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...

Enterohaemorrhagic Escherichia coli (EHEC) is a life-threatening human pathogen worldwide. The locus of enterocyte effacement (LEE) in EHEC encodes a type three secretion system and effector proteins, all of which are essential for bacterial adherence to host cells. When LEE expression is activated, flagellar gene expression is down-regulated because bacterial flagella induce the immune responses of host cells at the infection stage. Therefore, this inverse regulation is also important for EHEC infection. We report here that a small regulatory RNA (sRNA), Esr41, mediates LEE repression and flagellar gene activation. Multiple copies of esr41 abolished LEE expression by down-regulating the expression of ler and pch, which encode positive regulators of LEE. This regulation led to reduced EHEC adhesion to host cells. Translational gene-reporter fusion experiments revealed that Esr41 regulates ler expression at a post-transcriptional level, and pch transcription, probably via an unknown target of Esr41. Esr41-mediated ler and pch repression was not observed in cells lacking hfq, which encodes an RNA-binding protein essential for most sRNA functions, indicating that Esr41 acts in an Hfq-dependent manner. We previously reported an increase in cell motility induced by Esr41. This motility enhancement was also observed in EHEC lacking ler, showing that Esr41-mediated enhancement of cell motility is in a ler-independent manner. In addition, Esr41 activated the expression of flagellar Class 3 genes by indirectly inducing the transcription of fliA, which encodes the sigma factor for flagellar synthesis. These results suggest that Esr41 plays important roles in the inverse regulation of LEE and flagellar gene expression.