Mutant RNA polymerase can reduce susceptibility to antibiotics via ppGpp-independent induction of a stringent-like response

BACKGROUND: Mutations in RNA polymerase (RNAP) can reduce susceptibility to ciprofloxacin in Escherichia coli, but the mechanism of transcriptional reprogramming responsible is unknown. Strains carrying ciprofloxacin-resistant (Cip(R)) rpoB mutations have reduced growth fitness and their impact on clinical resistance development is unclear. OBJECTIVES: To assess the potential for Cip(R)rpoB mutations to contribute to resistance development by estimating the number of distinct alleles. To identify fitness-compensatory mutations that ameliorate the fitness costs of Cip(R)rpoB mutations. To understand how Cip(R)rpoB mutations reprogramme RNAP. METHODS: E. coli strains carrying five different Cip(R)rpoB alleles were evolved with selection for improved fitness and characterized for acquired mutations, relative fitness and MIC(Cip). The effects of dksA mutations and a ppGpp(0) background on growth and susceptibility phenotypes associated with Cip(R)rpoB alleles were determined. RESULTS: The number of distinct Cip(R)rpoB mutations was estimated to be >100. Mutations in RNAP genes and in dksA can compensate for the fitness cost of Cip(R)rpoB mutations. Deletion of dksA reduced the MIC(Cip) for strains carrying Cip(R)rpoB alleles. A ppGpp(0) phenotype had no effect on drug susceptibility. CONCLUSIONS: Cip(R)rpoB mutations induce an ppGpp-independent stringent-like response. Approximately half of the reduction in ciprofloxacin susceptibility is caused by an increased affinity of RNAP to DksA while the other half is independent of DksA. Stringent-like response activating mutations might be the most diverse class of mutations reducing susceptibility to antibiotics.