Characterization of the regulator TipR in Caulobacter crescentus in the context of copper tolerance

Abstract

Heavy metals are reaching toxic levels in the environment due to industrial usage. In order to maintain their fitness in these toxic environments, bacteria have evolved defence mechanisms including metal sequestration, metal oxidation, and efflux pumps. Metals like copper (Cu) are essential at low amount but toxic at higher concentrations, therefore requiring an efficient Cu homeostasis. In order to identify novel Cu homeostasis strategies in the aquatic alpha-proteobacterium Caulobacter crescentus, a genetic screen was undergone a few years ago in the lab from a mini-Tn5 mutant library seeking for Cu-sensitive mutants. Thereby, a mutation was identified in the tipR (CCNA_00852) gene. TipR is a homolog of the AcrR regulator in E. coli, which regulates the antibiotic efflux pump AcrABTolC. Interestingly, we were able to identify a hypothetical Cu binding site via in silico structural analysis. In accordance with the genetic screen, the ∆tipR mutant showed sensitivity to Cu in liquid culture, suggesting that TipR plays a role in Cu tolerance. A ChIP-Seq analysis together with a bioinformatic regulon prediction allowed us to identify potential TipR target genes such as the operon coding for acrABnodT (CCNA_00849: CCNA_00851), an efflux pump neighbouring the tipR gene. AcrABNodT was homologous to the AcrAB and CusC proteins from the AcrABTolC pump and CusCBA pump in E. coli, respectively. The homology analysis led to the hypothesis that AcrABNodT could have dual resistance to antibiotics and Cu. Surprisingly, no Cu sensitivity was observed upon deletion of this operon, suggesting (1) a potential redundancy with another Cu efflux pump or (2) that AcrABNodT has no role in Cu efflux. The CCNA_03857 gene predicted to encode a transporter was another promising TipR target candidate, which was also revealed by the genetic screen. Preliminary data seems to indicate that CCNA_03857 is not involved in Cu homeostasis, although a role in Cu redistribution or in the transport of another substrate cannot be ruled out. This is the first evidence of TipR implication in Cu tolerance, and further investigations are required to unravel its precise function.

Date of Award	20 Jan 2022
Original language	English
Awarding Institution	University of Namur
Supervisor	Jean-Yves Matroule (Supervisor)