growth rates were not significantly different, however, in low-pCO₂-acclimated cultures of WH0402 in comparison with higher pCO₂ treatments. Unlike previous reports for C. watsonii (strain WH8501), N₂-fixation rates did not increase further in cultures of WH0401 or WH0402 when acclimated to 750 ppm relative to those maintained at present-day pCO₂. Both light and pCO₂ had a significant negative effect on gross : net N₂-fixation rates in WH0402 and trends were similar in WH0401, implying that retention of fixed N was enhanced under elevated light and pCO₂. These data, along with previously reported results, suggest that C. watsonii may have wide-ranging, strain-specific responses to changing light and pCO₂, emphasizing the need for examining the effects of global change on a range of isolates within this biogeochemically important genus. In general, however, our data suggest that cellular N retention and CO₂-fixation rates of C. watsonii may be positively affected by elevated light and pCO₂ within the next 100 years, potentially increasing trophic transfer efficiency of C and N and thereby facilitating uptake of atmospheric carbon by the marine biota.     

Phenotypic and genotypic characterization of multiple strains of the diazotrophic cyanobacterium, Crocosphaera watsonii, isolated from the open ocean

Diazotrophic cyanobacteria have long been recognized as important sources of reduced nitrogen (N) and therefore are important ecosystem components. Until recently, species of the filamentous cyanobacterium Trichodesmium were thought to be the primary sources of fixed N to the open ocean euphotic zone. It is now recognized that unicellular cyanobacteria are also important contributors, with members of the oligotrophic genus Crocosphaera being the only cultured examples. Herein we genetically and phenotypically characterize 10 strains isolated from the tropical Atlantic and North Pacific Oceans, and show that although all of the strains are highly similar at the genetic level, with the internal transcribed sequence (ITS) region sequence varying by ∼2 bp on average, there are many unexpected phenotypic differences between the isolates (e.g. cell size, temperature optima and range, extracellular material excretion and variability in rates of nitrogen fixation). However based on the observed sequence similarity, we propose that all of these isolates are members of the genus Crocosphaera (type strain Crocosphaera watsonii WH8501), and that the phenotypic diversity we see may reflect ecologically important variation relevant for modelling N₂ fixation in the oligotrophic ocean.     

Growth,photosynthesis, nitrogen fixation and carbohydrate production by a unicellular cyanobacterium,Synechococcus sp. (Cyanophyta)

A polymer-producing strain of unicellular cyanobacteria, Synechococcus sp., was isolated from a coastal lagoon in Florida. This strain, designated BG0011, excreted a highly viscous polysaccharide. Maximum observed growth rates for BG0011 were 2.5 div. day^-2. BG0011 also exhibited nitrogen fixation (nitrogenase) activity under aerobic conditions and grew at near maximum rates in medium lacking reduced nitrogen. Growth and carbohydrate production were enhanced by carbon dioxide enrichment. Rheological study of the extracellular polysaccharide revealed a viscosity versus shear rate curve similar in shape to that of xanthan gum. Maximum observed rate of carbohydrate production was 1 g dry weight liter^-1 month^-1.     

Physical and gene maps of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome

A physical map of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome has been constructed with restriction endonucleases PmeI, SwaI, and an intron-encoded endonuclease I-CeuI. The estimated size of the genome is 2.7 Mb. On the genome 49 genes or operons have been mapped. Two rRNA operons are separated by 600 kb and transcribed oppositely.     

Temporal orchestration of glycogen synthase (GlgA) gene expression and glycogen accumulation in the oceanic picoplanktonic cyanobacterium Synechococcus sp. strain WH8103

Glycogen is accumulated during the latter half of the diel cycle in Synechococcus sp. strain WH8103 following a midday maximum in glgA (encoding glycogen synthase) mRNA abundance. This temporal pattern is quite distinct from that of Prochlorococcus and may highlight divergent regulatory control of carbon/nitrogen metabolism in these closely related picocyanobacteria.     

Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

Summary. Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells. Supplementary material to this paper is available in electronic form at Correspondence and reprints: Department of Biology, CB1137, Washington University, St. Louis, MO 63130, U.S.A.     

Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

Light-mediated regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechococcus sp. PCC 6301

Glutamine synthetase (GS; EC 6.3.1.2) activity from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 shows a short-term regulation by light-dark transitions. The enzyme activity declines down to 30% of the original level after 2 h of dark incubation, and can be fully reactivated within 15 min of re-illumination. The loss of activity is not due to protein degradation, but rather to a reversible change of the enzyme, as deduced from the GS-protein levels determined in dark-incubated cells using polyclonal antibodies raised against Synechococcus GS. Incubation with 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU) also provokes GS inactivation, indicating that an active electron flow between both photosystems is necessary to maintain GS in an active state. On the other hand, the light-mediated reactivation of GS in dark-incubated cells treated with dicyclohexyl-carbodiimide (DCCD) or carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicates that neither changes in the ATP synthesis nor the lack of an electrochemical proton gradient across the thylakoid membrane are directly involved in the regulation process. The inactive form of GS is extremely labile in vitro after disruption of the cells, and is not reactivated by treatment with dithiothreitol or spinach thioredoxin m. These results, taken together with the fact that dark-promoted GS inactivation is dependent on the growth phase, seem to indicate that GS activity is not regulated by a typical redox process and that some other metabolic signal(s), probably related to the ammonium-assimilation pathway, might be involved in the regulation process. In this regard, our results indicate that glutamine is not a regulatory metabolite of Synechococcus glutamine synthetase.Abbreviations CAP chloramphenicol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - GOGAT glutamate synthase - GS glutamine synthetase - PFD photon flux density This work has been financed by the Directión General de Investigación Científica y Técnica, (Grant PB88-0020) and by the Junta de Andalucía, Spain.     

Oxidative stress responses and shock proteins in the unicellular cyanobacterium Synechococcus R2 (PCC-7942)

Oxidative stress responses were tested in the unicellular cyanobacterium Synechococcus PCC 7942 (R2). Cells were exposed to hydrogen peroxide, cumene hydroperoxide and high light intensities. Activities of ascorbate peroxidase and catalase were correlated with the extent and time-course of oxidative stresses. Ascorbate peroxidase was found to be the major enzyme involved in the removal of hydrogen peroxide under the tested oxidative stresses. Catalase activity was inhibited in cells treated with high H₂O₂ concentrations, and was not induced under photo-oxidative stress. Regeneration of ascorbate in peroxide-treated cells was found to involve mainly monodehydroascorbate reductase and to a lesser extent dehydroascorbate reductase. The induction of the antioxidative enzymes was dependent on light and was inhibited by chloramphenicol. Peroxide treatment was found to induce the synthesis of eight proteins, four of which were also induced by heat shock.Abbreviations ASC ascorbate - DHA dehydroascorbate - MDA monodehydroascorbate - GSH reduced glutathione - GSSG oxidized glutathione - ASC Per ascorbate peroxidase - DHA red. dehydroascorbate reductase - MDA red. monodehydroascorbate reductase - GSSG red. glutathione reductase - HSP heat shock proteins - PSP peroxide shock proteins - C_m chloramphenicol     

Physical and genetic map of the chromosome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803.

A combined physical and genetic map of the cyanobacterium Synechocystis sp. strain PCC 6803 chromosome was constructed. An estimated genome size of 3.82 Mb was obtained by summing the sizes of 25 MluI or 40 NotI fragments seen by pulsed-field electrophoresis. The order of the restriction fragments was determined by using two independent experimental approaches: pulsed-field fragment hybridization and linking clone analysis. The relative positions of 30 known genes or gene clusters were localized.     

Analysis of the nifHDK operon and structure of the NifH protein from the unicellular, diazotrophic cyanobacterium, Cyanothece strain sp. ATCC 51142(1)

Cyanothece sp. ATCC 51142 is a unicellular, diazotrophic cyanobacterium that demonstrates diurnal rhythms for photosynthesis and N(2) fixation, with peaks of O(2) evolution and nitrogenase activity approximately 12 h out of phase. We cloned and sequenced the nifHDK operon, and determined that the amino acid sequences of all three proteins were highly conserved relative to those of other cyanobacteria and bacteria. However, the Fe-protein, encoded by the nifH gene, demonstrated two differences from the related protein in Azotobacter vinelandii, for which a 3-D structure has been determined. First, the Cyanothece Fe-protein contained a 37 amino acid extension at the N-terminus. This approximately 4 kDa addition to the protein appeared to fold as a separate domain, but remained a part of the active protein, as was verified by migration on acrylamide gels. In addition, the Cyanothece Fe-protein had amino acid differences at positions involved in formation of the Fe-protein dimer-dimer contacts in A. vinelandii nitrogenase. There were also changes in residues involved with interaction between the Fe-protein and the MoFe-protein when compared with A. vinelandii. Since the Cyanothece Fe-protein is quickly degraded after activity, it is suggested that the extension and the amino acid alterations were somehow involved in this degradative process.     

Potassium uptake in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 mainly depends on a Ktr-like system encoded by slr1509 (ntpJ)

The molecular basis of potassium uptake in cyanobacteria has not been elucidated. However, genes known from other bacteria to encode potassium transporters can be identified in the genome of Synechocystis sp. strain PCC 6803. Mutants defective in kdpA and ntpJ were generated and characterized to address the role of the Kdp and KtrAB systems in this strain. KtrAB is crucial for K(+) uptake, as the DeltantpJ mutant shows slowed growth, slowed potassium uptake kinetics, and increased salt sensitivity. The DeltakdpA mutant has the same phenotype as the wild type even at limiting potassium, but a DeltakdpADeltantpJ double mutant is not viable, indicating a role of Kdp for potassium uptake when the Ktr system is not functioning.