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Simultaneous inactivation of sigma factors B and D interferes with light acclimation of the cyanobacterium Synechocystis sp. strain PCC 6803.

ABSTRACT: In cyanobacteria, gene expression is regulated mainly at the level of transcription initiation, which is mediated by the RNA polymerase holoenzyme. The RNA polymerase core is catalytically active, while the sigma factor recognizes promoter sequences. Group 2 sigma factors are similar to the principal sigma factor but are nonessential. Group 2 sigma factors SigB and SigD are structurally the most similar sigma factors in Synechocystis sp. strain PCC 6803. Under standard growth conditions, simultaneous inactivation of sigB and sigD genes did not affect the growth, but the photosynthesis and growth of the DeltasigBD strain were slower than in the control strain at double light intensity. Light-saturated electron transfer rates and the fluorescence and thermoluminescence measurements showed that photosynthetic light reactions are fully functional in the DeltasigBD strain, but absorption and 77 K emission spectra measurements suggest that the light-harvesting system of the DeltasigBD strain does not acclimate normally to higher light intensity. Furthermore, the DeltasigBD strain is more sensitive to photoinhibition under bright light because impaired upregulation of psbA genes leads to insufficient PSII repair.

SUBMITTER: Pollari M

PROVIDER: S-EPMC2698387 | biostudies-other | 2009 Jun

REPOSITORIES: biostudies-other

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Simultaneous inactivation of sigma factors B and D interferes with light acclimation of the cyanobacterium Synechocystis sp. strain PCC 6803.

Pollari Maija M Ruotsalainen Virpi V Rantamäki Susanne S Tyystjärvi Esa E Tyystjärvi Taina T

Journal of bacteriology 20090410 12

In cyanobacteria, gene expression is regulated mainly at the level of transcription initiation, which is mediated by the RNA polymerase holoenzyme. The RNA polymerase core is catalytically active, while the sigma factor recognizes promoter sequences. Group 2 sigma factors are similar to the principal sigma factor but are nonessential. Group 2 sigma factors SigB and SigD are structurally the most similar sigma factors in Synechocystis sp. strain PCC 6803. Under standard growth conditions, simulta ...[more]

PMID: 19363110

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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

Simultaneous inactivation of sigma factors B and D interferes with light acclimation of the cyanobacterium Synechocystis sp. strain PCC 6803.

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Simultaneous inactivation of sigma factors B and D interferes with light acclimation of the cyanobacterium Synechocystis sp. strain PCC 6803.

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