Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Jul 17;19(14):3639-48.
doi: 10.1093/emboj/19.14.3639.

Coupling ion specificity of chimeras between H(+)- and Na(+)-driven motor proteins, MotB and PomB, in Vibrio polar flagella

Y Asai  1 , I Kawagishi, R E Sockett, M Homma
Affiliations

Coupling ion specificity of chimeras between H(+)- and Na(+)-driven motor proteins, MotB and PomB, in Vibrio polar flagella

Y Asai et al. EMBO J. .

Abstract

We have shown that a hybrid motor consisting of proton-type Rhodobacter sphaeroides MotA and sodium-type VIBRIO: alginolyticus PomB, MotX and MotY, can work as a sodium-driven motor in VIBRIO: cells. In this study, we tried to substitute the B subunits, which contain a putative ion-binding site in the transmembrane region. Rhodobacter sphaeroides MotB did not work with either MotA or PomA in Vibrio cells. Therefore, we constructed chimeric proteins (MomB), which had N-terminal MotB and C-terminal PomB. MomB proteins, with the entire transmembrane region derived from the H(+)-type MotB, gave rise to an Na(+) motor with MotA. The other two MomB proteins, in which the junction sites were within the transmembrane region, also formed Na(+) motors with PomA, but were changed for Na(+) or Li(+) specificity. These results show that the channel part consisting of the transmembrane regions from the A and B subunits can interchange Na(+)- and H(+)-type subunits and this can affect the ion specificity. This is the first report to have changed the specificity of the coupling ions in a bacterial flagellar motor.

PubMed Disclaimer

Figures

None
Fig. 1. (A) Amino acid alignments of V.alginolyticus PomB and R.sphaeroides MotB. White letters in black boxes and arrowheads show identical residues and the junction site of chimeric protein, respectively. Gray bars indicate putative transmembrane regions. (B) Restriction maps of plasmids. White boxes and the direction of solid arrowheads in the boxes indicate the vector part of pSU41 and the direction of transcription from the lac promoter, respectively. Inserted fragments from V.alginolyticus and R.sphaeroides, respectively, are indicated by a black or gray bold line. The hatched part of pRED1 is the region of the native R.sphaeroides promoter. The open arrows show the coding regions of pomA, pomB, motA and motB. VaPomB, V.alginolyticus PomB; RsMotB, R.sphaeroides MotB; H, HindIII; Xb, XbaI; B, BamHI; Sc, SacI; E, EcoRI; Sp, SpeI; Sh, SphI; X, XhoI; Sm, SmaI; Sl, SalI.
None
Fig. 2. Swarming abilities of transformants. Fresh colonies were inoculated in 0.3% agar VPG plates containing 100 µg/ml kanamycin and incubated at 30°C for 5 h. The pomAB mutant (NMB191) was used as the host strain. Proteins expressed in each strain, various combinations of A and B subunits of the proton-driven or sodium-driven motors (A), each MomB protein with proton-type MotA (B) and each MomB protein with sodium-type PomA (C), are noted around swarms.
None
Fig. 3. Detection of each protein by the anti-peptide antibody. NMB191 cells expressing each protein were cultured to exponential phase, harvested and suspended in distilled deionized water. Immunoblottings were carried out using anti-VaPomA (A), anti-VaPomB (B), anti-RsMotA (C) and anti-RsMotB antibody (D).
None
Fig. 4. Swimming speed of NMB191 cells expressing PomAB (pYA303), MomB5 with PomA or MomB6 with PomA. The cells were suspended in Tris-motility buffer (TMN50) and diluted 100-fold into buffers containing various concentration of Na+ (open squares) or Li+ (closed squares) as coupling ions, and the swimming speeds were measured.
None
Fig. 5. Competition of Na+ with Li+ for swimming speed of each strain. NMB191 cells expressing PomAB(pYA303), PomA+MomB6, RsMotA+PomB or RsMotA+MomB6 were suspended in buffers at various concentrations of Na+ with 200 mM K+ (open squares) or 200 mM Li+ (closed diamonds) and their swimming speeds were measured. The total salt concentration was adjusted to 300 mM with K+.
None
Fig. 6. The swimming speeds of each strain were measured at various concentrations of the specific inhibitor of sodium motor, phenamil, in TMN50. The results of MomB chimeras with RsMotA (upper panel) or with PomA (lower panel) are shown.
None
Fig. 7. Summary of the chimeric protein features. Black and gray boxes show regions that are derived from VaPomB and RsMotB, respectively. TM, transmembrane region; PGB, peptidoglycan-binding motif; W.T., wild type; MpaS, motility that is phenamil sensitive; N.F., non-functional; Mpar, motility that is phenamil resistant; Na+, motility coupling to sodium ion; Li+, motility coupling to lithium ion; ↓, impaired motility.
See this image and copyright information in PMC

References

    1. Armitage J.P. and Macnab,R.M. (1987) Unidirectional, intermittent rotation of the flagellum of Rhodobacter sphaeroides. J. Bacteriol., 169, 514–518. - PMC - PubMed
    1. Asai Y., Kojima,S., Kato,H., Nishioka,N., Kawagishi,I. and Homma,M. (1997) Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium. J. Bacteriol., 179, 5104–5110. - PMC - PubMed
    1. Asai Y., Kawagishi,I., Sockett,E. and Homma,M. (1999) Hybrid motor with the H+- and Na+-driven components can rotate Vibrio polar flagella by using sodium ions. J. Bacteriol., 181, 6332–6338. - PMC - PubMed
    1. Asai Y., Shoji,T., Kawagishi,I. and Homma,M. (2000) Cysteine-scanning mutagenesis of the periplasmic loop regions of PomA, a putative channel component of the sodium-driven flagellar motor in Vibrio alginolyticus. J. Bacteriol., 182, 1001–1007. - PMC - PubMed
    1. Atsumi T., Sugiyama,S., Cragoe,E.J.,Jr and Imae,Y. (1990) Specific inhibition of the Na+-driven flagellar motors of alkalophilic Bacillus strains by the amiloride analog phenamil. J. Bacteriol., 172, 1634–1639. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources