Peritrichous bacteria, which produce multiple flagella over the bacterial surface, have historically served as the model system for analyzing regulation of flagellar genes and flagellar biosynthesis. Investigations into polarly flagellated bacteria have uncovered variations in these pathways, indicating that bacteria have evolved diverse mechanisms to ensure proper expression of flagellar genes and biosynthesis of flagella. One of these differences has been the inclusion of FlhF in flagellar gene regulation and biosynthesis pathways in many polarly flagellated bacteria. With the studies conducted so far, it is evident that FlhF functions at different steps in flagellar organelle development in these bacteria.
Before this study, no experimental proof existed regarding whether FlhF proteins hydrolyze GTP or whether the GTPase activity is required for specific steps in production of flagella. By analyzing mutants of C. jejuni that either lacked FlhF or produced FlhF proteins with a reduced ability to hydrolyze GTP, we identified distinct GTPase-dependent and GTPase-independent steps in flagellar gene regulation and biosynthesis (Fig. ). We discovered that a mutant lacking FlhF expressed approximately 5- to 15-fold-lower levels of three σ54-dependent flagellar genes and more than 98% of the bacteria did not produce a flagellum, which contributed to the nonmotile phenotype. However, mutants producing FlhF(D321A) and FlhF(R324A), which in in vitro analysis demonstrated only 30 to 40% of the GTPase activity of wild-type FlhF, were not severely defective for σ54-dependent flagellar gene expression. These mutants were significantly defective only for proper flagellar biosynthesis, which is defined for C. jejuni as the production of a single flagellum at one or both bacterial poles. Whereas we feel that our results give credence to our conclusion that the GTPase activity is required only for proper flagellar biosynthesis, the possibility remains that the residual GTPase activity we observed with the FlhF(D321A) and FlhF(R324A) proteins was sufficient for expression of σ54-dependent flagellar genes analyzed in this study. Conclusive analysis that the GTPase activity of FlhF is not required for σ54-dependent flagellar gene expression will require the in vivo production of a stable FlhF mutant protein in C. jejuni that completely lacks GTPase activity and analysis of multiple σ54-dependent promoters. As was shown by this study, this approach may be difficult, since we were unable to generate such a stable, GTPase-deficient FlhF mutant protein, perhaps due to a requirement of GTP interactions for the proper folding or stability of FlhF. Nonetheless, our results demonstrate that proper flagellar biosynthesis is more sensitive to decreases in GTP hydrolysis by FlhF than σ54-dependent flagellar gene expression.
FIG. 6. Model for role of FlhF in activation of expression of σ54-dependent flagellar genes and the GTPase activity of FlhF in proper flagellar biosynthesis. Construction of the FEA is hypothesized to be required for the formation of a signal sensed by (more ...)
In our in vitro biochemical assays, we identified a requirement of residues K295, D321, and R324 of FlhF for full GTPase activity. K295 is located in the P loop (G1 region) of the GTPase domain, whereas D321 and R324 are in a conserved DXXR motif of the G2 region that has been postulated to assist in GTP hydrolysis (2
). However, these residues in any FlhF protein had not been analyzed to determine if they are required for GTPase activity until this study. Further characterization of these mutant FlhF proteins will be necessary to determine if these proteins are blocked only at the hydrolysis step or have defects in GTP binding as well. Because evidence from structural studies with FlhF of B. subtilis
suggests that FlhF may have an unusual mechanism for GTP hydrolysis compared to other SIMIBI family members, these mutant proteins we have created may have the potential to provide insights into the biochemical mechanism of GTP hydrolysis by FlhF (34
). In eukaryotic systems, GTPases are often regulated by GTPase-activating proteins (GAPs) and GTPase exchange factors (GEFs) (reviewed in reference 43
). Bacterial versions of these GAPs or GEFs have not been identified for the other SIMIBI members, FtsY and Ffh. Rather, heterodimer formation by FtsY and Ffh has been found to directly stimulate the GTPase activity of each individual protein (12
). More-extensive biochemical analysis will be required to determine if FlhF has an associated GAP- or GEF-like protein or if the GTPase activity of FlhF is influenced by possible homodimer formation or heterodimer formation with another GTPase.
Because the GTPase domain of FlhF proteins is most similar to those of the bacterial FtsY and Ffh SRP GTPases, some speculation has been made that FlhF may function as a SRP protein specific for flagellar proteins that compose the FEA (5
). The bacterial SRP system is required for targeting many proteins to the general secretory (Sec) system (25
). These proteins predominantly include those to be inserted in the inner membrane or some proteins that are to be secreted into the periplasm. Considering our analysis of the ΔflhF
mutant, we do not favor that FlhF functions exclusively in the targeting to the inner membrane of flagellar proteins, such as components of the FEA, that are essential for flagellar biosynthesis and flagellar gene expression. In the ΔflhF
mutant, we detected at least two FEA proteins localized to the membrane fraction. Furthermore, in a small minority of individual ΔflhF
mutant bacteria, a flagellum was detected, suggesting that C. jejuni
does not have an absolute dependence on FlhF for FEA formation and FEA-mediated flagellar protein secretion. Further indirect evidence that FlhF is likely not required to form the FEA is the observation that the ΔflhF
mutant shows 12- to 15-fold-reduced expression of σ54
-dependent flagellar genes but a mutant lacking a component of the FEA (such as a ΔflhB
mutant) generally demonstrates at least a 25- to 60-fold reduction in expression of these genes (20
). Thus, if a ΔflhF
mutant is lacking one of the FEA components in the inner membrane, we would have expected to see a deficiency in expression of σ54
-dependent flagellar genes equivalent to that typically seen in an FEA mutant.
Monitoring formation of the FEA by measuring FEA-dependent secretion of flagellar proteins in the ΔflhF mutant would be one method to study if FlhF affects formation of the FEA, but this approach would be difficult since many of the genes that encode the rod, hook, and flagellin proteins that are secreted by the FEA are part of the σ54-dependent regulon and by consequence are dependent on FlhF and the FEA for expression. Future studies will focus on characterizing FEA-mediated secretion in flhF mutants ectopically expressing genes for the rod, hook, and flagellin proteins from nonnative, FEA- and FlhF-independent promoters to determine if there is a functional relationship between FlhF and FEA formation.
It is possible that FlhF, and more specifically its GTPase activity, may be required for modulating the activity of the FEA. In support of this hypothesis, the flhF
) and flhF
) mutants were impaired for proper flagellar biosynthesis, since approximately 60 to 65% of these bacteria (compared to ~7% of wild-type bacteria) were unable to produce flagella, produced flagella at incorrect positions on the bacterial surface, or produced multiple flagella at a single pole. Since these mutants have a greater propensity for these phenotypes than wild-type bacteria, the GTPase activity of FlhF may be required at early initiation steps with respect to the FEA in flagellar biosynthesis (Fig. ). These steps may include one or more of the following: the proper positioning of the FEA at a pole, ensuring that only one FEA is formed at a pole, monitoring the FEA so that it properly secretes flagellar proteins in the correct order to build a flagellum, or assisting the FEA in more efficiently secreting flagellar proteins. Some evidence for the first hypothesis exists in V. cholerae
, since FlhF appears to assist in properly localizing at least one FEA component, the FliF MS ring, to the old pole in a bacterium (14
). If FlhF of C. jejuni
functions in an analogous process, it will be interesting to study this localization process, since C. jejuni
usually constructs a flagellum at both the old and new poles. Furthermore, it would be interesting to study the localization of the FEA components in the flhF
) and flhF
) mutants, which presumably mislocalize the FEA to produce nonpolar flagella or allow for multiple polar FEA formation to contribute to more than one flagellum at a single pole. All of these studies depend on visualization of proteins in C. jejuni
using fluorescence microscopy with labeled proteins, which has been difficult with some strains of C. jejuni
, including the strain used in this study, C. jejuni
strain 81-176 (33
; our unpublished observations). To study the other hypotheses, development of new and better reagents to characterize FEA formation and to monitor flagellar protein secretion in flhF
mutants is required to provide any insights into how FlhF may influence the activity of the FEA.
We found a more severe defect in flagellar gene regulation and biosynthesis with a mutant lacking flhF than with the GTP hydrolysis-hindered flhF(D321A) and flhF(R324A) mutants. The severity of the biosynthesis defect in the ΔflhF mutant is most likely related to the fact that expression of at least three σ54-dependent flagellar genes (and probably all other σ54-dependent flagellar genes) was reduced at least 5- to 15-fold. The decreased expression of multiple genes would greatly reduce the levels of the encoded proteins essential for construction of flagella. Since flhF(D321A) and flhF(R324A) were not significantly defective, if at all, for expression of σ54-dependent flagellar genes, these results indicate that other domains or activities of FlhF independent of GTP hydrolysis are required for processes leading to a full level of expression of σ54-dependent flagellar genes. These activities may be related to the B or N domain. To determine if the B and N domains of FlhF are specifically required for expression of σ54-dependent flagellar genes, we attempted to create an flhF mutant of C. jejuni lacking these domains, but this mutant protein was unstable in the bacterium. We continued our analysis of FlhF and revealed that the protein is not required for formation of the FEA or the FlgSR two-component system. Furthermore, we provided data that suggest that the FlgSR system does not function downstream of FlhF for activation of σ54 and that the lack of flhF and the FEA contributes to greater defects in expression of σ54-dependent flagellar genes than either mutation alone. Thus, our results indicate that FlhF may function in a separate pathway that converges with or acts downstream of the FEA-FlgSR pathway to stimulate σ54 (Fig. ). Future studies will determine if FlhF is required at the step of FlgR-dependent activation of σ54 or at a more downstream step, such as σ54-RNA polymerase initiation or stability of σ54-dependent mRNAs.
With continued investigation into the FlhF proteins of polarly flagellated bacteria, it is evident that these proteins are an essential component of regulatory systems for expression of flagellar genes, flagellar biosynthesis, or both. For instance, flhF
mutants of P. aeruginosa
are only slightly affected in expression of the major flagellin in P. aeruginosa
, but this defect is not detrimental to flagellar biosynthesis (34
). Instead, FlhF is more specifically required for the polar placement of flagella. Furthermore, expression of flhF
is dependent on σ54
, rather than being required for expression of the σ54
flagellar regulon, in P. aeruginosa
is also dependent on σ54
for expression in V. cholerae
, but the FlhF protein is required for expression of other σ54
-dependent flagellar genes and consequently flagellar biosynthesis (10
). In C. jejuni
and H. pylori
, no evidence exists to indicate that flhF
expression is σ54
dependent, and therefore, expression may be constitutive or regulated by a yet-unknown factor. However, FlhF is required for wild-type-level expression of the σ54
-dependent flagellar rod and hook genes in H. pylori
and C. jejuni
and flagellar biosynthesis (36
). Therefore, polarly flagellated bacteria have acquired flhF
and adapted the encoded protein to be involved at different specific steps in flagellar gene regulation and biosynthesis that behoove the individual species. Thus, exploration of the role of FlhF in diverse bacterial species will be required to fully understand the biochemical properties of FlhF and how these activities function to ensure proper flagellar biosynthesis. Our study has provided a foundation for the molecular characterization of FlhF and how biological properties of FlhF are linked to distinct steps in flagellar gene regulation and biosynthesis.