The identification of the mot gene product with Escherichia coli-lambda hybrids.

Abstract

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 ...

Analysis of the Polar Flagellar Gene System of Vibrio parahaemolyticus

“…Loss-of-function mutations in only two genes (motA and motB) yield the Mot phenotype, which is a flagellated but paralyzed cell. Insertion of the torque-generating components of the motor into the membrane is not required for assembly of the E. coli flagellar organelle, and expression of mot genes occurs at the final stage in the hierarchy of expression (57). The phenotypes of V. parahaemolyticus motility mutants differed from E. coli.…”

Downregulation of the motA gene delays the escape of the obligate predator Bdellovibrio bacteriovorus 109J from bdelloplasts of bacterial prey cells

“…The predatory lifecycle of B. bacteriovorus revolves around its ability to swim. In E. coli or Salmonella typhimurium, disruption of the motA or motB gene results in paralysis of bacterial motility despite the presence of an intact flagellum (Blair & Berg, 1988;Silverman et al, 1976). The MotA and MotB proteins constitute the stationary part of the flagellar motor (Chun & Parkinson, 1988;De Mot & Vanderleyden, 1994;Garza et al, 1995) and form a complex that facilitates the traffic of protons from the periplasmic space into the cytosol of the bacterial cell (Blair & Berg, 1990;Braun et al, 1999;Stolz & Berg, 1991;Zhou et al, 1998), which is required for flagellar rotation (Manson et al, 1977).…”

Co-overproduction and localization of the Escherichia coli motility proteins motA and motB

“…For a review see, for example, reference 14. What is actually known of the cellular location of the Mot proteins? They are normally present in amounts too low to be detected biochemically, but programmed expression of the genes cloned onto lambda hybrid phage (19) enabled the motA and motB gene products to be identified (as proteins with apparent molecular masses of 31 and 39 kilodaltons [kDa], respectively) and indicated that they are located in the cytoplasmic membrane (17), a location consistent with their function; a membrane location was also noted subsequently with plasmid-encoded MotA (22) and MotB (6).…”