The first protein map of Synechococcus sp. strain PCC 7942

The first protein map was developed of Synechococcus sp. strain PCC 7942, a model organism for studies of photosynthesis, prokaryotic circadian rhythms, cell division, carbon-concentrating mechanisms, and adaptive responses to a variety of stresses. The proteome was analyzed by two-dimensional gel electrophoresis with subsequent MALDI-TOF mass spectroscopy and database analysis. Of the 140 analyzed protein spots, 110 were successfully identified as 62 different proteins, many of which occurred as multiple spots on the gel. The identified proteins participate in the major metabolic and cellular processes in cyanobacterial cells during the exponential growth phase. In addition, 14 proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Synechococcus sp. strain PCC 7942. These results may be helpful for the annotation of the recently sequenced genome of this cyanobacterium, as well as for biochemical and physiological studies of Synechococcus.     

Analysis of glucose-6-phosphate dehydrogenase of the cyanobacterium Synechococcus sp. PCC 7942 in the zwf mutant Escherichia coli DF214 cells

The aim of this study was to express the zwf gene of Synechococcus sp. PCC 7942 in zwf mutant Escherichia coli DF214 cells and to analyse glucose-6-phosphate dehydrogenase (G6PDH) activity. Initially, mutant cells were transformed with plasmid pNUT1 containing a Synechococcus sp. PCC 7942 zwf gene with a 1 kb upstream region that is expected to contain promoter elements. Transformant DF214 cells were not complemented by this fragment in a glucose minimal medium, nor did they exhibit statistically meaningful G6PDH activity. Therefore, the zwf gene was cloned in the lac operon to express the Zwf as a fusion protein; this yielded the construct pSG162. The pSG162 transformant E. coli DF214 cells were complemented in a glucose minimal medium, indicating that cyanobacterial Zwf protein fused with the part of LacZ′ polypeptide, enabling the cells to utilize glucose via the oxidative pentose phosphate pathway. Compared with wild-type E. coli cells, approximately ten times more G6PDH activity was measured in transformant cells. This indicated that the Synechococcus sp. PCC 7942 zwf gene was expressed under the control of the E. coli lac promoter as a fusion protein and the zwf product was converted into an active G6PDH form. Analyses was also carried out to determine whether dithiothreitol (DTT) was an in vitro reducing agent affected the enzyme activity, as was previously reported for this cyanobacterial strain. The results showed no variation in enzyme activity in the reduced assay conditions. Therefore, the zwf mutant E. coli strain DF214 was found to provide a rapid system for analysis of cyanobacterial G6PDH enzymes, but not for the redox state analysis of this enzyme.     

The rpaC gene product regulates phycobilisome-photosystem II interaction in cyanobacteria

State transitions in cyanobacteria are a physiological adaptation mechanism that changes the interaction of the phycobilisomes with the Photosystem I and Photosystem II core complexes. A random mutagenesis study in the cyanobacterium Synechocystis sp. PCC6803 identified a gene named rpaC which appeared to be specifically required for state transitions. rpaC is a conserved cyanobacterial gene which was tentatively suggested to code for a novel signal transduction factor. The predicted gene product is a 9-kDa integral membrane protein. We have further examined the role of rpaC by overexpressing the gene in Synechocystis 6803 and by inactivating the ortholog in a second cyanobacterium, Synechococcus sp. PCC7942. Unlike the Synechocystis 6803 null mutant, the Synechococcus 7942 null mutant is unable to segregate, indicating that the gene is essential for cell viability in this cyanobacterium. The Synechocystis 6803 overexpressor is also unable to segregate, indicating that the cells can only tolerate a limited gene copy number. The non-segregated Synechococcus 7942 mutant can perform state transitions but shows a perturbed phycobilisome-Photosystem II interaction. Based on these results, we propose that the rpaC gene product controls the stability of the phycobilisome-Photosystem II supercomplex, and is probably a structural component of the complex.     

The IdiA protein of Synechococcus sp. PCC 7942 functions in protecting the acceptor side of Photosystem II under oxidative stress

Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from Q_A, gave a decreased O₂ evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage. This revised version was published online in June 2006 with corrections to the Cover Date.     

The heat shock response and acquired thermotolerance in three strains of cyanobacteria

The heat shock response of three cyanobacterial strains,Anabaena sp. Strain PCC (paris Culture Collection) 7120,Plectonema boryanum Strain PCC 6306, andSynechococcus sp. Strain PCC 7942, was characterized by polyacrylamide gel electrophoresis.Anabaena produced 33 heat shock proteins,P. boryanum 35 proteins, andSynechoccus 19 proteins. The rapid response to heat shock was consistent for all three strains, although the number of time-dependent proteins varied. All strains developed thermotolerance when first pretreated with a sublethal heat shock and then challenged with a previously lethal temperature. A 30-min 30°C incubation was required between the heat shock and challenge forSynechococcus, but not forAnabaena andP. boryanum. Synechococcus cells required a higher challenge temperature (51° vs. 49°C) than the other two strains to destroy control cells that were not pretreated with a heat shock.     

Inactivation of ccmO in Synechococcus sp. Strain PCC 7942 Results in a Mutant Requiring High Levels of CO2

Inactivation of ccmO in Synechococcus sp. strain PCC 7942 resulted in a mutant which possesses aberrant carboxysomes and a normal inorganic carbon uptake capability but a reduced ability to photosynthetically utilize the internal inorganic carbon pool. Consequently, it exhibits low apparent photosynthetic affinity for extracellular inorganic carbon and demands high levels of CO₂ for growth.     

Sequencing and Modification of the Gene Encoding the 42-Kilodalton Protein in the Cytoplasmic Membrane of Synechococcus PCC 7942

A 42-kilodalton cytoplasmic membrane protein is synthesized when high CO₂-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) are exposed to low CO₂. The structural gene for this protein (cmpA) has been cloned and sequenced and shown to encode a 450 amino acid polypeptide with a molecular mass of 49 kilodalton. A deletion mutant lacking the 42-kilodalton protein was obtained by transformation of Synechococcus PCC 7942 following in vitro mutagenesis of the cloned gene. There were no significant differences between the mutant and wild-type cells in their growth rates under either low or high CO₂ conditions. The activity of inorganic carbon (C_i) transport in the mutant was as high as that in the wild-type strain. In both types of cells, CO₂ was the main species of C_i transported and the activities of CO₂ and HCO₃⁻ transport increased when high CO₂-grown cells were exposed to low CO₂. We conclude that the 42-kilodalton protein is not directly involved in the C_i-accumulating mechanism of Synechococcus PCC 7942.     

Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp. PCC 6803

The gene products of sll0337 and slr0081 in Synechocystis sp. PCC 6803 have been identified as the homologues of the Escherichia coli phosphate-sensing histidine kinase PhoR and response regulator PhoB, respectively. Interruption of sll0337, the gene encoding the histidine protein kinase, by a spectinomycin-resistance cassette blocked the induction of alkaline phosphatase activity under phosphate-limiting conditions. A similar result was obtained when slr0081, the gene encoding the response regulator, was interrupted with a cassette conferring resistance to kanamycin. In addition, the phosphate-specific transport system was not up-regulated in our mutants when phosphate was limiting. Unlike other genes for bacterial phosphate-sensing two-component systems, sll0337 and slr0081 are not present in the same operon. Although there are three assignments for putative alkaline phosphatase genes in the Synechocystis sp. PCC 6803 genome, only sll0654 expression was detected by northern analysis under phosphate limitation. This gene codes for a 149 kDa protein that is homologous to the cyanobacterial alkaline phosphatase reported in Synechococcus sp. PCC 7942 [Ray, J.M., Bhaya, D., Block, M.A. and Grossman, A.R. (1991) J. Bact. 173: 4297–4309]. An alignment identified a conserved 177 amino acid domain that was found at the N-terminus of the protein encoded by sll0654 but at the C-terminus of the protein in Synechococcus sp. PCC 7942.     

Sensor and regulator proteins from the cyanobacterium Synechococcus species PCC7942 that belong to the bacterial signal-transduction protein families: implication in the adaptive response to phosphate limitation

A 1.2kb DNA fragment was cloned from Synechococcus sp. PCC7942, which is able phenotypicalty to complement a phoRcreC Escherichia coli mutant for the expression of alkaline phosphatase. A 2.5kb DNA fragment encompassing the putative gene was then cloned and its complete nucleotide sequence determined. Nucleotide sequencing revealed that the intact gene encodes a protein of 46389 Da, and that the deduced amino acid sequence shows a high degree of homology to those of the bacterial sensory kinase family. In the determined nucleotide sequence, another gene was adjacently located, which encodes a protein of 29012Da. This protein shows a high degree of homology to those of the response regulator family. Thus, we succeeded in the cloning of a pair of genes encoding the sensory kinase and response regulator, respectively, in a cyanobacterium. Mutant strains that lack these genes were constructed, and demonstrated to be defective in their ability to produce alkaline phosphatase and some inducible proteins in response to phosphate-limitation in the medium. These results imply that the gene products identified in this study are probably involved, either directly or indirectly, in the signal-transduction mechanism underlying regulation of the phosphate regulon in Synechococcus sp. PCC7942. Hence, the genes encoding the sensory kinase and response regulator were designated as sphS and sphR, respectively (S ynechococcusph osphate regulon). The SphS protein was demonstrated in vitro to undergo phosphorylation in the presence of ATP.     

The narA Locus of Synechococcus sp. Strain PCC 7942 Consists of a Cluster of Molybdopterin Biosynthesis Genes

The narA locus required for nitrate reduction in Synechococcus sp. strain PCC 7942 is shown to consist of a cluster of genes, namely, moeA, moaC, moaD, moaE, and moaA, involved in molybdenum cofactor biosynthesis. The product of the moaC gene of strain PCC 7942 shows homology in its N-terminal half to MoaC from Escherichia coli and in its C-terminal half to MoaB or Mog. Overexpression of the Synechococcus moaC gene in E. coli resulted in the synthesis of a polypeptide of 36 kDa, a size that would conform to a protein resembling a fusion of the MoaC and MoaB or Mog polypeptides of E. coli. Insertional inactivation of the moeA, moaC, moaE, and moaA genes showed that the moeA-moa gene cluster is required for growth on nitrate and expression of nitrate reductase activity in strain PCC 7942. The moaCDEA genes constitute an operon which is transcribed divergently from the moeA gene. Expression of the moeA gene and the moa operon was little affected by the nitrogen source present in the culture medium.     

Nucleotide sequence of the narB gene encoding assimilatory nitrate reductase from the cyanobacterium Oscillatoria chalybea

The nucleotide sequence of the structural gene of nitrate reductase (narB) has been determined from the filamentous, non-heterocystous cyanobacterium Oscillatoria chalybea. The narB gene encodes a protein of 737 amino acid residues, which shows 61% identity to nitrate reductase of the unicellular cyanobacterium Synechococcus sp. PCC 7942 and only weak homologies to different bacterial molybdoenzymes, such as nitrate reductases or formate dehydrogenases.     

Alleviation of NaCl toxicity in the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942 by exogenous calcium supplementation

Salinity (NaCl) is one of the major problems associated with irrigated agricultural lands, especially rice fields. Being the common inhabitants of rice fields, cyanobacteria frequently experience high concentration of NaCl which in turn causes cellular damage. Therefore, mitigation of NaCl stress in cyanobacteria, plant growth-promoting microorganisms, is of utmost importance. The present study was designed to investigate the role of calcium in the alleviation of NaCl stress-induced cellular in Synechococcus sp. PCC 7942. The cyanobacterium was subjected to sub-lethal concentration of NaCl (800 mM) with and without the supplementation of calcium (1 mM CaCl₂) for 8 days. The results showed a drastic reduction in growth due to excess NaCl, but supplementation of CaCl₂ reduced the salt stress damage and partially restored growth. Application of calcium increased pigment contents, photosynthetic efficiency, antioxidative enzyme activity, osmolyte contents and reduced the intracellular sodium ion concentration, MDA content, electrolyte leakage and free oxygen radical generation. Furthermore, proteins involved in photosynthesis, respiration, ATP synthesis and protein synthesis along with two hypothetical proteins were also observed to be upregulated in the cyanobacterium in presence of calcium. Furthermore, proteins related to oxidative stress defence, nitrogen metabolism, carbohydrate metabolism, fatty acid metabolism and secondary metabolism were found to be upregulated by several fold. Therefore, our study suggests that calcium suppresses salt toxicity in Synechococcus sp. PCC 7942 by restricting the entry of Na⁺ into the cell, increasing osmolyte production and upregulating defence-related proteins.     

Nucleotide sequence and homology comparison of two genes of the sulfate transport operon from the cyanobacterium Synechocystis sp. PCC 6803

The genome of Synechocystis sp. PCC 6803 contains an operon with homology to the sulfate permease of other prokaryotes. We used antibodies raised against cytoplasmic membrane protein to find three genes with strong homology to sbpA, orf81 and cysT genes of the cyanobacterium Synechococcus sp. PCC 7942, Escherichia coli, Salmonella typhymurium and Marchantia polymorpha. It is likely that the permease genes are expressed and the proteins are inserted into the cytoplasmic membrane.     

Role of the GlgX protein in glycogen metabolism of the cyanobacterium, Synechococcus elongatus PCC 7942

The putative glgX gene encoding isoamylase-type debranching enzyme was isolated from the cyanobacterium, Synechococcus elongatus PCC 7942. The deduced amino acid sequence indicated that the residues essential to the catalytic activity and substrate binding in bacterial and plant isoamylases and GlgX proteins were all conserved in the GlgX protein of S. elongatus PCC 7942. The role of GlgX in the cyanobacterium was examined by insertional inactivation of the gene. Disruption of the glgX gene resulted in the enhanced fluctuation of glycogen content in the cells during light–dark cycles of the culture, although the effect was marginal. The glycogen of the glgX mutant was enriched with very short chains with degree of polymerization 2 to 4. When the mutant was transformed with putative glgX genes of Synechocystis sp. PCC 6803, the short chains were decreased as compared to the parental mutant strain. The result indicated that GlgX protein contributes to form the branching pattern of polysaccharide in S. elongatus PCC 7942.     

A Synechococcus PglnA::luxAB Fusion for Estimation of Nitrogen Bioavailability to Freshwater Cyanobacteria

In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations. This situation may be due partly to the complexity in estimating its utilization and bioavailability. In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, P glnA, from Synechococcus sp. strain PCC7942 to the luxAB luciferase-encoding genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus chromosome to yield the reporter strain GSL. Light emission by this strain was dependent upon ambient nitrogen concentrations. The linear response range of the emitted luminescence was 1 mM to 1 μM for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea. When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen.     

Spectral Light Quality Affects Protein Profile of Synechococcus sp. PCC 7942: A Comparative 2-Dimensional Gel Electrophoresis (2-DGE) Analysis

We report here a comparative analysis of the effect of blue (450 nm), red (660 nm), and white light (400–700 nm) on the protein profile of cyanobacteria Synechococcus sp. PCC 7942. In vivo labeling of cells with [³⁵S] methionine and their subsequent analysis by two-dimensional gel electrophoresis (2-DGE) showed that eight polypeptides were unique to dark adapted cells, ten were blue light specific, and four were specifically induced in red light. The results show that Synechococcus sp. respond to various light treatments rapidly and synthesize new polypeptides in dark and blue/red light. Received: 12 October 1999 / Accepted: 16 November 1999