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Journal of Bacteriology, October 2008, p. 6660-6667, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00503-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Suppressor Analysis of the MotB(D33E) Mutation To Probe Bacterial Flagellar Motor Dynamics Coupled with Proton Translocation {triangledown} ,{dagger}

Yong-Suk Che,1,2 Shuichi Nakamura,1,2 Seiji Kojima,2,3 Nobunori Kami-ike,2 Keiichi Namba,1,2* and Tohru Minamino1,2*

Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan,1 Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan,2 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan3

Received 13 April 2008/ Accepted 9 July 2008

MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.

* Corresponding author. Mailing address: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-4625. Fax: 81-6-6879-4652. E-mail for T. Minamino: tohru{at}fbs.osaka-u.ac.jp. E-mail for K. Namba: keiichi{at}fbs.osaka-u.ac.jp

{triangledown} Published ahead of print on 22 August 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, October 2008, p. 6660-6667, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00503-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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