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.

The crystal structure of multidrug-resistance regulator RamR with multiple drugs

“…In this study, we identified five substrates of the RamR protein, including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G. Similar approaches in crystallizing the TetR family regulators with multiple drugs have been also reported in QacR 20 , TtgR 21 ARTICLE six amino acid substitutions in RamR that are related to bacterial drug resistance, including T18P, R46N, R46P, Y59H, M84I and E160D 15,17,23 . As shown in Supplementary Fig.…”

“…Its binding site covers essential features of the ramA promoter, including the À 10 conserved region, the transcriptional start site of ramA and two 7-bp inverted repeats 16 . Various types of mutations in ramR and the ramR-ramA intergenic region were identified in multidrug-resistant strains of S. typhimurium, other S. enterica serovars and K. pneumoniae, which result in increased expression of ramA and an increase in efflux-mediated multidrug resistance 15,17,18 .…”

“…Abouzeed et al 15 identified the ramR gene, which is located directly upstream of ramA and encodes a protein of the TetR family of transcriptional repressors (Fig. 1a).…”

“…1a). Its role as a local repressor of ramA was determined by inactivation and complementation experiments 15 . RamR binds to the region upstream of ramA.…”

Multiple Regulatory Pathways Associated with High-Level Ciprofloxacin and Multidrug Resistance in Salmonella enterica Serovar Enteritidis: Involvement of ramA and Other Global Regulators

“…Furthermore, little is known about rob and its contribution to antibiotic resistance in Salmonella. More recently, RamA, which displays close homology to MarA and is absent from E. coli, has been implicated in MDR in Salmonella and other bacteria (1,16,19,42). Overexpression of ramA has been associated with increased expression of acrB in Salmonella and other Enterobacteriaceae (1,19,42).…”