Effects of deficiency and overdose of group 2 sigma factors in triple inactivation strains of Synechocystis sp. strain PCC 6803

Acclimation of cyanobacteria to environmental changes includes major changes in the gene expression patterns partly orchestrated by the replacement of a particular σ subunit with another in the RNA polymerase holoenzyme. The cyanobacterium Synechocystis sp. strain PCC 6803 encodes nine σ factors, all belonging to the σ(70) family. Cyanobacteria typically encode many group 2 σ factors that closely resemble the principal σ factor. We inactivated three out of the four group 2 σ factors of Synechocystis simultaneously in all possible combinations and found that all triple inactivation strains grow well under standard conditions. Unlike the other strains, the ΔsigBCD strain, which contains SigE as the only functional group 2 σ factor, did not grow faster under mixotrophic than under autotrophic conditions. The SigB and SigD factors were important in low-temperature acclimation, especially under diurnal light rhythm. The ΔsigBCD, ΔsigBCE, and ΔsigBDE strains were sensitive to high-light-induced photoinhibition, indicating a central role of the SigB factor in high-light tolerance. Furthermore, the ΔsigBCE strain (SigD is the only functional group 2 σ factor) appeared to be locked in the high-fluorescence state (state 1) and grew slowly in blue but not in orange or white light. Our results suggest that features of the triple inactivation strains can be categorized as (i) direct consequences of the inactivation of a particular σ factor(s) and (ii) effects resulting from the higher probability that the remaining group 2 σ factors associate with the RNA polymerase core.     

The SigB sigma factor mediates high-temperature responses in the cyanobacterium Synechocystis sp. PCC6803

The sigma factors of RNA polymerase play central roles when bacteria adapt to different environmental conditions. We studied heat-shock responses in the cyanobacterium Synechocystis sp. PCC6803 using the sigma factor inactivation strains deltasigB, deltasigD and deltasigBD. The SigB factor was found to be important for short-term heat-shock responses and acquired thermotolerance. The normal high-temperature induction of the hspA gene depended on the SigB factor. The SigD sigma factor had a role in high-temperature responses as well, and the double inactivation strain deltasigBD grew more slowly at 43 degrees C than the deltasigB and deltasigD strains.     

SigB, SigC, and SigE from Myxococcus xanthus homologous to sigma32 are not required for heat shock response but for multicellular differentiation

Myxococcus xanthus has been known to have multiple sigma factors which are considered to play important roles in regulation of gene expression in development. A new gene encoding a putative sigma factor, sigE, was cloned by using a degenerate oligonucleotide corresponding to the conserved region 2.2 of M. xanthus SigA. In the 2.0-kb nucleotide sequence, an open reading frame consisting of 280 amino acid residues was identified. The amino acid sequence of SigE shows high similarity to heat shock sigma factors in bacteria. However, the sigE gene is not induced by heat shock and deletion of sigE does not affect production of heat shock proteins. SigE is expressed during both vegetative growth and fruiting body development. In the deletion mutant of the sigE gene fruiting body formation is initiated earlier and fewer spores are produced than in the parent strain. Interestingly, the deltasigE mutant shows defects in fruiting body formation at 37 degrees C. In addition to SigE, SigB and SigC show high sequence similarity to heat shock sigma factors. However, even if all three sigma factor genes are disrupted, heat shock proteins are still normally induced. A deltasigBdeltasigCdeltasigE triple deletion strain forms fruiting bodies earlier, but sporulats later than the parent strain. Spores from the triple deletion mutant are aberrant and their viability is less than 0.001% compared with that of the parent strain, suggesting that these sigma factors may have redundant functions in multicellular differentiation of M. xanthus.     

Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803

Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ?sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H₂O₂, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ?sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ?sigCDE than in the control strain. These results indicate that ?sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ?sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ?sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ?sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ?sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.     

Sucrose Synthesis in the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC 7120 Is Controlled by the Two-Component Response Regulator OrrA

The filamentous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 accumulates sucrose as a compatible solute against salt stress. Sucrose-phosphate synthase activity, which is responsible for the sucrose synthesis, is increased by salt stress, but the mechanism underlying the regulation of sucrose synthesis remains unknown. In the present study, a response regulator, OrrA, was shown to control sucrose synthesis. Expression of spsA, which encodes a sucrose-phosphate synthase, and susA and susB, which encode sucrose synthases, was induced by salt stress. In the orrA disruptant, salt induction of these genes was completely abolished. The cellular sucrose level of the orrA disruptant was reduced to 40% of that in the wild type under salt stress conditions. Moreover, overexpression of orrA resulted in enhanced expression of spsA, susA, and susB, followed by accumulation of sucrose, without the addition of NaCl. We also found that SigB2, a group 2 sigma factor of RNA polymerase, regulated the early response to salt stress under the control of OrrA. It is concluded that OrrA controls sucrose synthesis in collaboration with SigB2.