Pma1 is an alkali/alkaline earth metal cation ATPase that preferentially transports Na+ and K+ across the Mycobacterium smegmatis plasma membrane

Abstract

Mycobacterium smegmatis Pma1 is the orthologue of M. tuberculosis P-type ATPase cation transporter CtpF, which is activated under stress conditions, such as hypoxia, starvation and response to antituberculous and toxic substances. The function of Pma1 in the mycobacterial processes across the plasma membrane has not been characterised. In this work, bioinformatic analyses revealed that Pma1 likely contains potential sites for, Na(+), K(+) and Ca(2+) binding and transport. Accordingly, RT-qPCR experiments showed that M. smegmatis pma1 transcription is stimulated by sub-lethal doses of Na(+), K(+) and Ca(2+); in addition, the ATPase activity of plasma membrane vesicles in recombinant Pma1-expressing M. smegmatis cells is stimulated by treatment with these cations. In contrast, M. smegmatis cells homologously expressing Pma1 displayed tolerance to high doses of Na(+) and K(+) but not to Ca(2+) ions. Consistently, the recombinant protein Km embedded in plasma membrane demonstrated that Ca(2+) has more affinity for Pma1 than Na(+) and K(+) ions; furthermore, the estimation of Vmax/Km suggests that Na(+) and K(+) ions are more efficiently translocated than Ca(2+). Thus, these results strongly suggest that Pma1 is a promiscuous alkali/alkaline earth cation ATPase that preferentially transports Na(+) and/or K(+) across the mycobacterial plasma membrane.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis

“…This optimal temperature is similar to the average temperature of the human body, which in turn is the natural host of M. tuberculosis. Similarly, the observed optimal pH (7.4) is close to neutrality, as well as the observed for other mycobacterial P-type ATPases [35][36][37]. Finally, CtpB displayed a concentration-dependent Cu + ATPase activity when the enzymatic reaction was supplemented with Cys at concentrations below 0.5 mM.…”

“…For example, recombinant M. smegmatis cells overexpressing the putative M. tuberculosis cadmium transporter CtpG tolerate high doses of Cd 2+ compared with non-recombinant mycobacterial cells [36]. Similarly, M. smegmatis homologously expressing the putative alkali/alkaline earth cation P-type ATPase transporter Pma1 tolerates toxic concentrations of Na + , K + , and Na + /K + ions [37]. Regarding P-type ATPases associated with copper transport, we previously reported that mycobacterial cells expressing the putative M. tuberculosis CtpA, a P-type ATPase copper transporter, grow approximately twofold more than non-recombinant cells in the presence of toxic copper concentrations (from 1 to 4 mM), suggesting a possible role of CtpA in copper detoxification in mycobacteria [35].…”

“…Since there is a contribution to the ATPase activity from other cell-membrane ATPases, parallel enzymatic reactions using membranes from M. smegmatis mc 2 155 transformed with the empty vector pMV261 were used as control. Therefore, the enzymatic activity mediated by CtpB was calculated by subtracting the ATPase activity of the control membranes from the ATPase activity of the recombinant membranes [35][36][37]. Using this experimental approach, it was observed that Cu + was the only cation that significantly stimulated the ATPase activity of CtpB embedded in the mycobacterial plasma membrane, compared with other divalent heavy metal cations (Fig.…”

“…The integrity of pALT10 was confirmed by automated sequencing. pALT10 and pMV261 were separately transformed in M. smegmatis mc 2 155 cells by electroporation and, finally, expression of the recombinant protein was induced by incubation of mycobacterial cells at 45 °C for 3 h [37]. The induction of CtpB expression was assessed in cells used to isolate plasma membrane fractions for the ATPase activity assays, including the optimal enzymatic conditions and kinetic parameters of CtpB.…”

The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells

“…The ATPase activity of the plasma membrane vesicles was measured according to the Fiske-Subbarow method with modifications, as previously described [19, 20, 46]. Enzymatic reactions (final volume 50 μL) were performed in 96-well plates using 10 μg of protein in reaction buffer (3 mM MgCl 2 , 10 mM MOPS, pH = 7.4) and supplemented with 0.02 % Brij-58, 0.29 mM Ca 2+ and 0.25 mM EGTA (final concentrations) to control the amount of free calcium.…”