Diversification of Campylobacter jejuni Flagellar C-Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes

Abstract

Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species.IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.

Keywords: C ring; FlhG; FliI; FliN; FliY; flagellar motor; polar flagella; type III secretion.

FIG 1

Phylogenetic analysis of the SPOA domain-containing family of flagellar proteins. (A) Unrooted phylogenetic tree of aligned sequences of FliM, FliN, and FliY from 12 bacteria from the spirochetes, Firmicutes, Epsilonproteobacteria, and Gammaproteobacteria. Confidence values were generated through 1,000 bootstrap repeats. Branches are shaded red for FliM, green for FliY, and blue for FliN. (B) Genomic organization and conserved encoded domains of fliM, fliN, and fliY. The SPOA domain (Pfam domain pf01052) is shown in black, and the presence of true (yellow) and potential degenerate (gray) EIDAL motifs is indicated. Adjacent genes indicate adjacent genomic locations, whereas slashes indicate distant locations.

FIG 2

Flagellar protein stability and motility phenotypes of C. jejuni MS and C-ring mutants. (A) Immunoblot analysis of MS and C-ring protein levels in whole-cell lysates of WT C. jejuni and isogenic mutant strains. Specific antiserum to each MS and C-ring protein was used to detect each protein. The detection of RpoA served as a control to ensure equal loading of proteins across strains. (B) Motility phenotypes of WT C. jejuni and isogenic mutant strains. Motility was analyzed after 30 h of incubation at 37°C under microaerobic conditions in MH motility agar (0.4%).

FIG 3

Subtomogram average flagellar motors structures of WT C. jejuni and isogenic mutants. Imaging of WT C. jejuni and ΔfliI, ΔfliH, ΔfliN, ΔfliY, ΔfliM and ΔfliM ΔfliY mutant flagellar motors revealed the distal tip of the C ring in its in situ functional state. (A) Top row, unfiltered C17 symmetrized subtomogram averages at pixel size of 8.28 Å, 25-nm scale (bar); bottom row, cartoon schematic of C-ring structures, as follows: FliG (green), FliM (pink), FliY (gray), FliN (light blue), FliH and FliI (purple), axial flagellar components (blue), stator complexes (beige), and basal disk (gray disk). Column 1, WT top (7); column 2, ΔfliI mutant composed of 205 particles; column 3, ΔfliH mutant composed of 187 particles; column 4, ΔfliN mutant composed of 281 particles; column 5, ΔfliY mutant composed of 155 particles; column 6, ΔfliM mutant composed of 187 particles; column 7, ΔfliM ΔfliY mutant composed of 212 particles. All images are 100-nm by 100-nm boxes.

FIG 4

In vivo interactions between C. jejuni MS ring and C-ring proteins. N- or C-terminal FLAG-tagged proteins were expressed from plasmids in respective C. jejuni mutants and immunoprecipitated by FLAG tag antibody resin after cross-linking cells by formaldehyde. The position of the FLAG tag is indicated by the position of the asterisk (*) at the beginning or end of each protein that labels each lane at top. Each lane shows the detected proteins that coimmunoprecipitated with the FLAG-tagged protein indicated. Each protein was detected with specific antiserum. A whole-cell lysate (WCL) of WT C. jejuni was run alongside the coimmunoprecipitated proteins to indicate the size and position of the native protein. Arrows indicate the correct size of the monomer for each protein detected in the immunoblot. Arrowheads indicate bands that may represent a complex formed by the FLAG tag-immunoprecipitated protein with other proteins.

FIG 5

Interdependencies of C-ring proteins on each other for interactions. (A) Coimmunoprecipitation (Co-IP) analysis of C-ring-protein interactions in mutants lacking another C-ring protein. C-terminal FLAG-tagged FliM or N-terminal FLAG-tagged FliY, FliN, or FliH was expressed from a plasmid in C. jejuni single or double mutants lacking specific flagellar proteins and immunoprecipitated by FLAG tag antibody resin after cross-linking cells by formaldehyde. Immunoprecipitated proteins were detected with specific antisera. The top set of immunoblots (Co-IP) shows results from coimmunoprecipitation experiments. The bottom set of immunoblots (WCL) shows levels of proteins in whole-cell lysates of mutants. RpoA is included as a control protein that should not coimmunoprecipitate with a C-ring protein and as a control to ensure equal loading of whole-cell lysates. (B) Summary model of C. jejuni C-ring-protein interactions from coimmunoprecipitation experiments. Double arrows indicate interactions verified by reciprocal coimmunoprecipitation experiments. Single arrows indicate an interaction that could only be observed by immunoprecipitation of one of two interacting partners. Thinner arrows indicate relative weaker interactions. Pale-green arrows indicate that the FliY interactions with FliH and FliN are dependent on FliM.

FIG 6

Flagellation phenotypes and minicell production of C. jejuni C-ring mutants. WT C. jejuni and isogenic mutant strains were negatively stained with uranyl acetate and examined by transmission electron microscopy. Arrowheads indicate minicells. Scale bar = 1 μm.

FIG 7

Quantitative assessment of minicell production in C. jejuni MS and C-ring mutants. The lengths of cell bodies of WT C. jejuni and isogenic mutant strains from electron micrographs were measured to determine the percentage of cell populations that are normal size cells or are minicells. Two experiments were performed in which at least 100 individual bacteria were examined per strain. The data are reported as the percentage of bacterial populations with the following cell lengths: >2 μm (brown), 1 to 2 μm (yellow), 0.5 to 1 μm (blue), and <0.5 μm, minicells (red). The data represent the average of the results from the two experiments. Bars represent standard deviations.

FIG 8

FlhG polar localization in WT C. jejuni and isogenic flagellar mutants. Strains were analyzed by immunofluorescent microscopy after staining with both FlhG and whole C. jejuni antisera. Cells in which detection of FlhG was exclusively at poles were considered positive for polar localization of FlhG. Each strain was analyzed in triplicate, and at least 100 individual cells were counted per sample. After analysis, the percentage of cells with exclusive polar localization of FlhG were averaged, and the standard deviations were determined (bars). A Student's t test was performed to determine the statistical significance of differences in polar localization of FlhG between WT and mutant strains (*, P < 0.05).

FIG 9

Model of C-ring assembly, composition, and function in C. jejuni. Cartoon schematics show the architecture of the peritrichous C ring (top left) and the C. jejuni C ring (top right), the effect of deletion of fliN partially disrupting the C-ring and ATPase complex architecture (bottom left), and the effect of deletion of fliM further disrupting the C-ring architecture (bottom right).