The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide

Abstract

We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to theflaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that theflaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium Fliu and 29% identity to the 0 subunit of E. coli ATP synthase.Many bacterial species swim actively by means of flagella. Swimming tends to move the bacteria towards attractants and away from repellents. The flagellar organelle is made of three parts: the basal body, the hook, and the filament, with more than 10 different proteins involved in flagellar formation. In addition, there are the motor apparatus and the switch, necessary for rotation of the flagellar filaments and control of the sense (clockwise or counterclockwise) of rotation. A number of sensory elements and components for transduction of the signal to the motor switch are also part of the motility system. A considerable body of data is available regarding the structure and functioning of the flagella of Escherichia coli and Salmonella typhimurium (reviewed in references 21 and 22).About the gram-positive bacterium Bacillus subtilis much less is known. A limited number of mutants affected in motility have been isolated, and a few have been characterized (8, 32). More advanced are the studies related to the chemotactic mechanisms that operate in B. subtilis. A large number of mutants defective in chemotaxis have been isolated, 21 different complementation groups have been identified, and part of a chemotactic locus has been cloned (28, 29). Better knowledge of the genes and gene products of B. subtilis involved in flagellar assembly, motility, and taxis seemed desirable in view of the fact that B. subtilis is a gram-positive bacterium and that its chemotactic response is more complex than that of E. coli (37). From a B. subtilis chromosomal DNA library in XCharon 4A (5) we have isolated one clone that contains the wild-type alleles of two known flaA mutations, flaA4 and flaA15 (9).In this report, we describe the nucleotide sequence of 8,302 contiguous nucleotides of the flaA locus. Insertional inactivation with integrative plasmids showed that the region sequenced is part of a single operon involved in motility (10, 44). Nine of the deduced gene products show similarity to E. coli and S. typhimurium proteins known to be involved in * Corresponding author. flagellar structures and fu...

Purification and characterization of the flagellar hook–basal body complex of Bacillus subtilis

“…8). This sequence partially fits with that of Orf3 of the B. subtilis flaA locus, whose molecular mass is estimated to be 24 kDa (Albertini et al, 1991). Orf3 has similarity to FliHs (23% identity; Albertini et al, 1991), which is one of the putative export apparatuses (Macnab, 1996).…”

“…The acrylamide concentration of the gel was 12.5%. independently reported as the distal rod protein by two laboratories (Albertini et al, 1991;Zuberi et al, 1991). There were two strong pieces of evidence for this assignment: (i) the amino acid sequence of the gene product (Orf9 of the flaA locus) was 40% identical to the S. typhimurium FlgGs protein, and (ii) their molecular sizes were similar (264 amino acids deduced for Orf9 and 260 for FlgGs).…”

Characterization of the sigD transcription unit of Bacillus subtilis

“…1. Plasmids containing the 8.3-kb (previously thought to be 7.7-kb [1]), 4.0-kb, and 10.9-kb EcoRI fragments that make up the majority of the flaiche region were a gift of George Ordal (42) (Fig. 1).…”

“…In fact, uD holoenzyme is directly responsible for transcription of the flagellin structural gene (30), the motility genes motA and motB (31), and the flgM regulatory gene (30a). The specific role of CD in the expression of chemotaxis genes is still unknown, although several sequences which conform to the consensus promoter sequence for CD have been identified upstream of some chemotaxis genes (1,27). The uD regulon of B. subtilis is therefore thought to be composed of flagellar (fla), motility (mot), and chemotaxis (che) genes.…”

“…The fliM gene is located within a >26-kb region of DNA, originally identified as the chemotaxis (che) locus (34). This region of DNA is thought to be part of a single operon (42) and is now known to encode structural proteins required for hook-basal body formation (1,37,39,40) as well as several regulatory proteins required for chemotaxis (6,12,16). The che locus has therefore been renamed the fla/che region to more accurately represent the function of the proteins encoded therein (33).…”