Activation of the Campylobacter jejuni FlgSR Two-Component System Is Linked to the Flagellar Export Apparatus

University of Texas Southwestern Medical School, Department of Microbiology, 5323 Harry Hines Boulevard, Dallas, TX 75390-9048, USA.
Journal of bacteriology (Impact Factor: 2.81). 03/2009; 191(8):2656-67. DOI: 10.1128/JB.01689-08
Source: PubMed


Activation of sigma(54)-dependent gene expression essential for formation of flagella in Campylobacter jejuni requires the components of the inner membrane-localized flagellar export apparatus and the FlgSR two-component regulatory system. In this study, we characterized the FlgS sensor kinase and how activation of the protein is linked to the flagellar export apparatus. We found that FlgS is localized to the C. jejuni cytoplasm and that His141 of FlgS is essential for autophosphorylation, phosphorelay to the cognate FlgR response regulator, motility, and expression of sigma(54)-dependent flagellar genes. Mutants with incomplete flagellar export apparatuses produced wild-type levels of FlgS and FlgR, but they were defective for signaling through the FlgSR system. By using genetic approaches, we found that FlgSR activity is linked to and downstream of the flagellar export apparatus in a regulatory cascade that terminates in expression of sigma(54)-dependent flagellar genes. By analyzing defined flhB and fliI mutants of C. jejuni that form flagellar export apparatuses that are secretion incompetent, we determined that formation of the apparatus is required to contribute to the signal sensed by FlgS to terminate in activation of expression of sigma(54)-dependent flagellar genes. Considering that the flagellar export apparatuses of Escherichia coli and Salmonella species influence sigma(28)-dependent flagellar gene expression, our work expands the signaling activity of the apparatuses to include sigma(54)-dependent pathways of C. jejuni and possibly other motile bacteria. This study indicates that these apparatuses have broader functions beyond flagellar protein secretion, including activation of essential two-component regulatory systems required for expression of sigma(54)-dependent flagellar genes.

Download full-text


Available from: David Hendrixson, Aug 26, 2014
  • Source
    • "In vitro phosphotransfer from MBP-trCosS to rCosR_F, rCosR_J or CosRJ_N51D was monitored as described previously [25], [26]. Phosphorylation of 2 µM of rCosR_F, rCosR_J and CosRJ_N51D was achieved by adding the same amount of MBP-trCosS which had been autophosphorylated for 5 min in 20 µl of phosphorylation buffer as described above. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Two-component signal transduction systems are commonly composed of a sensor histidine kinase and a cognate response regulator, modulating gene expression in response to environmental changes through a phosphorylation-dependent process. CosR is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a major foodborne pathogenic species causing human gastroenteritis. Although CosR is a response regulator, its cognate sensor kinase has not been identified in C. jejuni. In this study, DNA sequence analysis of the cosR flanking regions revealed that a gene encoding a putative sensor kinase, which we named cosS, is prevalent in non-thermotolerant Campylobacter spp., but not in thermotolerant campylobacters. Phosphorylation assays indicated that C. fetus CosS rapidly autophosphorylates and then phosphorylates C. fetus CosR, suggesting that the CosRS system constitutes a paired two-component signal transduction system in C. fetus. However, C. fetus CosS does not phosphorylate C. jejuni CosR, suggesting that CosR may have different regulatory cascades between thermotolerant and non-thermotolerant Campylobacter species. Comparison of CosR homolog amino acid sequences showed that the conserved phosphorylation residue (D51), which is present in all non-thermotolerant Campylobacter spp., is absent from the CosR homologs of thermotolerant Campylobacter species. However, C. jejuni CosR was not phosphorylated by C. fetus CosS even after site-directed mutagenesis of N51D, implying that C. jejuni CosR may possibly function phosphorylation-independently. In addition, the results of cosS mutational analysis indicated that CosS is not associated with the temperature dependence of the Campylobacter spp. despite its unique divergent distribution only in non-thermotolerant campylobacters. The findings in this study strongly suggest that thermotolerant and non-thermotolerant Campylobacter spp. have different signal sensing mechanisms associated with the CosR regulation.
    PLoS ONE 02/2014; 9(2):e89774. DOI:10.1371/journal.pone.0089774 · 3.23 Impact Factor
  • Source
    • "The mechanism by which the export apparatus affects the RpoN regulon in H. pylori and other members of the Epsilonproteobacteria is not known, but it may do so by modulating FlgS activity. In support of this hypothesis, a variant of FlgR that functions independently of FlgS partially restores expression of RpoN-dependent reporter genes in C. jejuni mutants in which flhA, flhB or fliP is disrupted (Joslin & Hendrixson, 2009). It is possible that FlgS responds to conformational changes in the export apparatus during flagellar assembly. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Flagellar biogenesis in the gastric pathogen Helicobacter pylori involves a transcriptional hierarchy that utilizes all three sigma factors found in this bacterium (RpoD, RpoN, and FliA). Transcription of the RpoN-dependent genes requires the sensor kinase FlgS and response regulator FlgR. It is thought that FlgS senses some cellular cue to regulate transcription of the RpoN-dependent flagellar genes, but this signal has yet to be identified. Previous studies showed transcription of the RpoN-dependent genes is inhibited by mutations in flhA, which encodes a membrane-bound component of the flagellar protein export apparatus. We found that depending on the H. pylori strain used, insertion mutations in flhA had different effects on expression of RpoN-dependent genes. Mutations in flhA in H. pylori strains B128 and ATCC 434504 were generated by inserting a chloramphenicol resistance cassette so as to effectively eliminate expression of the gene (ΔflhA); or within the gene following codon 77 (designated flhA77) or codon 454 (designated flhA454) which could allow expression of truncated FlhA proteins. All three flhA mutations severely inhibited transcription of the RpoN-dependent genes flaB and flgE in H. pylori B128. In contrast, levels of flaB and flgE transcripts in H. pylori ATCC 43504 bearing either flhA77 or flhA454, but not ΔflhA, were ~60% of wild-type levels. The FlhA454 variant was detected in membrane fractions prepared from H. pylori ATCC 43504, but not H. pylori B128, which may account for the phenotypic differences in the flhA mutations of the two strains. Taken together, these findings suggest that only the N-terminal region of FlhA is needed for transcription of the RpoN regulon. Interestingly, expression of a flaB'-'xylE reporter gene in H. pylori ATCC 43504 bearing the flhA77 allele was ~8-fold higher than that of a strain with the wild-type allele, suggesting that expression of flaB is not only regulated at the level of transcription but is also regulated post-transcriptionally.
    Microbiology 11/2012; 159(Pt_1). DOI:10.1099/mic.0.059063-0 · 2.56 Impact Factor
  • Source
    • "Starting with wild-type Campylobacter jejuni 81-176 Sm R (DRH212) containing an rpsL allele conferring streptomycin resistance (Hendrixson et al, 2001), strain DRH2257 was constructed through in-frame deletions of fliI and marker gene astA (the enzyme arylsulphatase), which does not affect flagellar biosynthesis or motility (Joslin and Hendrixson, 2009). DRH212 and DRH2257 were grown on MH agar under microaerophilic conditions for 3 days, re-streaked and grown overnight. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.
    The EMBO Journal 06/2011; 30(14):2972-81. DOI:10.1038/emboj.2011.186 · 10.43 Impact Factor
Show more