Plasmid-borne transcriptional regulator RamAp modulates Salmonella genes for environmental and host adaptation

RamAp is a plasmid-borne homolog of the AraC family regulator RamA, commonly found on multidrug resistance (MDR) plasmids in Salmonella. Its expression is enhanced by a truncated ISEcp1 element, but the functional consequences of ramAp acquisition remain poorly characterized. In this study, we investigated whether ramAp modulates chromosomal gene expression in Salmonella enterica serovar Typhimurium LT2 and how its expression reshapes bacterial physiology and virulence-associated traits. We first assessed promoter activity using green fluorescent protein (GFP) reporter constructs. The truncated ISEcp1 element upstream of ramAp significantly increased the expression of downstream genes, supporting its role as a transcriptional enhancer. To identify the direct regulatory targets of RamAp, we focused on genes involved in membrane remodeling (micF), oxidative stress response (sodA), and motility (flhDC), with the efflux pump gene acrAB as a known positive control. Electrophoretic mobility shift assays (EMSAs) confirmed that RamAp directly binds to the upstream regulatory regions of all four genes. Functional assays revealed that ramAp expression increased SOD enzymatic activity, reduced swimming motility, and enhanced virulence-associated phenotypes in the Galleria mellonella infection model. Together, these findings demonstrate that ramAp reprograms host gene expression by directly engaging conserved regulatory targets, resulting in the activation of stress adaptation pathways and the repression of motility. Despite its plasmid-borne origin, RamAp functionally overlaps with chromosomal RamA while operating outside the native regulatory constraints, thereby integrating antibiotic resistance, stress resilience, and virulence-associated traits. These results highlight the adaptive potential of horizontally acquired transcriptional regulators under antibiotic pressure and host-associated stress conditions.