The RcsB-dependent Upregulation of rprA Contributes to the Intrinsic Antibiotic Resistance of Escherichia coli Exposed to Antibiotics Targeting Cell Wall Synthesis but not Protein Synthesis

SUMMARY The regulation of capsular synthesis (Rcs) phosphorelay system allows Escherichia coli to respond to stress resulting from disruption of the peptidoglycan layer. The cytosolic response regulator, RcsB, regulates the expression of various downstream genes including rprA. rprA is a small non-coding RNA that stabilizes the translation of RpoS, a regulator of general stress response in E. coli. Previous studies have shown that an E. coli K-12 strain bearing a deletion of rcsB displays enhanced susceptibility to β-lactams, which target peptidoglycan synthesis. This result correlated with a decrease in rprA expression. It is not known whether induction of rprA expression is limited to β-lactams. The objective of this study was to determine rprA expression levels using β-galactosidase assays in both wild-type and ΔrcsB, following treatment with sub-inhibitory concentrations of antibiotics targeting either cell wall or protein synthesis. We hypothesized that following treatment with antibiotics targeting cell wall synthesis, rprA expression would be lower in a strain bearing a deletion of rcsB in comparison to the wild-type strain. Furthermore, within a given strain, we expected rprA expression to be upregulated in response to cell wall synthesis stress and unchanged in response to protein synthesis stress in comparison to unstressed (no antibiotic) conditions. The antibiotics penicillin and phosphomycin target cell wall synthesis and would induce the Rcs phosphorelay system while tetracycline and streptomycin target protein synthesis and would not activate Rcs via peptidoglycan disruption. We observed that in the absence of rcsB, the minimum inhibitory concentration decreased two-fold with respect to treatment with penicillin and phosphomycin, but remained unchanged when treated with tetracycline and streptomycin. rprA expression was upregulated in wild-type cells in the presence of antibiotics targeting cell wall synthesis or protein synthesis and was suppressed in the absence of rcsB when treated with either type of antibiotic. This study provides insight into the intrinsic Rcs-mediated stress response mechanisms used by E. coli through the expression of RcsB, which appears to regulate rprA expression independently of the mechanistic class of antibiotic stressor used.