Na+ conductivity of the Na+-driven flagellar motor complex composed of unplugged wild-type or mutant PomB with PomA

Abstract

PomA and PomB form the stator complex, which functions as a Na(+) channel, in the Na(+)-driven flagellar motor of Vibrio alginolyticus. The plug region of PomB is thought to regulate the Na(+) flow and to suppress massive ion influx through the stator channel. In this study, in order to measure the Na(+) conductivity of the unplugged stator, we over-produced a plug-deleted stator of the Na(+)-driven flagellar motor in Escherichia coli. The over-production of the plug-deleted stator in E. coli cells caused more severe growth inhibition than in Vibrio cells and that growth inhibition depended on the Na(+) concentration in the growth medium. Measurement of intracellular Na(+) concentration by flame photometry and fluorescent analysis with a Na(+) indicator, Sodium Green, revealed that over-production of the plug-deleted stator increased the Na(+) concentration in cell. Some mutations in the channel region of PomB or in the cytoplasmic region of PomA suppressed both the growth inhibition and the increase in intracellular Na(+) concentration. These results suggest that the level of growth inhibition correlates with the intracellular Na(+) concentration, probably due to the Na(+) conductivity through the stator due to the mutations.

Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor

“…S1A). We previously showed that overproduction of the plug-deleted PomA/PomB complex inhibits cell growth (19), and this effect was more severely observed in E. coli than in Vibrio cells (34). We also showed that growth inhibition was correlated with the massive influx of sodium ion through the overproduced mutant stators (34); thus, we can evaluate the ion-conducting activity of the stator by this simple assay.…”

“…Localization of the stator was observed as described previously (30). Cell growth was monitored as described previously with slight modification (34). Full methods are provided in SI Materials and Methods.…”

Contribution of Many Charged Residues at the Stator-Rotor Interface of the Na + -Driven Flagellar Motor to Torque Generation in Vibrio alginolyticus

“…The PomA(R135D) mutation was not suppressed by any fliG mutations examined in this study. PomA(R135D) might cause the defect of ion conductivity previously reported in PomA(R135Q) (22,23). Regarding the pomA fliG double mutations, PomA(H136D) showed an inhibition of motility that was weakly synergistic with some fliG mutations (K284E, R301D, or D309K) ( Table 3; see also Fig.…”

“…In the Na ϩ -driven motor of V. alginolyticus, PomA-H136 was thought to be important for the stator-rotor interaction, because the PomA(H136Y) mutation inhibited the motor function without disrupting the Na ϩ influx through the stators (20,22,23). We introduced charge-reversed mutations into adjacent residues in PomA, R135D, and H136D.…”

“…Recently, it has been shown that mutations in the cytoplasmic region of PomA that disrupt the motor function also inhibit the assembly of the stator around the rotor (22). We found that among such mutations, R135Q, R207E, R215E, and D220K considerably inhibit the Na ϩ conductivity of the PomA/PomB (PomA/B) complex in an unplugged form whereas the H136Y mutation does not inhibit the Na ϩ conductivity of the PomA/B complex at all, although it results in a loss-of-function phenotype (22,23). Thus, it was suggested that the cytoplasmic region of PomA is involved not only in the interaction with FliG but also in the Na ϩ influx through the stator and that the region containing H136 is involved in the stator-rotor interaction, although this effect may be indirect.…”