A link between the assembly of flagella and lipooligosaccharide of the Gram-negative bacterium Campylobacter jejuni

Abstract

Campylobacter jejuni is the leading cause of acute bacterial diarrhea worldwide and is implicated in development of Guillain-Barré syndrome. Two major surface features, the outer membrane lipooligosaccharide and flagella, are highly variable and are often targets for modification. Presumably, these modifications provide a competitive advantage to the bacterium. In this work, we identify a gene encoding a phosphoethanolamine (pEtN) transferase (Cj0256) that serves a dual role in modifying not only the lipooligosaccharide lipid anchor lipid A with pEtN, but also the flagellar rod protein FlgG. Generation of a mutant in C. jejuni 81–176 by interruption of cj0256 resulted in the absence of pEtN modifications on lipid A as well as FlgG. The cj0256 mutant showed a 20-fold increase in sensitivity to the cationic antimicrobial peptide, polymyxin B, as well as a decrease in motility. Transmission EM of the cj0256 mutant revealed a population (approximately 95%) lacking flagella, indicating that, without pEtN modification of FlgG, flagella production is hindered. Most intriguing, this research identifies a pEtN transferase showing preference for two periplasmic substrates linking membrane biogenesis and flagellar assembly. Cj0256 is a member of a large family of mostly uncharacterized proteins that may play a larger role in the decoration of bacterial surface structures.

Campylobacter jejuni is a major cause of bacterial diarrhea worldwide (1). Infection with this pathogen results in significant acute illness as well as serious life-threatening consequences, such as Guillain-Barré syndrome (2). Like all pathogenic bacteria, C. jejuni synthesizes complex outer surface structures that are critical for pathogenesis. Two major surface features, the outer membrane lipooligosaccharide (LOS) and flagella, are highly variable and are often targets for modification, presumably to provide a competitive advantage to the bacterium. For example, the structural subunits in the flagella filament of epsilon proteobacteria are glycosylated. In the case of C. jejuni, these glycosylation events are required for assembly of flagellin subunits. Flagella components including the rod, hook, and structural filament are secreted from the cytoplasm through a growing narrow channel via the flagellar type III secretion system and polymerize at the distal end (3). To date, posttranslational modification of flagellar components has been shown to occur only in the cytoplasm before secretion and reported only in the filament flagellins.

Another well characterized bacterial surface structure, and the major molecule found on the surface of C. jejuni, is LOS. Like all Gram-negative bacteria, the hydrophobic anchor of LOS or lipopolysaccharide (LPS) is lipid A. Many Gram-negative bacteria modify their lipid A to provide protection against cationic antimicrobial peptides (CAMPs) and to avoid detection by the host Toll-like receptor 4/MD2 innate immune receptor (4, 5). The lipid A of C. jejuni is characterized by longer secondary acyl chains attached to the 2′ and 3′ positions of the molecule and by the addition of phosphoethanolamine (pEtN) to the phosphate groups attached at the 1 and 4′ positions of the disaccharide backbone (6) (Fig. 1C).

Fig. 1.

Structural comparison of the lipid A anchors found in E. coli (A and B) and C. jejuni (C). Dashed bonds indicate modifications of lipid A and the length of acyl chains is indicated. In WT E. coli, an additional phosphate group (in red) can be attached at the 1-position (11). Under conditions leading to activation of the transcriptional regulators PhoP and PmrA, E. coli lipid A can be further derivatized with pEtN (magenta), 4-amino-4-deoxy-L-arabionse (blue), and palmitate (black) (4, 5). In C. jejuni the glucosamine disaccharide backbone can be replaced with the analogue 2,3-diamino-2,3-dideoxy-D-glucopyranose, resulting in two additional amide linked (brown) acyl chains.

Here we report the identification of a pEtN transferase from C. jejuni, Cj0256, that unexpectedly modifies two periplasmic targets, a membrane lipid and a flagellar protein. Deletion of cj0256 results in the loss of pEtN modification of C. jejuni lipid A and sensitivity to CAMPs, polymyxin B. Suprisingly, cj0256 mutants showed decreased motility and greatly reduced flagella production. Our data indicate that Cj0256 also modifies the flagellar rod protein FlgG (7). Modfication of FlgG is a known periplasmic posttranslational modification of a bacterial flagellar component. Given that Cj0256 is member of a large family of proteins (COG2194) found in a number of pathogenic bacteria, periplasmic decoration of bacterial structures with phosphoryl substituents are likely to play an important role in pathogenesis.

Results

Cj0256 Is a Lipid A pEtN Transferase.

To identify the enzyme responsible for pEtN modification of C. jejuni lipid A (Fig. 1C), we performed a BLASTp search using a previously characterized pEtN transferase from Helicobacter pylori (hp0022) (8), revealing a single putative homologue, cj0256 (E-value <10⁻⁵⁵). To confirm that Cj0256 functions as a lipid A pEtN transferase, we heterologously expressed Cj0256 in E. coli K-12 strain W3110 (strain EC01). Cultures of EC01 were radiolabeled with ³²P_i and the purified lipid A species separated using TLC. E. coli K-12 W3110 produced the typical lipid A species seen in WT, 1,4´-bis-phosphate lipid A, and 1-diphosphate lipid A (Fig. 2A). As a control, an E. coli K-12 PmrA constitutive mutant WD101 (9), modified with pEtN at the 1 and 4´ positions, was used to identify the migratory patterns of pEtN modified lipid A (Fig. 2A). Expression of Cj0256 resulted in the production of pEtN-modified lipid A species (Fig. 2A). To confirm our findings, purified lipid A from strains W3110 and EC01 was analyzed by MALDI-TOF MS. Analysis of WT W3110 revealed major ion peaks at m/z 1,796.9 and 1,876.9 representing 1,4´-bis-phosphate lipid A and 1-diphosphate lipid A, respectively (Fig. 1A and Fig. S1). Analysis of EC01 revealed major ion peaks at m/z 1,918.8 and 2,041.5 representing lipid A species bearing a single pEtN or two pEtN residues, respectively (Fig. S1).

Fig. 2.

Cj0256 is a lipid A pEtN transferase. ³²P-labeled lipid A was isolated from the indicated strains, separated by TLC and visualized by phosphorimaging. WT (W3110) and pmrA^C (WD101) E. coli were used as controls to confirm the migratory patterns of pEtN modified lipid A. “1-PP” indicates 1-diphosphate lipid A produced by W3110. “Double” indicates lipid A species modified primarily with two pEtN groups as seen in WD101. (A) Heterologous expression of cj0256 in E. coli (EC01) results in modification of lipid A with pEtN. (B) Loss of cj0256 in C. jejuni (81-176A1 and 2) results in loss of pEtN modification compared with WT (81–176) and complemented (81–176B) C. jejuni.

To determine if phosphatidylethanolamine (PtdEtN) serves as the pEtN donor for Cj0256, we used an E. coli strain (AD90) harboring an insertion mutation in the gene encoding for phosphatidyl serine synthase (pss). These bacteria lack PtdEtN, and in this background, expression of Cj0256 had no effect on lipid A synthesis (Fig. S2). Complementation of the pss mutation with plasmid pDD72 restored the ability of Cj0256 to decorate E. coli lipid A with pEtN (Fig. S2). These data suggest that PtdEtN serves as the donor substrate for pEtN modification, but does not rule out the use of other putative donors such as lyso-PtdEtN.

Modification of lipid A most often occurs following its transport across the inner membrane by the dedicated flippase MsbA (4). In E. coli WD2, MsbA transport of lipid A across the inner membrane is lost following a temperature shift to 44 °C (10). To investigate the site of Cj0256-dependent modification, we examined pEtN transfer in WD2. WD2 containing pWcj0256 were cultured at the permissive temperature of 30 °C until midlog, followed by a shift to 44 °C. Cells were pulse labeled and de novo ³²P-labeled lipid A species separated by TLC as previously described (8). At 30 °C, Cj0256 was capable of modifying E. coli lipid A. However, inactivation of MsbA transport at 44 °C resulted in loss of pEtN modification, indicating that Cj0256-dependent pEtN occurs in the periplasm (Fig. S3). The production of the 1-diphosphate species of lipid A catalyzed by LpxT was lost upon inactivation of MsbA as previously shown (11) (Fig. S3).

Generation of a cj0256 Deletion Mutant in C. jejuni and Analysis of its Lipid A.

Many pathogen-associated virulence factors in C. jejuni are phase variable, including its LOS (12). Therefore, ³²P-labeled lipid A from three C. jejuni strains for which the genome sequences are available (strains 81–176, 11–168, and 81–116) and from a clinical isolate (strain VLAO) were compared. The migration pattern of lipid A species following TLC for all four strains was identical, thus indicating no variation in the lipid A domain of C. jejuni LOS. Also, a comparison of the lipid A migration to that of E. coli WD101 suggested the presence of both singly and doubly modified lipid A species (Fig. S4). To ascertain the role Cj0256 plays in lipid A modification in a C. jejuni background, deletion mutants were created in strain 81–176 by interruption of cj0256, to give strains 81–176A1 and 81–176A2. The mutation was later complemented by chromosomal insertion of a complete cj0256 gene into the arylsulfatase gene (astA) ORF as described previously (13), creating strain 81–176B.

The cj0256 deletion mutants revealed one major lipid A species similar to the 1,4´-bis-phosphate lipid A of E. coli W3110, devoid of pEtN modification (Fig. 2B). The lipid A modification was recovered by complementation in strain 81–176B showing lipid A species identical to that of WT (Fig. 2B). To confirm our findings, purified lipid A from C. jejuni strains was analyzed by MALDI-TOF MS. Analysis of WT 81–176 and complemented strain 81–176B revealed a major ion peak at m/z 1921, indicating the addition of a pEtN residue to the disaccharide backbone of hexa-acylated lipid A and the loss of a phosphate group at the 1-position (Fig. 3 A and C). The latter often occurs during MS of lipid A species (8). Analysis of cj0256 deletion mutant 81–176A1 revealed a major ion peak at m/z 1798, indicative of 1-dephosphorylated C. jejuni lipid A showing no pEtN modification (Fig. 3B). Additionally, MS indicated that the disaccharide backbone of C. jejuni lipid A is not composed solely of glucosamine residues, but can be replaced with the analogue 2,3-diamino-2,3-dideoxy-D-glucopyranose (Fig. 1) as previously reported (6). Altogether, our results confirm that Cj0256 is responsible for catalyzing the transfer of pEtN to C. jejuni lipid A.

Fig. 3.

MALDI-TOF MS analysis of C. jejuni lipid A. Analysis of of lipid A from strains 81–176, 81–176A1, and 81–176B revealed major ion peaks of m/z 1,921.2, 1,798.3, and 1,921.1, respectively, indicating the addition of a pEtN residue to the disaccharide backbone in strains with an active copy of cj0256 and the loss of a phosphate group at the 1-position in all strains. The latter often occurs during MS of lipid A species.

pEtN Modification Is Required for Antimicrobial Resistance in C. jejuni.

In the related organism H. pylori, it was demonstrated that decoration of lipid A with pEtN was critical for resistance to polymyxin B (PMB), a positively charged lipopeptide that binds to the phosphate groups of lipid A, killing Gram-negative bacteria in a manner similar to CAMPs of the innate immune system (14). Strain 81–176A1 (∆cj0256) showed a striking decrease in resistance to PMB (MIC of 0.8 ± 0.2 μg/mL) compared with WT (MIC of 17.3 ± 3.3 μg/mL) and cj0256 complemented strain (17.6 ± 3.6 μg/mL; Table S1). Additionally, PMB resistance (MIC of 15.0 ± 2.0 μg/mL) was restored in strain 81–176C, which was created by chromosomal complementation of 81–176A1 with E. coli eptA. EptA was previously shown to be involved in the modification of E. coli lipid A with pEtN (15). These results confirm that the addition of pEtN to the lipid A backbone in C. jejuni provides a 20-fold increase in resistance to PMB and is likely important for resistance to CAMPs found within the intestinal mucosa.

Cj0256 Is Required for Efficient Motility and Flagella Production.

In C. jejuni, the flagellum or flagella motility is required for effective adherence and invasion into intestinal epithelial cells for the colonization of chickens and humans, demonstrating the importance of this virulence factor (16–18). Interestingly, a previous screen of C. jejuni transposon mutants identified Cj0256 as an unknown protein involved in motility (19). To test the role Cj0256 plays in motility, we used soft agar assays. Strain 81–176A1 (∆cj0256) revealed a decrease in motility to approximately 50% relative to 81–176 or 81–176B (∆cj0256, cj0256⁺; Fig. 4 A and B). To explain the loss of motility, bacterial strains were examined by TEM. Strains 81–176 and 81–176B were indistinguishable from each other, showing normal C. jejuni morphology, spiral or curved rods with bipolar flagella. Unexpectedly, strain 81–176A1 revealed a population (approximately 95%) lacking flagella but otherwise unremarkable with regard to cell morphology (Fig. 4C and Table S2). Surprisingly, motility was not recovered in strain 81–176C (∆cj0256, eptA⁺), although E. coli EptA was capable of restoring polymyxin resistance. The latter suggested that the observed motility phenotype was not the result of changes in the LOS lipid A anchor, but rather an unrelated role played by Cj0256 in flagella production (Fig. 4B).

Fig. 4.

Cj0256 is required for efficient motility and flagella production. (A and B) Quantitative and qualitative analysis of motility of indicated C. jejuni strains was determined using semisolid MH agar. (C) Transmission EM of select C. jejuni strains.

To confirm that our motility phenotype was not the result of phase variation, cj0256 was deleted in the following backgrounds: 81–116, VLAO, and 11–168. All cj0256 deletion mutants showed reduced motility relative to its respective parent, similar to that found in 81–176A1, confirming our previous results and ruling out random phase variation as the cause (Fig. 4B and Fig. S5).

Cj0256 Modifies FlgG with pEtN.

Discovery of an unexpected motility phenotype and the loss of flagella production raised unanswered questions about the role played by Cj0256 in C. jejuni. Identification of the PptA, a protein involved in the decoration of the Neisseria gonorrheae Type IV pili with phosphoryl substituents that is structurally related to enzymes involved in pEtN modification of LPS, raised the possibility of phosphoryl modification of bacterial proteins (20). Therefore, we reasoned that C. jejuni use Cj0256 to modify a flagellar structural component with pEtN promoting flagella assembly. Following an exhaustive search of the literature, we found a published report detailing a whole-genome screen of C. jejuni using two-hybrid arrays looking for protein–protein interactions involving known flagellar proteins (21). The screen identified Cj0256 as a motility protein showing direct interaction with FlgG (21). To test for FlgG modification, we created a chromosomal in-frame C-terminal histidine-tagged (His₆) flgG in strains 81–176 and 81–176A1, creating 81–176D (flgG-His₆⁺) and 81–176E (∆cj0256, flgG-His₆⁺), respectively, giving us the ability to easily purify FlgG from C. jejuni.

The His₆ fusion to FlgG did not alter motility when compared to its wild type parent (Fig. 4B). Cultures of 81–176D and 81–176E were labeled with ³²P_i and FlgG-purified via affinity chromatography followed by SDS/PAGE. FlgG-His₆ was easily purified from both strains and detectable by Western blotting (Fig. 5A). However, only FlgG-His₆ purified from 81 to 176D revealed ³²P-modified protein (Fig. 5A). ³²P-modification of FlgG-His₆ was absent in 81–176E, implicating Cj0256 in phosphoryl modification of FlgG (Fig. 5A). To confirm this finding and identify the type of modification, FlgG purified from both strains 81–176D and 81–176E was subjected to electrospray ionization (ESI) MS. Strain 81–176D deconvoluted spectra revealed a major ion peak at m/z 28,670 (Fig. 6A), whereas strain 81–176E deconvoluted spectra revealed a major ion peak at m/z 28,546.9 (Fig. 6B). The resulting mass difference of 123.4 Da is the predicted size of a single pEtN residue, thus confirming the Cj0256-dependent modification of FlgG.

Fig. 5.

Cj0256 modifies FlgG with pEtN. ³²P-labeled FlgG-His₆ was purified via affinity chromatography from C. jejuni, Western blotted using anti-His antibody, and visualized by phosphorimaging. ³²P-labeled cell free extracts of E. coli strains were used directly for all analyses. (A) Purified FlgG from C. jejuni strains reveal pEtN modification in strains with an active copy of cj0256. (B) Coheterologous expression of Cj0256 with FlgG-His₆ or FlgF-His₆ in E. coli reveals the addition of pEtN to only FlgG. Coheterologous expression of eptA with FlgG-His₆ reveals no pEtN modification.

Fig. 6.

ESI-MS confirmation of pEtN modification of FlgG in C. jejuni background. C. jejuni FlgG-His₆ purified via affinity chromatography was subjected to ESI-MS for mass analysis and are shown as deconvoluted spectra. (A) FlgG-His₆ purified from strain 81–176D reveals a major ion peak at m/z 28,670.3. (B) FlgG-His₆ purified from strain 81–176E reveals a major ion peak at m/z 28,546.9, a difference of 123.4 Da, the approximate mass of a single pEtN.

In C. jejuni, the distal portion of the flagella rod is made of two major structural proteins, FlgF (annotated as FlgG2) and FlgG (22). We reasoned that, if FlgG is modified with pEtN, then perhaps other structural components, such as the structurally similar protein FlgF, are modified with pEtN. Furthermore, in light of our current findings, we believed it necessary to test if other identified lipid A pEtN transferases could modify FlgG. To test this theory, we generated plasmid constructs capable of expressing C. jejuni flgG-His₆ or flgF-His₆ along with cj0256 or eptA. The plasmids were transformed into strain W3110 creating strains EC06 (flgF-His₆⁺, cj0256⁺), EC08 (flgG-His₆⁺, cj0256⁺), and EC09 (flgG-His₆⁺, eptA⁺). Briefly, the strains were labeled with ³²P_i and the proteins separated by SDS/PAGE followed by Western blotting (Fig. 5B). ³²P-labeled Flg proteins were visualized by exposure to a phosphorimaging screen and only strain EC08 (flgG-His₆⁺, cj0256⁺) revealed the addition of phosphoryl substituents. The other two test strains, EC06 (flgF-His₆⁺, cj0256⁺) and EC09 (flgG-His₆⁺, eptA⁺), showed no modification of Flg proteins.

Protein FlgG-His₆ was later purified by affinity chromatography from strain EC08 and a control strain not expressing cj0256, EC07 (flgG-His₆⁺), and subjected to ESI-MS. The deconvoluted spectra of EC08 revealed the addition of pEtN to FlgG; this modification was absent in EC07 (Fig. S6). These results confirm that Cj0256 is responsible for catalyzing the transfer of pEtN to FlgG and not the rod flagellar component FlgF. Furthermore, E. coli EptA was not capable of modifying C. jejuni FlgG, illustrating an unexpected promiscuous activity for Cj0256 in catalyzing not only lipid A modification with pEtN, but also FlgG. A model for Cj0256-dependent pEtN modification is shown in Fig. 7.

Fig. 7.

Proposed model for pEtN modifications catalyzed by Cj0256. The model illustrates the bifunctional nature of Cj0256 in the periplasmic modification of lipid A and the flagellar rod of C. jejuni with pEtN. The model indicates that PtdEtN serves as the pEtN donor resulting in the production of diacylglycerol. Lyso-PtdEtN could also serve as a substrate for pEtN decoration. The TMHMM program (34) was used to predict the membrane topology of Cj0256. Organization of the flagellum is based on what is described for Salmonella (35) and what has been proposed for C. jejuni (36).

Discussion

Biogenesis of bacterial surface glycans and flagella assembly are two of the most dynamic processes observed in prokaryotes. The outer leaflet of the outer membrane in all medically important pathogenic Gram-negative bacteria is composed of LOS or LPS that is anchored to the outer membrane by lipid A. Not surprisingly, great variability is seen in the composition of this outer membrane component when comparing different pathogens (4, 5). The lipid A of C. jejuni differs from E. coli K-12 by increases in acyl-chain length and acyl-chain linkage, and by modification of the lipid A phosphate groups with pEtN (6). Our data indicates that the Campylobacter protein Cj0256 is responsible for pEtN addition to C. jejuni lipid A and provides resistance to antimicrobial peptides. Furthermore, the lipid A domain of C. jejuni’s LOS is not variable, but constant, as illustrated by examination of the lipid A profiles from multiple isolates (Fig. S4).

Flagella production is a metabolically costly endeavor for a growing bacterial population, but necessary for adaptation to the surrounding environment. Flagella assembly is tightly regulated and has been well characterized in organisms such as Salmonella enterica serovar Typhimurium; however, the process is highly variable and much less is known about the regulatory and structural characteristics of C. jejuni flagella (22). Flagellins from many polarly flagellated bacteria such as Campylobacter and Helicobacter spp. are glycosylated (23). The function of posttranslational glycan modification of flagellin subunits is not fully understood, but without these modifications, members of the ε-proteobacteria (e.g., C. jejuni) show motility defects and in some cases are unable to assemble flagella (24). Here we identify an unexpected flagellar modification occurring on the rod assembly in C. jejuni. Purified FlgG from cj0256 deletion strains reveals a change in mass of approximately 123.4 Da compared with WT, representing a single pEtN modification. This illustrates the bifunctional nature of Cj0256, linking flagella assembly and LOS biogenesis. Deletion of Cj0256 results in decreased motility and an approximately 95% reduction in flagella production, suggesting that modification of FlgG with pEtN may impart structural stability between the FlgG subunits. Similarly, it has been proposed that glycosylation of filament proteins may provide additional stability to the flagellar filament (25, 26). Still, it is not possible to rule out that Cj0256 modifies other targets that play a role in flagellar assembly.

Flagellin glycosylation in C. jejuni occurs on the cytoplasmic side of the inner membrane before export through the flagella export apparatus (27). The active site of Cj0256 is localized on the periplasmic side of the inner membrane (Fig. S3), demonstrating that modification of FlgG occurs during subunit assembly representing a posttranslational periplasmic modification of a flagellar component. Interestingly, proteins involved in glycosylation of C. jejuni flagellin subunits are shown to localize to the poles of the bacterium where the growing flagella subunits are exported, suggesting that Cj0256 may be localized to the poles in a similar manner (27). A small subpopulation (approximately 5%) retained the ability to assemble mature flagella in the absence of an active copy of Cj0256, suggesting a possible second site suppressor mutation. Perhaps FlgF, a structurally related rod protein can play a similar role in distal rod assembly, compensating for the loss of properly assembled FlgG. However, this merits further investigation.

Members of COG2194 and other distantly related proteins (e.g., PptA) have been shown to catalyze the transfer of pEtN from a conveniently located phospholipid donor, PtdEtN, to membrane-associated components (28, 29). Phosphoryl substituents are increasingly being recognized as important membrane associated structural components (20). A well documented example is modification of various LPS components with pEtN by CptA (28), EptB (30), and EptA (15, 29) in E. coli and Salmonella typhimurium. All three proteins presumably use PtdEtN as a substrate donor but show specificity in the pEtN recipient. Here, we demonstrate that Cj0256 requires PtdEtN for lipid A modification in whole cells (Fig. S2). A BLAST search of the genomes of E. coli and S. typhimurium reveals multiple proteins homologous to pEtN transferases, whereas C. jejuni contains only one, Cj0256. Our research shows that Cj0256 functions to modify both lipid A and FlgG. Complementation of a cj0256 deletion mutant with cj0256 or eptA reveals, in both cases, recovery of pEtN-modified lipid A. Only complementation with cj0256 showed recovery of motility, suggesting that Cj0256 diverged from other identified pEtN transferases, acquiring a bifunctional promiscuous role in pEtN modification, perhaps in response to the specialization of C. jejuni to a specific environmental niche and a “slimming down” of C. jejuni’s genomic size. An LOS core sugar of C. jejuni is modified by the addition of pEtN (31) similar to that of S. typhimurium (28). Considering Cj0256s identified promiscuous pEtN transferase activity, a third role in modification of core LOS is possible. The LOS from the cj0256 mutant showed no differences compared with WT by SDS/PAGE analysis (Fig. S7); however, this does not rule out possible changes in phosphorylation patterns.

This research identifies an unexpected link between the assemblies of two of the most important surface-associated virulence factors. We have identified a promiscuous pEtN transferase showing preference for two substrates, a membrane lipid and a structural protein. In light of our current findings, more research is needed to elucidate the role played by this family of mostly uncharacterized transferases in the assembly of bacterial surface structures and pathogenesis.

Materials and Methods

Bacterial Strains and Growth.

A complete list of bacterial strains can be found in Table S3. E. coli strains were grown routinely at 37 °C in Luria–Bertani (LB) broth or on LB agar. C. jejuni strains were grown routinely at 37 °C in Mueller–Hinton (MH) broth, on MH agar, or on tryptic-soy agar supplemented with 5% blood under microaerophilic conditions.

Construction of cj0256 Deletion Mutants.

The cj0256 gene and 1,000-bp flanking sequence upstream and downstream were amplified by PCR (primers: 1, 2) from 81 to 176 genomic DNA. The amplicon was digested with KpnI and SacI and inserted into vector pBluescript II SK(+) creating pBcj0256. The vector pBcj0256 was then used as template for an inverse PCR (primers: 3, 4) engineered to remove 1211 bps from the center of cj0256 and insert two restriction sites, XbaI and XhoI. A chloramphenicol resistance cassette (cam), obtained by PCR (primers: 5, 6) from cloning vector pRY111 (32), was inserted into the XbaI and XhoI sites on the inverse PCR amplicon creating pBcj0256KO:Cam^R. The resulting vector, pBcj0256KO:Cam^R, was transformed into C. jejuni strains by natural transformation, and resistant colonies were selected on blood agar plates containing 10 μg/mL of chloramphenicol.

Complementation of the 81–176 cj0256 mutant (81–176A1) was achieved by insertion of WT cj0256 or eptA into the arylsulfatase gene atsA (13). Vector pGEMatsAKO:Kan^R, a gift from S.A. Thompson (Medical College of Georgia, Augusta, GA), containing atsA interrupted with a kanamycin resistance cassette (aph3) on original vector pGEM-T Easy (32, 33) was digested with AgeI. AgeI cuts the vector in a noncoding region of the aph3 cassette, upstream from its promotor. The cj0256 and eptA genes plus 100 bp upstream sequence were amplified by PCR (primers: 7–10) from 81 to 176 and W3110 genomic DNA, respectively, and inserted into the AgeI cut site. The resulting vectors pAtsAKO::cj0256:Kan^R and pAtsAKO::epta:Kan^R were used to transform 81–176A1 for complementation studies. Kanamycin-resistant colonies were screened for loss of AstA activity as previously described (13).

Construction of C. jejuni Chromosomal FlgG-His₆ Mutants.

The coding sequence 500 bp upstream and downstream of the flgG stop codon was amplified by PCR (primers: 11, 12) from 81 to 176 genomic DNA. The amplicon was digested with KpnI and SacI, and inserted into vector pBluescript II SK(+) creating pBflgGHis. The vector pBflgGHis was then used as template for an inverse PCR (primers: 13, 14) engineered to add an in-frame His₆ coding sequence before the stop codon of flgG and insert the restriction site NdeI to both ends of the amplicon. A aph3 cassette, obtained by PCR (primers: 15, 16) from cloning vector pRY107 (32), was inserted into the NdeI sites on the inverse PCR amplicon creating pFlgGHISKO:Kan^R. The resulting vector, pFlgGHISKO:Kan^R, was transformed into C. jejuni strain 81–176 allowing for expression of C. jejuni FlgG-His₆.

Visualization of ³²P_i-Labeled Flagellar Components.

E. coli (50 mL) and C. jejuni (200 mL) cultures were grown in media supplemented with 1.5 μCi/mL ³²P_i. Chromosomally expressed FlgG-His₆ from ³²P-labeled C. jejuni was concentrated for further analysis from approximately 8.0 mg of cell-free extracts (CFEs) using the ProFound Pull-Down PolyHis Protein:Protein Interaction Kit (Thermo Scientific) in the presence of 6 M urea. For E. coli, no further processing of the CFE sample was required as C. jejuni flagella components were expressed from inducible plasmids. Protein samples (approximately 2 μg of purified FlgG or 15 μg of E. coli CFE) were resolved by SDS/PAGE (NuPAGE 4–12% Bis-Tris Gels; Invitrogen), western blotted using anti–polyHis-alkaline phosphatase antibody (Sigma), and analyzed by phosphorimaging. A more detailed description of each step can be found in SI Materials and Methods.

Other Methods.

Methods describing motility assays, lipid A isolation and analysis, large-scale purification of C. jejuni FlgG-His₆, protein MS, primer sequences (Table S4), and general cloning techniques are described in SI Materials and Methods.