ramR mutations involved in efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium

Abstract

In the sequenced genome of Salmonella enterica serovar Typhimurium strain LT2, an open reading frame (STM0580) coding for a putative regulatory protein of the TetR family is found upstream of the ramA gene. Overexpression of ramA results in increased expression of the AcrAB efflux pump and, consequently, multidrug resistance (MDR) in several bacterial species. The inactivation of the putative regulatory protein gene upstream of ramA in a susceptible serovar Typhimurium strain resulted in an MDR phenotype with fourfold increases in the MICs of unrelated antibiotics, such as quinolones/fluoroquinolones, phenicols, and tetracycline. The inactivation of this gene also resulted in a fourfold increase in the expression of ramA and a fourfold increase in the expression of the AcrAB efflux pump. These results indicated that the gene encodes a local repressor of ramA and was thus named ramR. In contrast, the inactivation of marR, marA, soxR, and soxS did not affect the susceptibilities of the strain. In quinolone- or fluoroquinolone-resistant strains of serovar Typhimurium overexpressing AcrAB, several point mutations which resulted in amino acid changes or an in-frame shift were identified in ramR; in addition, mutations interrupting ramR with an IS1 element were identified in high-level fluoroquinolone-resistant serovar Typhimurium DT204 strains. One serovar Typhimurium DT104 isolate had a 2-nucleotide deletion in the putative RamR binding site found upstream of ramA. These mutations were confirmed to play a role in the MDR phenotype by complementing the isolates with an intact ramR gene or by inactivating their respective ramA gene. No mutations in the mar or sox region were found in the strains studied. In conclusion, mutations in ramR appear to play a major role in the upregulation of RamA and AcrAB and, consequently, in the efflux-mediated MDR phenotype of serovar Typhimurium.

FIG. 1.

Sequence analysis of the ramR-ramA region in serovar Typhimurium strains. (A) Features of the 288-bp-long ramA-ramR intergenic region in serovar Typhimurium strain LT2. The predicted −10 and −35 promoter regions are underlined. The inverted repeat sequences are bold and indicated by arrows. The putative ribosome-binding site (RBS) is indicated with a dotted line. (B) Sequence alignment of the putative promoter region of serovar Typhimurium strains LT2 and BN10055 showing two nucleotide deletions in the putative RamR binding site of the latter strain. (C) Mutations found in the ramR gene in serovar Typhimurium strains BN18/21, BN18/41, BN18/71, BN9945, and 543SA98. nt, nucleotide. (D) Interruption by an IS1 element of the ramR gene in serovar Typhimurium DT204 strains 102SA00 and 902SA92.

FIG. 2.

RT-PCR analysis of ramA and gyrB (control) expression in serovar Typhimurium wild-type strain S/921495 (lane 1), deletion mutant strains S/921495 ΔramR::kan (lane 2) and S/92/1495 ΔramR (lane 3), and the complemented strain S/921495 ΔramR(pRamR) (lane 4).