MrdH, a Novel Metal Resistance Determinant of Pseudomonas putida KT2440, Is Flanked by Metal-Inducible Mobile Genetic Elements

Abstract

We report here the identification and characterization of mrdH, a novel chromosomal metal resistance determinant, located in the genomic island 55 of Pseudomonas putida KT2440. It encodes for MrdH, a predicted protein of ϳ40 kDa with a chimeric domain organization derived from the RcnA and RND (for resistance-nodulation-cell division) metal efflux proteins. The metal resistance function of mrdH was identified by the ability to confer nickel resistance upon its complementation into rcnA mutant (a nickel-and cobalt-sensitive mutant) of Escherichia coli. However, the disruption of mrdH in P. putida resulted in an increased sensitivity to cadmium and zinc apart from nickel. Expression studies using quantitative reverse transcription-PCR showed the induction of mrdH by cadmium, nickel, zinc, and cobalt. In association with mrdH, we also identified a conserved hypothetical gene mreA whose encoded protein showed significant homology to NreA and NreA-like proteins. Expression of the mreA gene in rcnA mutant of E. coli enhanced its cadmium and nickel resistance. Transcriptional studies showed that both mrdH and mreA underwent parallel changes in gene expression. The mobile genetic elements Tn4652 and IS1246, flanking mrdH and mreA were found to be induced by cadmium, nickel, and zinc, but not by cobalt. This study is the first report of a single-component metal efflux transporter, mrdH, showing chimeric domain organization, a broad substrate spectrum, and a location amid metal-inducible mobile genetic elements.Bacterial efflux systems for inorganic metal cations and anions play an imperative role in the regulatory network governing metal homeostasis. These efflux systems are also important for the environmental adaptability of the bacteria thriving in metal-rich serpentine environments. Bioinformatic and functional genomic analyses have revealed that the efflux systems belonging to the resistance-nodulation-cell division (RND), cation diffusion facilitator (CDF), and P-type ATPases constitute the majority of the multiple layers of heavy metal resistance in an organism (36). Members of the RND protein family include group of bacterial transport proteins involved in heavy metal resistance, nodulation, and cell division. The bestcharacterized RND family members include the efflux systems CzcCBA (Cd 2ϩ , Zn 2ϩ , and Co 2ϩ resistance), CnrCBA (Co 2ϩ

Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clusters within proteobacteria

“…This confirms a common association of these genes to mobile genetic elements, as discussed for other bacteria [7,8]. …”

Metal Response in Cupriavidus metallidurans

“…All mer genes except merR are induced by Hg 2+ and to a lesser extent by Cd 2+ , Zn 2+ and Pb 2+ ). In addition, the transposition modules are induced by Hg 2+ (without an unequivocal discrimination between Tn4738, Tn4380 and Tn6050) , which is consistent with the increased transposition frequency of mercury transposons in the presence of mercury (Sherratt et al 1981;Kitts et al 1982;Haritha et al 2009). The urf-2 gene is not observed outside of the mer gene cluster and has not been shown to be directly involved in resistance to mercury.…”

The Escherichia coli metallo-regulator RcnR represses rcnA and rcnR transcription through binding on a shared operator site: Insights into regulatory specificity towards nickel and cobalt

“…At both functional and regulatory levels, this system is dedicated to the detoxification of Ni and Co. Until recently, E. coli RcnA was the only described member of this family of efflux pumps. However a new member, MdrH, has recently been described in Pseudomonas putida which confers resistance to Cd, Zn and Ni; its expression is induced by Cd, Ni, Zn and Co [15]. The mdrH gene is thought to be regulated by the upstream gene mreA, which has the same expression pattern as the target gene.…”