RESUMO
Genes homologous to hydrogenase accessory genes are scattered over the whole genome in the cyanobacterium Synechocystis sp. PCC 6803. Deletion and insertion mutants of hypA1 (slr1675), hypB1 (sll1432), hypC, hypD, hypE and hypF were constructed and showed no hydrogenase activity. Involvement of the respective genes in maturation of the enzyme was confirmed by complementation. Deletion of the additional homologues hypA2 (sll1078) and hypB2 (sll1079) had no effect on hydrogenase activity. Thus, hypA1 and hypB1 are specific for hydrogenase maturation. We suggest that hypA2 and hypB2 are involved in a different metal insertion process. The hydrogenase activity of DeltahypA1 and DeltahypB1 could be increased by the addition of nickel, suggesting that HypA1 and HypB1 are involved in the insertion of nickel into the active site of the enzyme. The urease activity of all the hypA and hypB single- and double-mutants was the same as in wild-type cells. Therefore, there seems to be no common function for these two hyp genes in hydrogenase and urease maturation in Synechocystis. Similarity searches in the whole genome yielded Slr1876 as the best candidate for the hydrogenase-specific protease. The respective deletion mutant had no hydrogenase activity. Deletion of hupE had no effect on hydrogenase activity but resulted in a mutant unable to grow in a medium containing the metal chelator nitrilotriacetate. Growth was resumed upon the addition of cobalt or methionine. Because the latter is synthesized by a cobalt-requiring enzyme in Synechocystis, HupE is a good candidate for a cobalt transporter in cyanobacteria.
Assuntos
Hidrogenase/genética , Synechocystis/genética , Sequência de Bases , Hidrogenase/fisiologia , Dados de Sequência Molecular , Família Multigênica , Mutagênese , Níquel/farmacologia , Ácido Nitrilotriacético/farmacologia , Synechocystis/enzimologiaRESUMO
To elucidate influences on the tocopherol biosynthesis in cyanobacteria, wild type and mutant cells of a putative methyltransferase in tocopherol and plastoquinone biosynthesis of Synechocystis sp. PCC 6803 were grown under different conditions. The vitamin E content of cells grown under different light regimes, photomixotrophic or photoautotrophic conditions and varying carbon dioxide supplies were compared by HPLC measurements. The tocopherol levels in wild type cells increased under higher light conditions and low carbon dioxide supply. Photomixotrophic growth led to lower vitamin E amounts in the cells compared to those grown photoautotrophically. We were able to segregate a homozygous deltasll0418 mutant under photoautotrophic conditions. In contrast to former suggestions in the literature the deletion of this gene is not lethal under photomixotrophic conditions and the influence on tocopherol and plastoquinone amounts is diminutive. The methyltransferase encoded by the gene sll0418 is not essential either for tocopherol or plastoquinone synthesis.
Assuntos
Synechocystis/metabolismo , Tocoferóis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dióxido de Carbono/fisiologia , Glucose/metabolismo , Luz , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Químicos , Estrutura Molecular , Mutação , Plastoquinona/metabolismo , Synechocystis/genética , Tocoferóis/químicaRESUMO
The disruption of the Synechocystis open reading frame Deltaslr0090 encoding a gene with high homology to plant genes encoding 4-hydroxyphenylpyruvate dioxygenase results in an impairment of tocopherol biosynthesis without affecting levels of plastoquinone, carotenoids and chlorophyll as well as cell growth and photosynthesis. Our results indicate that in Synechocystis in contrast to the situation in higher plants the 4-hydroxyphenylpyruvate dioxygenase is not required for the synthesis of plastoquinone.
Assuntos
4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Cianobactérias/enzimologia , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Plastoquinona/metabolismo , 4-Hidroxifenilpiruvato Dioxigenase/genética , Cianobactérias/genética , Cianobactérias/metabolismo , Deleção de Genes , Complexos de Proteínas Captadores de Luz , Mutação , Plastoquinona/análise , Tocoferóis/análise , Vitamina K 1/análiseRESUMO
Hydrogenases are important enzymes in the energy metabolism of microorganisms. Therefore, they are widespread in prokaryotes. We analyzed the occurrence of hydrogenases in cyanobacteria and deduced a FeFe-hydrogenase in three different heliobacterial strains. This allowed the first phylogenetic analysis of the hydrogenases of all five major groups of photosynthetic bacteria (heliobacteria, green nonsulfur bacteria, green sulfur bacteria, photosynthetic proteobacteria, and cyanobacteria). In the case of both hydrogenases found in cyanobacteria (uptake and bidirectional), the green nonsulfur bacterium Chloroflexus aurantiacus was found to be the closest ancestor. Apart from a close relation between the archaebacterial and the green sulfur bacterial sulfhydrogenase, we could not find any evidence for horizontal gene transfer. Therefore, it would be most parsimonious if a Chloroflexus-like bacterium was the ancestor of Chloroflexus aurantiacus and cyanobacteria. After having transmitted both hydrogenase genes vertically to the different cyanobacterial species, either no, one, or both enzymes were lost, thus producing the current distribution. Our data and the available data from the literature on the occurrence of cyanobacterial hydrogenases show that the cyanobacterial uptake hydrogenase is strictly linked to the occurrence of the nitrogenase. Nevertheless, we did identify a nitrogen-fixing Synechococcus strain without an uptake hydrogenase. Since we could not find genes of a FeFe-hydrogenase in any of the tested cyanobacteria, although strains performing anoxygenic photosynthesis were also included in the analysis, a cyanobacterial origin of the contemporary FeFe-hydrogenase of algal plastids seems unlikely.
Assuntos
Proteínas de Bactérias/genética , Cianobactérias/enzimologia , Eucariotos/enzimologia , Hidrogenase/genética , Fotossíntese , Hidrogênio/metabolismo , FilogeniaRESUMO
The bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 is encoded by five genes (hoxEFUYH) which are transcribed as one unit. The transcription of the hox-operon is regulated by a promoter situated upstream of hoxE. The transcription start point was located at -168 by 5'Race. Several promoter probe vectors carrying different promoter fragments revealed two regions to be essential for the promoter activity. One is situated in the untranslated 5'leader region and the other is found -569 to -690 nucleotides upstream of the ATG. The region further upstream was shown to bind a protein. Even though an imperfect NtcA binding site was identified, NtcA did not bind to this region. The protein binding to the DNA was purified and found to be LexA by MALDI-TOF. The complete LexA and its DNA binding domain were overexpressed in Escherichia coli. Both were able to bind to two sites in the examined region in band-shift-assays. Accordingly, the hydrogenase activity of a LexA-depleted mutant was reduced. This is the first report on LexA acting not as a repressor but as a transcriptional activator. Furthermore, LexA is the first transcription factor identified so far for the expression of bidirectional hydrogenases in cyanobacteria.