FlhF and its GTPase activity are required for distinct processes in flagellar gene regulation and biosynthesis in Campylobacter jejuni

Abstract

FlhF proteins are putative GTPases that are often necessary for one or more steps in flagellar organelle development in polarly flagellated bacteria. In Campylobacter jejuni, FlhF is required for sigma(54)-dependent flagellar gene expression and flagellar biosynthesis, but how FlhF influences these processes is unknown. Furthermore, the GTPase activity of any FlhF protein and the requirement of this speculated activity for steps in flagellar biosynthesis remain uncharacterized. We show here that C. jejuni FlhF hydrolyzes GTP, indicating that these proteins are GTPases. C. jejuni mutants producing FlhF proteins with reduced GTPase activity were not severely defective for sigma(54)-dependent flagellar gene expression, unlike a mutant lacking FlhF. Instead, these mutants had a propensity to lack flagella or produce flagella in improper numbers or at nonpolar locations, indicating that GTP hydrolysis by FlhF is required for proper flagellar biosynthesis. Additional studies focused on elucidating a possible role for FlhF in sigma(54)-dependent flagellar gene expression were conducted. These studies revealed that FlhF does not influence production of or signaling between the flagellar export apparatus and the FlgSR two-component regulatory system to activate sigma(54). Instead, our data suggest that FlhF functions in an independent pathway that converges with or works downstream of the flagellar export apparatus-FlgSR pathway to influence sigma(54)-dependent gene expression. This study provides corroborative biochemical and genetic analyses suggesting that different activities of the C. jejuni FlhF GTPase are required for distinct steps in flagellar gene expression and biosynthesis. Our findings are likely applicable to many polarly flagellated bacteria that utilize FlhF in flagellar biosynthesis processes.

FIG. 1.

Domain organization and sequence alignments of FlhF proteins. (A) The FlhF proteins of C. jejuni 81-176 (GenBank accession number YP_999790) and B. subtilis strain 168 (GenBank accession number CAA47062) can be divided into three domains: an N-terminal basic domain (B; black rectangles), a central N domain (N; white rectangles), and a C-terminal GTPase domain (G; gray rectangles). Within the GTPase domain, multiple conserved subdomains are evident, including G1 containing the P-loop domain (striped box) and G2 containing the DXXR motif (dotted box). Numbers above proteins indicate boundaries of domains. (B) ClustalW alignment of the G1 and G2 domains of the FlhF proteins from C. jejuni 81-176 (Cj), H. pylori 26695 (Hp) (GenBank accession number NP_207825), V. cholerae O395 (Vc) (GenBank accession number YP_001217595), V. alginolyticus12G01 (Va) (GenBank accession number ZP_01258825), P. aeruginosa PAO1 (Pa) (GenBank accession number AAG04842), P. putida MK1 (Pp) (GenBank accession number AF67042), and B. subtilis (Bs). Numbers flanking each sequence indicate positions of amino acids in the respective proteins. Arrowheads indicate conserved amino acids changed to alanine residues in the C. jejuni FlhF(K295A), FlhF(D321A), and FlhF(R324A) mutant proteins analyzed in this study.

FIG. 2.

Nucleotide hydrolysis activity of wild-type (WT) and mutant FlhF proteins. Released phosphate from [γ-³²P]GTP or [γ-³²P]ATP was monitored in reactions with purified wild-type and FlhF mutant proteins over 30 min. FlhF proteins and nucleotides were used at final concentrations of 0.5 μM and 0.1 μM, respectively. Hydrolysis activity is expressed as the percentage of labeled phosphate released compared to the amount of labeled phosphate in nonhydrolyzed nucleotides remaining at each time point. Data presented are from a representative assay. Closed symbols indicate phosphate released from GTP with addition of WT FlhF (♦), FlhF(K295A) (▴), FlhF(D321A) (×), FlhF(R324A) (▪), and FlhF_ΔG (•). Open symbols indicate phosphate released from ATP with addition of WT FlhF (⋄).

FIG. 3.

Analysis of flhF mutants for flagellar gene expression, motility, and protein production. (A) Immunoblot analysis of production of wild-type (WT) and FlhF mutant proteins in C. jejuni. FlhF proteins from WCL were detected with murine anti-FlhF M1 antiserum (top panel), and RpoA from WCL was detected with murine anti-RpoA M59 antiserum to verify equal loading of samples (bottom panel). Arrows indicate FlhF or FlhF_ΔG. Strains include DRH212 (wild-type 81-176 Sm^r), DRH1056 (81-176 Sm^r ΔflhF), MB630 [81-176 Sm^r flhF(D321A)], MB628 [81-176 Sm^r flhF(R324A)], DRH1302 [81-176 Sm^r flhF(K295A)], and SNJ206 [81-176 Sm^r flhF_ΔG]. (B) Motility phenotypes of wild-type (WT) and mutant C. jejuni strains in MH semisolid agar. Strains include DRH212 (wild-type 81-176 Sm^r), DRH1056 (81-176 Sm^r ΔflhF), MB630 [81-176 Sm^r flhF(D321A)], and MB628 [81-176 Sm^r flhF(R324A)]. (C) Arylsulfatase assays measuring expression of the σ⁵⁴-dependent transcriptional fusions, flgDE2::astA and flaB::astA, in C. jejuni strains. Results are from a typical assay with each strain tested in triplicate. Values reported for each strain are average arylsulfatase activity ± standard deviation relative to the amount of expression of each transcriptional fusion in wild-type 81-176 Sm^r ΔastA, which was set to 100 arylsulfatase units. For expression of flgDE2::astA (black bars), strains include wild-type DRH533 (WT), SNJ150, MB657, and MB662. For expression of flaB::astA (white bars), strains include wild-type DRH665, SNJ155, MB659, and MB666. (D) Immunoblot analysis of production of FlgG in C. jejuni strains. Anti-FlgG M69 murine antiserum was used to detect FlgG in WCL in the top panel. The lower panel shows an immunoblot for RpoA detected with anti-RpoA M59 murine antiserum to verify equal loading of samples. Strains include DRH212 (wild-type 81-176 Sm^r [WT]), SNJ925 (81-176 Sm^r ΔastA ΔflgG), DRH1056 (81-176 Sm^r ΔflhF), MB630 [81-176 Sm^r flhF(D321A)], and MB628 [81-176 Sm^r flhF(R324A)]. (E) Arylsulfatase assays measuring expression of flaA::astA in C. jejuni strains. Results are from a typical assay with each strain tested in triplicate. Values reported for each strain are average arylsulfatase activity ± standard deviation relative to the amount of expression of each transcriptional fusion in wild-type 81-176 Sm^r ΔastA, which was set to 100 arylsulfatase units. Strains include DRH655, SNJ154, MB704, and MB706.

FIG. 4.

Flagellar biosynthesis phenotypes of C. jejuni wild-type (WT) and flhF mutant strains. Transmission electron microscopy of negatively stained bacteria was performed. All micrographs are between magnification ×11,500 and ×20,500. Bars = 1 μm. (A) DRH212 (WT 81-176 Sm^r); (B) DRH1056 (81-176 Sm^r ΔflhF); (C to F) MB630 [81-176 Sm^r flhF(D321A)]; (G to J) MB628 [81-176 Sm^r flhF(R324A)]. Arrowheads indicate truncated flagella produced by some bacteria in panels C, G, and J.

FIG. 5.

Formation of the FEA-FlgSR signaling pathway components and activity of the FEA in C. jejuni wild-type (WT) and flhF mutant strains. (A) Immunoblot analysis of FlgS and FlgR production in WCL of C. jejuni strains. Immunoblots were performed with anti-FlgS Rab11 rabbit antiserum and anti-FlgR Rab13 rabbit antiserum. Control immunoblots were performed with anti-RpoA M59 antisera to verify equal loading of protein samples. Strains include DRH212 (WT 81-176 Sm^r), DRH460 (81-176 Sm^r ΔflgS) (20), DRH737 (81-176 Sm^r ΔflgR) (20), and DRH1056 (81-176 Sm^r ΔflhF). (B) Immunoblot analysis of FliF and FlhB production and localization to the membrane fraction in C. jejuni strains. Total membranes were isolated from wild-type and mutant C. jejuni strains and then analyzed by immunoblot analysis. Immunoblots were performed with anti-FliF M1 murine antiserum and anti-FlhB Rab476 rabbit antiserum. Control immunoblots were performed with anti-AtpF M3 and anti-RpoA M59 murine antisera to verify the presence of AtpF and absence of RpoA in the membrane preparations. The membrane proteins from 500 μl of bacterial culture at equivalent densities were analyzed for FliF, AtpF, and RpoA, and those from 5 ml of bacterial culture at equivalent densities was analyzed for FlhB. Strains include DRH212 (wild-type 81-176 Sm^r), DRH2074 (81-176 Sm^r ΔfliF), SNJ471 (81-176 Sm^r ΔflhB), and DRH1056 (81-176 Sm^r ΔflhF).

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 the FlgS sensor kinase for phosphorelay to the cognate FlgR response regulator to activate σ⁵⁴ for expression of a subset of flagellar genes (20, 23, 24). An activity of FlhF independent of GTP hydrolysis is hypothesized to be required for a late step in regulating the expression of σ⁵⁴-dependent flagellar genes which may include coactivation mechanisms with FlgR to stimulate σ⁵⁴ in RNA polymerase, activation of transcription initiation by σ⁵⁴-RNA polymerase holoenzyme, or stability of σ⁵⁴-dependent mRNA transcripts. The GTP hydrolysis activity of FlhF is hypothesized to be required at an early step in flagellar biosynthesis by possibly influencing the FEA so that a flagellum is constructed and only a single flagellum forms at each pole.