Identification of the genome‐wide distribution of cyanobacterial group‐2 sigma factor SigE, accountable for its regulon

Abstract

Sigma factors are the subunits of bacterial RNA polymerase that govern the expression of genes by recognizing the promoter sequence. Cyanobacteria, which are oxygenic phototrophic eubacteria, have multiple alternative sigma factors that respond to various environmental stresses. The subgroup highly homologous to the primary sigma factor (SigA) is called the group-2 sigma factor. The model cyanobacteriumSynechocystis sp. PCC 6803 has four group-2 sigma factors (SigB-SigE) conserved within the phylum Cyanobacteria. Among the group-2 sigma factors in Synechocystis sp. PCC 6803, SigE is unique because it alters metabolism by inducing the expression of genes related to sugar catabolism and nitrogen metabolism. However, the features of the promoter sequences of the SigE regulon remain elusive. Here, we identified the direct targets of SigA and SigE by chromatin immunoprecipitation sequencing (ChIP-seq). We then showed that the binding sites of SigE and SigA overlapped substantially, but that SigE localized exclusively to SigEdependent promoters. We also found consensus sequences from SigE-dependent promoters and confirmed their importance. ChIP-seq analysis showed both the redundancy and the specificity of SigE, compared with SigA, integrating information obtained from a previously adopted genetic approach and in vitro assays. The features of SigE elucidated in our study indicate its similarity with group-2 sigma factors of other bacteria, even though they are evolutionarily irrelevant. Our approach is also applicable to other organisms and organelles, such as plant plastids, which have multiple group-2 sigma factors.

CyAbrB2 is a nucleoid-associated protein inSynechocystiscontrolling hydrogenase expression during fermentation

“…We searched binding sites of SigE and SigA under aerobic and microoxic conditions (Figures 4A and 4B,top and bottom). Although our peak-calling threshold ignored many minor binding summits, SigE and SigA peaks identified in this study predominantly cover the previously identified peaks (Figure S1D), reproducing the previous study's conclusion (Kariyazono and Osanai, 2022). In other words, SigE and primary sigma factor SigA share localization on the promoters of housekeeping genes, but SigE exclusively binds to the promoters of SigE-dependent genes.…”

“…Specifically, we generated a Synechocystis strain in which CyAbrB2 was epitope-tagged and performed a ChIP-seq assay under aerobic and microoxic conditions (Figures S2B and S2C). SigE-tagged strains previously constructed and analyzed elsewhere were also employed (24). The primary sigma factor SigA was also analyzed to determine SigE-specific binding.…”

“…Sample preparation for the immunoblotting was performed as previously described (Kariyazono and Osanai, 2022), and FLAG-tagged proteins were detected by alkaline-phosphatase-conjugated anti-FLAG IgG (A9469, Sigma Aldrich) and 1-Step™ NBT/BCIP substrate solution (Thermo Fisher Scientific).…”

“…ChIP and qPCR analyses were conducted as described in a previous study with a few modifications (Kariyazono and Osanai, 2022); FLAG-tagged proteins were immunoprecipitated with FLAG-M2 antibody (F1804 Sigma-Aldrich) conjugated to protein G dynabeads (10003D Invitrogen).…”

“…SigE shows high amino acid sequence homology to primary sigma factor SigA, which transcribes housekeeping and photosynthetic genes (Imamura and Asayama, 2009). ChIP-seq study revealed that most SigE binding sites are the same as SigA, but promoters of glycogen catabolism and glycolysis are exclusively occupied by SigE (Kariyazono and Osanai, 2022).…”