The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle and protect against antibiotics

Abstract

How DNA-dependent RNA polymerase (RNAP) acts on bacterial cell cycle progression during transcription elongation is poorly investigated. A forward genetic selection for Caulobacter crescentus cell cycle mutants unearthed the uncharacterized DUF1013 protein (TrcR, transcriptional cell cycle regulator). TrcR promotes the accumulation of the essential cell cycle transcriptional activator CtrA in late S-phase but also affects transcription at a global level to protect cells from the quinolone antibiotic nalidixic acid that induces a multidrug efflux pump and from the RNAP inhibitor rifampicin that blocks transcription elongation. We show that TrcR associates with promoters and coding sequences in vivo in a rifampicin-dependent manner and that it interacts physically and genetically with RNAP. We show that TrcR function and its RNAP-dependent chromatin recruitment are conserved in symbiotic Sinorhizobium sp. and pathogenic Brucella spp. Thus, TrcR represents a hitherto unknown antibiotic target and the founding member of the DUF1013 family, an uncharacterized class of transcriptional regulators that track with RNAP during the elongation phase to promote transcription during the cell cycle.

Adaptive β-lactam resistance from an inducible efflux pump that is post-translationally regulated by the DjlA co-chaperone

“…We discovered that TipR significantly binds 4 chromosomal targets in vivo. Importantly, TipR occupancy of these sites is reduced by ≈500% after exposing cells to NAL (for 30 minutes; Figs 1F and S2A and S3 Data ), indicating that NAL antagonizes repression of P acrA and P tipR by TipR in vivo, allowing recruitment of RNAP (and TrcR [ 10 ]). The coordinated induction of AcrAB-NodT and TipR by NAL points towards a homeostatic control mechanism in which the adaptive and transient resistance to β-lactam antibiotics is conferred by induction of AcrAB-NodT, followed by TipR synthesis to reestablish the repressed ground state once the inducers have been expelled by AcrAB-NodT.…”

“…To search for determinants conferring β-lactam resistance in cells lacking MBL, we conducted a transposon (Tn) mutagenesis of Δbla mutant cells, delivering the Tn by intergeneric conjugation from an E. coli donor. Initially, we counterselected the donor on plates containing NAL (20 μg/mL, NAL 20 ), but we unexpectedly observed that Δbla cells grew on PIR 40 or CEF 10 plates supplemented with NAL 20 . NAL is not known to affect β-lactam resistance directly, as it targets the A subunit of DNA gyrase (GyrA) in susceptible bacteria.…”

“…We selected resistant mutants on plates containing KAN 20 and PIR 40 and mapped the Tn insertions of 2 different clones to the tipR gene (Fig 2A and 2C) [9,13]. Next, we backcrossed the tipR::Tn mutation into Δbla cells and compared the adaptive β-lactam resistance conferred by NAL in Δbla;tipR::Tn double-mutant cells to that of Δbla parental cells by Kirby-Bauer antibiotic disc diffusion assays (Fig 1B ), as well as by growth on plates containing CEF 10 or PIR 40 (Fig 2C). These tests revealed an identical β-lactam resistance spectrum between the Δbla strain grown in the presence of NAL and…”

“…Thus, these transcriptional repressors act as single-component chemical sensors for the expulsion of toxic molecules that are encountered and produced, for example, by competing microbes. Although induction of AcrAB-NodT pumps rids the cell from toxic molecules, the massive synthesis and assembly of new trans -envelope complexes can also pose (sometimes even lethal) stress to the envelope [ 9 , 10 ]. Thus, protective measures are needed in anticipation of such imbalances, deformations, and/or prolonged stress of the envelope.…”

“…We recently described the RND-type efflux pump, AcrAB-NodT, which confers adaptive resistance in the oligotrophic α-proteobacterium Caulobacter crescentus, a model system for cell cycle and polarity studies. C. crescentus AcrAB-NodT is expressed from a promoter (P acrA ) that is inducible by the first-generation quinolone nalidixic acid (NAL) and related molecules [9,13], and this AcrAB-NodT induction by NAL can be lethal to certain envelope mutants, for example, mutants lacking the TipN polarity factor [9] or the conserved TrcR protein, an RNA polymerase (RNAP)-associated factor [10]. P acrA is repressed by TipR, a TetRlike DNA-binding protein that is divergently encoded and expressed from a shared promoter region that spans 167 nucleotides between the predicted start codons of AcrA and TipR (Fig 1A).…”