Genome Of Synechocystis Sp Research Articles

SpkH (Sll0005) from Synechocystis sp. PCC 6803 is an active Mn2+-dependent Ser kinase

Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and “activity of bc1 complex” kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previously annotated as a potential STPK of ABC1-type (SpkH, Sll0005) was expressed and purified to homogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [γ-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest activation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase – X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors.

Post-translational Modifications of Serine/Threonine and Histidine Kinases and Their Roles in Signal Transductions in Synechocystis Sp. PCC 6803.

Cyanobacterium Synechocystis sp. PCC 6803, a popular model organism for researches in photosynthesis and biofuel production, contains plant-like photosynthetic machineries which significantly contribute to global carbon fixation. There are 12 eukaryotic-type Ser/Thr kinases (SpkA-L) and 49 His kinases (Hik1-49) of two-component systems in the genome of Synechocystis sp. PCC 6803. They are the key regulators in sensing and transmitting stimuli including light- and glucose-mediate signal transduction. Proteomic studies were able to identify all the kinases. The majority of kinases no matter whether they have a predicted transmembrane domain were identified in the membrane fractions. Six Ser/Thr kinases (SpkA-D, F and G) and ten His kinases (Hik4, 12, 14, 21, 26-27, 29, 36, 43, and 46) were identified to have one or more of the three types of post-translational modifications: phosphorylation, acetylation, and thiol oxidation. Interestingly, SpkG has the phosphorylatable threonine residue that was aligned with the phosphorylated threonine residue in the activation loop of human CDK7, demonstrating conserved phosphorylation between cyanobacterial and human kinases. Transcriptomics and proteomics revealed differential expression of the kinases in heterotrophic and photoheterotrophic compared with photoautotrophic conditions, indicating their roles in regulating the growth modes of cyanobacteria. In summary, this review focuses on the discussions on post-transcriptional modifications, transcriptomic, and proteomic studies of Ser/Thr and His kinases. This together with our published review in 2019 present a complete story of an overview of sequences, domain architectures, and biochemical and physiological functions of cyanobacterial kinases with adequate details in the context of high throughput systems. We also emphasize the importance of discovering upstream molecules and substrates to understand the exact functions of the kinases in vivo. As an attempt, a model is proposed in which Hik31, His33, Sll1334, and IcfG are hypothesized to be critical for switching between autotrophic and heterotrophic modes based on the results from the phenotypes of the gene knockout strains combined with their post-translational modifications, and gene expression profiles.

Deep Sequencing-Based Identification of Small Regulatory RNAs in Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 is a genetically tractable model organism for photosynthesis research. The genome of Synechocystis sp. PCC 6803 consists of a circular chromosome and seven plasmids. The importance of small regulatory RNAs (sRNAs) as mediators of a number of cellular processes in bacteria has begun to be recognized. However, little is known regarding sRNAs in Synechocystis sp. PCC 6803. To provide a comprehensive overview of sRNAs in this model organism, the sRNAs of Synechocystis sp. PCC 6803 were analyzed using deep sequencing, and 7,951,189 reads were obtained. High quality mapping reads (6,127,890) were mapped onto the genome and assembled into 16,192 transcribed regions (clusters) based on read overlap. A total number of 5211 putative sRNAs were revealed from the genome and the 4 megaplasmids, and 27 of these molecules, including four from plasmids, were confirmed by RT-PCR. In addition, possible target genes regulated by all of the putative sRNAs identified in this study were predicted by IntaRNA and analyzed for functional categorization and biological pathways, which provided evidence that sRNAs are indeed involved in many different metabolic pathways, including basic metabolic pathways, such as glycolysis/gluconeogenesis, the citrate cycle, fatty acid metabolism and adaptations to environmentally stress-induced changes. The information from this study provides a valuable reservoir for understanding the sRNA-mediated regulation of the complex physiology and metabolic processes of cyanobacteria.

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO(2) has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an l-lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to l-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single ldh mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.

Regulation of the cyanobacterial CO2-concentrating mechanism involves internal sensing of NADP+ and α-ketogutarate levels by transcription factor CcmR.

Inorganic carbon is the major macronutrient required by organisms utilizing oxygenic photosynthesis for autotrophic growth. Aquatic photoautotrophic organisms are dependent upon a CO2 concentrating mechanism (CCM) to overcome the poor CO2-affinity of the major carbon-fixing enzyme, ribulose-bisphosphate carboxylase/oxygenase (Rubisco). The CCM involves the active transport of inorganic forms of carbon (Ci) into the cell to increase the CO2 concentration around the active site of Rubisco. It employs both bicarbonate transporters and redox-powered CO2-hydration enzymes coupled to membranous NDH-type electron transport complexes that collectively produce Ci concentrations up to a 1000-fold greater in the cytoplasm compared to the external environment. The CCM is regulated: a high affinity CCM comprised of multiple components is induced under limiting external Ci concentrations. The LysR-type transcriptional regulator CcmR has been shown to repress its own expression along with structural genes encoding high affinity Ci transporters distributed throughout the genome of Synechocystis sp. PCC 6803. While much has been learned about the structural genes of the CCM and the identity of the transcriptional regulators controlling their expression, little is known about the physiological signals that elicit the induction of the high affinity CCM. Here CcmR is studied to identify metabolites that modulate its transcriptional repressor activity. Using surface plasmon resonance (SPR) α­ketoglutarate (α-KG) and the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) have been identified as the co-repressors of CcmR. Additionally, ribulose­1,5­bisphosphate (RuBP) and 2­phosphoglycolate (2­PG) have been confirmed as co-activators of CmpR which controls the expression of the ABC-type bicarbonate transporter.

The search for new chlorophyll-binding proteins in the cyanobacterium Synechocystis sp. PCC 6803

Light harvesting provides a major challenge in the production of biofuels from microorganisms; while sunlight provides the energy necessary for biomass/biofuel production, at the same time it damages the cells. The genome of Synechocystis sp. PCC 6803 was searched for open reading frames that might code for yet unidentified chlorophyll-binding proteins with low molecular mass that could be involved in stress-adaptation. Amongst 9167 hypothetical ORFs corresponding to potential polypeptides of 100 amino acids or less, two were identified that had the potential to be pigment-binding, because they (i) encoded a potential transmembrane region, (ii) showed sequence similarity with known chlorophyll-binding domains, (iii) were conserved in other cyanobacterial species, and (iv) their codon adaptation index indicated significant translation probability. The two ORFs were located complementary (antisense) and internal to the ferrochelatase (hemH) and the pyruvate dehydrogenase (pdh) genes and therefore were named a-fch and a-pdh, respectively. Transcription of both genes was confirmed; however, no translated proteins could be detected immunologically. Whereas mutations within a-pdh or a-fch did not lead to any obvious phenotype, it is clear that transcripts and proteins over and above the currently known set may play a role in defining the physiology of cyanobacteria and other organisms.

Gene expression study of the flavodi-iron proteins from the cyanobacterium Synechocystis sp. PCC6803

The flavodi-iron proteins, also named FDPs, are an extensive family of enzymes able to reduce dioxygen to water and/or nitric oxide to nitrous oxide. These proteins are formed by a metallo-β-lactamase-like module with a di-iron catalytic site fused to a flavodoxin-like module bearing an FMN. However, in cyanobacteria, which are oxygenic photosynthetic organisms widespread in Nature, FDPs have an extra NAD(P)H:flavin reductase-like domain as a C-terminal extension. Interestingly, cyanobacteria contain more than one gene encoding FDP-like proteins, with the genome of Synechocystis sp. PCC6803 containing four genes coding for putative FDPs. However, the function of those proteins remains unclear. In the present study, we have analysed the expression profile of these genes under oxidative and nitrosative stress conditions. The results indicate that one of the flavodi-iron genes, the so-called flv1, is induced in cells exposed to nitrosative stress. By conducting a broad analysis on the primary sequences of FDPs, we have identified that the FDPs of cyanobacteria and oxygenic photosynthetic eukaryotes may be divided into multiple types (1-12), according to the amino acid residues of the di-iron catalytic site.

An alternative methionine aminopeptidase, MAP-A, is required for nitrogen starvation and high-light acclimation in the cyanobacterium Synechocystis sp. PCC 6803

Methionine aminopeptidases (MetAPs or MAPs, encoded by map genes) are ubiquitous and pivotal enzymes for protein maturation in all living organisms. Whereas most bacteria harbour only one map gene, many cyanobacterial genomes contain two map paralogues, the genome of Synechocystis sp. PCC 6803 even three. The physiological function of multiple map paralogues remains elusive so far. This communication reports for the first time differential MetAP function in a cyanobacterium. In Synechocystis sp. PCC 6803, the universally conserved mapC gene (sll0555) is predominantly expressed in exponentially growing cells and appears to be a housekeeping gene. By contrast, expression of mapA (slr0918) and mapB (slr0786) genes increases during stress conditions. The mapB paralogue is only transiently expressed, whereas the widely distributed mapA gene appears to be the major MetAP during stress conditions. A mapA-deficient Synechocystis mutant shows a subtle impairment of photosystem II properties even under non-stressed conditions. In particular, the binding site for the quinone Q(B) is affected, indicating specific N-terminal methionine processing requirements of photosystem II components. MAP-A-specific processing becomes essential under certain stress conditions, since the mapA-deficient mutant is severely impaired in surviving conditions of prolonged nitrogen starvation and high light exposure.

Construction of bioluminescent cyanobacterial reporter strains for detection of nickel, cobalt and zinc

Two whole-cell bioluminescent reporters were constructed by fusing the reporter genes luxAB with the Co(2+) and Zn(2+) inducible coaT promoter or the Ni(2+)-inducible nrsBACD promoter, respectively, in the genome of Synechocystis sp. PCC 6803. The obtained reporters, designated coaLux and nrsLux, respectively, responded quantitatively to metal ions. After 3 h incubation at 40 micromol m(-2) s(-1) visible light, the detection range of coaLux was 0.3-6 microM for Co(2+) and 1-3 microM for Zn(2+). Incubation in darkness increased the detection range by about four times. The nrsLux reporter was specific to Ni(2+), with a detection range of 0.2-6 microM. However, its activity was inhibited by Zn(2+) with a half maximal inhibitory concentration c. 6 microM, and totally inhibited by darkness. This is the first whole-cell Ni(2+)-specific reporter with a clear dose-signal relationship. In a soil-like mixture of different chemical and oil industry wastes, the coaLux reporter strain detected about 90% of the zinc content of the sample. This study demonstrates the potential for development of a rapid, simple and economical field assay for nickel, cobalt and zinc detection using the coaLux and nrsLux reporters.

The functional divergence of two glgP homologues in Synechocystis sp. PCC 6803

Glycogen phosphorylase (GlgP, EC 2.4.1.1) catalyzes the cleavage of glycogen into glucose-1-phosphate (Glc-1-P), the first step in glycogen catabolism. Two glgP homologues are found in the genome of Synechocystis sp. PCC 6803, a unicellular cyanobacterium: sll1356 and slr1367. We report on the different functions of these glgP homologues. sll1356, rather than slr1367, is essential for growth at high temperatures. On the other hand, when CO2-fixation and the supply of glucose are both limited, slr1367 is the key factor in glycogen metabolism. In cells growing autotrophically, sll1356 plays a more important role in glycogen digestion than slr1367. This functional divergence is also supported by a phylogenetic analysis of glgP homologues in cyanobacteria.

The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy.

BackgroundDNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein). We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project.ResultsChimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress.ConclusionsThe technique detailed here minimizes the cost and effort to replicate a PCR-generated DNA gene fragment library and facilitates several downstream processes (e.g. directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling.

Chlorophyll b Expressed in Cyanobacteria Functions as a Light-harvesting Antenna in Photosystem I through Flexibility of the Proteins

Photosynthetic pigments bind to their specific proteins to form pigment-protein complexes. To investigate the pigment-binding activities of the proteins, chlorophyll b was for introduced the first time to a cyanobacterium that did not synthesize that pigment, and expression of its function in the native pigment-protein complex of cyanobacterium was confirmed by energy transfer. Arabidopsis CAO (chlorophyll a oxygenase) cDNA was introduced into the genome of Synechocystis sp. PCC6803. The transformant cells accumulated chlorophyll b, with the chlorophyll b content being in the range of 1.4 to 10.6% of the total chlorophyll depending on the growth phase. Polyacrylamide gel electrophoresis analysis of the chlorophyll-protein complexes of transformant cells showed that chlorophyll b was incorporated preferentially into the P700-chlorophyll a-protein complex (CP1). Furthermore, chlorophyll b in CP1 transferred light energy to chlorophyll a, indicating a functional transformation. We also found that CP1 of Chlamydomonas reinhardtii, believed to be a chlorophyll a protein, bound chlorophyll b with a chlorophyll b content of approximately 4.4%. On the basis of these results, the evolution of pigment systems in an early stage of cyanobacterial development is discussed in this paper.

Purification and characterization of UDP-glucose:tetrahydrobiopterin glucosyltransferase from Synechococcus sp. PCC 7942

Tetrahydrobiopterin (BH4)-glucoside was identified from Synechococcus sp. PCC 7942 by HPLC analysis and the enzymatic activity of a glycosyltransferase producing the compound from UDP-glucose and BH4. The novel enzyme, named UDP-glucose:BH4 glucosyltransferase, has been purified 846-fold from the cytosolic fraction of Synechococcus sp. PCC 7942 to apparent homogeneity on SDS-PAGE. The native enzyme exists as a monomer having a molecular mass of 39.2 kDa on SDS-PAGE. The enzyme was active over a broad range of pH from 6.5 to 10.5 but most active at pH 10.0. The enzyme required Mn 2+ for maximal activity. Optimum temperature was 42°C. Apparent K m values for BH4 and UDP-glucose were determined as 4.3 μM and 188 μM, respectively, and V max values were 16.1 and 15.1 pmol min −1 mg −1, respectively. The N-terminal amino acid sequence was Thr-Ala-His-Arg-Phe-Lys-Phe-Val-Ser-Thr-Pro-Val-Gly-, sharing high homology with the predicted N-terminal sequence of an unidentified open reading frame slr1166 determined in the genome of Synechocystis sp. PCC 6803, which is known to produce a pteridine glycoside cyanopterin.

Two Types of Functionally Distinct NAD(P)H Dehydrogenases inSynechocystis sp. Strain PCC6803

The ndhD gene encodes a membrane protein component of NAD(P)H dehydrogenase. The genome of Synechocystis sp. PCC6803 contains 6 ndhD genes. Three mutants were constructed by disrupting highly homologous ndhD genes in pairs. Only the DeltandhD1/DeltandhD2 (DeltandhD1/D2) mutant was unable to grow under photoheterotrophic conditions and exhibited low respiration rate, although the mutant grew normally under photoautotrophic conditions in air. The DeltandhD3/DeltandhD4 (DeltandhD3/D4) mutant grew very slowly in air and did not take up CO(2). The results demonstrated the presence of two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis PCC6803 cells. TheDeltandhD5/DeltandhD6 (DeltandhD5/D6) mutant grew like the wild-type strain. Under far-red light (>710 nm), the level of P700(+) was high in DeltandhD1/D2 and M55 (ndhB-less mutant) at low intensities. The capacity of Q(A) (tightly bound plastoquinone) reduction by plastoquinone pool, as measured by the fluorescence increase in darkness upon addition of KCN, was much less in DeltandhD1/D2 and M55 than in DeltandhD3/D4 and DeltandhD5/D6. We conclude that electrons from NADPH are transferred to the plastoquinone pool mainly by the NdhD1.NdhD2 type of NAD(P)H dehydrogenases.

Salt-induced Sucrose Accumulation is Mediated by Sucrose-phosphate-synthase in Cyanobacteria

In cyanobacteria sucrose-phosphate-synthase (SPS) represents the main sucrose-synthesizing enzyme, which is involved in sucrose accumulation of salt-stressed cells. The enzyme was found to be dependent on UDP-glucose. In Synechococcus sp. PCC 6301, SPS activity was stimulated by adding NaCl to the assay buffers, while in Anabaena sp. PCC 7120 enhanced NaCl concentrations in vitro did not change the SPS activity. In salt-adapted cells of these two cyanobacterial strains, osmotically significant amounts of sucrose were accumulated and the SPS activity was clearly increased compared to cells grown in basal medium. In salt-loaded cells of Synechocystis sp. PCC 6803 small amounts of sucrose were found beside the main osmolyte glucosylglycerol. A putative sps gene homolog found in the genome of Synechocystis sp. PCC 6803 was mutated by insertion of an antibiotic resistance gene cartridge. This mutation led to a complete absence of sucrose biosynthesis and SPS activity confirming the proposed function of the gene.

The icfG gene cluster of Synechocystis sp. strain PCC 6803 encodes an Rsb/Spo-like protein kinase, protein phosphatase, and two phosphoproteins.

A set of open reading frames (ORFs) potentially encoding signal transduction proteins are clustered around icfG, a gene implicated in the regulation of carbon metabolism, in the genome of Synechocystis sp. strain PCC 6803. slr1860 is the ORF for icfG, whose predicted product resembles the protein phosphatases SpoIIE, RsbU, and RsbX from Bacillus subtilis. Bracketing slr1860/icfG are (i) ORF slr1861, whose predicted product resembles the SpoIIAB, RsbT, and RsbW protein kinases from B. subtilis, and (ii) ORFs slr1856 and slr1859, whose predicted products resemble the respective phosphoprotein substrates for the B. subtilis protein kinases: SpoIIAA, RsbS, and RsbV. In order to determine whether the protein products encoded by these ORFs possessed the functional capabilities suggested by sequence comparisons, each was expressed in Escherichia coli as a histidine-tagged fusion protein and analyzed for its ability to participate in protein phosphorylation-dephosphorylation processes in vitro. It was observed that ORF slr1861 encoded an ATP-dependent protein kinase capable of phosphorylating Slr1856 and, albeit with noticeably lower efficiency, Slr1859. Site-directed mutagenesis suggests that Slr1861 phosphorylated these proteins on Ser-54 and Ser-57, respectively. Slr1860 exhibited divalent metal ion-dependent protein-serine phosphatase activity. It catalyzed the dephosphorylation of Slr1856, but not Slr1859, in vitro.

Quinol and cytochrome oxidases in the cyanobacterium Synechocystis sp. PCC 6803.

The genome of Synechocystis sp. PCC 6803 contains three sets of genes for terminal respiratory oxidases: the previously identified cytochrome aa3-type cytochrome c oxidase (CtaI), a second putative oxidase (CtaII) that we interpret to be a cytochrome bo-type quinol oxidase, and a putative cytochrome bd quinol oxidase (Cyd). Genes for the two putative oxidases were cloned, and deletion constructs were made. Strains that lack one, two, or all three of the oxidases were generated. Deletion of the respiratory oxidases had no effect on photoautotrophic or photomixotrophic growth. Strains that lack one oxidase respire at near-wild-type rates, whereas those that lack both CtaI and Cyd do not respire. Thus, CtaII does not play a significant role in cellular metabolism under the conditions tested. An expression construct containing cydAB from Synechocystis sp. PCC 6803 was able to restore aerobic growth in a strain of Escherichia coli that lacks the cytochrome bo oxidase and the cytochrome bd oxidase encoded by cydAB. These results show that the cydAB operon from Synechocystis sp. PCC 6803 encodes a functional quinol oxidase. Deletion of Cyd and/or CtaII in strains lacking photosystem I did not change the fluorescence decay kinetics after illumination, and therefore, these oxidases do not significantly utilize reducing equivalents in the thylakoid membrane. This, combined with our inability to delete CtaI from strains lacking photosystem I, suggests that CtaI is the major oxidase on the thylakoid membrane and that Cyd is localized mostly on the cytoplasmic membrane. Transcripts for ctaDI were detected under all growth conditions tested, while transcripts for cydA and ctaEII could only be detected in cells grown at low light intensity (5 microE m(-2) s(-1)).

Prediction of translation initiation sites on the genome of Synechocystis sp. strain PCC6803 by Hidden Markov model.

We developed a computer program, GeneHackerTL, which predicts the most probable translation initiation site for a given nucleotide sequence. The program requires that information be extracted from the nucleotide sequence data surrounding the translation initiation sites according to the framework of the Hidden Markov Model. Since the translation initiation sites of 72 highly abundant proteins have already been assigned on the genome of Synechocystis sp. strain PCC6803 by amino-terminal analysis, we extracted necessary information for GeneHackerTL from the nucleotide sequence data. The prediction rate of the GeneHackerTL for these proteins was estimated to be 86.1%. We then used GeneHackerTL for prediction of the translation initiation sites of 24 other proteins, of which the initiation sites were not assigned experimentally, because of the lack of a potential initiation codon at the amino-terminal position. For 20 out of the 24 proteins, the initiation sites were predicted in the upstream of their amino-terminal positions. According to this assignment, the processed regions represent a typical feature of signal peptides. We could also predict multiple translation initiation sites for a particular gene for which at least two initiation sites were experimentally detected. This program would be effective for the prediction of translation initiation sites of other proteins, not only in this species but also in other prokaryotes as well.

A second groEL-like gene, organized in a groESL operon is present in the genome of Synechocystis sp. PCC 6803.

Using a groEL gene of Synechococcus sp. PCC 7942 as a DNA probe, a 4.8-kilobase pair (kbp) BamHI fragment of chromosomal DNA of Synechocystis sp. PCC 6803 was cloned. Sequencing of 3.25 kbp of the BamHI fragment revealed three open reading frames. The amino acid sequences deduced from the nucleotide sequences of the two open reading frames are identical to those gained from N-terminal sequencing of purified groEL and groES proteins. This finding demonstrates that these two open reading frames correspond to groEL and groES genes of Synechocystis sp. PCC 6803. groEL of Synechocystis sp. PCC 6803 is remarkably homologous to groEL proteins of other organisms. Southern blot analysis indicates that only one groESL operon is present in the genomic DNA of Synechocystis sp. PCC 6803, whereas the existence of at least two copies of groEL-analogous genes are anticipated. The level of the bicistronic, 2.2-kb transcript of groESL operon increased 100-fold within 15 min upon heat stress. A 9-base pair inverted repeat revealed around the groESL promoter might be involved in regulation of the heat shock response.