RamA, a Member of the AraC/XylS Family, Influences Both Virulence and Efflux inSalmonella entericaSerovar Typhimurium

Abstract

The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 lacking a functional ramA or ramR or with plasmid-mediated high-level overexpression of ramA were compared to those of the wild-type parental strain. Inactivation of ramA led to increased expression of 14 SPI-1 genes and decreased expression of three SPI-2 genes, and it altered expression of ribosomal biosynthetic genes and several amino acid biosynthetic pathways. Furthermore, disruption of ramA led to decreased survival within RAW 264.7 mouse macrophages and attenuation within the BALB/c ByJ mouse model. Highly overexpressed ramA led to increased expression of genes encoding multidrug resistance (MDR) efflux pumps, including acrAB, acrEF, and tolC. Decreased expression of 34 Salmonella pathogenicity island (SPI) 1 and 2 genes, decreased SipC production, decreased adhesion to and survival within macrophages, and decreased colonization of Caenorhabditis elegans were also seen. Disruption of ramR led to the increased expression of ramA, acrAB, and tolC, but not to the same level as when ramA was overexpressed on a plasmid. Inactivation of ramR had a more limited effect on pathogenicity gene expression. In silico analysis of a suggested RamA-binding consensus sequence identified target genes, including ramR, acrA, tolC, sipABC, and ssrA. This study demonstrates that the regulation of a mechanism of MDR and expression of virulence genes show considerable overlap, and we postulate that such a mechanism is dependent on transcriptional activator concentration and promoter sensitivity. However, we have no evidence to support the hypothesis that increased MDR via RamA regulation of AcrAB-TolC gives rise to a hypervirulent strain.Multidrug efflux pumps, such as the resistance-nodulationdivision (RND) superfamily member AcrAB-TolC in the Enterobacteriaceae, export many substrates, including antibiotics (9,27,29,49,51,64). AcrAB-TolC and homologous efflux systems are important in the pathogenicity of Salmonella enterica serovar Typhimurium (10, 14) and other bacterial species (11,15,16). Recent work by our laboratory in S. Typhimurium has shown that following the disruption of acrA, acrB, or tolC, a phenotype of decreased pathogenicity was associated with global changes in expression of genes involved in virulence (such as those in SPI-1), chemotaxis, and motility (70).Regulation of RND efflux pumps has been widely studied in Escherichia coli, with the global AraC/XylS family regulators marA, soxS, and rob having been shown to activate expression of efflux pumps (5,8,46,52,71). Mechanistically, this is achieved via the binding, and subsequent activation, of discrete but degenerate nucleotide sequences within regulon genes, known as mar-, rob-, or soxboxes (30,35,42,43). E. coli marA is part of the marRAB operon, linked to another operon, marCD, of unknown function. Expression of both is controlled from the same operator site, marO (20). Regulation of E. coli marA is provided through the local repressor marR (62), which controls the transcription of marA b...

Regulation of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium in response to indole and paraquat

“…RamA affects not only efflux pump gene expression of Salmonella but also the virulence genes, and there are some binding sequences of RamA in the upstream region of both SPI-1 and -2 genes (Bailey et al, 2010). Furthermore, RamA and SoxS are global regulators that affect the expression levels of various genes.…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…Another gene locus of ramRA that is widespread in Enterobacteriaceae except E. coli also significantly influences the expression of not only AcrAB but also AcrEF and MdtABC in Salmonella (16,229,773,775,776). ramRA are transcribed divergently, with RamR repressing ramA expression (775)(776)(777)(778)(779).…”

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

“…Despite the fact that these activators are able to induce the expression of acrAB under stress conditions in E. coli, they appear to be less important in regulation of multidrug resistance in S. enterica (39). Interestingly, in S. enterica, RamA (resistance antibiotic multiple), another member of the AraC/XylS family, was found to be involved in conferring multidrug resistance by directly increasing the expression of acrAB (39). Furthermore, it has also been demonstrated that mutational inactivation of the AcrAB-TolC pump leads to increased expression of ramA in S. enterica (40).…”