Identification of a U/Zn/Cu responsive global regulatory two‐component system in Caulobacter crescentus

Abstract

Despite the well-known toxicity of uranium (U) to bacteria, little is known about how cells sense and respond to U. The recent finding of a U-specific stress response in Caulobacter crescentus has provided a foundation for studying the mechanisms of U- perception in bacteria. To gain insight into this process, we used a forward genetic screen to identify the regulatory components governing expression of the urcA promoter (P ) that is strongly induced by U. This approach unearthed a previously uncharacterized two-component system, named UzcRS, which is responsible for U-dependent activation of P . UzcRS is also highly responsive to zinc and copper, revealing a broader specificity than previously thought. Using ChIP-seq, we found that UzcR binds extensively throughout the genome in a metal-dependent manner and recognizes a noncanonical DNA-binding site. Coupling the genome-wide occupancy data with RNA-seq analysis revealed that UzcR is a global regulator of transcription, predominately activating genes encoding proteins that are localized to the cell envelope; these include metallopeptidases, multidrug-resistant efflux (MDR) pumps, TonB-dependent receptors and many proteins of unknown function. Collectively, our data suggest that UzcRS couples the perception of U, Zn and Cu with a novel extracytoplasmic stress response.

The UzcRS two‐component system in Caulobacter crescentus integrates regulatory input from diverse auxiliary regulators

“…Well‐characterized sensory regulatory pathways in C. crescentus include the general stress response, which is governed by a extracytoplasmic family sigma factor σ T , an antisigma factor and the PhyK‐PhyR and LovK‐LovR TCS that coordinately control σ T activity (Foreman et al ., ; Lourenço and Gomes, ), the PhoBR TCS, which mediates the response to phosphate limitation (Lubin et al ., ) and the oxygen sensory pathway FixLJ‐FixK‐FixT, which enables growth under hypoxic conditions (Crosson et al ., ). Recently, the UzcRS TCS, comprised of the HK UzcS and the RR UzcR, was identified as an activator of ~40 operons (Park et al ., ). UzcRS activity is strongly stimulated by the metals uranium, zinc and copper, but not by several other tested divalent metals (Park et al ., ).…”

“…Recently, the UzcRS TCS, comprised of the HK UzcS and the RR UzcR, was identified as an activator of ~40 operons (Park et al ., ). UzcRS activity is strongly stimulated by the metals uranium, zinc and copper, but not by several other tested divalent metals (Park et al ., ). Upon activation, UzcR binds extensively across the genome, activating transcription of genes which predominately encode uncharacterized proteins with predicted extracytoplasmic localization, including metallopeptidases (9), multidrug‐resistant efflux pumps (3), TonB‐dependent receptors (2), an outer membrane S‐layer transporter (RsaF b ) and numerous hypothetical proteins (Park et al ., ).…”

“…Based on the data presented herein, these genes have been named urtAP (UzcRS Regulating Transporter Atpase aminoPeptidase). Three transposons were mapped within CCNA_02866 (hereafter uzcX ), encoding a putative membrane protein of unknown function (DUF805 domain) that is located within a prophage region of the genome (Scott and Ely, ) and is part of the UzcR regulon (Park et al ., ). An additional three transposons either mapped to CCNA_03498 (1) or CCNA_02289 (2), both encoding uncharacterized transcription factors of the MarR family (hereafter given the names marR 1 and marR 2 ).…”

“…Upon activation, UzcR binds extensively across the genome, activating transcription of genes which predominately encode uncharacterized proteins with predicted extracytoplasmic localization, including metallopeptidases (9), multidrug‐resistant efflux pumps (3), TonB‐dependent receptors (2), an outer membrane S‐layer transporter (RsaF b ) and numerous hypothetical proteins (Park et al ., ). Despite the central role of UzcRS in mediating U‐, Zn‐ and Cu‐dependent gene expressions in Caulobacter , Δ uzcR and Δ uzcS mutants lack an obvious U/Zn/Cu tolerance phenotype (Park et al ., ), raising questions about the physiological role of UzcRS…”

“…During the course of screening for transposons that abolished metal‐dependent activation of the UzcR‐regulated P urcA – a process that lead to the identification of uzcR and uzcS (Park et al ., ) – 14 transposons were isolated that caused high P urcA expression in the absence of exogenously supplied metal (Table ), raising the possibility that additional cellular processes are involved in the regulation of the UzcRS activity. Eight mutants had transposons located within CCNA_03681 or CCNA_03680 that encode an ABC transporter ATPase and an ABC‐2 family transporter fused to a C‐terminal aminopeptidase domain respectively.…”