Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601.

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.     

Molecular cloning and nucleotide sequence of a developmentally regulated gene from the cyanobacterium Calothrix PCC 7601: a gas vesicle protein gene.

Since the gas vesicle protein (GVP) is highly conserved among the different gas-vacuolate prokaryotes, a 29-mer oligonucleotide corresponding to a portion of the Anabaena flos-aquae GVP gene was synthesized and used to isolate the GVP structural gene from Calothrix PCC 7601 (= Fremyella diplosiphon). Gas vacuole production in this filamentous cyanobacterium is restricted to hormogonia which occur at a specific stage during the developmental cell cycle. The GVP gene (gvpA) was localized on a 709 bp HindIII-HincII fragment. Nucleotide sequence analysis revealed a 213 bp open reading frame whose deduced amino-acid sequence shows a very high homology with that of the Anabaena flos-aquae GVP. Assuming that the first methionine residue is proteolytically processed, the molecular mass of the Calothrix GVP is 7375 daltons. Sequences resembling the Escherichia coli consensus promoter were found upstream from the gvpA gene. The initiator codon of the gvpA gene is preceded by a polypurine sequence assumed to be the ribosome binding site. Southern hybridizations with a probe specific for the gvpA gene indicated that this gene is not plasmid-borne, and that another homologous gene is present in the Calothrix genome.     

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.     

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.     

The amino-acid sequence of three proteins of photosystem I of the cyanobacterium Fremyella diplosiphon (Calothrix sp PCC 7601)

Three proteins containing 138 amino acids (psaD protein), 80 amino acids (psaC protein) and 66 amino acids (psaE protein) of the photosystem I (PS I) complex of the cyanobacterium Fremyella diplosiphon (Calothrix sp PCC 7601) were isolated and sequenced. Comparison with previously known sequences showed a close relationship to homologous proteins of Nostoc, another filamentous cyanobacterium.     

Three C-phycoerythrin-associated linker polypeptides in the phycobilisome of green-light-grown Calothrix sp. PCC 7601 (cyanobacteria).

Microanalyses by SDS-PAGE and microsequencing demonstrate that, under green-light conditions, 3 C-phycoerythrin associated rod-linker polypeptides with different N-terminal amino acid sequences are present in phycobilisomes (PBS) from Calothrix sp. 7601 cells. Two of these polypeptides, corresponding to SDS-PAGE bands at 36 and 37 kDa, could be assigned, respectively, to the cpeC and cpcD genes found on a separate cpeCD-operon in Calothrix sp. 7601 (Federspiel, N.A. and Grossman, A.R. (1990) J. Bacteriol, 172, 4072-4081). The third C-PE rod-linker polypeptide, LR,2PE,33, requires, therefore, a third gene with the suggested locus designation 'cpeE'. A C-PE (alpha beta)6-LR,2PE,33 complex containing this third rod-linker polypeptide could be isolated from phycobilisomes and characterized. PBS from both green- and red-light cells of Calothrix contain a single, unique LRC28 rod-core linker polypeptide which is not altered during chromatic adaptation.     

Molecular cloning and characterization of the recA gene from the cyanobacterium Synechococcus sp. strain PCC 7002.

The recA gene of Synechococcus sp. strain PCC 7002 was detected and cloned from a lambda gtwes genomic library by heterologous hybridization by using a gene-internal fragment of the Escherichia coli recA gene as the probe. The gene encodes a 38-kilodalton polypeptide which is antigenically related to the RecA protein of E. coli. The nucleotide sequence of a portion of the gene was determined. The translation of this region was 55% homologous to the E. coli protein; allowances for conservative amino acid replacements yield a homology value of about 74%. The cyanobacterial recA gene product was proficient in restoring homologous recombination and partial resistance to UV irradiation to recA mutants of E. coli. Heterologous hybridization experiments, in which the Synechococcus sp. strain PCC 7002 recA gene was used as the probe, indicate that a homologous gene is probably present in all cyanobacterial strains.