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

Abstract

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 (CipR) rpoB mutations have reduced growth fitness and their impact on clinical resistance development is unclear. Objectives To assess the potential for CipRrpoB mutations to contribute to resistance development by estimating the number of distinct alleles. To identify fitness-compensatory mutations that ameliorate the fitness costs of CipRrpoB mutations. To understand how CipRrpoB mutations reprogramme RNAP. Methods E. coli strains carrying five different CipRrpoB alleles were evolved with selection for improved fitness and characterized for acquired mutations, relative fitness and MICCip. The effects of dksA mutations and a ppGpp0 background on growth and susceptibility phenotypes associated with CipRrpoB alleles were determined. Results The number of distinct CipRrpoB mutations was estimated to be >100. Mutations in RNAP genes and in dksA can compensate for the fitness cost of CipRrpoB mutations. Deletion of dksA reduced the MICCip for strains carrying CipRrpoB alleles. A ppGpp0 phenotype had no effect on drug susceptibility. Conclusions CipRrpoB 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.

Deep mutational scanning reveals the molecular determinants of RNA polymerase-mediated adaptation and tradeoffs

“…Therefore, they may also activate the expression of other maintenance functions to again alter global traits. In line with our proposition that RNAP mutations select for altered global traits, it is important to note here that, stringent mutations of the RNAP have also been selected for maintenance-associated adaptation to stresses such as long-term starvation, tolerance, and resistance to antibiotics and chemicals 12 – 14 .…”

“…Alternatively, the mutations are also often involved in trade-offs 27 . While improved growth can increase pH sensitivity, decrease motility, and antibiotic persistence 3 ; RNAP mutation-mediated improved stress tolerance can decrease growth 12 , 14 . Due to the pleiotropy, it is difficult to identify the specific cellular traits under selection and delineate the true systems’ impact of adaptive RNAP mutations.…”

“…The global rewiring of transcription could be linked to molecular changes in the RNAP which favors the expression of one class of genes over the others. For instance, a few mutations selected for improved stress tolerance and long-term starvation fall into a category of RNAP mutations called “stringent” mutations 12 , 14 . Some RNAP stringent mutations are known to impact the stability of one of the open complex intermediates to favor the expression of stress-associated genes over growth 28 .…”

mSphere of Influence: Targeting bacterial signaling and metabolism to overcome antimicrobial resistance

“…Moreover, this research shows how the presence of antibiotics in our environment may open up opportunities for tolerance and resistance mechanisms to emerge. Intriguingly, mutations in rpoB have been shown to lead to a stringent-like response that also increases resistance to rifampicin and ciprofloxacin ( 18 , 19 ). The stringent response alarmone (p)ppGpp is required for high-level beta-lactam resistance and represses the activity of the c-di-AMP phosphodiesterase GdpP ( 6 , 15 , 18 – 23 ).…”

Genomic Analysis of Ciprofloxacin-Resistant Salmonella enterica Serovar Kentucky ST198 From Spanish Hospitals

“…Such change, however, does not appear to be associated with quinolone resistance, as it was also identified in isolates susceptible to nalidixic acid (Weill et al, 2006). Although alterations in the target topoisomerases are the main cause of ciprofloxacin resistance in S. Kentucky, the involvement of the major multidrug AcrAB-TolC efflux pump, and of mutations affecting the rpoB gene coding for the β-subunit of the enzyme RNA polymerase, have also been reported (Weill et al, 2006;Baucheron et al, 2013;Brandis et al, 2021). Many other efflux pumps were identified in S. enterica (Li et al, 2018), but their contribution to high level CIP R in S. Kentucky has yet to be demonstrated.…”