Multiple signaling systems target a core set of transition metal homeostasis genes using similar binding motifs

Abstract

Bacterial response to metals can require complex regulation. We report an overlapping regulation for copper and zinc resistance genes in the denitrifying bacterium, Pseudomonas stutzeri RCH2, by three two-component regulatory proteins CopR1, CopR2 and CzcR. We conducted genome-wide evaluations to identify gene targets of two paralogous regulators, CopR1 and CopR2, annotated for copper signaling, and compared the results with the gene targets for CzcR, implicated in zinc signaling. We discovered that the CopRs and CzcR have largely common targets, and crossregulate a core set of P. stutzeri copper and zinc responsive genes. We established that this crossregulation is enabled by a conserved binding motif in the upstream regulatory regions of the target genes. The crossregulation is physiologically relevant as these regulators synergistically and antagonistically target multicopper oxidases, metal efflux and sequestration systems. CopR1 and CopR2 upregulate two cop operons encoding copper tolerance genes, while all three regulators downregulate a putative copper chaperone, Psest_1595. CzcR also upregulated the oprD gene and the CzcIABC Zn efflux system, while CopR1 and CopR2 downregulated these genes. Our study suggests that crossregulation of copper and zinc homeostasis can be advantageous, and in P. stutzeri this is enabled by shared binding motifs for multiple response regulators.

A Novel Legionella Genomic Island Encodes a Copper-Responsive Regulatory System and a Single Icm/Dot Effector Protein Transcriptionally Activated by Copper

“…5B). To determine the specificity of LciS, we also exposed the bacteria to other similar metals (57,64), and as seen in Fig. 5C, the LciRS TCS was found to be specific to copper and no induction was obtained with any of the other metals examined.…”

Tools to map target genes of bacterial two‐component system response regulators

“…The purified tagged protein is added and allowed to bind (along with any effectors needed), and after washing unbound protein, the DNA‐bound protein is detected by enzyme‐conjugated antibodies that recognize the affinity tag. This assay was originally developed to study plant TFs (Brand et al ., ) but has also been applied to TCS RRs (Garber et al ., ). The DPI‐ELISA provides quantitative information and can also be automated easily (Brand et al ., ).…”

“…In the environmental denitrifying microbe P . stutzeri , the overlap in the regulons of copper‐ and zinc‐responsive RRs was revealed through DAP‐seq and may have remain hidden when examining only single RRs of interest (Garber et al ., ).…”

“…In some of these studies, the challenges associated with heterologous expression and purification of the TF protein have been addressed by employing in vitro translations, which further assists in the automation of this approach. Among bacterial TCSs, DAP‐seq has been applied to map the target genes for metal‐responsive TCSs in Pseudomonas stutzeri , enabling the discovery of co‐regulated networks (Garber et al ., ), and to determine the targets for the RR NarP of Actinobacillus pleuropneumoniae (Zhang et al ., ). Application of DAP‐seq to more than one RR often reveals aspects of signal integration.…”

Fluorescent amplification for next generation sequencing (FA-NGS) library preparation

“…Pseudomonas stutzeri RCH2 was grown overnight in 5 mL UGA media [20]. UGA media contained 4.7 mM ammonium chloride, 1.3 mM potassium chloride, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 0.3 mM sodium chloride, 5 mM sodium dihydrogen phosphate, 20 mM sodium lactate, and 25 mM MOPS.…”