Activation of the Campylobacter jejuni FlgSR two-component system is linked to the flagellar export apparatus.
ABSTRACT 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.
Full-textDOI: · Available from: David Hendrixson, Aug 26, 2014
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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; DOI:10.1099/mic.0.059063-0 · 2.84 Impact Factor
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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 · 10.75 Impact Factor
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ABSTRACT: Campylobacter jejuni is a leading cause of bacterial diarrheal disease and a frequent commensal of the intestinal tract of poultry and other animals. For optimal growth and colonization of hosts, C. jejuni employs two-component regulatory systems (TCSs) to monitor environmental conditions and promote proper expression of specific genes. We analyzed the potential of C. jejuni Cjj81176_1484 (Cjj1484) and Cjj81176_1483 (Cjj1483) to encode proteins of a cognate TCS that influences expression of genes possibly important for C. jejuni growth and colonization. Transcriptome analysis revealed that the regulons of the Cjj1484 histidine kinase and the Cjj1483 response regulator contain many common genes, suggesting that these proteins likely form a cognate TCS. We found that this TCS generally functions to repress expression of specific proteins with roles in metabolism, iron/heme acquisition, and respiration. Furthermore, the TCS repressed expression of Cjj81176_0438 and Cjj81176_0439, which had previously been found to encode a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract. However, the TCS and other specific genes whose expression is repressed by the TCS were not required for colonization of chicks. We observed that the Cjj1483 response regulator binds target promoters both in unphosphorylated and phosphorylated forms and influences expression of some specific genes independently of the Cjj1484 histidine kinase. This work further expands the signaling mechanisms of C. jejuni and provides additional insights regarding the complex and multifactorial regulation of many genes involved in basic metabolism, respiration, and nutrient acquisition that the bacterium requires for optimal growth in different environments. Bacterial two-component regulatory systems (TCS) link environmental cues to expression of specific genes that enable optimal bacterial growth or colonization of hosts. We found that the Campylobacter jejuni Cjj1484 histidine kinase and Cjj1483 response regulator function as a cognate TCS to largely repress expression of target genes encoding a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract plus other genes encoding proteins for heme or iron acquisition, metabolism, and respiration. We also discovered different modes by which Cjj1483 may mediate repression with and without Cjj1484. This work provides insight into the signal transduction mechanisms of a leading cause of bacterial diarrheal disease and emphasizes the multifactorial and complex regulation of specific biological processes in C. jejuni. Copyright © 2015, American Society for Microbiology. All Rights Reserved.Journal of Bacteriology 02/2015; 197(9). DOI:10.1128/JB.02564-14 · 2.69 Impact Factor