Characterization of the Effects of an rpoC Mutation That Confers Resistance to the Fst Peptide Toxin-Antitoxin System Toxin

Abstract

cOverexpression of the Fst toxin in Enterococcus faecalis strain OG1X leads to defects in chromosome segregation, cell division and, eventually, membrane integrity. The M7 mutant derivative of OG1X is resistant to most of these effects but shows a slight growth defect in the absence of Fst. Full-genome sequencing revealed two differences between M7 and its OG1X parent. First, OG1X contains a frameshift mutation that inactivates the etaR response regulator gene, while M7 is a wild-type revertant for etaR. Second, the M7 mutant contains a missense mutation in the rpoC gene, which encodes the ␤= subunit of RNA polymerase. Mutagenesis experiments revealed that the rpoC mutation was primarily responsible for the resistance phenotype. Microarray analysis revealed that a number of transporters were induced in OG1X when Fst was overexpressed. These transporters were not induced in M7 in response to Fst, and further experiments indicated that this had a direct protective effect on the mutant cells. Therefore, exposure of cells to Fst appears to have a cascading effect, first causing membrane stress and then potentiation of these effects by overexpression of certain transporters.

Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range

“…OG1RF Δpar EF0409 , with a deletion of the chromosomal copy of the par EF0409 locus, was constructed utilizing the protocol of Kristich et al (5), modified as in the work of Brinkman et al (44). Primers used to construct deletions of alleles and verify deletions are shown in Table 1.…”

“…Genomic DNA was purified from OG1RF as described previously (44). Plasmid DNA was purified from E. coli strains utilizing the Quantum Prep plasmid miniprep kit (Bio-Rad, Hercules, CA) or using the Qiagen midiprep kit (Qiagen, Valencia, CA).…”

“…DNA was introduced into competent E. coli DH5␣ cells according to the manufacturer's instructions (Invitrogen). PCR was performed using either PCR-HiFi Supermix or Platinum PCR kits from Invitrogen as previously described (44). PCR products were prepared for cloning or sequencing using Wizard Plus SV miniprep columns (Promega) according to the manufacturer's instructions.…”

“…DNA sequencing was performed at the DNA sequencing facility at Iowa State University (Ames, IA). Electroporation of E. faecalis cells was performed using glycine-treated electrocompetent cells prepared and electroporated as previously described (44,45) or using lysozyme-treated cells as previously described (46). DNA was introduced into competent E. coli DH5␣ cells according to the manufacturer's instructions (Invitrogen).…”

Mutations in the Primary Sigma Factor σ A and Termination Factor Rho That Reduce Susceptibility to Cell Wall Antibiotics

“…Mutations affecting bacterial RNA polymerase (RNAP) arise during many selections (2,(19)(20)(21)(22)(23), suggesting that alterations to RNAP can facilitate adaptation. Bacterial RNAP is an essential, highly conserved, multisubunit enzyme that is responsible for transcription (24).…”