Suppression of capsule expression in Δlon strains of Escherichia coli by two novel rpoB mutations in concert with HNS: possible role for DNA bending at rcsA promoter

Abstract

Analyses of mutations in genes coding for subunits of RNA polymerase always throw more light on the intricate events that regulate the expression of gene(s). Lon protease of Escherichia coli is implicated in the turnover of RcsA (positive regulator of genes involved in capsular polysaccharide synthesis) and SulA (cell division inhibitor induced upon DNA damage). Failure to degrade RcsA and SulA makes lon mutant cells to overproduce capsular polysaccharides and to become sensitive to DNA damaging agents. Earlier reports on suppressors for these characteristic lon phenotypes related the role of cochaperon DnaJ and tmRNA. Here, we report the isolation and characterization of two novel mutations in rpoB gene capable of modulating the expression of cps genes in Δlon strains of E. coli in concert with HNS. clpA, clpB, clpY, and clpQ mutations do not affect this capsule expression suppressor (Ces) phenotype. These mutant RNA polymerases affect rcsA transcription, but per se are not defective either at rcsA or at cps promoters. The results combined with bioinformatics analyses indicate that the weaker interaction between the enzyme and DNA::RNA hybrid during transcription might play a vital role in the lower level expression of rcsA. These results might have relevance to pathogenesis in related bacteria.

A putative curved DNA region upstream of rcsA in Escherichia coli plays a key role in transcriptional regulation by H‐NS

“…Detailed analyses showed that the elicitation of this Ces phenotype by these rif mutations primarily stems from the downregulation of rcsA transcription. Our study also revealed the requirement of functional H-NS in the elicitation of Ces phenotype [32]. oligomerization [8].…”

“…They have suggested that the upstream region of rcsA promoter might possess bending/curved DNA region [25]. In our earlier study, we have provided evidence for the occurrence of bendable DNA region upstream of rcsA promoter through bioinformatics analyses [32]. In this study, perhaps for the first time we have given the genetic evidence that supports the presence of putative curved DNA region upstream of rcsA promoter.…”

“…Genetic and molecular analyses clearly showed that downregulation of rcsA transcription is the primary reason for the elicitation of this capsule expression suppression (Ces) phenotype by these two rif alleles. Furthermore, our study clearly indicated that the presence of functional H‐NS is mandatory for both the rpoB mutations to function as capsule expression suppressors in the ∆ lon strain of E. coli . Sledjesky and Gottesman have shown that H‐NS functions as a repressor for rcsA transcription, and their study also revealed the involvement of a small RNA, namely DsrA located downstream of rcsA , in the regulation of rcsA by H‐NS.…”

“…In our earlier study pertaining to the isolation and characterization of novel rpoB mutations capable of suppressing the overproduction of colanic acid Cps in lon mutant of E. coli, we have substantiated the role of functional H-NS in the elicitation of Ces phenotype by the two rpoB mutations, namely rpoB12 (C 1576 to T 1576 ; His526 to Tyr526) and rpoB77 (C 1535 to T 1535 ; Ser512 to Tyr512) [32]. As a continuation to this aspect, the effect of dominant-negative alleles of hns in Ces strains (Δlon rpoB12 and Δlon rpoB77) and in parental strains (Δlon and lon + ) was studied (strains bearing the dominant-negative hns alleles were procured from Gowrishankar, CDFD, India).…”

Glu571 of PheT plays a pivotal role in the thermal stability of Escherichia coli PheRS enzyme

“…Subsequently, allele specificity of rif‐nal mutations in the elicitation of this unorthodox phenotype was also reported . Most recently we have shown that one of the characteristic phenotype of Δlon mutant viz capsule over‐expression can be suppressed by two novel rif alleles ( rpoB12 and rpoB77 ) . These two rif alleles selectively down regulate the expression of rcsA coding for the positive regulator of cps genes and thus elicit capsule expression suppression (Ces) phenotype .…”