Genetic inactivation of acrAB or inhibition of efflux induces expression of ramA

Abstract

ObjectivesThe transcriptional activator RamA regulates production of the multidrug resistance efflux AcrAB–TolC system in several Enterobacteriaceae. This study investigated factors that lead to increased expression of ramA.MethodsIn order to monitor changes in ramA expression, the promoter region of ramA was fused to a gfp gene encoding an unstable green fluorescence protein (GFP) on the reporter plasmid, pMW82. The ramA reporter plasmid was transformed into Salmonella Typhimurium SL1344 and a ΔacrB mutant. The response of the reporter to subinhibitory concentrations of antibiotics, dyes, biocides, psychotropic agents and efflux inhibitors was measured during growth over a 5 h time period.ResultsOur data revealed that the expression of ramA was increased in a ΔacrB mutant and also in the presence of the efflux inhibitors phenylalanine-arginine-β-naphthylamide, carbonyl cyanide m-chlorophenylhydrazone and 1-(1-naphthylmethyl)-piperazine. The phenothiazines chlorpromazine and thioridazine also increased ramA expression, triggering the greatest increase in GFP expression. However, inducers of Escherichia coli marA and soxS and 12 of 17 tested antibiotic substrates of AcrAB–TolC did not induce ramA expression.ConclusionsThis study shows that expression of ramA is not induced by most substrates of the AcrAB–TolC efflux system, but is increased by mutational inactivation of acrB or when efflux is inhibited.

MarA, SoxS and Rob of Escherichia coli – Global Regulators of Multidrug Resistance, Virulence and Stress Response

“…However, if AcrAB is inhibited or disrupted and the strain subjected to these antibiotics, RamA expression increases. It is suggested that internal metabolites or stress signals resulting from the absence of the pump or a decrease in efflux, rather than the antibiotics themselves, causes the increased ramA transcription [196]. This effect is similar to the mechanism proposed for MarA, SoxS and Rob activation in E. coli in a tolC strain (see section 5.2.1).…”

Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella enterica Serovar Typhimurium

“…We have previously reported that ramA and homologous RND genes acrD and acrF were significantly upregulated in an Δ acrB mutant (53–55). However, we did not observe upregulation of these genes in the D408A mutant.…”

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

“…ramRA are transcribed divergently, with RamR repressing ramA expression (775)(776)(777)(778)(779). Induced by a variety of environmental signals, including bile salts, indole, and phenothiazines, RamA serves as a small activator protein to contribute to increased AcrAB production (776,(780)(781)(782). Bile binds to RamR (778) and in this way is thought to increase the expression of AcrAB.…”

“…Indole induces AcrAB production by also increasing RamA expression (780). RamA expression can be also increased by the inactivation of AcrAB and is regulated by the ATP-dependent Lon protease (782,783). (The ramA sequence was present, but escaped notice, within the E. cloacae genomic fragment that was found to downregulate E. coli OmpF in 1990 [784] and was correctly identified in K. pneumoniae later [785].)…”