Two Redundant Sodium-Driven Stator Motor Proteins Are Involved in Aeromonas hydrophila Polar Flagellum Rotation

Abstract

Motility is an essential characteristic for mesophilic Aeromonas strains. We identified a new polar flagellum region (region 6) in the A. hydrophila AH-3 (serotype O34) chromosome that contained two additional polar stator genes, named pomA 2 and pomB 2. A. hydrophila PomA 2 and PomB 2 are highly homologous to other sodium-conducting polar flagellum stator motors as well as to the previously described A. hydrophila AH-3 PomA and PomB. pomAB and pomA 2 B 2 were present in all the mesophilic Aeromonas strains tested and were independent of the strains' ability to produce lateral flagella. Unlike MotX, which is a stator protein that is essential for polar flagellum rotation, here we demonstrate that PomAB and PomA 2 B 2 are redundant sets of proteins, as neither set on its own is essential for polar flagellum motility in either aqueous or high-viscosity environments. Both PomAB and PomA 2 B 2 are sodium-coupled stator complexes, although PomA 2 B 2 is more sensitive to low concentrations of sodium than PomAB. Furthermore, the level of transcription in aqueous and high-viscosity environments of pomA 2 B 2 is reduced compared to that of pomAB. The A. hydrophila AH-3 polar flagellum is the first case described in which two redundant sodium-driven stator motor proteins (PomAB and PomA 2 B 2 ) are found.The bacterial flagellum usually is powered by a reversible rotator motor at the base of the flagellum structure, which uses energy from either the proton or sodium ion gradient to drive the rotation of the flagellum filament. The flagellum motor is divided into two substructures: the stator and the rotor. The rotor is composed of the FliM and FliN proteins, which form the C ring structure, and the FliG protein. These three proteins act as a switch that controls the direction of flagellum rotation, clockwise or counterclockwise. The stator is the stationary component of the motor and consists of different proteins surrounding the rotor, which constitute proton or sodium ion channels that couple the flow of ions to flagellum rotation (8,10

Aeromonas hydrophila motY is essential for polar flagellum function, and requires coordinate expression of motX and Pom proteins

“…B. subtilis MotAB and S. oneidensis MotAB-MotXY are proton-driven, whereas B. subtilis MotPS and S. oneidensis PomAB-MotXY are sodium-driven. In contrast, both A. hydrophila polar flagellum stator complexes are sodium-driven, although they have different sensitivities to sodium variations (Wilhelms et al, 2009).…”

“…Previously, we demonstrated that A. hydrophila has two redundant sodium-dependent polar stators, PomAB and PomA 2 B 2 , which allow bacteria to swim in liquid medium and soft agar (Wilhelms et al, 2009). Since both polar flagellum stators are sodium-dependent, and subunits PomA/PomA 2 and PomB/PomB 2 are homologous proteins, they might constitute functional natural hybrid motors.…”

“…Since both polar flagellum stators are sodium-dependent, and subunits PomA/PomA 2 and PomB/PomB 2 are homologous proteins, they might constitute functional natural hybrid motors. To analyse this we transferred the pomB, pomB 2 , pomAB and pomA 2 B 2 genes to three mutants: AH-4473, unable to express pomAB-A 2 B 2 (Wilhelms et al, 2009); AH-4452, which expresses pomA 2 alone (Wilhelms et al, 2009); and AH4471 : : pomB, which expresses pomA alone (see Methods). The three mutants had a polar flagellum, but were unable to swim in liquid medium and showed reduced radial expansion in soft agar in relation to the wild-type, AH-3 (Fig.…”

“…3). Furthermore, the motility of the AH-3 : : motY and a non-polar flagellum mutant, flhA : : Km (AH-4442) (Wilhelms et al, 2009), was assayed in soft agar with the sodium-driven flagellar inhibitor amiloride or the protonophore CCCP. These assays showed that the motility of both mutants was strongly inhibited with an increase in the CCCP concentration, whereas no decrease in radial expansion was induced by amiloride treatment (Fig.…”

“…Triparental mating with the mobilizing strain HB101/pRK2073 was used to transfer the recombinant plasmid into the A. hydrophila AH-405 rifampicin-resistant (Rif r ) strain to obtain the defined insertion mutant AH-3 : : motY, selecting for Rif r and Km r . To obtain the AH-4471 : : pomB mutant, the recombinant plasmid pFS-MOTB (Canals et al, 2006a) was transferred by triparental mating with the mobilizing strain HB101/ pRK2073 into the A. hydrophila AH-4471 rifampicin-resistant mutant (AH-405DpomA 2 B 2 ) (Wilhelms et al, 2009) to obtain the AH-4471 : : pomB mutant (AH-405DpomA 2 B 2 : : pomB), selecting for Rif r and Km r . The correct construction of mutants was verified by Southern blot hybridization.…”