Mutations alter the sodium versus proton use of a Bacillus clausii flagellar motor and confer dual ion use on Bacillus subtilis motors

Abstract

Bacterial flagella contain membrane-embedded stators, Mot complexes, that harness the energy of either transmembrane proton or sodium ion gradients to power motility. Use of sodium ion gradients is associated with elevated pH and sodium concentrations. The Mot complexes studied to date contain channels that use either protons or sodium ions, with some bacteria having only one type and others having two distinct Mot types with different ion-coupling. Here, alkaliphilic Bacillus clausii KSM-K16 was shown to be motile in a pH range from 7 to 11 although its genome encodes only one Mot (BCl-MotAB). Assays of swimming as a function of pH, sodium concentration, and ion-selective motility inhibitors showed that BCl-MotAB couples motility to sodium at the high end of its pH range but uses protons at lower pH. This pattern was confirmed in swimming assays of a statorless Bacillus subtilis mutant expressing either BCl-MotAB or one of the two B. subtilis stators, sodium-coupled Bs-MotPS or proton-coupled BsMotAB. Pairs of mutations in BCl-MotB were identified that converted the naturally bifunctional BCl-MotAB to stators that preferentially use either protons or sodium ions across the full pH range. We then identified trios of mutations that added a capacity for dual-ion coupling on the distinct B. subtilis Bs-MotAB and Bs-MotPS motors. Determinants that alter the specificity of bifunctional and single-coupled flagellar stators add to insights from studies of other ion-translocating transporters that use both protons and sodium ions.alkaliphile ͉ MotAB ͉ MotPS ͉ motility ͉ bacteria T he bacterial flagella that support swimming in liquid and on surfaces are rigid, propeller-like structures. Their rotation is powered by electrochemical gradients of either protons or sodium ions across the cytoplasmic membrane (1, 2). Ion channels are part of membrane-embedded stator structures at the base of the flagella. They play a critical role in the transduction of chemiosmotic energy into mechanical energy. The stators of the flagellar motor are most often called Mot complexes. Mot complexes are thought to contain four MotA-like proteins, each of which has four transmembrane segments (TMS), and two MotB-like proteins, each of which has a single TMS (1-3). The MotB-like proteins have a major role in determining ioncoupling specificity (4, 5). Initially, only proton-coupled motility was documented in bacteria, e.g., in Escherichia coli (1, 2). However, work of Imae and colleagues in the 1980s showed that extremely alkaliphilic Bacillus species use only sodium ion gradients for motility (6, 7). This was followed by evidence that Vibrio alginolyticus and Bacillus subtilis possess two distinct stators with different ion specificities that make contributions to swimming under particular physical-chemical conditions of pH, salinity, and viscosity (8-12).To date, only a limited number of additional flagellar stators have been directly probed for a capacity to use sodium as the coupling ion. The MotAB-like system of alkaliphilic Bacillus clausi...

Evolution of Na+ and H+ bioenergetics in methanogenic archaea

“…This question was solved recently for the marine organism M. acetivorans [44]. Ion promiscuity has been observed before for other Na + /H + -coupled transporters, such as the flagellar motor [45] or solute transporter [46,47], but this was the first demonstration of a promiscuous ATP synthase. Under these conditions, an ATPdependent primary and electrogenic Na + transport could be observed.…”

Promiscuous archaeal ATP synthase concurrently coupled to Na + and H + translocation

“…It is also conceivable, however, that ATP synthesis may in some cases be driven directly by both Na + and H + gradients (20). This phenomenon has never been reported for any ATP synthase in physiological conditions, but such promiscuity has been observed in other biological systems for example, some cation/ solute symporters (22,23) and motility channels (24).…”

The Function of the Na + -Driven Flagellum of Vibrio cholerae Is Determined by Osmolality and pH

“…They are composed of the subunits MotA and MotB in H ϩ -dependent flagella or PomA and PomB in Na ϩ -dependent flagella (5). Stator complexes which, depending on the environment of the cell, may use both Na ϩ and H ϩ (6) or Na ϩ and K ϩ (7) have also been described. A stator complex, or force-generating unit, is composed of four PomA (MotA) and two PomB (MotB) subunits (PomA 4 B 2 or MotA 4 B 2 ) and is anchored to peptidoglycan in the periplasm by a specific binding region of PomB (1,8).…”