The complex flagellar torque generator of Pseudomonas aeruginosa

Abstract

Flagella act as semirigid helical propellers that are powered by reversible rotary motors. Two membrane proteins, MotA and MotB, function as a complex that acts as the stator and generates the torque that drives rotation. The genome sequence of Pseudomonas aeruginosa PAO1 contains dual sets of motA and motB genes, PA1460-PA1461 (motAB) and PA4954-PA4953 (motCD), as well as another gene, motY (PA3526), which is known to be required for motor function in some bacteria. Here, we show that these five genes contribute to motility. Loss of function of either motAB-like locus was dispensable for translocation in aqueous environments. However, swimming could be entirely eliminated by introduction of combinations of mutations in the two motAB-encoding regions. Mutation of both genes encoding the MotA homologs or MotB homologs was sufficient to abolish motility. Mutants carrying double mutations in nonequivalent genes (i.e., motA motD or motB motC) retained motility, indicating that noncognate components can function together. motY appears to be required for motAB function. The combination of motY and motCD mutations rendered the cells nonmotile. Loss of function of motAB, motY, or motAB motY produced similar phenotypes; although the swimming speed was only reduced to approximately 85% of the wild-type speed, translocation in semisolid motility agar and swarming on the surface of solidified agar were severely impeded. Thus, the flagellar motor of P. aeruginosa represents a more complex configuration than the configuration that has been studied in other bacteria, and it enables efficient movement under different circumstances.

FIG. 1.

P. aeruginosa flagellar motor genes. Open reading frames are indicated by arrows, and the directions of transcription are indicated by the arrowheads. The solid triangles indicate the positions of the Tn5 (Tet^r) insertions. Restriction sites that were used to create drug resistance cassette mutations are indicated as follows: st, StuI; rv, EcoRV; sm, SmaI; and m, MscI. The motAB locus, the rpmAB locus (which we renamed motCD due to its role in motor function), and the motY gene were cloned from strain PAK by using high-fidelity PCR. A gentamicin resistance cassette was inserted into the StuI site to make motA2::Gen^r, into the EcoRV site to make motB2::Gen^r, into the StuI and EcoRV sites to make ΔmotAB3::Gen^r, and into the SmaI sites to make ΔmotCD6. A tetracycline cassette was introduced into StuI sites to create ΔmotC7::Tet^r, into SmaI sites to create ΔmotCD8, and into MscI sites to create ΔmotY2::Tet^r. In-frame deletions that removed the coding regions for motAB, motCD, and motY in strain PAO1 were also created.

FIG. 2.

Transposon insertions in either the motAB or motCD locus affect movement in semisolid motility agar. The strains and normalized rates of radial expansion were as follows: LMP4 (motA1::Tn5Tet^r), 0.66 ± 0.10; LMP7 (motC1::Tn5Tet^r), 0.51 ± 0.05; and LMP8 (motD2::Tn5Tet^r), 0.53 ± 0.06. The rates of expansion were normalized to the rate for reference strain LMP1, which contained Tn5 (Tet^r) and showed wild-type swimming motility. Motility plates contained 50 μg of tetracycline per ml and were incubated at 30°C for 20 h. Similar results were observed in plates without an antibiotic.

FIG. 3.

Deletion of either the motAB or motCD locus affects motility in semisolid agar. (A) Motility of strains LMP7, LMP8, LMP13, and wild-type strain PAK. The normalized rates of radial expansion were as follows: LMP7 (motC1::Tn5Tet^r), 0.51 ± 0.05; LMP8 [motD2::Tn5 (Tet^r)], 0.53 ± 0.06; and LMP13 (ΔmotCD6::Gen^r), 0.55 ± 0.03. (B) Motility of strains LMP5 (motA2::Gen^r), LMP6 (motB3::Gen^r), LMP9 (ΔmotAB4::Gen^r), and LMP2. The reference strain LMP2 contained Tn5 (Gen^r) and showed wild-type swimming motility. Radial expansions rates were not calculated because the strains failed to significantly migrate in the motility plate. (C) Motility of strains LMP13, LMP9, and LMP2. The plate in panel A contained no antibiotic and was incubated for 15 h at 30°C. The plates in panels B and C contained 50 μg of gentamicin per ml and were incubated for 20 h at 30°C.

FIG. 4.

Swimming speeds of the wild type and motor mutants. The average swimming rates were determined by using the Hobson Tracker for cells growing exponentially in liquid LB medium. Strains PAK, LMP44 (ΔmotC7), LMP7 (motC1), LMP8 (motD2), LMP13 (ΔmotCD6), LMP5 (motA2), LMP6 (motB3), LMP9 (ΔmotAB4), LMP54 (ΔmotY2), LMP55 (ΔmotAB4 ΔmotY2), LMP60 (ΔmotCD6 ΔmotY2), LMP18 (motA2 motD2), LMP49 (motB3 motC7), LMP17 (motA2 motC1), and LMP20 (motB3 motD2) were examined.

FIG. 5.

Disruption of genes in both motAB-like loci abolishes swimming motility. (Plate A) Triple gene knockouts. Strains LMP15 (ΔmotAB4 motC1), LMP16 (ΔmotAB4 motD2), LMP57 (flgC1; nonmotile Fla⁻ control), LMP48 (motA2 ΔmotCD8), LMP50 (motB3 ΔmotCD8), and PAK were included. (Plate B) Double gene knockouts. Strains LMP57 (flgC1; nonmotile control), LMP47 (motA2 ΔmotC7), LMP20 (motB3 motD2), LMP18 (motA2 motD2), LMP49 (motB3 ΔmotC7), and PAK were included. The motility plates were incubated overnight at room temperature.

FIG. 6.

The fifth motor gene, motY. Strains LMP9 (ΔmotAB), LMP54 (ΔmotY), and LMP55 (ΔmotAB ΔmotY) were included in plate A, and strains LMP13 (ΔmotCD), LMP54 (ΔmotY), and LMP60 (ΔmotCD ΔmotY) were included in plate B. The motility plates were incubated overnight at room temperature.

FIG. 7.

Swimming and swarming phenotypes of mot mutants in PAK and PAO1 backgrounds. Swimming motility plates contained 0.325% semisolid agar, and solidified swarming plates contained 0.5% agar for PAK strains and 0.6% agar for PAO1 strains. The PAK strains used (top row of plates) were PAK, LMP9 (ΔmotAB), LMP13 (ΔmotCD), LMP54 (ΔmotY), LMP47 (motA2 motC7), LMP20 (motB3 motD2), LMP57 (flgC1), LMP18 (motA2 motD2), and LMP49 (motB3 motC7). The PAO1 strains used (bottom row of plates) were MPAO1, PAO1001 (ΔmotAB in MPAO1), PAO1002 (ΔmotCD in MPAO1), PAO2000 (ΔmotY in IgPAO1), and IgPAO1. Swimming plates were incubated overnight at room temperature, and swarming plates were incubated for 2 days at 37°C.

FIG. 8.

Chemotactic responses of PAK, PAO1, and isogenic mutants in the presence of an agarose plug containing arginine (ARG) or chemotaxis buffer (CB) containing no chemoattractant. A positive response resulted in a light ring around the dark plug.

FIG. 9.

Movement in motility agar solidified with different concentrations of agar. Tryptone motility agar was solidified with the concentrations of agar indicated. Strains LMP57 (flgC1), LMP47 (motA2 motC7), LMP18 (motA2 motD2), LMP49 (motB3 motC7), LMP9 (ΔmotAB), LMP54 (ΔmotY), LMP55 (ΔmotAB ΔmotY), LMP60 (ΔmotCD ΔmotY), and PAK were used. The plates were incubated overnight at room temperature.