Molecular cloning techniques were used to construct lambda-E. coli hybrid bacteriophage carrying genes involved in bacterial flagellar motility (mot) and chemotaxis (cheA). A series of hybrid bacteriophage without each of these genes was also prepared. When paralyzed mutants of E. coli were infected with lambda that carried the mot gene, the ability of the bacterium to swim was rapidly restored. The restoration of motility was the result of the synthesis and insertion into the cell membrane of a protein with an apparent molecular weight of 31,000 (the Mot protein).Another polypeptide with a mobility on acrylamide gel electrophoresis which corresponded to a molecular weight of 39,000 was associated with the cheA gene. The presence of this polypeptide alone was not sufficient to restore chemotactic activity to mutant cheA strains. It was suggested that only a portion of the cheA gene was cloned, and thus the 39,000 protein may be a partial product of the cheA gene, or the product of a second mot gene.Bacterial flagella function by rotating, and their rotation is regulated so that it responds to information provided by specific chemoreceptors on the surface of the cell (1-3). The genetic basis for flagellar structure, function, and regulation has been analyzed in detail (4-6). However, very little is known about the biochemistry involved in these processes. The flagellar organelle can be isolated from Escherichia coli and Bacillus subtilis (7,8). It is composed of a long helical filament and a complex basal structure which is tightly bound to the outer membrane of the cell. The basal structure has been purified (9) and its components were characterized by gel electrophoresis. It was found to contain eleven different polypeptide chains (10). It is clear, however, that these components do not account for all of the gene products involved in flagellar function. This conclusion is based on a comparison of the proteins obtained from flagella isolated from mot amber mutants and wild-type strains. The mot gene appears to control the synthesis of a product which is directly involved in flagellar activity. Mutations in (16,17).To understand the mechanisms involved in motility, and in the assembly and function of flagella, it is necessary to identify the products of all of the genes that contribute to these processes.One approach involves an isolation of the genes and detection of their products directly. In a previous report (18), we showed that hybrid E. coli-lambda phage carrying genes that control flagellin synthesis could be obtained and used to determine the regulation of flagellin production. We will now show how hybrid lambda bacteriophage can be used to assay for other flagella-specific proteins which include the mot gene product.
MATERIALS AND METHODSConstruction and Propagation of Hybrid Lambda. Hybrid lambda (X) E. coli DNA molecules were constructed using XgtXc DNA as the vehicle and fragments of DNA obtained by EcoRI endonuclease digestion of F factor carrying flagellar genes. The isolation of the F ...