| Abstract: | Pseudorevertants (second-site suppressor mutants) were isolated from a set of parental mutants of Salmonella with defects in the flagellar switch genes fliG and fliM. Most of the suppressing mutations lay in flagellar region IIIb of the chromosome. One fliG mutant, SJW2811, gave rise to a large number of suppressor mutations in the motility genes motA and motB, which are in flagellar region II. SJW2811, which has a three-amino-acid deletion (delta Pro-Ala-Ala) at positions 169 to 171 of FliG, had an extreme clockwise motor bias that produced inverse smooth swimming (i.e., swimming by means of clockwise rotation of a hydrodynamically induced right-handed helical bundle), and formed Mot(-)-like colonies on semisolid medium. Unlike previously reported inverse-swimming mutants, it did not show a chemotactic response to serine, and it remained inverse even in a delta che background; thus, its switch is locked in the clockwise state. The location of the mutation further underscores the conclusion from a previous study of spontaneous missense 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 important for switching. All of the second-site mutations in motA and motB caused 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 considerable degree as follows: in transmembrane helices 3 and 4 of MotA and the sole transmembrane helix of MotB, at helix-membrane interfaces, in the cytoplasmic domains of MotA, and in the vicinity of the peptidoglycan binding region of the periplasmic domain of MotB. The potential importance of Lys28 and Asp33 of the MotB sequence for proton delivery to the site of torque generation is discussed. |
