Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.
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.
Analyses of mutations in rpoB subunit of Escherichia coli that lead to resistance to rifampicin have been invaluable in providing insight into events during transcription continue to be discovered. Earlier we reported that rpoB12 suppresses over-expression of cps genes in Δlon mutant of E. coli, by interfering with the transcription of rcsA. Here we report Microarray based Transcriptome profile of Δlon and Δlon rpoB12 strains. The data analyses clearly reveal that rpoB12 mutation results in the differential expression of ∼450 genes. The transcription profiles of some of the genes namely, rcsA, gadE, csgD, bolA, ypdI, dnaJ, clpP, csrA and hdeA are significantly altered, particularly the genes implicated in virulence. Some of the phenotypic traits namely, biofilm formation, motility, curli synthesis and ability to withstand acidic stress in a lonrpoB12 strain were assessed. The results clearly indicate that rpoB12 up-regulates biofilm formation and curli synthesis while it makes the cells sensitive for growth in acidic medium and inhibits motility almost completely. Furthermore, rpoB12 modulates the expression profile of a significant number of genes involved in stress responses, genes encoding small RNAs. Thus, this study reveals the versatile role of the rpoB12 mutation, especially its impact on the regulation of genes related to virulence and highlights its medical importance.
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