Previous studies in our laboratory have found that the proteins of the FEA, the putative FlhF GTPase, and the FlgSR two-component system are required for full expression of σ54
-dependent flagellar genes in C. jejuni
). In the current study, we obtained evidence that links the FEA to stimulation of the FlgSR two-component regulatory system. We found that activation rather than production of the FlgSR system is dependent on the FEA. Furthermore, we believe that formation of the apparatus rather than the secretory function of the apparatus is key to producing the signal detected by FlgS leading to its activation and subsequent expression of σ54
-dependent flagellar genes. Analysis of the genomic sequences of various C. jejuni
strains indicates that the consensus σ54
-binding site is in the promoters of most genes that encode the flagellar proteins that are external to the cytoplasm and likely secreted by the FEA (20
). Because gene expression and protein production are energetically expensive processes, it is likely that the introduction of a level of transcriptional control by the FEA allows C. jejuni
to ensure that σ54
-dependent flagellar genes are expressed and the secreted proteins are produced only after the apparatus has formed.
The flagellar regulatory cascade of C. jejuni
appears to bear some resemblance to the cascades utilized by species of Helicobacter, Vibrio
, and Pseudomonas
). First, all the cascades are known to require σ54
and a two-component regulatory system with functional similarity to FlgSR for expression of a subset of flagellar genes. In addition, Vibrio
species require the activity of a master regulator protein to initiate transcription of genes encoding FEA proteins and these flagellar two-component regulatory systems (2
). However, in C. jejuni
and Helicobacter pylori
, no master regulator of flagellar biosynthesis has been found, and one current hypothesis is that the expression of genes encoding components of the FEA and FlgSR is largely constitutive (26
). In all these bacteria, activation of the flagellar two-component regulatory system leads to the σ54
-dependent expression of genes encoding flagellar proteins that are secreted by the FEA (16
). Considering the similarity of the compositions of these flagellar regulatory cascades, our findings may suggest that the formation of the FEA could influence σ54
-dependent flagellar gene expression in a number of bacterial species. Further analysis of each of these organisms is required to determine if this relationship is shared across multiple genera of motile bacteria.
The analysis presented in this work allowed us to more precisely clarify the relationship between the FEA and the FlgSR system in σ54
-dependent flagellar gene expression. We constructed C. jejuni
mutants whose mutations impaired FEA-mediated secretion to determine if formation of the export apparatus or its secretory activity was required for FlgS activation. Based on our finding that three of four flhB
mutations and a fliI
mutation reduced or blocked secretion of the FlaA flagellin but did not negatively affect σ54
-dependent gene expression, we concluded that the formation of the FEA in the inner membrane could be the signal detected by FlgS that directly leads to activation of the kinase. Alternatively, formation of the FEA may be indirectly involved by being required for the production of a downstream activating signal. Although the data alone do not define the nature of the communication between the FEA and FlgSR, we have provided a foundation for future studies to understand activation of the system. Characterization of additional FEA proteins and structures such as the inner membrane MS ring and the cytoplasmic C ring that are associated with the FEA (43
), along with better reagents to detect complete FEA formation, may allow us to further define the activating signal emanating from this secretory apparatus.
If our hypothesis that FlgS detects formation of the FEA for autoactivation is correct, the cytoplasmic localization of FlgS may provide insight into the origin of the signal relative to the FEA structure. Since FlgS is a cytoplasmic protein, FlgS may detect a signal originating on the cytoplasmic face of the inner membrane-localized FEA complex. For instance, FlgS may detect a completed FEA structure by monitoring whether certain proteins with large cytoplasmic domains are in the FEA. Possible candidates for this type of signal include the cytoplasmic domains of FlhA and FlhB. To find evidence supporting this hypothesis, we attempted to use numerous approaches to directly detect interactions that may occur between FlgS and FEA proteins, including affinity chromatography, affinity blotting, and in vivo chemical cross-linking. However, the results of these assays were inconsistent and inconclusive. New and better reagents and protocols have to be developed to extend these types of analyses. In vivo detection of an FlgS interaction with a member of the FEA may be difficult, due to the fact that flagellated C. jejuni assembles only one or two of these secretory apparatuses per bacterium. Thus, the number of interactions of FlgS with the FEA or an FEA component may be small and the interactions may be temporally transient.
As mentioned above, our results strongly support the hypothesis that formation of the FEA either directly comprises the signal or is required to produce the signal to activate FlgSR and expression of σ54-dependent flagellar genes. An alternative hypothesis that we considered suggested that the secretory activity of the FEA could be the activating signal, with a cytoplasmic repressor hindering the FlgSR regulatory cascade prior to formation of and secretion by the FEA. However, four of the five flhB or fliI mutants whose mutations were shown to hinder or block secretion of flagellar proteins were not affected for σ54-dependent expression of flagellar genes. Only the flhBΔ226-230 mutant showed decreased expression of these genes, but analysis of this mutant suggested that it behaved most like a ΔflhB mutant, which does not form a complete FEA. Thus, we cannot confidently conclude that the flhBΔ224-228 mutant makes a fully formed but secretion-incompetent apparatus. Second, our transposon mutagenesis screen did not reveal any transposon insertions in FEA mutants that relieved repression of expression of σ54-dependent flagellar genes. These combined results greatly weaken the hypothesis that the secretory activity of the FEA alone forms the FlgS-activating signal. Thus, the results of this study strongly favor the hypothesis that that formation of the FEA is a requirement for and quite possibly a component of the essential signal for activating the FlgSR system that results in expression of σ54-dependent flagellar genes.
Our work also suggests a new function in the signaling mediated by the FEA in flagellar regulatory cascades. In the well-characterized pathways observed in E. coli
, formation of the FEA ultimately controls the activity of the alternative sigma factor σ28
involved in expression of genes encoding the major flagellins and some motor proteins (41
). The FEA is responsible for secretion of flagellar proteins and the anti-σ factor, FlgM, which represses the activity of σ28
until the cell has completed formation of the FEA, basal body, and hook structures required to secrete flagellins to build a filament (32
). In this study, we found that the FEA is intimately involved in creating a signal that activates the FlgSR two-component system, leading to activation of σ54
. Therefore, the FEA plays a different role in influencing signaling for σ54
-dependent expression of flagellar genes in C. jejuni
. This finding may also be applicable to other motile bacteria that utilize σ54
in flagellar gene regulation and biosynthesis, including species of Vibrio, Pseudomonas
, and Helicobacter
. This work expands the known mechanisms of regulating flagellar gene expression and suggests that there are more complex functions associated with the FEA beyond protein secretion.
Future analyses of FlgS will involve determining the domain and residues of the protein required for sensing an autoactivating signal. In analyzing the sequence of FlgS, we found that the central and C-terminal portions of the protein contain the histidine-containing phosphotransfer domain and the ATP-catalytic domain (61
). These domains are required for accepting a phosphate group on a conserved histidine and for ATP hydrolysis, respectively, for autophosphorylation. Indeed, we found that H141 in the phosphotransfer domain is required for modification by phosphorylation and for functioning of the active FlgS to stimulate expression of σ54
-dependent flagellar gene expression. In a comparison of the amino acid sequence of FlgS to those of other sensor kinases, the predominant homology with the latter kinases is localized almost exclusively to the phosphoacceptor and ATP hydrolysis domains. Only limited homology between the initial 130 amino acids of FlgS and other sensor kinases is apparent. The sensor kinases that share the most homology to this region of the C. jejuni
FlgS protein are other FlgS homologues in Campylobacter
species (almost 100% identity), the FlgS orthologue in Helicobacter
species (31 to 37% identity and 57 to 66% similarity), and the FlrB sensor kinase of Vibrio cholerae
(26% identity and 54% similarity). The N-terminal regions of these proteins have no obvious motifs that suggest a function or how they may sense a specific factor. Since these N-terminal domains are unique to the group of FlgS orthologues, it is likely that this region of these proteins may function in specifically recognizing the signal necessary to culminate in expression of σ54
-dependent flagellar genes. Future studies will focus on further characterizing this domain of the protein.
Previous work in our laboratory focused on understanding the activation and function of the FlgR response regulator (25
). In this study, we describe work that provides a foundation for understanding the activation of the cognate sensor kinase, FlgS, and how the FEA influences activation of this two-component regulatory system. To date, we have linked activation of the FlgSR system to the FEA and have characterized a previously undescribed mechanism for controlling activation of flagellar gene expression. In addition, FlgSR appears to be an unusual two-component system in which expression of both components is controlled by phase-variable mechanisms (25
), a trait unique among well-characterized bacterial two-component systems. Thus, there appears to be at least two mechanisms for controlling σ54
-mediated expression via the FlgSR proteins. Future analyses will focus on further defining the nature of the activating signal emanating from the FEA and how it influences expression of σ54
-dependent flagellar genes.