The eubacterial RNA polymerase core, a transcription machinery performing DNA-dependent RNA polymerization, consists of two α subunits and β, β′ and ω subunits. An additional σ subunit is recruited for promoter recognition and transcription initiation. Cyanobacteria, a group of eubacteria characterized by oxygenic photosynthesis, have a unique composition of the RNA polymerase (RNAP) core due to splitting of the β′ subunit to N-terminal γ and C-terminal β′ subunits. The physiological roles of the small ω subunit of RNAP, encoded by the rpoZ gene, are not yet completely understood in any bacteria. We found that although ω is non-essential in cyanobacteria, it has a major impact on the overall gene expression pattern. In ΔrpoZ strain, recruitment of the primary σ factor into the RNAP holoenzyme is inefficient, which causes downregulation of highly expressed genes and upregulation of many low-expression genes. Especially, genes encoding proteins of photosynthetic carbon concentrating and carbon fixing complexes were down, and the ΔrpoZ mutant showed low light-saturated photosynthetic activity and accumulated photoprotective carotenoids and α-tocopherol. The results indicate that the ω subunit facilitates the association of the primary σ factor with the RNAP core, thereby allowing efficient transcription of highly expressed genes.

SummaryIn eubacteria, replacement of one σ factor in the RNA polymerase (RNAP) holoenzyme by another one changes the transcription pattern. Cyanobacteria are eubacteria characterized by oxygenic photosynthesis, and they typically encode numerous group 2 σ factors that closely resemble the essential primary σ factor. A mutant strain of the model cyanobacterium Synechocystis sp. PCC 6803 without functional group 2 σ factors (named as ΔsigBCDE) could not acclimate to heat, high salt or bright light stress, but in standard conditions ΔsigBCDE grew only 9% slower than the control strain. One-fifth of the genes in ΔsigBCDE was differently expressed compared with the control strain in standard growth conditions and several physiological changes in photosynthesis, and pigment and lipid compositions were detected. To directly analyze the σ factor content of RNAP holoenzyme in vivo, a His-tag was added to the γ subunit of RNAP in Synechocystis and RNAPs were collected. The results revealed that all group 2 σ factors were recruited by RNAP in standard conditions, but recruitment of SigB and SigC increased in heat stress, SigD in bright light, SigE in darkness and SigB, SigC and SigE in high salt, explaining the poor acclimation of ΔsigBCDE to these stress conditions.

Acclimation of cyanobacteria to environmental conditions is mainly controlled at the transcriptional level, and σ factors of the RNA polymerase have a central role in this process. The model cyanobacterium Synechocystis sp. PCC 6803 has four non-essential group 2 σ factors (SigB, SigC, SigD and SigE) that regulate global metabolic responses to various adverse environmental conditions. Here we show that although none of the group 2 σ factors is essential for the major metabolic realignments induced by a short period of nitrogen starvation, the quadruple mutant without any group 2 σ factors and triple mutants missing both SigB and SigD grow slowly in BG-11 medium containing only 5% of the nitrate present in standard BG-11. These ΔsigBCDE, ΔsigBCD and ΔsigBDE strains lost PSII activity rapidly in low nitrogen and accumulated less glycogen than the control strain. An abnormally high glycogen content was detected in ΔsigBCE (SigD is active), while the carotenoid content became high in ΔsigCDE (SigB is active), indicating that SigB and SigD regulate the partitioning of carbon skeletons in low nitrogen. Long-term survival and recovery of the cells after nitrogen deficiency was strongly dependent on group 2 σ factors. The quadruple mutant and the ΔsigBDE strain (only SigC is active) recovered more slowly from nitrogen deficiency than the control strain, and ΔsigBCDE in particular lost viability during nitrogen starvation. Nitrogen deficiency-induced changes in the pigment content of the control strain recovered essentially in 1 d in nitrogen-replete medium, but little recovery occurred in ΔsigBCDE and ΔsigBDE.

Adjustment of gene expression during acclimation to stress conditions, such as bright light, in the cyanobacterium Synechocystis sp. PCC 6803 depends on four group 2 σ factors (SigB, SigC, SigD, SigE). A ΔsigCDE strain containing the stress-responsive SigB as the only functional group 2 σ factor appears twice as resistant to photoinhibition of photosystem II (PSII) as the control strain. Microarray analyses of the ΔsigCDE strain indicated that 77 genes in standard conditions and 79 genes in high light were differently expressed compared with the control strain. Analysis of possible photoprotective mechanisms revealed that high carotenoid content and up-regulation of the photoprotective flavodiiron operon flv4-sll0218-flv2 protected PSII in ΔsigCDE, while up-regulation of pgr5-like, hlipB or isiA genes in the mutant strain did not offer particular protection against photoinhibition. Photoinhibition resistance was lost if ΔsigCDE was grown in high CO2, where carotenoid and Flv4, Sll0218, and Flv2 contents were low. Additionally, photoinhibition resistance of the ΔrpoZ strain (lacking the omega subunit of RNA polymerase), with high carotenoid but low Flv4-Sll0218-Flv2 content, supported the importance of carotenoids in PSII protection. Carotenoids likely protect mainly by quenching of singlet oxygen, but efficient nonphotochemical quenching in ΔsigCDE might offer some additional protection. Comparison of photoinhibition kinetics in control, ΔsigCDE, and ΔrpoZ strains showed that protection by the flavodiiron operon was most efficient during the first minutes of high-light illumination.