In Salmonella typhimurium, nearly 50 genes are involved in flagellar formation and function and constitute at least 13 different operons. In this study, we examined the transcriptional interaction among the flagellar operons by combined use of Mu dl(Apr Lac) cts62 and TnlO insertion mutants in the flagellar genes. The results showed that the flagellar operons can be divided into three classes: class I contains only theflhD operon, which is controlled by the cAMP-CAP complex and is required for expression of all of the other flagellar operons; class U contains seven operons, flgA, flgB, flhB, fliA, fliE, fliF, and fliL, which are under control of class I and are required for the expression of class Ill; class III contains five operons,flgK,fliDfliC, motA, and tar. This ordered cascade of transcription closely paralels the assembly of the flagellar structure. In addition, we found that the fliD defect enhanced expression of the class III operons. This suggests that the fliD gene product may be responsible for repression of the class Ill operons in the mutants in the class II genes. These results are compared with the cascade model of the flagellar regulon ofEscherichia coli proposed previously (Y. Komeda, J. Bacteriol. 170:1575-1581, 1982.The bacterial flagellum is composed of three structural components, a basal body, a hook, and a filament. The filament extends into the extracellular space and is connected by the hook to the basal body embedded in the cell membrane. Genetic analysis of flagellar mutants have revealed that there are nearly 50 genes involved in flagellar formation and function in both Salmonella typhimurium and Escherichia coli (24,25). Intergeneric complementation analysis revealed functional homology in the flagellar genes between these two organisms (8,21,39). Genes responsible for flagellar formation are calledflg,flh,fli, orflj. Except for a few genes, mutants defective in these genes are nonflagellate and some of them produce presumptive precursor structures of the flagellum (35, 36). There are three kinds of genes responsible for flagellar function, including flagellar rotation (mot), chemotaxis (che), and transmembrane signal transduction of chemotactic stimuli (tar, trg, tsr, etc.) Mutants defective in these genes produce flagellar structures indistinguishable from those of the wild-type strain. Most of the flagellar genes are clustered in three regions of the bacterial chromosome, termed regions I, II, and III. These clustered genes constitute 14 and 13 different operons in E. coli (16, 32) and S. typhimurium (22), respectively. Flagellar genes of S. typhimurium are summarized in Fig. 1.In E. coli, Komeda (14) constructed fusions of most of the flagellar operons to the lac genes by using Mu dl(Apr Lac) bacteriophage developed by Casadaban.and Cohen (5). This phage contains the lac genes with no promoter, and its integration in a gene can result in rescue of expression of the lac genes due to the promoter of that gene. By using these operon fusions, he examined the transcriptional interac...

Through genetic studies, the fliA gene product has been shown to regulate positively gene expression in late operons of the flagellar regulon in Salmonella typhimurium. In the present study, the fliA gene was cloned and sequenced. The fliA coding region consisted of 717 nucleotides beginning with the GTG initiation codon and the conserved sequence specific to promoters for flagellar operons was found to exist upstream of the coding region. The fliA gene product deduced from the nucleotide sequence was a protein with 239 amino acid residues and the calculated molecular mass was 27,470 dalton. The deduced amino acid sequence was homologous with that of sigma 28, a flagellar specific sigma factor of Bacillus subtilis. The fliA gene product was identified as a protein of molecular mass 29 kDa in the in vitro transcription-translation system, while three proteins of 29 kDa, 31 kDa and 32 kDa were found in the products programmed by the fliA gene in minicells and in maxicells. The 29 kDa FliA protein was purified from the FliA overproducing strain which carried the ptac-fliA fusion. This protein activated the in vitro synthesis of flagellin, the fliC gene product. RNA polymerase containing the purified FliA protein was shown to transcribe the fliC gene. These results indicate that FliA protein functions as an alternative sigma factor specific for S. typhimurium flagellar operons.

In the flagellar regulon of Salnonella typhimurium, the flagellar operons are divided into three classes, 1, 2, and 3, with respect to transcriptional hierarchy. The class 2 operons are controlled positively by the class 1 genes,flhD andflhC. The class 3 operons are controlled positively byfliA and negatively byflgM. It has been shown that FliA is a sigma factor specific for class 3, whereas FlgM is an anti-sigma factor which binds FliA to prevent its association with RNA polymerase core enzyme. Therefore, the FliA-FlgM regulatory system has been believed to control specifically the class 3 operons. In the present study, we showed that theflgM mutation enhanced the expression of class 2 by more than fivefold. When afliAL mutation was present simultaneously, this enhancement was not observed.These results indicate that the FliA-FlgM regulatory system is involved not only in the expression of class 3 but also in that of class 2. However, though neitherflhD norflhC mutants could express the class 2 operons, thefli4 mutants permitted the basal-level expression of those operons. Therefore, FlhD and FlhC are indispensable for the expression of class 2, whereas FliA is required only for its enhancement in the FlgM-depletion condition. Furthermore, we showed that theflgM mutation resulted in a two-to threefold increase in flagellar number. On the basis of these results, we propose that the relative concentration of FliA and FlgM may play an important role in the determination of flagellar numbers produced by a single cell.Salmonella typhimurium and Escherichia coli cells have 5 to 10 flagella which are responsible for motility and chemotaxis. An individual flagellum is composed of three structural parts, a basal body, a hook, and a filament. More than 50 genes are required for formation of functional flagellum (21). These flagellar genes constitute at least 15 different operons in the chromosome (Fig. 1), and each flagellar operon is named after the gene which is transcribed first in that operon (14,18). Examination of transcriptional interaction of the flagellar operons revealed that they are organized into a regulon (13,17). According to the flagellar regulon model of S. typhimurium, the flagellar operons are divided into three classes, 1, 2, and 3, with respect to transcriptional hierarchy (17). Class 1 contains only the flhD operon, which has been called the master operon and is required for expression of the class 2 operons. Class 2 contains seven operons, fig4, flgB, flhB, fliA, fliE,fliF, and fliL, which are under the positive control of class 1 and required for expression of class 3. Class 3 contains at least five operons, flgK, tar, motA, fliC, and fliD. The striking feature of the flagellar regulon is the coupling of the sequential expression of the flagellar operons with the assembly process of flagellar structures; that is, all the genes involved in hook-basal body assembly belong to class 2, and those involved in filament assembly belong to class 3. Mutations in any one of the hook-basal body genes inhibit ...

FlgD is known to be absolutely required for hook assembly, yet it has not been detected in the mature flagellum. We have overproduced and purified FlgD and raised an antibody against it. By using this antibody, we have detected FlgD in substantial amounts in isolated basal bodies fromflgA,flgE,flgH,flgI,flgK, andfliK mutants, in much smaller amounts in those from the wild type andflgL,fli4,fliC,fliD, andfliE mutants, and not at all in those fromflgB,flgD,flgG, andflgj mutants. In terms of the morphological assembly pathway, these results indicate that FlgD is first added to the structure when the rod is completed and is discarded when the hook, having reached its mature length, has the first of the hook-filament junction proteins, FlgK, added to its tip. Immunoelectron microscopy established that FlgD initially is located at the distal end of the rod and eventually is located at the distal end of the hook. Thus, it appears to act as a hook-capping protein to enable assembly of hook protein subunits, much as another flagellar protein, FliD, does for the flagellin subunits of the filament. However, whereas FliD is associated with the filament tip indefinitely, FlgD is only transiently associated with the hook tip; i.e., it acts as a scaffolding protein. When FlgD was added to the culture medium of aflgD mutant, cells gained motility; thus, although the hook cap is normally added endogenously, it can be added exogenously. When culture media were analyzed for the presence of hook protein, it was found only with the flgD mutant and, in smaller amounts, the fliK (polyhook) mutant. Thus, although FlgD is needed for assembly of hook protein, it is not needed for its export.The bacterial flagellum is a complicated structure composed of the basal body, the hook, and the filament (see, e.g., reference 19), as well as more labile structures, such as the motor, switch, and export apparatus. Flagella, under the control of the associated sensory apparatus, provide the cell with the ability to move to favorable environments. The flagellar basal body consists of subunits of at least eight different proteins, which form two outer rings (the L and P rings), an inner ring (the MS ring), and the rod (Fig. 1). The hook and the filament are homopolymers of hook protein and flagellin, respectively. The morphological pathway of flagellar formation is well characterized in both Escherichia coli and Salmonella typhimurium (13,15,26,27) and is coordinated with flagellar gene expression (17). The flagellum is sequentially constructed from simpler to more complex structures. At the earliest stage, the MS ring complex is formed from subunits of the FliF protein. It is thought that the flagellar switch and the flagellar export apparatus are then added (13, 15). Basal body assembly continues with formation of the rod and addition of the outer (P and L) rings. After the basal body is completed, the hook is assembled and finally polymerization of the filament, the major external structure and the propeller for the cell, commences and continues in...

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