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. 2000 Aug;182(15):4234-40.
doi: 10.1128/JB.182.15.4234-4240.2000.

Requirements for conversion of the Na(+)-driven flagellar motor of Vibrio cholerae to the H(+)-driven motor of Escherichia coli

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Requirements for conversion of the Na(+)-driven flagellar motor of Vibrio cholerae to the H(+)-driven motor of Escherichia coli

K K Gosink et al. J Bacteriol. 2000 Aug.

Abstract

Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H(+) or Na(+) into mechanical work. In Escherichia coli, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of Vibrio species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions in pomA, pomB, motX, or motY in Vibrio cholerae resulted in a nonmotile phenotype, whereas deletion of fliG gave a nonflagellate phenotype. fliG genes on plasmids complemented fliG-null strains of the parent species but not fliG-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A V. cholerae strain deleted of pomA, pomB, motX, and motY became weakly motile when the E. coli motA and motB genes were introduced on a plasmid. Like E. coli, but unlike wild-type V. cholerae, motility of some V. cholerae strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the E. coli proton motor components MotA and MotB can function in place of the motor proteins of V. cholerae and that the hybrid motors are driven by the proton motive force.

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Figures

FIG. 1
FIG. 1
Analyses of mutants for motility and flagellum production. Swarms in soft agar plates incubated for 8 h at 37°C (A) and electron micrographs (B) of the V. cholerae strain O395N1 (WT [wild-type]) and the motX (VcΔX), motY (VcΔY), pomAB (VcΔAB), and fliG (VcΔG) mutant derivatives as well as the motX motY pomAB (VcΔXYAB) quadruple mutant strain are shown.
FIG. 2
FIG. 2
Complementation of fliG mutants by plasmids carrying various fliG genes. (A) Amino acid sequence alignment of the E. coli and V. cholerae FliG proteins. The arrow indicates the position of the junction between the two domains in the fusion proteins. (B) Diagram of the chimeric FliG proteins. Hatched boxes indicate V. cholerae sequence, and open boxes indicate E. coli sequence. Numbers correspond to amino acid residues. (C) Swarming abilities in the presence or absence of arabinose (ara) of the E. coli (EcΔG) or V. cholerae (VcΔG) fliG deletion strains complemented by plasmids carrying the E. coli (pBAD-EcG), V. cholerae (pBAD-VcG), or chimeric (pBAD-FP1, pBAD-FP2) fliG genes. pBAD-24 is the parent vector and contains no flagellar genes. Plates were incubated for 8 h at 37°C.
FIG. 2
FIG. 2
Complementation of fliG mutants by plasmids carrying various fliG genes. (A) Amino acid sequence alignment of the E. coli and V. cholerae FliG proteins. The arrow indicates the position of the junction between the two domains in the fusion proteins. (B) Diagram of the chimeric FliG proteins. Hatched boxes indicate V. cholerae sequence, and open boxes indicate E. coli sequence. Numbers correspond to amino acid residues. (C) Swarming abilities in the presence or absence of arabinose (ara) of the E. coli (EcΔG) or V. cholerae (VcΔG) fliG deletion strains complemented by plasmids carrying the E. coli (pBAD-EcG), V. cholerae (pBAD-VcG), or chimeric (pBAD-FP1, pBAD-FP2) fliG genes. pBAD-24 is the parent vector and contains no flagellar genes. Plates were incubated for 8 h at 37°C.
FIG. 3
FIG. 3
Complementation of V. cholerae VcΔXYABG by plasmids carrying the E. coli motAB and different fliG genes. (A) Swarm circles of the quintuple deletion strain carrying the pMotAB or pACYC184 control plasmid as well as either pBAD-24, pBAD-EcG, pBAD-VcG, or pBAD-FP1. (B) Swarming behavior of the parental strain (P) and of spontaneous hypermotile derivatives (HM-1, HM-2, and HM-3) of strain VcΔXYABG carrying pMotAB and pBAD-VcG. Both soft agar plates contain arabinose. Plates were incubated overnight at 37°C.
FIG. 4
FIG. 4
Linking of the hypermotile phenotype to the pMotAB plasmid. Swarm circles in an arabinose-containing soft agar plate of V. cholerae strain VcΔXYABG carrying plasmids pMotAB and pBAD-VcG from different origins. Shown are the parental strain (P) and one of the spontaneous hypermotile derivatives (HM). Both plasmids, pMotAB and pBAD-VcG, isolated from the HM strain were transformed back into the host strain either together or with the original nonmutated plasmids. An asterisk indicates that the plasmid was derived from the hypermotile strain. Plates were incubated overnight at 37°C.
FIG. 5
FIG. 5
Effects of different medium pHs on swarm circles. Motility of the V. cholerae (VcΔG, pBAD-VcG) and E. coli (EcΔG, pBAD-EcG) control strains as well as several spontaneous hypermotile derivatives of the V. cholerae hybrid motor strain (HM-1, HM-2, and HM-3) were assayed in arabinose-containing soft agar plates with a pH of 6.5 or 8.5.

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