Properties of motility in Bacillus subtilis powered by the H+-coupled MotAB flagellar stator, Na+-coupled MotPS or hybrid stators MotAS or MotPB

Abstract

Bacillus subtilis has a single set of flagellar rotor proteins that interact with two distinct stator-force generators, the H+-coupled MotAB complex and the Na+-coupled MotPS complex, that energize rotation. Here, motility on soft agar plates and in liquid was assayed in wild-type B.subtilis and strains expressing only one stator, either MotAB, MotPS or hybrid MotAS or MotPB. The strains expressing MotAB or MotAS had an average of 11 flagella/cell while those expressing MotPS or MotPB had an average of seven flagella/cell, and a Mot-less double mutant had three to four flagella/cell. MotAB had a more dominant role in motility than MotPS under most conditions, but MotPS supported comparable motility to MotAB on malate-containing soft agar plating media at elevated pH and Na+. MotAB supported much faster swimming speeds in liquid than MotPS, MotAS or MotPB under all conditions, but a contribution of MotPS to wild-type swimming was discernible from differences in swimming speeds of wild-type and MotAB at elevated viscosity, pH and Na+. Swimming supported by MotPS and MotAS was stimulated by Na+ and elevated pH whereas the converse was true of MotAB and MotPB. This suggests that MotAS is Na+-coupled and MotPB is H+-coupled and that MotB and MotS are major determinants of ion-coupling. However, the swimming speed supported by MotPB, as well as MotPS and MotAS, was inhibited severely at Na+ concentrations above 300 mM whereas MotAB-dependent swimming was not. The presence of either the MotP or MotS component in the stator also conferred sensitivity to inhibition by an amiloride analogue. These observations suggest that MotP contributes to Na+-coupling and inhibition by Na+ channel inhibitors. Similarly, a role for MotA in H+-dependent stator properties is indicated by the larger effects of pH on the Na+-response of MotAS versus MotPS. Finally, optimal function at elevated viscosity was found only in MotPS and MotPB and is therefore conferred by MotP.

Figure 1

Schematic diagram of the stators of MotAB, MotPS, hybrid MotAS and hybrid MotPB highlighting the question of ion-coupling of the hybrids and indicating, with dotted arrows, the results of this study.

Figure 2

The motility behavior on soft agar plate of up-motile derivatives of MotAS and MotPB. A. Spreading of Δ ABΔ PS (negative control), MotAS-Spac (initial isolate), and MotAS-M (up-motile derivative) after 36 h of incubation at 37 C on TY medium plus 200mM NaCl, pH 8.5, without (left) or with (right) 0.6 mM IPTG, and solidified with 0.3% agar. B. Spreading of Δ ABΔ PS (negative control), MotPB-Spac (original isolate), and MotPB-M (up-motile derivative) after 24 h of incubation at 37 C on TY, pH 6.0, without (left) or with (right) 0.6 mM IPTG and solidified with 0.3% agar.

Figure 3

Effect of PVP on the swimming speed of wild-type and single Mot-bearing strains. Each strain was grown overnight on TY medium, pH 7.0, at 37 C. A 20 μl sample was used to inoculate 1ml of a fresh TY medium, and growth was continued at 37 C for 6 hours. For growth of MotAS-M and MotPB-M, 0.6mM IPTG was added during this period. After 6 hours, each cell culture (at an A₆₀₀ of about 0.45-0.50) was diluted by 50-fold with TY medium, pH 7.0, with a range of PVP contents. The swimming speed was assayed by dark field microscopy, as described under Materials and Methods. The results represent the averages of three independent experiments in each of which the swimming speed of twenty independent cells was calculated.

Figure 4

The swimming speed of the wild-type and four single Mot-bearing strains in liquid media as a function of the Na⁺ concentration and pH. Each strain was pre-grown overnight on TY medium, pH 7.0, and then inoculated into fresh TY medium and grown for 6 more hours as described in the legend to Figure 3, except that the TY medium for the 6 hour growth period contained several Na⁺ concentrations at four different pH values, pH 6.0 (open circles), 7.0 (closed circles), 8.0 (open triangles), and 8.5 (closed triangles). For the growth of MotAS-M and MotPB-M, 0.6mM IPTG was added in the each culture medium during this period; data for assays at pH 8.0 without added IPTG are shown by the gray triangles. After 6 hours, each cell culture (at A₆₀₀ of 0.45-0.50) was diluted by 50 times by same growth medium. When PVP was added to the MotPB-M medium, it was added to 1.5% wt/vol. The swimming speed was analyzed as described under Materials and Methods. Note that the swimming speed scale of the bottom four panels is different from that of the top two panels.

Figure 5

Dependence of the swimming speed on the pH at the optimal Na⁺ concentration and the dependence of swimming speed on the Na⁺ concentration at pH 7.0. (a) Relationship between the swimming speed ant the pH at the optimal Na⁺ concentration. The swimming speed of wild-type, MotAB and MotPB-M were measured at the indicated pH values in the absence of added Na⁺; MotPB-M was assayed in the presence and absence of 1.5% PVP. The swimming speed of MotPS and MotAS-M was measured in the presence of 180 mM Na⁺. The data are extracted from the data shown in Figure 4b. Relationship between the swimming speed and [Na⁺] at pH 7.0. The data are extracted from the data shown in Figure 4c. The swimming speed for the single stator strains was determined at pH 6.0, for MotAB and MotPB-M (in the presence of 1.5% PVP), and at pH 8.5 for MotPS and MotAS-M in assays conducted in 1/20TY at the indicated concentrations of Na⁺.

Figure 6

Effect of the Na⁺ channel inhibitor EIPA on motility. Wild-type, MotAB and MotPB-M strains were grown overnight on TY medium, pH 6.0, at 37 C and the MotPS and MotAS-M strains were grown overnight on TY medium, pH 8.5, at 37 C. Each strain was then inoculated into fresh TY medium at the same pH and grown for 6 hours at 37 C. During this growth period 0.6 mM IPTG was added to the culture medium for MotAS-M and MotPB-M. After 6 hours, each culture was dilute 50-fold into TY medium that was prepared as follows for particular strains: MotAB, TY medium, pH 6.0; MotPB-M, TY medium, pH 6.0, with/without 1.5% (w/v) PVP; MotPS, TY medium, pH 8.5, containing 50 mM NaCl; and MotAS-M, TY medium, pH 8.5, containing 100 mM NaCl. The swimming speed of each strain was measured in the presence of EIPA at concentrations over a range from 0-100 μM. The results represent the averages of three independent experiments in which the swimming speed of twenty independent cells was calculated by each experiment.