Selective Alleviation of Mitomycin C Sensitivity in lexA3 Strains of Escherichia coli Demands Allele Specificity of rif-nal Mutations: A Pivotal Role for rpoB87-gyrA87 Mutations

Abstract

Very recently, we have reported about an unconventional mode of elicitation of Mitomycin C (MMC) specific resistance in lexA3 (SOS repair deficient) mutants due to a combination of Rif-Nal mutations (rpoB87-gyrA87). We have clearly shown that UvrB is mandatory for this unconventional MMC resistance in rpoB87-gyrA87-lexA3 strains and uvrB is expressed more even without DNA damage induction from its LexA dependent promoter despite the uncleavable LexA3 repressor. The rpoB87 allele is same as the rpoB3595 which is known to give rise to a fast moving RNA Polymerase and gyrA87 is a hitherto unreported NalR allele. Thus, it is proposed that the RNA Polymerase with higher elongation rate with the mutant DNA Gyrase is able to overcome the repressional hurdle posed by LexA3 to express uvrB. In this study we have systematically analysed the effect of three other rpoB (rif) mutations-two known to give rise to fast moving RNAP (rpoB2 and rpoB111) and one to a slow moving RNAP (rpoB8) and four different alleles of gyrA NalR mutations (gyrA199, gyrA247, gyrA250, gyrA259) isolated spontaneously, on elicitation of MMC resistance in lexA3 strains. Our results indicate that in order to acquire resistance to 0.5 µg/ml MMC cells require both rpoB87 and gyrA87 but resistance to 0.25 µg/ml of MMC can be brought about by either rpoB87, gyrA87, fast moving rpoB mutations or other nal mutations also. We have also depicted increased constitutive uvrB expression in strains carrying fast moving RNAP (rpoB2 and rpoB111) with gyrA87 and another nal mutation with rpoB87 and expression level in these strains is lesser than rpoB87-gyrA87 strain. These results evidently suggest an allele specific role for the rif-nal mutations to acquire MMC resistance in lexA3 strains via increased constitutive uvrB expression and a pivotal role for rpoB87-gyrA87 combination to elicit higher levels of resistance.

The SOS and RpoS Regulons Contribute to Bacterial Cell Robustness to Genotoxic Stress by Synergistically Regulating DNA Polymerase Pol II

“…Interstrand cross-links, which are one type of DNA damage caused by MMC, are in E. coli repaired by proteins involved in the HR, NER, and TLS pathways ( Van Houten et al 1986 ; Sladek et al 1989 ; Slater and Maurer 1993 ; Berardini et al 1999 ), all of which are upregulated as part of the SOS regulon ( Radman 1975 ; Little and Mount 1982 ; Walker et al 1982 ; Friedberg et al 2005). Indeed, we observed that the impossibility of inducing the SOS response in the lexA (Ind − ) genetic background resulted in a very high sensitivity of the cells to MMC (data not shown), as previously reported ( Shanmughapriya et al 2014 ).…”

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

“…This phenotype was originally called as SOS Independent Repair (SIR) and now called as SOS Interdependent Repair. 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 ) .…”