Cytoplasmic component of the bacterial flagellar motor

Abstract

Bacterial flagellar basal bodies isolated using standard protocols do not possess the proteins required for motor function. Five motor proteins have been identified to date based on genetic analysis in Escherichia coli and Salmonella typhimurium. Two of these, MotA and MotB are required for motor rotation only. These form a transmembrane complex. Three others, FliG, FliM and FliN are all required not only for motor rotation but also for flagellar synthesis and switching. These have been postulated to form a cytoplasmically based structural complex. In this study, novel bell-like cytoplasmic flagellar basal structures were revealed upon electron microscopic examination of freeze-substituted preparations of cytoplasm-free bacterial cell envelopes. An isolation protocol was then developed that has enabled routine preparations of flagella possessing such basal structures. Once isolated, these structures dissociated rapidly under various conditions. Their lability was exploited in examining the basal bodies from point mutants of fliG, fliM and fliN genes. In mutants with lesions giving rise to paralysed flagella ({dollar}Mot\sp-{dollar} phenotype) in each of the three genes, the bell structures did not co-isolate with the basal bodies. But the co-isolation of bell was unaffected in a mutant deleted for motA and motB genes, and a mutant deleted for all the chemotaxis genes. Thus FliG, FliM and FliN proteins play a crucial role in the integrity of the bell shaped structures. However, these structures were unaffected in point mutants of fliM with switch defects ({dollar}Che\sp-{dollar} phenotype) including lesions in the immediate vicinity of {dollar}Mot\sp-{dollar} mutations. This demonstrates a specific effect of the {dollar}Mot\sp-{dollar} mutations. Furthermore, bell structures isolated from a temperature sensitive {dollar}Mot\sp-{dollar} mutant in fliG dissociated more rapidly at non-permissive temperature compared to those from wild type, indicating that FliG is an integral component of the bell structures. In immunoblots, the isolated flagellar preparations with belled basal bodies reacted positively with anti-FliG antibody. In immuno-gold labelling experiments, the same antibody decorated the bell. Taken together these results suggest the bell is at least in part comprised of FliG protein.