Resistance to microcin J25 by target modification. Is there a second-step mutation?

LACHENICHT, J. A.; ARIAS, V. J.; VALLEJOS A. C.; SALOMÓN, R. A.

Mar del Plata

Congreso; 51 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2015

SAIB

Bacterial RNA polymerase is the target of the peptide antibiotic microcin J25 (MccJ25). Long ago, we isolated a strain (SBG231) with a point mutation in the rpoC gene of RNA polymerase (RNAP), which confers high-level resistance to the antibiotic. The mutated residue is located at the entry of the RNAP secondary channel. A plasmid, called pDJJ12, carrying the rpoB and rpoC genes of RNAP, complements the mutation and turns SBG231 cells again sensitive to MccJ25. A vector with rpoB only has no effect. To our surprise, in the present study we found that a plasmid bearing the rpoC gene alone does not complement the mutation. We also determined that RNAP mutants emerge much less frequently than non-target mutants. An attractive hypothesis to explain these observations is that there is a need for two mutations in RNAP for MccJ25 resistance development. Perhaps the second hit is in the rpoB gene. A closer look at the pDJJ12 plasmid used in complementation assays showed that in addition to the RNAP genes it includes a short ORF of unknown function called yjaZ. This could be a likely candidate for the second hit. However, against this possibility there is the fact that a deletion of the entire ORF did not affect the sensitivity to MccJ25. We are currently searching for the putative second mutation by using transduction analysis and nucleotide sequencing of the rpoB gene in SBG231.