Roles of the intramolecular disulfide bridge in MotX and MotY, the specific proteins for sodium-driven motors in Vibrio spp

Abstract

The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na+-driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented the formation of an intramolecular disulfide bond, which is presumably important for protein stability. Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge for proper function.

FIG. 1.

Characterization of MotY mutants. (A) VPG (1% polypeptone, 0.4% K₂HPO₄, 3% NaCl, and 0.5% [wt/vol] glycerol) plates containing 0.25% agar and 100 μg/ml kanamycin were inoculated with fresh colonies of the wild-type strain (VIO5) or the ΔmotY mutant strain (GRF2) expressing the indicated proteins from plasmids. They were incubated at 30°C for 4 h or 15 h. The plasmids and strains used are listed in Table 1. (B) Whole-cell lysates and the membrane fractions of GRF2 cells (ΔmotY) expressing the indicated proteins from plasmids and the wild-type strain [VIO5; wt (genomic)] were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as described previously (35) using anti-MotY antibodies (MotYB0079) in the absence (left) or the presence (right) of 2-mercaptoethanol (2ME). The asterisks (upper left and upper right) indicate that those samples were 1/10 the volume of the other samples. The anti-MotY antibody was raised against purified MotY produced from pKJ503 (25), which encoded C-terminally histidine-tagged MotY.

FIG. 2.

Detection of MotX in strains coexpressing various MotY mutant proteins. Whole-cell lysates of the GRF2 strain (ΔmotY) expressing the indicated proteins from plasmids and the wild-type strain [VIO5; wt (genomic)] were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as described previously (35) using anti-MotX antibodies (MotXB0080).

FIG. 3.

Characterization of the MotX mutants. (A) VPG plates containing 0.25% agar, kanamycin, and various concentrations of DTT were inoculated with fresh colonies of motX mutant cells (NMB94) expressing the indicated proteins from plasmids and incubated at 30°C for 4 h. (B) Whole-cell lysates of the NMB94 strain expressing the indicated proteins from plasmids were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as described previously (35) using anti-MotX antibodies (MotXB0080) in the absence (−) and the presence (+) of 2-mercaptoethanol (2ME). The anti-MotX antibody was raised against purified MotX produced from pKJ402, which encoded C-terminally histidine-tagged MotX.

FIG. 4.

Biotin maleimide labeling of MotX cysteine residues. NMB94 harboring pMO401 (MotX wild type) was treated with biotin maleimide (BM) and DTT or CuCl₂. The immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Biotinylated MotX was detected with streptavidin-conjugated horseradish peroxidase and chemiluminescence (A) or immunoblotting using anti-MotX antibodies (B).

FIG. 5.

Schematic diagram of MotX and MotY. MotX and MotY contain two cysteine residues and the conserved tetrapeptide CQLV. The arrowheads indicate the cleavage sites of the signal sequences (gray sections). The double-headed arrow demarcates the region containing the putative motif for peptidoglycan binding (PG motif). a.a., amino acids.