Mutations of Bacterial RNA Polymerase Leading to Resistance to Microcin J25

Julia Yuzenkova,Monica Delgado,Sergei Nechaev,Dhruti Savalia,Vitaly Epshtein,Irina Artsimovitch,Rachel A Mooney,Robert Landick,Ricardo N Farias,Raul Salomon,Konstantin Severinov

doi:10.1074/jbc.m209425200

Abstract

A mutation in the conserved segment of the rpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, beta', was found to make Escherichia coli cells resistant to microcin J25 (MccJ25), a bactericidal 21-amino acid peptide active against Gram-negative bacteria (Delgado, M. A., Rintoul, M. R., Farias, R. N., and Salomon, R. A. (2001) J. Bacteriol. 183, 4543-4550). Here, we report that mutant RNAP prepared from MccJ25-resistant cells, but not the wild-type RNAP, is resistant to MccJ25 in vitro, thus establishing that RNAP is a true cellular target of MccJ25. We also report the isolation of additional rpoC mutations that lead to MccJ25 resistance in vivo and in vitro. The new mutations affect beta' amino acids in evolutionarily conserved segments G, G', and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center. We also report that previously known rpoB (RNAP beta subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25. We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center.

Highlights

Bacterial RNA polymerase (RNAP)1 is the central enzyme of gene expression and a target of genetic regulation
A mutation in the conserved segment of the rpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, ␤؅, was found to make Escherichia coli cells resistant to microcin J25 (MccJ25), a bactericidal 21amino acid peptide active against Gram-negative bacteria
RNA Polymerase from Microcin-resistant E. coli Cells Is Resistant to MccJ25 in Vitro—Earlier, one of our groups [18] reported that E. coli cells harboring the sjmA1 mutation, but not the wild-type E. coli, were able to grow on selective medium containing MccJ25

Summary

Introduction

Bacterial RNA polymerase (RNAP)1 is the central enzyme of gene expression and a target of genetic regulation. A mutation in the conserved segment of the rpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, ␤؅, was found to make Escherichia coli cells resistant to microcin J25 (MccJ25), a bactericidal 21amino acid peptide active against Gram-negative bacteria

Results

Conclusion