Role in metal homeostasis of CtpD, a Co²⁺transporting P_1B4‐ATPase ofMycobacterium smegmatis

Abstract

Summary Genetic studies in the tuberculosis mouse model have suggested that mycobacterial metal efflux systems, such as the P1B4-ATPase CtpD, are important for pathogenesis. The specificity for substrate metals largely determines the function of these ATPases; however, various substrates have been reported for bacterial and plant P1B4-ATPases leaving their function uncertain. Here we describe the functional role of the CtpD protein of Mycobacterium smegmatis. An M. smegmatis mutant strain lacking the ctpD gene was hypersensitive to Co2+ and Ni2+ and accumulated these metals in the cytoplasm. ctpD transcription was induced by both Co2+ and superoxide stress. Biochemical characterization of heterologously expressed, affinity purified CtpD showed that this ATPase is activated by Co2+, Ni2+ and to a lesser extend Zn2+ (20% of maximum activity). The protein was also able to bind one Co2+, Ni2+ or Zn2+ to its transmembrane transport site. These observations indicate that CtpD is important for Co2+ and Ni2+ homeostasis in M. smegmatis, and that M. tuberculosis CtpD ortholog could be involved in metal detoxification and resisting cellular oxidative stress by modulating the intracellular concentration of these metals.

Fine-tuning of Substrate Affinity Leads to Alternative Roles of Mycobacterium tuberculosis Fe2+-ATPases

“…This medium contained less than 1 M residual iron as determined by atomic absorption spectroscopy (AAS) (PerkinElmer Life Sciences PinAAcle 900z). Construction of Ms⌬ctpJ (MSMEG_5403), Mt⌬ctpD (Rv1469), Mt⌬ctpJ (Rv3743), and Mt⌬ctpD:⌬ctpJ mutant and complemented strains was described previously (15,16).…”

“…Streptonigrin (15,16). Cells were grown at 37°C in ZYP-505 autoinduction medium supplemented with 0.05% arabinose, 100 mg ml Ϫ1 ampicillin, and 50 mg ml Ϫ1 kanamycin (33).…”

“…In particular, activation of P 1B4 -ATPases by Co 2ϩ , Ni 2ϩ , Ca 2ϩ , Cu ϩ , Zn 2ϩ , and Cd 2ϩ has been proposed (15-17, 28 -30). We previously reported in vivo and in vitro functional studies directed at understanding the metal selectivity and consequent physiological roles of mycobacterial P 1B4 -ATPases (15,16). The presence of one or two P 1B4 -ATPasecoding genes in mycobacterial species enabled comparative studies of Mycobacterium smegmatis CtpJ and Mycobacterium * This work was supported by National Institutes of Health Grant DK068139 (to T. L. S.).…”

“…PfeT is expressed under the control of PerR in response to peroxide exposure (14). Initial biochemical characterization showed that Fe 2ϩ activates isolated PfeT ATPase, leading to a higher V max than generated by Co 2ϩ , which is the proposed substrate of P 1B4 -ATPases (13,(15)(16)(17). Interestingly, phenotypic analysis of Listeria monocytogenes lacking the P 1B4 -ATPase FrvA showed a role of this ATPase in resistance to heme toxicity (18).…”

“…These observations suggest a significant role of this subfamily of P-type ATPases in Fe 2ϩ homeostasis (13,14). P 1B4 -ATPases present in prokaryotes and plant chloroplasts are part of the large family of P-type ATPases (15,19,20). P-type ATPases are polytopic membrane proteins that transport a variety of ions using the energy provided by ATP hydrolysis (21)(22)(23).…”