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.     

Cloning and Characterization of the nrtA Gene That Encodes a 45-kDa Protein Involved in Nitrate Transport in the Cyanobacterium Synechococcus PCC 7942

A 45-kDa protein in the cytoplasmic membrane of the cyanobacteriumSynechococcus PCC 7942 is involved in the active transport ofnitrate [Omata et al. (1989) Proc. Natl. Acad. Sci. USA 86:6612]. The gene coding for this protein (designated herein asnrtA) has been cloned and sequenced. The nrtA gene encodes aprotein of 443 amino acids with a calculated molecular weightof 48424. The deduced amino acid sequence of the protein is46.5% homologous to that of a 42-kDa cytoplasmic membrane proteinthat is synthesized under carbon-limited conditions in SynechococcusPCC 7942. (Received July 16, 1990; Accepted December 5, 1990)     

Isolation and characterization of the ccmM gene required by the cyanobacterium Synechocystis PCC6803 for inorganic carbon utilization

A high CO₂-requiring mutant of Synechocystis PCC6803 (G3) capable of C_i transport but unable to utilize the intracellular C_i pool for photosynthesis was constructed. A DNA clone of 6.1 kbp that transforms the G3 mutant to the wild-type phenotype was isolated from a Synechocystis PCC6803 genomic library. Complementation test with subclones allocated the mutation site within a DNA fragment of 674 bp nucleotides. Sequencing analysis of the mutation region elucidated an open reading frame encoding a 534 amino-acid protein with a significant sequence homology to the protein coded by the ccmN gene of Synechococcus PCC7942. The ccmM-like gene product of Synechocystis PCC6803 contains four internal repeats with a week similarity to the rbcS gene product. An open reading frame homologous to the ccmN gene of Synechococcus PCC7942 was found downstream to the ccmM-like gene. As opposed to the Synechococcus PCC7942 ccmM and ccmN genes located 2 kbp upstream to, and oriented in the same direction as, the rbc operon, the ccm-like genes in Synechocystis PCC6803 are not located within 22 kbp upstream to the rbcL gene of the Rubisco operon. Thus, despite the resemblance in clustering of the ccmM and ccmN genes in both cyanobacterial species, the difference in their genomic location relative to the rbc genes demonstrates variability in structural organization of the genes involved in inorganic carbon acquisition.Abbreviations CCM CO₂-concentrating mechanism - C_i inorganic carbon - HCR high CO₂-requiring - kbp kilobase pair - ORF open reading frame - Rubisco ribulose 1,5-bisphosphate carboxylase-oxygenase gene - SSC sodium chloride and sodium citrate - WT wild-type     

Heterologous expression of the gene for the ethylene-forming enzyme fromPseudomonas syringae in the cyanobacteriumSynechococcus

Summary Bioconversion of atmospheric carbon dioxide to ethylene was studied in a recombinant cyanobacterium. The gene for the ethylene-forming enzyme ofPseudomonas syringae pv.phaseolicola PK2 was cloned and expressed in the cyanobacteriumSynechococcus PCC7942 R2-SPc by use of a shuttle vector pUC303. The ethylene-forming activityin vivo ofSynechococcus PCC7942 R2-SPc that carried the gene for the ethylene-forming enzyme ofP. syringae pv.phaseolicola PK2 was one-fifth of that ofE. coli JM109 that harbored the same plasmid. The enzyme accounted for 0.021% by weight of the total soluble protein inSynechococcus PCC7942 R2-SPc.     

The gap1 Operon of the Cyanobacterium SynechococcusPCC 7942 Carries a Gene Encoding Glycogen Phosphorylase and Is Induced under Anaerobic Conditions

The cloning and sequencing of the gap1 operon, which encodes the glycolytic NAD-specific glyceraldehyde-3-phosphate dehydrogenase in the cyanobacterium Synechococcus PCC 7942, showed that the gap1 gene is closely linked to the glgP gene encoding glycogen phosphorylase (an enzyme that catalyzes the first step of glycogen degradation). Northern blotting experiments showed that the gap1 and glgP genes are coexpressed and organized in a bicistronic operon, whose expression is enhanced under anaerobic conditions. The nucleotide sequence of the operon has been submitted to GenBank under accession number AF428099.     

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.     

Multiple rpoD-Related Genes of Cyanobacteria

Genomes of many eubacterial strains have been shown to encode for multiple rpoD-related genes. In this report, we describe the identification of the multiple rpoD-related genes of cyanobacterial strains. DNAs of three cyanobacterial strains, Anabaena sp. PCC7120, Synechococcus sp. PCC7942, and Synechocystis sp. PCC6803, were examined by Southern hybridization, using a synthetic probe designed for detecting rpoD or rpoD-related genes. Four or five hybridization signals were found in each DNA. Four DNA regions of Synechococcus sp. PCC7942 corresponding to the hybridization signals were cloned and partially sequenced. The sequence data indicate the presence of genes, named rpoDl, rpoD2, rpoD3, and rpoD4, whose products are highly similar to the basic structure of the principal σ factors of eubacterial strains. The rpoDl gene showed the greatest similarity to the sigA gene of Anabaena sp. PCC7120.     

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.     

Identification of a genomic region that complements a temperature-sensitive, high CO2-requiring mutant of the cyanobacterium, Synechococcus sp. PCC7942

Summary In a temperature-sensitive, high CO₂-requiring mutant of Synechococcus sp. PCC7942, the ability to fix intracellularly accumulated inorganic carbon was severely impaired at non-permissive temperature (41° C). In contrast, inorganic carbon uptake and ribulose-1,5-bisphosphate carboxylase activity in the mutant were comparable to the respective values obtained with the wild-type strain. The mutant was transformed to the wild-type phenotype (ability to form colonies at non-permissive temperature under ordinary air) with the genomic DNA of the wild-type strain. A clone containing a 36 kb genomic DNA fragment of the wild-type strain complemented the mutant phenotype. The complementing activity region was associated with internal 17 kb SmaI, 15 kb HindIII, 3.8 kb BamHI and 0.87 kb Pstl fragments. These 4 fragments overlapped only in a 0.4 kb HindIII-PstI region. In the transformants obtained with total genomic DNA or a plasmid containing the 3.8 kb BamHI fragment, the ability to fix intracellular inorganic carbon was restored. Southern hybridization and partial nucleotide sequence analysis indicated that the cloned genomic region was located approximately 20 kb downstream from the structural genes for subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase. The cloned region was transcribed into a 0.5 kb mRNA. These results indicate that the cloned genomic region of Synechococcus sp. PCC7942 is involved in the efficient utilization of intracellular inorganic carbon for photosynthesis.     

Expression of mouse metallothionein in the cyanobacteriumSynechococcus PCC7942

A cDNA encoding mouse metallothionein was cloned into the shuttle vector pUC303, creating a translational fusion with the bacterial chloramphenicol acetyltransferase gene. The resulting fusion protein has been expressed in the cyanobacteriumSynechococcus PCC7942. Cyanobacterial transformants expressed mouse metallothionein-specific mRNA species as detected by RNA slot blots. In addition, the transformants expressed a unique cadmium ionbinding protein corresponding to the predicted size of the mouse metallothionein fusion protein. Expression of this fusion protein conferred a two-to five-fold increase in cadmium ion tolerance and accumulation onSynechococcus PCC7942.     

Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation

Summary The phycobilisome rod linker genes in the two closely related cyanobacteria Synechococcus sp. PCC 6301 and Synechococcus sp. PCC 7942 were studied. Southern blot analysis showed that the genetic organization of the phycobilisome rod operon is very similar in the two strains. The phycocyanin gene pair is duplicated and separated by a region of about 2.5 kb. The intervening region between the duplicated phycocyanin gene pair was cloned from Synechococcus sp. PCC 6301 and sequenced. Analysis of this DNA sequence revealed the presence of three open reading frames corresponding to 273, 289 and 81 amino acids, respectively. Insertion of a kanamycin resistance cassette into these open reading frames indicated that they corresponded to the genes encoding the 30, 33 and 9 kDa rod linkers, respectively, as judged by the loss of specific linkers from the phycobilisomes of the insertional mutants. Amino acid compositions of the 30 and 33 kDa linkers derived from the DNA sequence were found to deviate from those of purified 33 and 30 kDa linkers in the amounts of glutamic acid/glutamine residues. On the basis of similarity of the amino acid sequence of the rod linkers between Synechococcus sp. PCC 6301 and Calothrix sp. PCC 7601 we name the genes encoding the 30, 33 and 9 kDa linkers cpcH, cpcI and cpcD, respectively. The three linker genes were found to be co-transcribed on an mRNA of 3700 nucleotides. However, we also detected a smaller species of mRNA, of 3400 nucleotides, which would encode only the cpcH and cpcI genes. The 30 kDa linker was still found in phycobilisome rods lacking the 33 kDa linker and the 9 kDa linker was detected in mutants lacking the 33 or the 30 kDa linkers. Free phycocyanin was found in the mutants lacking the 33 or the 30 kDa linkers, whereas no free phycocyanin could be found in the mutant lacking the 9 kDa linker.Abbreviations PCC Pasteur Culture Collection - UTEX University of Texas Culture Collection The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank Nucleotide Sequence Databases under the accession number M94218     

The molecular basis of resistance to the herbicide norflurazon

We have cloned and sequenced a gene, pds, from the cyanobacterium Synechococcus PCC7942 that is responsible for resistance to the bleaching herbicide norflurazon. A point mutation in that gene, leading to an amino acid substitution from valine to glycine in its polypeptide product, was found to confer this resistance. Previous studies with herbicide-resistant mutants have indicated that this gene encodes phytoene desaturase (PDS), a key enzyme in the biosynthesis of carotenoids. A short amino acid sequence that is homologous to conserved motifs in the binding sites for NAD(H) and NADP(H) was identified in PDS, suggesting the involvement of these dinucleotides as cofactors in phytoene desaturation.     

Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.     

A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization,cloning and targeted mutagenesis

DNA probes from the narG gene of Escherichia coli, which encodes the large polypeptide of respiratory nitrate reductase, show cross-hybridization at low stringency to a single region of the genome of the cyanobacterium Synechococcus PCC6301. This segment of cyanobacterial DNA was cloned as the insert of plasmid pDN1 and characterized. RNA complementary to pDN1 was shown to be substantially more abundant in nitrate grown cells of Synechococcus PCC6301 than in ammonium grown cells, thus parallelling the nitrate induction and ammonium repression of nitrate reductase activity in cultures of this cyanobacterium. A mutant of Synechococcus PCC6301 deficient in nitrate reductase activity was obtained after a potentially mutagenic transformation treatment using pDN1 as a donor. This mutant was restored to the wild type phenotype following stable integrative transformation with pDN1 DNA. Taken together these data suggest that pDN1 might encode a polypeptide of nitrate reductase. pDN1 is distinct from three clones of genes involved in nitrate assimilation that were isolated previously from the related cyanobacterium Synechococcus PCC7942 (Kuhlemeier et al., 1984a, J.Bact. 159, 36–41, and 1984b, Gene 31, 109–116).     

Isolation and purification of siderophores produced by cyanobacteria,Synechococcus sp. PCC 7942 andAnabaena variabilis ATCC 29413

New siderophores were isolated and purified from the spent growth medium of the cyanobacteriaSynechococcus sp. PCC 7942 (Anacystis nidulans R2) andAnabaena variabilis ATCC 29413 by solvent extraction and thin-layer chromatography. For each species the siderophore was released into the medium when the cells were grown at low iron concentrations and was not found in the medium of cells grown in iron-sufficient medium. Through a series of biological and chemical tests, combined with spectral analysis, the dihydroxamate nature of each siderophore was confirmed. The siderophores produced bySynechococcus sp. PCC 7942 andA. variabilis had distinct relative molecular masses of 310–313 Da and 520–525 Da, respectively. Neither of the two strains produced Arnow-positive extracellular organics, which indicate the excretion of extracellular catechol-type siderophores.     

Nitrite reductase gene from Synechococcus sp. PCC 7942: homology between cyanobacterial and higher-plant nitrite reductases

The gene encoding nitrite reductase (nir) from the cyanobacterium Synechococcus sp. PCC 7942 has been identified and sequenced. This gene comprises 1536 nucleotides and would encode a polypeptide of 56506 Da that shows similarity to nitrite reductase from higher plants and to the sulfite reductase hemoprotein from enteric bacteria. Identities found at positions corresponding to those amino acids which in the above-mentioned proteins hold the Fe₄S₄-siroheme active center suggest that nitrite reductase from Synechococcus bears an active site much alike that present in those reductases. The fact that the Synechococcus and higher-plant nitrite reductases are homologous proteins gives support to the endosymbiont theory for the origin of chloroplasts.