A Chaperone for the Stator Units of a Bacterial Flagellum

Abstract

The stator units of the flagellum supply power to the flagellar motor via ion transport across the cytoplasmic membrane and generate torque on the rotor for rotation. Flagellar motors across bacterial species have evolved adaptations that impact and enhance stator function to meet the demands of each species, including producing stator units using different fuel types or various stator units for different motility modalities. Campylobacter jejuni produces one of the most complex and powerful flagellar motors by positioning 17 stator units at a greater radial distance than in most other bacteria to increase power and torque for high velocity of motility. We report another evolutionary adaptation impacting flagellar stators by identifying FlgX as a chaperone for C. jejuni stator units to ensure sufficient power and torque for flagellar rotation and motility. We discovered that FlgX maintains MotA and MotB stator protein integrity likely through a direct interaction with MotA that prevents their degradation. Suppressor analysis suggested that the physiology of C. jejuni drives the requirement for FlgX to protect stator units from proteolysis by the FtsH protease complex. C. jejuni ΔflgX was strongly attenuated for colonization of the natural avian host, but colonization capacity was greatly restored by a single mutation in MotA. These findings suggest that the likely sole function of FlgX is to preserve stator unit integrity for the motility required for host interactions. Our findings demonstrate another evolved adaptation in motile bacteria to ensure the equipment of the flagellar motor with sufficient power to generate torque for motility.IMPORTANCE The bacterial flagellum is a reversible rotating motor powered by ion transport through stator units, which also exert torque on the rotor component to turn the flagellum for motility. Species-specific adaptations to flagellar motors impact stator function to meet the demands of each species to sufficiently power flagellar rotation. We identified another evolutionary adaptation by discovering that FlgX of Campylobacter jejuni preserves the integrity of stator units by functioning as a chaperone to protect stator proteins from degradation by the FtsH protease complex due to the physiology of the bacterium. FlgX is required to maintain a level of stator units sufficient to power the naturally high-torque flagellar motor of C. jejuni for motility in intestinal mucosal layers to colonize hosts. Our work continues to identify an increasing number of adaptations to flagellar motors across bacterial species that provide the mechanics necessary for producing an effective rotating nanomachine for motility.

Keywords: Campylobacter jejuni; FlgX; MotA; MotB; chaperone; flagellar motility; stator.

FIG 1

Requirement of FlgX for motility and stability of MotA and MotB stator proteins. For panels A and B, the C. jejuni ΔflgX mutant contained a deletion of codons 6 to 153 of the larger predicted coding sequence and all complementing plasmids expressed flgX encoding the predicted 165-amino-acid-long protein. (A) Motility phenotypes of WT C. jejuni and isogenic mutants. Motility was analyzed at 30 h of incubation in MH motility agar (0.4% agar) at 37°C under microaerobic conditions. Each strain contained vector alone (vec) or vector used to produce FlgX, FLAG-FlgX, or FlgX-FLAG as indicated. (B) Immunoblot analysis of MotA and MotB stator proteins in whole-cell lysates of WT C. jejuni and isogenic mutants. Specific antiserum to MotA and MotB was used to detect each protein. Detection of RpoA served as a control to ensure equal loading of proteins across strains. C. jejuni ΔflgX mutants were complemented with vector alone or plasmid to produce FlgX, FLAG-FlgX, or FlgX-FLAG as indicated.

FIG 2

Detection of in vivo interactions between FlgX and MotA or MotB in C. jejuni. (A) Analysis of FlgX coimmunoprecipitated (Co-IP) C. jejuni proteins. WT C. jejuni with vector alone and C. jejuni ΔflgX complemented with vector alone or vector to produce untagged WT FlgX, FLAG-FlgX, or FlgX-FLAG were included for analysis. Briefly, C. jejuni cells were cross-linked with formaldehyde to trap complexes and then quenched to stop the cross-linking reaction. Cell were pelleted, osmotically lysed, and then solubilized with a solution containing Triton X-100. After centrifugation, FlgX was immunoprecipitated overnight with anti-FLAG M2 affinity gel resin (see Materials and Methods for full details). MotA, MotB, FliF, and RpoA were detected by specific antisera after immunoprecipitation. FLAG-tagged FlgX was detected by a monoclonal anti-FLAG antibody. The first lane contains proteins from whole-cell lysates of WT C. jejuni for reference. Note that, although the FLAG-FlgX and FlgX-FLAG proteins are predicted to be the same size, a difference in the levels of mobility during SDS-PAGE is responsible for the apparent size shift. This possible structural alteration does not appear to impact function as both proteins coimmunoprecipitate with the stator proteins. (B) Analysis of FlgX coimmunoprecipitated proteins in C. jejuni motA and motB mutants. C. jejuni ΔflgX, ΔflgX ΔmotA, or ΔflgX ΔmotB contained a vector to express FLAG-FlgX. MotA and MotB were detected by specific antisera whereas FLAG-FlgX was detected by a monoclonal anti-FLAG antibody after immunoprecipitation with FLAG-FlgX (left) or in whole-cell lysates of the strains analyzed (right).

FIG 3

Localization of FlgX in C. jejuni. Data represent results of immunoblot analysis of FLAG-FlgX localization in different cellular fractions of WT C. jejuni or C. jejuni ΔmotAB. C. jejuni cultures were standardized to similar cellular densities and then fractionated to recover proteins from the cytoplasmic (C), inner membrane (IM), periplasmic (P), and outer membrane (OM) fractions. Samples from whole-cell lysates (WCL) were also recovered. Equal amounts of proteins were loaded for all strains. FLAG-FlgX was detected by a monoclonal anti-FLAG antibody. Specific antisera were used to detect proteins as specific markers for different fractions, including RpoA (cytoplasm), PflB (inner membrane), Cjj81176_0382 (periplasm), and FlgP (outer membrane).

FIG 4

FlgX-dependent stability of MotA and MotB stator proteins in flagellated amotile mutants. Data represent results of immunoblot analysis of MotA and MotB stator proteins in whole-cell lysates of WT C. jejuni and isogenic single or double mutants that result in flagellated but amotile mutants. Specific antiserum to MotA and MotB was used to detect each protein. Detection of RpoA served as a control to ensure equal loading of proteins across strains.

FIG 5

Analysis of C. jejuni ΔflgX suppressor mutants with restored motility. (A to C) DAR4803, representing strain 81-176 rpsL^Sm ΔflgX with deletion of codons 101 to 153 of flgX, and derived isogenic suppressor mutants (annotated as “S1” through “S10”) are shown. (A) Motility phenotypes of WT C. jejuni, the parental ΔflgX mutant, and isolated motile ΔflgX suppressor mutants. Motility was analyzed after 30 h of incubation in MH motility agar (0.4% agar) at 37°C under microaerobic conditions. (B) Immunoblot analysis of MotA and MotB stator protein levels in whole-cell lysates of WT C. jejuni, the parental ΔflgX mutant, and isolated motile ΔflgX suppressor mutants. Specific antiserum to MotA and MotB was used to detect each protein. Detection of RpoA served as a control to ensure equal loading of proteins across strains. (C) Semiquantitative real-time PCR analysis of transcription of ftsH in the parental ΔflgX mutant and selected motile ΔflgX suppressor mutants. Suppressor mutants S4, S8, and S9 contain frameshift mutations in Cjj81176_1135. Suppressor mutant S10 with the motA_H138Y missense mutation served as a control. The level of expression of ftsH in C. jejuni ΔflgX as measured by qRT-PCR is set to 1. Expression of ftsH suppressor mutants is shown relative to that seen with C. jejuni ΔflgX. Error bars indicate standard deviations. Statistically significant differences in ftsH expression levels between C. jejuni ΔflgX and suppressor mutants (*, P < 0.05) as performed by Student's t test are indicated.

FIG 6

Sufficiency of MotA_H138Y to suppress C. jejuni ΔflgX phenotypes. (A) Motility phenotypes of WT C. jejuni and isogenic mutants. Motility was analyzed after 30 h of incubation in MH motility agar (0.4% agar) at 37°C under microaerobic conditions. (B) Immunoblot analysis of MotA and MotB stator protein levels in whole-cell lysates of WT C. jejuni and isogenic mutants. Specific antiserum to MotA and MotB was used to detect each protein. Detection of RpoA served as a control to ensure equal loading of proteins across strains. In panels A and B, C. jejuni strains contained WT motA or an in-frame deletion of motA (ΔmotA) or the WT motA gene was replaced on the chromosome with motA_H138Y. (C) Analysis of FlgX coimmunoprecipitation of WT MotA and MotA_H138Y in C. jejuni. C. jejuni ΔflgX and ΔflgX motA_H138Y contained a vector to express FLAG-FlgX. MotA and MotB were detected by specific antisera, whereas FLAG-FlgX was detected by a monoclonal anti-FLAG antibody in proteins after immunoprecipitation with FLAG-FlgX (left) or in whole-cell lysates of the strains analyzed (right).

FIG 7

Stability of stator units upon heterologous expression. For panels A and B, the coding sequences of the motAB loci from C. jejuni and S. enterica serovar Typhimurium were expressed from the same constitutive promoter and plasmid backbone in C. jejuni strain ΔmotAB with production of WT FlgX (+) or without production of FlgX (−) (A) or in E. coli (B). A FLAG tag was fused to the C terminus of the MotB proteins to monitor stator production with the same anti-FLAG antibody in whole-cell lysates (WCL) or in the total membrane fraction. Detection of C. jejuni RpoA or E. coli RpoD served as a control to ensure equal loading of proteins across strains and as a control for fractionation.

FIG 8

Commensal colonization capacity of WT C. jejuni and ΔflgX and and ΔflgX motA_H138Y suppressor mutants in avian hosts. Day-of-hatch chicks were orally gavaged with WT C. jejuni 81-176 Sm^r (red), the ΔflgX mutant (blue), or the reconstructed ΔflgX motA_H138Y suppressor mutant (yellow). The measured levels of inocula for the chicks as determined by dilution plating were as follows: 2.1 × 10⁴ CFU for WT C. jejuni; 2.06 × 10⁴ CFU for theΔflgX mutant; and 9.7 × 10³ CFU for the ΔflgX motA_H138Y mutant. Chicks were sacrificed at day 14 postinfection, and the levels of all C. jejuni strains in the ceca (reported as CFU per gram of content) were determined. Each closed circle represents the level of C. jejuni in a single chick. The black horizontal bars represent the geometric mean for each group. Statistical analysis was performed using the Mann-Whitney U test. The asterisks (*) indicate a statistically lower level of colonization of mutants than of WT C. jejuni (P < 0.05).