Identification and Characterization of a Major Zn(II) Resistance Determinant of Mycobacterium smegmatis

Abstract

A zinc ion-sensitive mutant of Mycobacterium smegmatis was isolated. The transposon insertion was located in zitA (MSMEG0750), a gene coding for a cation diffusion facilitator family protein. Zinc ions specifically induced expression of zitA. In silico analysis revealed that environmental and opportunistic pathogenic species contain higher numbers of cation diffusion facilitator genes than do obligate pathogens.Zinc is an essential trace element. However, a high concentration of zinc ions causes toxicity. Therefore, cells require homeostasis mechanisms to control the intracellular zinc ion concentration. In bacteria, the zinc ion concentration is controlled by the activity of membrane transporters belonging to different families that mediate its uptake and efflux. The cation diffusion facilitators (CDFs) are integral membrane proteins that transport various divalent cations, including Zn(II) (19). Some of the CDF proteins have been shown to be specific for a single metal ion substrate whereas others possess broad substrate specificity (8,15).The genus Mycobacterium encompasses various environmental and pathogenic species. Zinc acts as an important component of many mycobacterial enzymes such as superoxide dismutase, alcohol dehydrogenase, carbonic anhydrase, etc. (5,7,25). Sufficient acquisition of zinc ions may therefore be important for production of zinc-containing enzymes in active form. Moreover, both pathogenic and environmental mycobacteria survive in various inhospitable conditions in external habitats (6,12,16). Resistance mechanisms become important for survival and growth in zinc ion-contaminated habitats. Studies aiming to understand zinc ion homeostasis mechanisms in mycobacteria have recently been initiated. Two transcriptional regulators shown to be induced by zinc ions have been identified from Mycobacterium tuberculosis and Mycobacterium smegmatis (13), and their role in regulation of genes involved in zinc ion homeostasis was later suggested (3). However, molecular determinants for zinc ion acquisition and resistance in mycobacteria are yet to be identified. Here we report the identification and characterization of a chromosomal zinc ion resistance determinant of M. smegmatis.Isolation of a zinc ion-sensitive mutant. Strains, plasmids, and oligonucleotides used in this study are summarized in Table 1. A transposon mutant library of M. smegmatis mc 2 155 was prepared using a previously described protocol with minor modifications (22). The plasmid pMycoMar was transformed by electroporation (23), and the transformants were allowed to grow at 28°C for 24 h without antibiotic selection. The mutants were selected at 42°C on Luria-Bertani (LB) agar plates supplemented with 5% sucrose and kanamycin (20). The mutant library, consisting of more than 2,500 mutants, was screened for zinc ion sensitivity, and mutant K516, which failed to grow on 400 M zinc acetate-containing LB agar, was identified. The MIC assays were performed by growing M. smegmatis strains for 24 h in LB broth with 0.05% Tween 80 and f...

Defining the Roles of the Cation Diffusion Facilitators in Fe2+/Zn2+ Homeostasis and Establishment of Their Participation in Virulence in Pseudomonas aeruginosa

“…We also included the only Zn 2+ -transporting CDF member present in M. smegmatis ZitA (Grover and Sharma, 2006; Raimunda et al, 2012). The same alignment shows that ZitA is phylogenetically related to CzcD.…”

Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage

“…Another advantage of replacing the Zn 2+ motif in topoI could be related to the intracellular scarcity of Zn 2+ ions. The free Zn 2+ ions have been shown to cause metallotoxicity, leading to the death of mycobacteria (38,39). Studies with various mycobacteria have revealed the protective mechanisms used by them to overcome the Zn 2+ cytotoxicity (38,39).…”

“…The free Zn 2+ ions have been shown to cause metallotoxicity, leading to the death of mycobacteria (38,39). Studies with various mycobacteria have revealed the protective mechanisms used by them to overcome the Zn 2+ cytotoxicity (38,39). Mycobacterium tuberculosis uses the CtpC (P-type ATPases) transporters to pump out the Zn 2+ from the cell, thus circumventing the host defense system that exploits the Zn 2+ ions to kill the pathogen (38).…”

Role in metal homeostasis of CtpD, a Co2+transporting P1B4‐ATPase ofMycobacterium smegmatis

“…It could be postulated that other redundant Zn 2+ ‐ATPases (P IB2 ‐ATPases) might mask the CtpD Zn 2+ ‐ATPase activity, although M. smegmatis genome encodes no predicted P IB2 ‐ATPases. On the other hand, the cation diffusion facilitator (CDF) transporter ZitA appears responsible for Zn 2+ homeostasis in M. smegmatis (Grover and Sharma, 2006). To further assess a possible role of CtpD in Zn 2+ homeostasis a double‐deletion Δ ctpD Δ zitA mutant strain was constructed and its Zn 2+ sensitivity was compared with that of a Δ zitA strain.…”