Expression of Bacillus subtilis ABCF antibiotic resistance factor VmlR is regulated by RNA polymerase pausing, transcription attenuation, translation attenuation and (p)ppGpp

Abstract

Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.

Regulation of the macrolide resistance ABC-F translation factor MsrD

“…2c and Supplementary fig. 2a), as previously described for vmlR 52,53 . In order to test this possible transcription termination activity, we generated a mutant by removing the putative terminator (referred as pMMB-msrDL (no_term) -msrD (1-3) : yfp ).…”

“…5f and 5g), as a functional site within the ribosome and we propose to name it proximal crevice. ARE ABC-F genes tend to be transcriptionally regulated as illustrated by msrD, vmlR, vgaA, lmo0919 and lmrC 52,73,[76][77][78] . However, MsrD can regulated its own expression by creating a negative feedback-loop which is not the case for VmlR 52 .…”

“…ARE ABC-F genes tend to be transcriptionally regulated as illustrated by msrD, vmlR, vgaA, lmo0919 and lmrC 52,73,[76][77][78] . However, MsrD can regulated its own expression by creating a negative feedback-loop which is not the case for VmlR 52 . The gene msrD forms an operon with the efflux pump mefA (Fig.…”

Complementary Ribo-seq approaches map the translatome and provide a small protein census in the foodborne pathogenCampylobacter jejuni

“…In line with this, we identified the Met-codon enriched (MMYQMR) CJsORF19, a potentially new leader peptide that could regulate expression of the downstream Cj0772c (Dmethionine transport system substrate-binding protein). Likewise, CJsORF15 (MAFY) is located upstream of a potential multidrug efflux protein (Cj0560), and could be related to translation-related control of expression of the downstream gene in the presence of translation inhibitors, as reported in, e.g., Bacillus (Takada et al, 2022).…”

uORF4u: a tool for annotation of conserved upstream open reading frames

“…This involves retrieval of the 5' upstream regions of protein coding genes of interest from sequence databases, and manual inspection of the region, with or without the aid of a sequence alignment to indicate functional conservation. Tell-tale signatures of potential uORFs are the presence of Shine-Dalgarno elements (in the case of prokaryotes), along with start and stop codons in the right context and frame (12)(13)(14). However, sequence gazing is a time-consuming and tedious task.…”