Identification of promoter PcadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter PcadR from Pseudomonas aeruginosa BC15.

Abstract

Cadmium (Cd) remediation in Pseudomonas aeruginosa is achieved through the function of two vital genes, cadA and cadR, that code for P-type ATPase (CadA) and transcription regulatory protein (CadR), respectively. Although numerous studies are available on these metal-sensing and regulatory proteins, the promoter of these genes, metal sensing and binding ability, are poorly understood. The present work is aimed at the characterization of the CadR protein, identification of the PcadR promoter and protein-promoter-metal binding affinity using bioinformatics and to validate the results by cloning the PcadR promoter in Escherichia coli DH5α. The promoter regions and its curvature were identified and analysed using PePPER software (University of Groningen, The Netherland) and the Bendit program (Version: v.1.0), respectively. Using Phyre, the three-dimensional structure of CadR was modelled, and the structure was validated by Ramachandran plots. The DNA-binding domain was present in the N-terminal region of CadR. A dimeric interface was observed in helix-turn-helix and metal ion-binding sites at the C-terminal. Docking studies showed higher affinity of Cd to both CadR (Atomic contact energy = -15.04 kcal/Mol) and PcadR (Atomic contact energy = -40.18 kcal/Mol) when compared to other metal ions. CadR with PcadR showed the highest binding affinity (Atomic contact energy= -250.40 kcal/Mol) when compared with PcadA. In vitro studies using green fluorescent protein tagged with PcadR (gfp-PcadR) cloned in E. coli-expressed gfp protein in a concentration-dependent manner upon Cd exposure. Based on our in silico studies and in vitro molecular cloning analysis, we conclude that PcadR and CadR are active only in the presence of Cd. The CadR protein has the highest binding affinity with PcadR. As it became apparent that the cadR gene regulates the PcadR activity in the presence of Cd with high specificity, and the cadR and PcadR can be used as a biological tool for development of a microbial biosensor.