Mutations of bacterial RNA polymerase leading to resistance to microcin j25.
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.
Yuzenkova
Julia
J
Department of Genetics, Waksman Institute, Piscataway, New Jersey 08854, USA.
Delgado
Monica
M
Nechaev
Sergei
S
Savalia
Dhruti
D
Epshtein
Vitaly
V
Artsimovitch
Irina
I
Mooney
Rachel A
RA
Landick
Robert
R
Farias
Ricardo N
RN
Salomon
Raul
R
Severinov
Konstantin
K
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects* / Q000201:enzymology; D001430:Bacteriocins / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides; D010957:Plasmids; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic / Q000187:drug effects
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; C020411:microcin; D012321:DNA-Directed RNA Polymerases