The Systematic Substitutions Around the Conserved Charged Residues of the Cytoplasmic Loop of Na 1 -driven Flagellar Motor Component PomA Tomohiro Yorimitsu 1 , Yoshiyuki Sowa 2 , Akihiko Ishijima 3 Toshiharu Yakushi 1 and Michio Homma 1 * 1 Division of Biological Science Graduate School of Science Nagoya University Chikusa-Ku, Nagoya 464-8602 Japan 2 Department of Biophysical Engineering, Osaka University Toyonaka, Osaka 560-8531 Japan 3 Department of Applied Physics School of Engineering Nagoya University Chikusa-Ku, Nagoya 464-8603 Japan PomA, a homolog of MotA in the H þ -driven flagellar motor, is an essential component for torque generation in the Na þ -driven flagellar motor. Previous studies suggested that two charged residues, R90 and E98, which are in the single cytoplasmic loop of MotA, are directly involved in this process. These residues are conserved in PomA of Vibrio alginolyticus as R88 and E96, respectively. To explore the role of these charged residues in the Na þ -driven motor, we replaced them with other amino acids. However, unlike in the H þ -driven motor, both of the single and the double PomA mutants were functional. Several other positively and negatively charged residues near R88 and E96, namely K89, E97 and E99, were neutralized. Motility was retained in a strain producing the R88A/K89A/E96Q/E97Q/E99Q (AAQQQ) PomA protein. The swimming speed of the AAQQQ strain was as fast as that of the wild- type PomA strain, but the direction of motor rotation was abnormally counterclockwise-biased. We could, however, isolate non-motile or poorly motile mutants when certain charged residues in PomA were reversed or neutralized. The charged residues at positions 88–99 of PomA may not be essential for torque generation in the Na þ -driven motor and might play a role in motor function different from that of the equivalent residues of the H þ -driven motor. q 2002 Elsevier Science Ltd. All rights reserved Keywords: energy transduction; flagella; motor protein; sodium-driven motor; Vibrio alginolyticus *Corresponding author Introduction Bacterial flagella derive their energy for rotation from the electrochemical potential of a specific ion, either H þ or Na þ , across the cell membrane. 1,2 This energy is harnessed by a motor complex located at the base of the flagellum. From studies of Salmonella typhimurium and Escherichia coli, which have H þ -driven flagellar motors, three proteins, MotA, MotB, and FliG, are thought to be involved directly in the energy conversion to generate the rotation from the ion flux. 3–6 MotA has four transmembrane segments and a single large cytoplasmic loop, 7 and MotB has a single transmembrane segment. 8 The C-terminal domain of MotB appears to be bound to peptido- glycan, and the MotA/B complex is proposed to act as a stator. 8 – 11 The MotA/B complex functions as a H þ channel, converting the H þ flux into mechanical power for flagellar rotation. 12 – 16 It is speculated that conformational changes are induced by ion flow and are transferred to the cytoplasmic region of MotA, and that interactions between this region of MotA and FliG of the rotor complex drive rotation. FliG is part of the switch complex, which includes the FliM and FliN proteins, which together make up the C-ring. 6,17,18 Unlike E. coli and S. typhimurium, some Vibrio species, including Vibrio alginolyticus, V. parahaemo- lyticus, and V. cholerae, and alkalophilic Bacillus species are known to have Na þ -driven flagella. 19,20 In Vibrio, PomA and PomB, which are homologs of MotA and MotB, respectively, are essential for the function of the Na þ -driven motor. Like MotA and MotB, PomA and PomB have been shown to 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: g44416a@nucc.cc.nagoya-u.ac.jp Abbreviations used: CCW, counterclockwise; CW, clockwise. doi:10.1016/S0022-2836(02)00426-6 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 320, 403–413