The presence of glutamate dehydrogenase is a selective advantage for the Cyanobacterium synechocystis sp. strain PCC 6803 under nonexponential growth conditions

The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 has two putative pathways for ammonium assimilation: the glutamine synthetase-glutamate synthase cycle, which is the main one and is finely regulated by the nitrogen source; and a high NADP-dependent glutamate dehydrogenase activity (NADP-GDH) whose contribution to glutamate synthesis is uncertain. To investigate the role of the latter, we used two engineered mutants, one lacking and another overproducing NADP-GDH. No major disturbances in the regulation of nitrogen-assimilating enzymes or in amino acids pools were detected in the null mutant, but phycobiline content, a sensitive indicator of the nutritional state of cyanobacterial cells, was significantly reduced, indicating that NADP-GDH plays an auxiliary role in ammonium assimilation. This effect was already prominent in the initial phase of growth, although differences in growth rate between the wild type and the mutants were observed at this stage only at low light intensities. However, the null mutant was unable to sustain growth at the late stage of the culture at the point when the wild type showed the maximum NADP-GDH activity, and died faster in ammonium-containing medium. Overexpression of NADP-GDH improved culture proliferation under moderate ammonium concentrations. Competition experiments between the wild type and the null mutant confirmed that the presence of NADP-GDH confers a selective advantage to Synechocystis sp. strain PCC 6803 in late stages of growth.     

Sucrose-phosphate synthase from Synechocystis sp. strain PCC 6803: identification of the spsA gene and characterization of the enzyme expressed in Escherichia coli

The first identification and characterization of a prokaryotic gene (spsA) encoding sucrose-phosphate synthase (SPS) is reported for Synechocystis sp. strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.     

Analysis of the psbU gene encoding the 12-kDa extrinsic protein of photosystem II and studies on its role by deletion mutagenesis in Synechocystis sp. PCC 6803

The gene encoding the 12-kDa extrinsic protein of photosystem II from Synechocystis sp. PCC 6803 was cloned based on N-terminal sequence of the mature protein. This gene, named psbU, encodes a polypeptide of 131 residues, the first 36 residues of which were absent in the mature protein and thus served as a transit peptide required for its transport into the thylakoid lumen. A psbU gene deletion mutant grew photoautotrophically in normal BG11 medium at almost the same rate as that of the wild type strain. This mutant, however, grew apparently slower than the wild type did upon depletion of Ca2+ or Cl- from the growth medium. Photosystem II oxygen evolution decreased to 81% in the mutant as compared with that in the wild type, and the thermoluminescence B- and Q-bands shifted to higher temperatures accompanied by an increase in the Q-band intensity. These results indicate that the 12-kDa protein is not essential for oxygen evolution but may play a role in optimizing the ion (Ca2+ and Cl-) environment and maintaining a functional structure of the cyanobacterial oxygen-evolving complex. In addition, a double deletion mutant lacking cytochrome c-550 and the 12-kDa protein grew photoautotrophically with a phenotype identical to that of the single deletion mutant of cytochrome c-550. This supports our previous biochemical results that the 12-kDa protein cannot bind to photosystem II in the absence of cytochrome c-550 (Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832).     

Kinematic analysis of patients with spinal muscular atrophy during spontaneous breathing and mechanical ventilation

Dinoseb is a herbicide known to inhibit photosystem II electron transfer like DCMU, triazine and phenolic-type herbicides. The mutant Din7 of the cyanobacterium Synechocystis sp. PCC 6803, selected for resistance to dinoseb, and the mutant Ins2, constructed by the insertion of the kanamycin resistance cassette into the drgA gene, were cross-resistant to other nitrophenolic herbicides (DNOC, 2,4-dinitrophenol) and to the cell inhibitor metronidazole but not to the photosystem II inhibitors DCMU or ioxynil. The Din7 mutant had the same characteristics of photosystem II inhibition by dinoseb as the wild type. This result suggested the existence of another site for dinoseb inhibition. The wild type cells modified dinoseb to a non-toxic product that gave an absorption spectrum similar to that of dithionite treated dinoseb containing reduced nitro groups. In contrast, the Din7 mutant did not modify dinoseb. These phenomena were controlled by the drgA gene encoding a protein which showed similarity to several enzymes having nitroreductase activity. The addition of superoxide dismutase to the medium relieved the toxic effect of dinoseb in wild type cells but not in Din7. It is proposed that in wild type cells of Synechocystis sp. PCC 6803 the DrgA protein is involved in detoxification of dinoseb via the reduction of the nitro group(s) and this process is accompanied by the formation of toxic superoxide anions. Mutations blocking the activity of the DrgA protein lead to the development of resistance to nitrophenolic herbicides and metronidazole.     

Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits

The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.     

An aroA homologue from Synechocystis sp. PCC 6803

In this study, we report the entire nucleotide sequence of an aroA homologue encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), isolated from the cyanobacterium Synechocystis sp. PCC 6803. The proposed coding region is an open reading frame of 447 amino acids. The deduced sequence of the gene product is particularly similar to the Gram+ EPSPS sequences available to date, in particular to that in Bacillus subtilis. Analysis of the Synechocystis putative EPSPS sequence does not lead to an obvious explanation for the natural tolerance of this cyanobacterium to glyphosate.     

The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli

The Escherichia coli ispB gene encoding octaprenyl diphosphate synthase is responsible for the synthesis of the side chain of isoprenoid quinones. We tried to construct an E. coli ispB-disrupted mutant but could not isolate the chromosomal ispB disrupted mutant unless the ispB gene or its homolog was supplied on a plasmid. The chromosomal ispB disruptants that harbored plasmids carrying the ispB homologs from Haemophilus influenzae and Synechocystis sp. strain PCC6803 produced mainly ubiquinone 7 and ubiquinone 9, respectively. Our results indicate that the function of the ispB gene is essential for normal growth and that this function can be substituted for by homologs of the ispB gene from other organisms that produce distinct forms of ubiquinone.     

Directed inactivation of the psbI gene does not affect photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

PsbI is a small, integral membrane protein component of photosystem II (PSII), a pigment-protein complex in cyanobacteria, algae and higher plants. To understand the function of this protein, we have isolated the psbI gene from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and determined its nucleotide sequence. Using an antibiotic-resistance cartridge to disrupt and replace the psbI gene, we have created mutants of Synechocystis 6803 that lack the PsbI protein. Analysis of these mutants revealed that absence of the PsbI protein results in a 25-30% loss of PSII activity. However, other PSII polypeptides are present in near wild-type amounts, indicating that no significant destabilization of the PSII complex has occurred. These results contrast with recently reported data indicating that PsbI-deficient mutants of the eukaryotic alga Chlamydomonas reinhardtii are highly light-sensitive and have a significantly lower (80-90%) titer of the PSII complex. In Synechocystis 6803, PsbI-deficient cells appear to be slightly more photosensitive than wild-type cells, suggesting that this protein, while not essential for PSII biogenesis or function, plays a role in the optimization of PSII activity.     

The cyanobacterium Synechocystis sp. strain PCC 6803 expresses a DNA methyltransferase specific for the recognition sequence of the restriction endonuclease PvuI

By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a dam-like site including N6-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain. slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C5-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in slr0214 were generated by the insertion of an aphII gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5'-CGATCG-3', corresponding to the recognition sequence of PvuI. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis. The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro. We propose the designation M.Ssp6803I [corrected] (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.     

Origin of a chloroplast protein importer

During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system. A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares approximately 22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the Synechocystis PCC6803 genome. Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of Synechocystis PCC6803 and that SynToc75 forms a voltage-gated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes. These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane. Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.     

Circadian rhythm of the cyanobacterium Synechocystis sp. strain PCC 6803 in the dark

The cyanobacterium Synechocystis sp. strain PCC 6803 exhibited circadian rhythms in complete darkness. To monitor a circadian rhythm of the Synechocystis cells in darkness, we introduced a PdnaK1::luxAB gene fusion (S. Aoki, T. Kondo, and M. Ishiura, J. Bacteriol. 177:5606-5611, 1995), which was composed of a promoter region of the Synechocystis dnaK1 gene and a promoterless bacterial luciferase luxAB gene set, as a reporter into the chromosome of a dark-adapted Synechocystis strain. The resulting dnaK1-reporting strain showed bioluminescence rhythms with a period of 25 h (on agar medium supplemented with 5 mM glucose) for at least 7 days in darkness. The rhythms were reset by 12-h-light-12-h-dark cycles, and the period of the rhythms was temperature compensated for between 24 and 31 degrees C. These results indicate that light is not necessary for the oscillation of the circadian clock in Synechocystis.     

The evolutionary origin of the protein-translocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog

The known envelope membrane proteins of the chloroplastic protein import apparatus lack sequence similarity to proteins of other eukaryotic or prokaryotic protein transport systems. However, we detected a putative homolog of the gene encoding Toc75, the protein-translocating channel from the outer envelope membrane of pea chloroplasts, in the genome of the cyanobacterium Synechocystis sp. PCC 6803. We investigated whether the low sequence identity of 21% reflects a structural and functional relationship between the two proteins. We provide evidence that the cyanobacterial protein is also localized in the outer membrane. From this information and the similarity of the predicted secondary structures, we conclude that Toc75 and the cyanobacterial protein, referred to as SynToc75, are structural homologs. synToc75 is essential, as homozygous null mutants were not recovered after directed mutagenesis. Sequence analysis indicates that SynToc75 belongs to a family of outer membrane proteins from Gram-negative bacteria whose function is not yet known. However, we demonstrate that these proteins are related to a specific group of prokaryotic secretion channels that transfer virulence factors, such as hemolysins and adhesins, across the outer membrane.