AN EXTREME CLOCKWISE SWITCH BIAS MUTATION IN FLIG OF SALMONELLA-TYPHIMURIUM AND ITS SUPPRESSION BY SLOW-MOTILE MUTATIONS IN MOTA AND MOTB

Pseudorevertants (second-site suppressor mutants) were isolated from a set of parental mutants of Salmonella with defects in the flagellar s witch genes fliG and fliM. Most of the suppressing mutations lay in fl agellar region IIIb of the chromosome, One fliG mutant, SJW2511, gave rise to a large number of suppressor mutations in the motility genes m otA and motB, which are in flagellar region IL, SJW2811, which has a t hree-amino-acid deletion (Delta Pro-Ala-Ala) at positions 169 to 171 o f FliG, had an extreme clockwise motor bias that produced inverse smoo th swimming (i,e,, swimming by means of clockwise rotation of a hydrod ynamically induced right-handed helical bundle), and formed Mot(-)-lik e colonies on semisolid medium. Unlike previously reported inverse-swi mming mutants, it did not shaw a chemotactic response to serine, and i t remained inverse even in a Delta che background; thus, its switch is locked in the clockwise state, The location of the mutation further u nderscores the conclusion from a previous study of spontaneous missens e mutants (V.M. Irikura, M, Kihara, S, Yamaguchi, H, Sockett, and R, M , Macnab, J, Bacteriol, 175:802-810, 1993) that a relatively localized region in the central part of the FliG sequence is critically importa nt for switching, All of the second-site mutations in motA and motB ca used some impairment of motility, both in the pseudorevertants and in a wild-type fliG background, The mechanism of suppression of the fliG mutation by the mot mutations is complex, involving destabilization of the right-handed flagellar bundle as a result of reduced motor speed, The mutations in the MotA and MotB sequences were clustered to a cons iderable degree as follows: in transmembrane helices 3 and 4 of MotA a nd the sole transmembrane helix of MotB, at helix-membrane interfaces, in the cytoplasmic domains of MotA, and in the vicinity of the peptid oglycan binding region of the periplasmic domain of MotB. The potentia l importance of Lys28 and Asp33 of the MotB sequence for proton delive ry to the site of torque generation is discussed.