In this study, we demonstrated the peptidoglycan-hydrolyzing activity of the flgJ gene product. Mutations which affected this enzymatic activity also affected the motility of the cell, indicating that this enzymatic activity is involved in the process of flagellar formation. Because FlgJ is essential for the formation of the rod and because the rod must penetrate and assemble through the peptidoglycan layer, our results strongly suggest that the peptidoglycan-hydrolyzing activity of FlgJ is responsible for the penetration of the peptidoglycan layer by the rod.
On the basis of the above results and other available information, we propose a model for the flagellar morphogenetic pathway with special emphasis on the processes of crossing three envelope barriers by the rod (Fig. ). First, the FliF protein integrates into the inner membrane and self-assembles to form the MS ring (20
), which together with the flagellum-specific export apparatus makes a selective pore through the inner membrane. Second, the muramidase activity of FlgJ hydrolyzes the peptidoglycan layer to make a hole for rod elongation, which allows the rod to penetrate the peptidoglycan. This process may also be involved in P-ring formation, which is postulated to occur in the peptidoglycan layer (6
). Third, formation of the L ring in the outer membrane around the tip of the rod makes a hole through the outer membrane (16
). Through these processes, the rod can cross all the envelope barriers. Finally, the hook and the filament can be formed in the extracellular space to make a mature flagellum.
FIG. 6 Model of the flagellar assembly pathway with special emphasis on the processes involved in the crossing of three envelope barriers by the rod structure. The rod is composed of at least four proteins, FlgB, FlgC, FlgF, and FlgG, which are exported into (more ...)
We showed that the domain for the peptidoglycan-hydrolyzing activity is confined to the C-terminal half, which has homology to known muramidase enzymes. Mutations which replaced amino acids at the putative active center of the muramidase reduced the peptidoglycan-hydrolyzing activity, suggesting that the peptidoglycan-hydrolyzing activity of FlgJ is due to its muramidase activity.
Although the N-terminal half of FlgJ is dispensable for the peptidoglycan-hydrolyzing activity, the truncated FlgJ protein lacking the N-terminal half could not support flagellation of the cells. This suggests that the N-terminal region is also required for FlgJ to function in vivo.
Because the peptidoglycan layer resides in the periplasmic space, FlgJ must be transported across the inner membrane before or at the time of the rod formation. However, unlike other known muramidases (4
), FlgJ has no signal sequence at its N terminus (16
). Most of the flagellar proteins lack signal sequence and are believed to be exported via the flagellum-specific export pathway which resides within the flagellar structure (12
). Their export signals are postulated to exist in their N-terminal regions (15
). In this study, we showed evidence suggesting that FlgJ may be exported into the periplasmic space via the flagellum-specific export pathway. Therefore, its N-terminal region is likely to contain information essential for its own export. This may be one of the reasons why the N-terminal half is indispensable. However, we cannot exclude the possibility that the N-terminal half plays an additional essential role in the flagellar assembly process.
We would like to consider regulatory roles of the N-terminal half of FlgJ on the muramidase activity. To avoid undesirable cell lysis caused by random hydrolysis of peptidoglycan, muramidases such as autolysin are known to contain information directing themselves to the sites where they should act (3
). By analogy to this, the action of FlgJ should be restricted to the area where the flagellum will be formed. In the known muramidases, amino acid sequences conveying this information are located in their C-terminal regions (3
). However, FlgJ lacks regions homologous to these amino acid sequences and instead contains a large N-terminal region (17
). We suppose that the N-terminal region of FlgJ may contain information for this localized hydrolysis. Specific binding of the N-terminal region of FlgJ to the tip of the MS ring or the growing rod may ensure the localization of the FlgJ protein. Interestingly, the truncated FlgJ protein lacking its N-terminal half showed slightly greater peptidoglycan-hydrolyzing activity than did the intact FlgJ protein in the zymogram analysis (Fig. B). This suggests that the N-terminal portion may play some inhibitory role on the muramidase activity. Binding of the N-terminal region of FlgJ to the tip of the MS ring or the growing rod may relieve this inhibition to promote the muramidase activity.