Mutations conferring resistance to phenamil and amiloride, inhibitors of sodium-driven motility of Vibrio parahaemolyticus

Abstract

The bacterial flagellum is powered by a rotary motor capable of turning the helical flagellar propeller at very high speeds. Energy to drive rotation is derived from the transmembrane electrochemical potential of specific ions. Ions passing through a channel component are thought to generate the force to power rotation. Two kinds of motors, dependent on different coupling ions, have been described: proton-driven and sodium-driven motors. There are four known genes encoding components of the sodium-powered polar flagellar motor in Vibrio parahaemolyticus. Two, which are characterized here, are homologous to genes encoding constituents of the proton-type motor (motA and motB), and two encode components unique to the sodium-type motor (motX and motY). The sodium-channel-blocking drugs phenamil and amiloride inhibit rotation of the polar flagellum and therefore can be used to probe the architecture of the motor. Mutants were isolated that could swim in the presence of phenamil or amiloride. The majority of the mutations conferring phenamil-resistant motility alter nucleotides in the motA or motB genes. The resultant amino acid changes localize to the cytoplasmic face of the torque generator and permit identification of potential sodium-interaction sites. Mutations that confer motility in the presence of amiloride do not alter any known component of the sodium-type flagellar motor. Thus, evidence supports the existence of more than one class of sodium-interaction site at which inhibitors can interfere with sodium-driven motility.

Figure 1

Topology of MotA and MotB, showing conserved residues within the predicted transmembrane (TM) domains and sites conferring phenamil resistance. Shading denotes conservation of amino acids observed in MotA and MotB sequences from E. coli, Rhodobacter sphaeroides, Bacillus subtilis, and V. parahaemolyticus (sequences for lateral and polar Mot proteins). Darkly shaded circles indicate well conserved amino acids (consensus >3), and lightly shaded circles indicate a match of the V. parahaemolyticus residue with at least one other as identified by the clustal w program (40). Invariant residues, conserved among organisms, are designated by the dark boxes. Nonshaded amino acids circled in black are nonconserved and nonpolar, whereas those within diamonds are nonconserved and polar. Filled black symbols indicate amino acids altered in phenamil-resistant mutants.

Figure 2

Motility of wild-type and phenamil-resistant strains in M agar containing 300 mM NaCl and 40 μM phenamil after 10-hr incubation. Relative rates of expansion were normalized to the expansion rate of LM4609. The normalized rates are: LM4616 (0.8 ± .07), LM4641 (1.04 ± .03), LM4647 (0.90 ± .03), LM4629 (1.8 ± .18), LM4613 (1.23 ± .09), LM4606 (1.27 ± .03), LM4603 (1.11 ± .06), LM4614 (.81 ± .05), LM4609 (1.0 ± .05) LM4639 (1.0 ± .07), and LM4598 (1.0 ± .12).

Figure 3

Motility conferred by plasmid pLM2059 in M agar with 300 mM NaCl: (A) without phenamil after 6 hr; (B) with 40 μM phenamil after 12 hr. Strains were inoculated in duplicate in each row. From top to bottom: row 1 is LM1017; row 2 is LM4660 (motA∷TnphoA)/pLM2058; row 3 is LM4660 (motA∷TnphoA)/pLM2059; and row 4 is LM4467.

Figure 4

Relative expansion rates of selected phenamil-resistant mutants normalized to the expansion rate of LM1017 in M agar supplemented with the indicated salt. Rates are the mean of three determinations, and error bars show standard deviation.

Figure 5

Motility of wild-type and amiloride-resistant strains in the presence and absence of amiloride. (A) M agar with 50 mM NaCl, 250 mM KCl, and 2.25 mM amiloride after 12 hr; (B) M agar with 300 mM NaCl after 7 hr; and (C) M agar with 75 mM LiCl after 21 hr. Expansion rates measured in the presence of amiloride were normalized to LM4303 and in the absence of amiloride were normalized to LM1017. Strains (and expansion rates in A, B, and C) are indicated. Top row, from left to right: LM4267 (0.42 ± .06, 0.94 ± .03, 0.76 ± .16), LM4268 (1.09 ± .04, 1.26 ± .07, 2.63 ± .14), and LM4601 (1.56 ± .02, 1.52 ± .08, 2.0 ± .13). Bottom row: LM4303 (1.0, 1.46 ± .13, 1.28 ± .02), LM4300 (1.64 ± .08, 1.91 ± .21, 1.29 ± .06), and LM1017 (ND, 1.0, 1.0).