Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans in developed countries throughout the world. This bacterium frequently promotes a commensal lifestyle in the gastrointestinal tracts of many animals including birds and consumption or handling of poultry meats is a prevalent source of C. jejuni for infection in humans. To understand how the bacterium promotes commensalism, we used signature-tagged transposon mutagenesis and identified 29 mutants representing 22 different genes of C. jejuni strain 81–176 involved in colonization of the chick gastrointestinal tract. Among the determinants identified were two adjacent genes, one encoding a methyl-accepting chemotaxis protein (MCP), presumably required for proper chemotaxis to a specific environmental component, and another gene encoding a putative cytochrome c peroxidase that may function to reduce periplasmic hydrogen peroxide stress during in vivo growth. Deletion of either gene resulted in attenuation for growth throughout the gastrointestinal tract. Further examination of 10 other putative MCPs or MCP-domain containing proteins of C. jejuni revealed one other required for wild-type levels of caecal colonization. This study represents one of the first genetic screens focusing on the bacterial requirements necessary for promoting commensalism in a vertebrate host.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72403/1/j.1365-2958.2004.03988.x.pd

Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73261/1/j.1365-2958.2001.02376.x.pd

Structural diversity of bacterial flagellar motors

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

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes

Architecture of the major component of the type III secretion system export apparatus

Type III secretion systems (T3SSs) are bacterial membrane–embedded nanomachines designed to export specifically targeted proteins from the bacterial cytoplasm. Secretion through T3SS is governed by a subset of inner membrane proteins termed the 'export apparatus'. We show that a key member of the Shigella flexneri export apparatus, MxiA, assembles into a ring essential for secretion in vivo. The ring-forming interfaces are well-conserved in both nonflagellar and flagellar homologs, implying that the ring is an evolutionarily conserved feature in these systems. Electron cryo-tomography revealed a T3SS-associated cytoplasmic torus of size and shape corresponding to those of the MxiA ring aligned to the secretion channel located between the secretion pore and the ATPase complex. This defines the molecular architecture of the dominant component of the export apparatus and allows us to propose a model for the molecular mechanisms controlling secretion

Polar Flagellar Biosynthesis and a Regulator of Flagellar Number Influence Spatial Parameters of Cell Division in Campylobacter jejuni

Spatial and numerical regulation of flagellar biosynthesis results in different flagellation patterns specific for each bacterial species. Campylobacter jejuni produces amphitrichous (bipolar) flagella to result in a single flagellum at both poles. These flagella confer swimming motility and a distinctive darting motility necessary for infection of humans to cause diarrheal disease and animals to promote commensalism. In addition to flagellation, symmetrical cell division is spatially regulated so that the divisome forms near the cellular midpoint. We have identified an unprecedented system for spatially regulating cell division in C. jejuni composed by FlhG, a regulator of flagellar number in polar flagellates, and components of amphitrichous flagella. Similar to its role in other polarly-flagellated bacteria, we found that FlhG regulates flagellar biosynthesis to limit poles of C. jejuni to one flagellum. Furthermore, we discovered that FlhG negatively influences the ability of FtsZ to initiate cell division. Through analysis of specific flagellar mutants, we discovered that components of the motor and switch complex of amphitrichous flagella are required with FlhG to specifically inhibit division at poles. Without FlhG or specific motor and switch complex proteins, cell division occurs more often at polar regions to form minicells. Our findings suggest a new understanding for the biological requirement of the amphitrichous flagellation pattern in bacteria that extend beyond motility, virulence, and colonization. We propose that amphitrichous bacteria such as Campylobacter species advantageously exploit placement of flagella at both poles to spatially regulate an FlhG-dependent mechanism to inhibit polar cell division, thereby encouraging symmetrical cell division to generate the greatest number of viable offspring. Furthermore, we found that other polarly-flagellated bacteria produce FlhG proteins that influence cell division, suggesting that FlhG and polar flagella may function together in a broad range of bacteria to spatially regulate division

A phase-variable mechanism controlling the Campylobacter jejuni FlgR response regulator influences commensalism

Summary Phase variation of genes in bacteria enables phenotypic alteration to modulate interactions within a host as conditions change. To promote commensalism in animals and disease in humans, Campylobacter jejuni produces a flagellar organelle for motility. In addition to tight transcriptional regulation of flagellar genes, C. jejuni also controls flagellar biosynthesis by phase variation. In this study, an unusual phasevariable mechanism controlling production of FlgR, the response regulator of the FlgSR two-component system required for transcription of s 54 -dependent flagellar genes, is identified. Phase variation of FlgR production is due to loss or gain of a nucleotide in homopolymeric adenine or thymine tracts within flgR. This mechanism occurs during commensalism in poultry to alter the colonization capacity of C. jejuni, presumably by influencing the motility phenotype of the bacterium. These findings provide more understanding into the genetic and colonization strategies C. jejuni employs to achieve commensalism in a natural host. Second, due to the richness of the C. jejuni genome in adenine or thymine residues and the apparent lack of the normal set of mismatch repair enzymes, the results from this study may suggest that the C. jejuni genome is more unstable and variable than previously realized. Furthermore, phase variation of flagellar motility by targeting flgR may be a phenomenon specific to C. jejuni that is absent in other Campylobacter species and contribute to reasons why C. jejuni is more frequently found as a commensal organism in poultry and as the cause of disease in humans

Transcription of σ 54 -dependent but not σ 28 -dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatus

We performed a genetic analysis of flagellar regulation in Campylobacter jejuni , from which we elucidated key portions of the flagellar transcriptional cascade in this bacterium. For this study, we developed a reporter gene system for C. jejuni involving astA , encoding arylsulphatase, and placed astA under control of the σ 54 -regulated flgDE2 promoter in C. jejuni strain 81-176. The astA reporter fusion combined with transposon mutagenesis allowed us to identify genes in which insertions abolished flgDE2 expression; genes identified were on both the chromosome and the plasmid pVir. Included among the chromosomal genes were genes encoding a putative sensor kinase and the σ 54 -dependent transcriptional activator, FlgR. In addition, we identified specific flagellar genes, including flhA , flhB , fliP , fliR and flhF , that are also required for transcription of flgDE2 and are presumably at the beginning of the C. jejuni flagellar transcriptional cascade. Deletion of any of these genes reduced transcription of both flgDE2 and another σ 54 -dependent flagellar gene, flaB , encoding a minor flagellin. Transcription of the σ 28 -dependent gene flaA , encoding the major flagellin, was largely unaffected in the mutants. Further examination of flaA transcription revealed significant σ 28 -independent transcription and only weak repressive activity of the putative anti-σ 28 factor FlgM. Our study suggests that σ 54 -dependent transcription of flagellar genes in C. jejuni is linked to the formation of the flagellar secretory apparatus. A key difference in the C. jejuni flagellar transcriptional cascade compared with other bacteria that use σ 28 for transcription of flagellar genes is that a mechanism to repress significantly σ 28 -dependent transcription of flaA in flagellar assembly mutants is absent in C. jejuni .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73376/1/j.1365-2958.2003.03731.x.pd

Analysis of the Roles of FlgP and FlgQ in Flagellar Motility of Campylobacter jejuni

Flagellar motility is an important determinant of Campylobacter jejuni that is required for promoting interactions with various hosts to promote gastroenteritis in humans or commensal colonization of many animals. In a previous study, we identified a nonmotile mutant of C. jejuni 81-176 with a transposon insertion in Cj1026c, but verification of the role of the encoded protein in motility was not determined. In this study, we have determined that Cj1026c and the gene immediately downstream, Cj1025c (here annotated as flgP and flgQ, respectively), are both required for motility of C. jejuni but not for flagellar biosynthesis. FlgP and FlgQ are not components of the transcriptional regulatory cascades to activate σ(28)- or σ(54)-dependent expression of flagellar genes. In addition, expression of flgP and flgQ is not largely dependent on σ(28) or σ(54). Immunblot analyses revealed that the majority of FlgP in C. jejuni is associated with the outer membrane. However, in the absence of FlgQ, the amounts of FlgP in the outer membrane of C. jejuni are greatly reduced, suggesting that FlgQ may be required for localization or stability of FlgP at this location. This study provides insight into features of FlgP and FlgQ, two proteins with previously undefined functions that are required for the larger, multicomponent flagellar system of C. jejuni that is necessary for motility

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

Activation of σ54-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 σ54-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 σ54-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 σ54-dependent flagellar genes. Considering that the flagellar export apparatuses of Escherichia coli and Salmonella species influence σ28-dependent flagellar gene expression, our work expands the signaling activity of the apparatuses to include σ54-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 σ54-dependent flagellar genes