MOLECULAR CHARACTERIZATION OF THE SALMONELLA-TYPHIMURIUM FLHB OPERON AND ITS PROTEIN PRODUCTS

The flhB and flhA genes constitute an operon called flhB operon on the Salmonella typhimurium chromosome. Their gene products are required f or formation of the rod structure of flagellar apparatus. Furthermore, several lines of evidence suggest that they, together with FliI and F liH, mag constitute the export apparatus of flagellin, the component p rotein of flagellar filament. In this study, we determined the nucleot ide sequence of the entire flhB operon from S. typhimurium. It was sho wn that the flhB and flhA genes encode highly hydrophobic polypeptides with calculated molecular masses of 42,322 and 74,848 Da, respectivel y. Both proteins have several potential membrane-spanning segments, su ggesting that they may be integral membrane proteins. The flhB operon was found to contain an additional open reading frame capable of encod ing a polypeptide with a calculated molecular mass of 14,073 Da. We de signated this open reading frame flhE. The N-terminal 16 amino acids o f FlhE displays a feature of a typical signal sequence. A maxicell lab eling experiment enabled us to identify the precursor and mature forms of the flhE gene products. Insertion of a kanamycin-resistant gene ca rtridge into the chromosomal flhE gene did not affect the motility of the cells, indicating that the flhE gene is not essential for flagella r formation and function. We have overproduced and purified N-terminal ly truncated FlhB and FlhA proteins and raised antibodies against them . By use of these antibodies, localization of the FlhB and FlhA protei ns was analyzed by Western blotting (immunoblotting) with the fraction ated cell extracts. The results obtained indicated that both proteins are localized in the cytoplasmic membrane.