Direct repression of D-galactonate metabolism by the stationary phase and stress-responsive sigma factor, σ S , in Escherichia coli

Abstract

Abstract Transcription initiation requires specific binding of RNA Polymerase (RNAP) to the promoters of target genes. In bacteria, σ factors associate with RNAP to form holoenzyme (Eσ) that recognizes promoter elements, initiates promoter melting and DNA duplex unwinding, and assists early stages in transcript formation and promoter escape. In Escherichia coli, besides the housekeeping and essential σ factor, σ 70 , there are alternative σ factors that direct the transcription of specific subsets of genes under adaptive conditions. σ S , an alternative σ factor, is a central regulator of adaptive response during stationary phase and environmental stress. Because σ S and σ 70 show a high degree of sequence similarity, their Eσ’s bind optimally to similar promoters in vitro; however, variable combinations of the cis- acting promoter features and trans-acting factors determine whether a promoter will be recognized by Eσ S or Eσ 70 , or by both Eσ’s in vivo. σ factors by their nature should act as positive regulators of transcription, however, due to an extensive overlap between σ 70 and σ S binding sites on promoter DNA, antagonism between σ factors at the promoter DNA can result in direct transcriptional interference and thus σ factor-mediated direct repression. In the present thesis, we established that the promoter of D-galactonate operon (dgo), involved in the metabolism of a sugar acid, D-galactonate, in E. coli, is directly repressed by σ S . D-galactonate is widely used by enteric bacteria as a carbon and energy source. The genes responsible for the metabolism of D-galactonate are organized in an operon and are under the negative regulation of DgoR and positive regulation by cAMP-CRP. Sequence analysis of the dgo promoter revealed the presence of several σ S - selective and activity stimulating features. Interestingly, we observed that σ S negatively regulates D-galactonate metabolism. By employing σ S mutants proficient for Eσ S formation but defective in binding to DNA, we established that σ S directly represses the dgo promoter. However, unlike the two known instances of direct repression of promoters by σ S (promoters of sdhC in SalmonellaTyphimurium and esrB in Edwardsiella piscida) where an unfavorable discriminator (promoter element involved in promoter escape) is the reason for σ S -mediated repression, in the dgo promoter, besides unfavorable discriminator, a sub-optimally placed -35 element prevents its transcription by σ S . Whereas CRP acting as a class II activator supports Eσ 70 - mediated transcription of the dgo promoter, σ S represses the promoter by inhibiting CRP- dependent Eσ 70 -mediated transcription. We suggest that both Eσ 70 and Eσ S likely get recruited by CRP at the dgo promoter, but due to the unsuitable interacting interface between CRP and σ S , the recruitment of Eσ S at the promoter remains unproductive and offers competition to Eσ 70 . In a genome-wide study to map σ factor- transcription units in E. coli, an extensive overlap was reported between Eσ S and Eσ 70 binding sites in the genome (Cho et al., BMC Biology 2014). Further, of ~1100 genes with σ S -specific promoters, ~200 genes were up-regulated in a strain lacking σ S , the majority of which were bound more strongly by σ 70 . Thus, there are certainly more instances of direct repression of σ 70 -driven promoters by σ S that call for further investigation of this mode of negative regulation by the stationary phase σ factor.