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

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Previous work from our laboratory involved the description of the Neurospora metal transportome, which included seven hypothetical zinc transporters belonging to the ZIP family. The aim of the present study was to make a comparative functional evaluation of two hypothetical zinc transporters named tzn1 (NCU07621.3) and tzn2 (NCU11414.3). Phenotypic analysis of tzn1 and tzn2 mutants and a double mutant (tzn1tzn2) revealed that the deletion of tzn1 causes aconidiation and a greater defect in growth than the single deletion of tzn2. Supplementation with zinc restores growth but not conidiation in tzn1 and tzn1tzn2. TZN1 complemented a zinc-uptake-deficient Saccharomyces cerevisiae mutant (zrt1zrt2) in zinc-deficient conditions, while tzn2 restored growth upon supplementation with zinc (0.05 mM). Furthermore, the Deltatzn1 mutant was found to have severely reduced zinc content indicating that tzn1 functions as a key regulator of intracellular zinc levels in Neurospora crassa. Zinc uptake studies indicate tzn1 is a specific transporter of zinc, while tzn2 transports both zinc and cadmium. Quantitative RT-PCR showed up-regulation of tzn1 (128-fold) under zinc-depleted conditions and down-regulation (>1,000-fold) in zinc-replete conditions. The present study indicates that the zinc transport proteins encoded by tzn1 and tzn2 are members of the zinc uptake system regulated by zinc status in N. crassa.

The Neurospora crassa gene NcZrg-17 encodes a membrane protein with homology to the cation diffusion facilitator (CDF) family of transporters. We analyzed the phenotypic and functional characteristics of ΔNcZrg-17 and the implications of these characteristics in vivo. The ΔNcZrg-17 mutant showed several phenotypes that are zinc suppressible such as reduced growth rate, short aerial hyphae, increased hyphal branching, early and enhanced conidiation and delayed conidial germination. Furthermore, the NcZrg-17 gene was found to be crucial for survival in the presence of endoplasmic reticulum (ER) stress inducing chemical agents. In addition, we found that ΔNcZrg-17 mutant is defective in protein secretion on cellulose media under low zinc conditions, pointing towards a physiological role for NcZrg-17 in N. crassa. A gradual and delayed transcriptional upregulation (~ threefold) of NcZrg-17 on exposure to low zinc suggests its role in adaptation to low zinc rather than zinc homeostasis. Together our findings support a function of NcZrg-17 in normal vegetative growth, tolerance to ER stress and degradation of cellulose under low zinc conditions in N. crassa.

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