mrp, a Multigene, Multifunctional Locus in Bacillus subtilis with Roles in Resistance to Cholate and to Na(+) and in pH Homeostasis

A 5.9-kb region of the Bacillus subtilis chromosome is transcribed as a single transcript that is predicted to encode seven membrane-spanning proteins. Homologues of the first gene of this operon, for which the designation mrp (multiple resistance and pH adaptation) is proposed here, have been suggested to encode an Na(+)/H(+) antiporter or a K(+)/H(+) antiporter. In the present studies of the B. subtilis mrp operon, both polar and nonpolar mutations in mrpA were generated. Growth of these mutants was completely inhibited by concentrations of added Na(+) as low as 0.3 M at pH 7.0 and 0.03 M at pH 8.3; there was no comparable inhibition by added K(+). A null mutant that was constructed by full replacement of the mrp operon was even more Na(+) sensitive. A double mutant with mutations in both mrpA and the multifunctional antiporter-encoding tetA(L) gene was no more sensitive than the mrpA mutants to Na(+), consistent with a major role for mrpA in Na(+) resistance. Expression of mrpA from an inducible promoter, upon insertion into the amyE locus, restored significant Na(+) resistance in both the polar and nonpolar mrpA mutants but did not restore resistance in the null mutant. The mrpA disruption also resulted in an impairment of cytoplasmic pH regulation upon a sudden shift in external pH from 7.5 to 8.5 in the presence of Na(+) and, to some extent, K(+) in the range from 10 to 25 mM. By contrast, the mrpA tetA(L) double mutant, like the tetA(L) single mutant, completely lost its capacity for both Na(+)- and K(+)-dependent cytoplasmic pH regulation upon this kind of shift at cation concentrations ranging from 10 to 100 mM; thus, tetA(L) has a more pronounced involvement than mrpA in pH regulation. Measurements of Na(+) efflux from the wild-type strain, the nonpolar mrpA mutant, and the complemented mutant indicated that inducible expression of mrpA increased the rate of protonophore- and cyanide-sensitive Na(+) efflux over that in the wild-type in cells preloaded with 5 mM Na(+). The mrpA and null mutants showed no such efflux in that concentration range. This is consistent with MrpA encoding a secondary, proton motive force-energized Na(+)/H(+) antiporter. Studies of a polar mutant that leads to loss of mrpFG and its complementation in trans by mrpF or mrpFG support a role for MrpF as an efflux system for Na(+) and cholate. Part of the Na(+) efflux capacity of the whole mrp operon products is attributable to mrpF. Neither mrpF nor mrpFG expression in trans enhanced the cholate or Na(+) resistance of the null mutant. Thus, one or more other mrp gene products must be present, but not at stoichiometric levels, for stability, assembly, or function of both MrpF and MrpA expressed in trans. Also, phenotypic differences among the mrp mutants suggest that functions in addition to Na(+) and cholate resistance and pH homeostasis will be found among the remaining mrp genes.

枯草芽孢杆菌（Bacillus subtilis）染色体上一段5.9 kb的区域可转录为单一转录本，该转录本预计编码7个跨膜蛋白。该操纵子的首个基因的同源物，本文暂将其命名为mrp（multiple resistance and pH adaptation，多重耐药与pH适应），此前被认为编码Na(+)/H(+)反向转运蛋白（Na(+)/H(+) antiporter）或K(+)/H(+)反向转运蛋白（K(+)/H(+) antiporter）。本研究针对枯草芽孢杆菌mrp操纵子展开，构建了mrpA的极性突变与非极性突变株。在pH 7.0条件下，添加0.3 M Na(+)即可完全抑制这些突变株的生长；pH 8.3条件下，仅需0.03 M Na(+)即可达到相同抑制效果，而添加K(+)则无类似抑制作用。通过完全替换mrp操纵子构建的无效突变株对Na(+)的敏感性更高。在mrpA与多功能反向转运蛋白编码基因tetA(L)双突变株中，其Na(+)敏感性与单株mrpA突变株无显著差异，这与mrpA在Na(+)耐药性中发挥主要作用的结论一致。将mrpA置于诱导型启动子（inducible promoter）控制下并插入amyE位点（amyE locus）进行表达，可显著恢复极性与非极性mrpA突变株的Na(+)耐药性，但无法恢复无效突变株的耐药性。在Na(+)存在且胞外pH从7.5骤升至8.5的条件下，mrpA破坏突变株的胞质pH调控能力受损；当K(+)浓度处于10~25 mM范围内时，该突变株的胞质pH调控也会受到一定程度的损害。相比之下，mrpA tetA(L)双突变株与单株tetA(L)突变株一样，在阳离子浓度10~100 mM范围内经上述pH骤变后，完全丧失了依赖Na(+)和K(+)的胞质pH调控能力，这表明tetA(L)在pH调控中的作用比mrpA更为显著。对野生型菌株、非极性mrpA突变株及互补突变株的Na(+)外流进行检测，结果显示：在预负载5 mM Na(+)的细胞中，诱导表达mrpA可提升对质子载体（protonophore）和氰化物（cyanide）敏感的Na(+)外流速率，高于野生型菌株；而mrpA突变株与无效突变株在该Na(+)浓度范围内未表现出此类外流活性。这与MrpA编码依赖质子动力势（proton motive force）供能的次级Na(+)/H(+)反向转运蛋白的结论相符。针对导致mrpFG表达缺失的极性突变株，以及通过反式表达mrpF或mrpFG对其进行互补的实验结果，进一步证实MrpF可作为Na(+)和胆酸盐（cholate）的外流系统。整个mrp操纵子编码产物的部分Na(+)外流能力归因于mrpF的表达。单独反式表达mrpF或mrpFG均无法增强无效突变株对胆酸盐或Na(+)的耐药性，这表明需要其他一种或多种mrp基因产物的参与（而非仅达到化学计量水平），才能维持反式表达的MrpF与MrpA的稳定性、组装或功能。此外，各mrp突变株间的表型差异提示，除Na(+)耐药、胆酸盐耐药与pH稳态外，剩余mrp基因还可能参与其他生物学功能。