Identification and analysis of Rob, a transcriptional regulator from Serratia marcescens
Serratia marcescens, a member of Enterobacteriaceae family, is a causative agent of nosocomial and opportunistic infections. Numerous reports show that the multidrug resistance among S. marcescens is growing. This organism has high-level intrinsic resistance to a variety of antimicrobial agents, which makes the treatment of infections caused by this bacterium very difficult. The major mechanism for antibiotic resistance, especially to fluoroquinolones, in Gram-negative organisms is the active efflux of the antibiotic molecule mediated by efflux pumps belonging to the Resistance-Nodulation-Cell Division (RND) family. It was previously shown that the SdeAB and SdeXY multidrug efflux pumps are important for conferring the intrinsic drug resistance in S.marcescens. In Escherichia coli, the up-regulation of transcriptional activators, such as MarA, SoxS and Rob, affect transcription of acrAB, tolC and micF. Over-expression of Rob results in increased expression of the E. coli AcrAB-TolC efflux pump and decreases outer membrane permeability through up-regulation of micF, resulting in multidrug, organic solvent and heavy metal resistance. In the present study, we report the identification of a rob gene in S. marcescens which has a 70% identity at the DNA level and 71% identity at the amino acid level to that of E. coli. Moreover, the S. marcescens rob demonstrated similar properties to the E. coli rob including having an effect on expression of outer membrane protein F (OmpF) and over-expression of SdeAB and SdeXY, conferring antibiotic resistance to divergent antibacterial agents and tolerance to organic solvents. We performed rob promoter evaluations using transcriptional fusions to the Green Fluorescence Protein (GFP) in the vector pGlow-TOPO and constructed a rob knock-out using the TargeTron Gene Knockout System. Promoter activity assessment, using the pGlow-TOPO reporter plasmid, showed that rob had higher promoter activity at 37°C than 30°C. In the presence of 2,2’-dipyridyl, rob promoter activity was observed to be slightly increased in the early and mid-log phase by 1.4 and 1.1 fold, respectively. We also showed that sodium decanoate and sodium salicylate can reduce the transcription of rob at 30°C and 37°C. This reduction was observed more potently when rob was exposed to sodium decanoate at 30°C. Minimum inhibitory concentration (MIC) for various antibiotics of the S. marcescens rob knock-out demonstrated a decrease in susceptibility to nalidixic acid, tetracycline, chloramphenicol, ciprofloxacin, norfloxacin, and ofloxacin. Over-expression of rob resulted in an increased resistance by 4, 2, and 2-fold to nalidixic acid, tetracycline and chloramphenicol, respectively. In addition, rob over-production displayed 8, 4, and 4-fold increase in resistance to ciprofloxacin, norfloxacin, ofloxacin, respectively. To discover the role of rob in the efflux mechanism, we performed ethidium bromide accumulation assays on over-expressing and knock-out strains. Organic solvent tolerance assays were carried out using n-hexane to determine if rob is involved in expression of efflux pumps. We found the rob null mutant to be sensitive to n-hexane while the over-expression of rob resulted in resistance to n-hexane. RT-PCR of the rob knock-out strain showed a decrease in expression of micF, ompC, sdeXY, sdeAB and tolC, respectively, and an increase in the expression of ompF. To conclude, we identified a rob homolog in S. marcescens which contributes to resistance to multiple antibiotics and tolerance to organic solvent.
S. marcescens, multi-drug resistance, efflux pumps, porins, transcriptional regulator