The <i>Vibrio cholerae</i> Mrp System: Cation/Proton Antiport Properties and Enhancement of Bile Salt Resistance in a Heterologous Host

Abstract

The mrp operon from Vibrio cholerae encoding a putative multisubunit Na⁺/H⁺ antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na⁺ and Li⁺ as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li⁺, Na⁺, and K⁺ ions in pH-dependent manner with maximal activity at pH 9.0–9.5. Exchange was electrogenic (more than one H⁺ translocated per cation moved in opposite direction). The apparent K_m at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li⁺, Na⁺, and K⁺, respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems.

Structure-based Functional Study Reveals Multiple Roles of Transmembrane Segment IX and Loop VIII–IX in NhaA Na+/H+ Antiporter of Escherichia coli at Physiological pH

“…2C). Notably a counterion (150 mM Cl Ϫ ) exists in this experimental system so that no ⌬ exists across the membrane (43). (b) When the ⌬pH-driven antiport activity was measured by 22 Na uptake in proteoliposomes in the absence of a counterion, the activity of the electrogenic wild type NhaA, due to its stoichiometry of 2 H ϩ /1 Na ϩ , increased dramatically in the presence of a counterion (K ϩ in the presence of valinomycin); its rate and steady state level of Na ϩ uptake drastically increased (4 -5-fold) (Fig.…”

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation

“…Mrp antiporters are also broadly distributed among non-alkaliphilic Gram-positive and Gram-negative bacteria and play roles in diverse physiological processes that may depend upon pH and Na ϩ homeostasis (2). In addition to Na ϩ /H ϩ antiport, several of these Mrp antiporters support resistance to anions, such as arsenite and bile salts (13)(14)(15). We hypothesize that Mrp complexes are consortiums of more than one transporter type (2).…”

pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti

“…In contrast, antiport activities of Mrp antiporters from Bacillus species (Bs-Mrp and BpOF4-Mrp), S. aureus (Mnh) and V. cholerae (VcMrp) were rather constant or increased as pH was shifted to above pH 9.0 (DziobaWinogrodzki et al, 2009;Swartz et al, 2007). These Mrp antiporters are implicated in the tolerance of constant or transient extreme alkaline conditions in these organisms (Dzioba-Winogrodzki et al, 2009;Hiramatsu et al, 1998;Ito et al, 1999Ito et al, , 2001Kosono et al, 1999). Taken together, our results indicate that the Pha1 system has a role in the tolerance of mild alkaline conditions, rather than extreme alkaline tolerance as observed in the above-mentioned Mrp antiporters.…”

“…Mrp antiporters typically consist of six or seven membrane proteins encoded in a single operon. The mrp operons are categorized into two major groups (Swartz et al, 2005): group 1, which includes mrp from Bacillus subtilis, contains seven genes (mrpABCDEFG); and group 2, which includes Vibrio cholerae Vc-mrp, contains six genes (mrpA9CDEFG) (Dzioba-Winogrodzki et al, 2009). It has been shown that all of the components are required for the Mrp antiporter to be fully functional (Ito et al, , 2000Putnoky et al, 1998;Yoshinaka et al, 2003).…”

“…Detailed functional analysis of group 1 Mrp antiporters from Staphylococcus aureus and several Bacillus species (Swartz et al, 2007) determined that these antiporters are electrogenic Na + (Li + )/H + antiporters with no detectable K + transport activity. In comparison, a group 2 Mrp antiporter, VcMrp, was shown to be a Na + (Li + )/H + antiporter with low-affinity K + /H + antiport activity (Dzioba-Winogrodzki et al, 2009). …”