THE SALMONELLA-TYPHIMURIUM MAR LOCUS - MOLECULAR AND GENETIC ANALYSESAND ASSESSMENT OF ITS ROLE IN VIRULENCE

The marRAB operon is a regulatory locus that controls multiple drug re sistance in Escherichia coli. marA encodes a positive regulator of the antibiotic resistance response, acting by altering the expression of unlinked genes. marR encodes a repressor of marRAB transcription and c ontrols the production of MarA in response to environmental signals. A molecular and genetic study of the homologous operon in Salmonella ty phimurium was undertaken, and the role of marA in virulence in a murin e model was assessed. Expression of E. coli marA (marA(Ec)) present on a multicopy plasmid in S. typhimurium resulted in a multiple antibiot ic resistance (Mar) phenotype, suggesting that a similar regulon exist s in this organism. A genomic plasmid library containing S. typhimuriu m chromosomal sequences was introduced into an E. coli strain that was deleted for the mar locus and contained a single-copy marR'-'lacZ tra nslational fusion. Plasmid clones that contained both S. typhimurium m arR (marR(St)) and marA (marA(St)) genes were identified as those that were capable of repressing expression of the fusion and which resulte d in a Mar phenotype. The predicted amino acid sequences of MarR(St), MarA(St), and MarB(St), were 91, 86, and 42% identical, respectively, to the same genes from E. coli, while the operator/promoter region of the operon was 86% identical to the same 98-nucleotide-upstream region in E. coli. The marRAB transcriptional start sites for both organisms were determined by primer extension, and a marRAB(St) transcript of s imilar to 1.1 kb was identified by Northern blot analysis. Its accumul ation was shown to be inducible by sodium salicylate. Open reading fra mes flanking the marRAB operon were also conserved. An S. typhimurium marA disruption strain was constructed by an allelic exchange method a nd compared to the wild-type strain for virulence in a murine BALB/c i nfection model. No effect on virulence was noted. The endogenous S. ty phimurium plasmid that is associated with virulence played no role in marA-mediated multiple antibiotic resistance. Taken together, the data show that the S. typhimurium mar locus is structurally and functional ly similar to marRAB(Ec) and that a lesion in marA(Ec) has no effect o n S. typhimurium virulence for BALB/c mice.