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

Vinod Shanmughapriya,M Hussain Munavar,Shanmugaraja Meenakshi,Farook Thameem

doi:10.1371/journal.pone.0087702

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.

Highlights

Mitomycin C (MMC) is a DNA intercalating agent that gives rise to DNA interstrand crosslinks
The DNA crosslinking by MMC leads to the induction of the SOS response in Escherichia coli by activating the RecA protein to its activated form called as RecA* and subsequent cleavage of the LexA repressor [2,3]
The process of transcription has been linked to DNA repair mechanisms for a long time

Summary

Introduction

Mitomycin C (MMC) is a DNA intercalating agent that gives rise to DNA interstrand crosslinks. A mutant form of the repressor due to the mutation lexA3, renders the repressor nonreactive to the activated RecA* and fails to cleave itself This mutation makes E. coli strains sensitive to all DNA damaging agents including MMC as the expression of all the SOS induced genes are turned off in lexA3 strains [5]. An alternate path for MMC repair in SOS deficient strains (lexA3 or recA) was identified by Kumaresan and Jayaraman in 1988 and was termed as ‘‘SOS Independent Repair’’ or SIR This SIR effect increased the MMC resistance of a lexA3 strain by two additional spontaneously acquired mutations – A Rif resistant mutation, named rpoB87, mapped to the rpoB gene that codes for the b subunit of RNA Polymerase and a Nal resistant mutation, named gyrA87, mapped to gyrA gene that codes for the DNA Gyrase A subunit [6]. RNAP with RpoB3595 b subunit was shown to have increased termination

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Conclusion