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[NiFe]-hydrogenase is essential for cyanobacterium Synechocystis sp. PCC 6803 aerobic growth in the dark.

ABSTRACT: The cyanobacterium Synechocystis sp. PCC 6803 has a bidirectional [NiFe]-hydrogenase (Hox hydrogenase) which reversibly reduces protons to H2. This enzyme is composed of a hydrogenase domain and a diaphorase moiety, which is distinctly homologous to the NADH input module of mitochondrial respiratory Complex I. Hox hydrogenase physiological function is still unclear, since it is not required for Synechocystis fitness under standard growth conditions. We analyzed the phenotype under prolonged darkness of three Synechocystis knock-out strains, lacking either Hox hydrogenase (?HoxE-H) or one of the proteins responsible for the assembly of its NiFe active site (?HypA1 and ?HypB1). We found that Hox hydrogenase is required for Synechocystis growth under this condition, regardless of the functional status of its catalytic site, suggesting an additional role beside hydrogen metabolism. Moreover, quantitative proteomic analyses revealed that the expression levels of several subunits of the respiratory NADPH/plastoquinone oxidoreductase (NDH-1) are reduced when Synechocystis is grown in the dark. Our findings suggest that the Hox hydrogenase could contribute to electron transport regulation when both photosynthetic and respiratory pathways are down-regulated, and provide a possible explanation for the close evolutionary relationship between mitochondrial respiratory Complex I and cyanobacterial [NiFe]-hydrogenases.

SUBMITTER: De Rosa E

PROVIDER: S-EPMC4517062 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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[NiFe]-hydrogenase is essential for cyanobacterium Synechocystis sp. PCC 6803 aerobic growth in the dark.

De Rosa Edith E Checchetto Vanessa V Franchin Cinzia C Bergantino Elisabetta E Berto Paola P Szabò Ildikò I Giacometti Giorgio M GM Arrigoni Giorgio G Costantini Paola P

Scientific reports 20150728

The cyanobacterium Synechocystis sp. PCC 6803 has a bidirectional [NiFe]-hydrogenase (Hox hydrogenase) which reversibly reduces protons to H2. This enzyme is composed of a hydrogenase domain and a diaphorase moiety, which is distinctly homologous to the NADH input module of mitochondrial respiratory Complex I. Hox hydrogenase physiological function is still unclear, since it is not required for Synechocystis fitness under standard growth conditions. We analyzed the phenotype under prolonged dark ...[more]

PMID: 26215212

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Project description:Two cAMP receptor proteins (CRPs), Sycrp1 (encoded by sll1371) and Sycrp2 (encoded by sll1924), exist in the cyanobacterium Synechocystis sp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report that sycrp2 disruption results in the loss of motility of Synechocystis sp. PCC 6803, and that the phenotype can be recovered by reintroducing the sycrp2 gene into the genome of sycrp2-disrupted mutants. Electron microscopy showed that the sycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of the pilA9-pilA11 operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased in sycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region of pilA9 Together, these findings indicate that in Synechocystis sp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCE cAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacterium Synechocystis sp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.

Project description:BACKGROUND: Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H+ + 2e- <--> H2 (g). RESULTS: Hydrogen yields were enhanced up to 41-fold by cloning the bidirectional hydrogenase (encoded by hoxEFUYH) from the cyanobacterium into E. coli. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well-studied Enterobacter aerogenes HU-101, and hydrogen gas bubbles are clearly visible from the cultures. Overexpression of HoxU alone (small diaphorase subunit) accounts for 43% of the additional hydrogen produced by HoxEFUYH. In addition, hydrogen production in E. coli mutants with defects in the native formate hydrogenlyase system show that the cyanobacterial hydrogenase depends on both the native E. coli hydrogenase 3 as well as on its maturation proteins. Hydrogen absorption by cells expressing hoxEFUYH was up to 10 times lower than cells which lack the cloned cyanobacterial hydrogenase; hence, the enhanced hydrogen production in the presence of hoxEFUYH is due to inhibition of hydrogen uptake activity in E. coli. Hydrogen uptake by cells expressing hoxEFUYH was suppressed in three wild-type strains and in two hycE mutants but not in a double mutant defective in hydrogenase 1 and hydrogenase 2; hence, the active cyanobacterial locus suppresses hydrogen uptake by hydrogenase 1 and hydrogenase 2 but not by hydrogenase 3. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system; instead, biofilm-related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2-fold elevated biofilm formation. This appears to be the first enhanced hydrogen production by cloning a cyanobacterial enzyme into a heterologous host. CONCLUSION: Enhanced hydrogen production in E. coli cells expressing the cyanobacterial HoxEFUYH is by inhibiting hydrogen uptake of both hydrogenase 1 and hydrogenase 2.

Project description:BackgroundCyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock.ResultsHere, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC-MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.ConclusionsOur best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.

Project description:Every cyanobacterial species contains gene encoding site-2-protease (S2P) homolog. The studied prokaryotic S2P homologs play essential roles in regulating stress response through intramembrane proteolysis of membrane-bound anti-sigma factors. Here, gene of Slr0643, one of four S2P homologs in Synechocystis sp. PCC 6803, was insertionally disrupted to explore its physiological role. Only a partially segregated mutant was obtained, indicating its indispensability for growth. The partially disrupted mutant could not grow at pH 6.5, while wild type acclimated to pH 6.5 quickly. The slr0643 gene expression was transiently induced after pH transfer from 7.5 to 6.5. Both evidences demonstrated the pivotal role of fully functional Slr0643 in acid acclimation. DNA microarray and quantitative RT-PCR analyses decoded genes involved in early acid acclimation and revealed differentially expressed genes due to slr0643 disruption at both pH conditions. Early acid acclimation to pH 6.5 included upregulation of sigH, hik16 and hik35, and downregulation of pcrR and sigG; as well as downregulation of porins and upregulation of inorganic carbon and nitrogen transporters. Defective photosynthesis and excess expression of NADH dehydrogenase, together with over upregulation of carbon transporter and repression of nitrogen transporter and metabolism gene contributed to the acid lethality of mutant at pH 6.5. Most interestingly, analysis of microarray data revealed the close relationship between slr0643 disruption and expression of sigH operon. Therefore it was implied that Slr0643/Sll0857/SigH might acts through S2P/anti-Sigma factor/Sigma factor mechanism to play a role in acid acclimation. M-bM-^@M-^C Loop design was used to compare differential expression of wild type and mutant at pH 7.5 and pH 6.5 respectively, including dye-swape. Biological variation was sampled by extracting RNA from three independent experiments and pooling them together before hybridizations. Two or four replicate chips for each comparison and two replicate printings per chip represent technical repeats.

Dataset Information

[NiFe]-hydrogenase is essential for cyanobacterium Synechocystis sp. PCC 6803 aerobic growth in the dark.

Publications

[NiFe]-hydrogenase is essential for cyanobacterium Synechocystis sp. PCC 6803 aerobic growth in the dark.

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