JOURNAL OF BACTERIOLOGY, Aug. 1991, p. 4757-4764 Vol. 173, No. 150021-9193/91/154757-08$02.00/0Copyright © 1991, American Society for Microbiology

Role of Two Flagellin Genes in Campylobacter MotilityPATRICIA GUERRY,1* RICHARD A. ALM,2 MARY E. POWER,2 SUSAN M. LOGAN,2 AND TREVOR J. TRUST2Enteric Diseases Program, Naval Medical Research Institute, 12300 Washington Avenue, Rockville, Maryland 20852,'

and Department ofBiochemistry and Microbiology, University of Victoria, Victoria,British Columbia, V8W 3P6, Canada2

Received 1 February 1991/Accepted 27 May 1991

Campylobacter coli VC167 T2 has two flagellin genes, flaA and flaB, which share 91.9% sequence identity.TheflaA gene is transcribed from a r28 promoter, and theflaB gene from a cS4 promoter. Gene replacementmutagenesis techniques were used to generate flaA flaB and flaA flaB+ mutants. Both gene products arecapable of assembling independently into functional filaments. A flagellar filament composed exclusively of theJaA gene product -is indistinguishable in length from that of the wild type and shows a slight reduction inmotility. The flagellar filament composed exclusively of theflaB gene product is severely truncated in length andgreatly reduced in motility. Thus, while both flagellins are not necessary for motility, both products arerequired for a fully active flageilar filament. Although the wld-type flageliar filament is a heteropolymer of theflaA and flaB gene products, immunogold electron microscopy suggests that flaB epitopes are poorly surfaceexposed along the length of the wild-type filament.

Campylobacterjejuni and Campylobacter coli are amongthe most frequently isolated causative agents of bacterialdiarrhea worldwide (7, 9, 37). While little is understoodabout the mechanisms by which campylobacters cause en-teric disease, it has been established that the motility im-parted by the polar flagellum is required for the establish-ment of infection in both experimental animal systems and inhuman volunteer studies (6, 8, 30). Moreover, flagellin is animmunodominant antigen of campylobacters, and data areaccumulating suggesting that it may be a protective antigen(1, 2, 41).

In the case of one strain, C. coli VC167, variants VC167type 1 (Ti) and T2, which produce two antigenically distin-guishable flagellar filaments, have been isolated (14). Theseantigenic variants have previously been termed phase 1 andphase 2, respectively, but the nomenclature has beenchanged to avoid confusion with the phenomenon of phasevariation of Campylobacter flagella (8). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)analysis of these antigenically different flagellar filamentsfrom VC167 Ti and T2 suggested that each was composed ofa single species of flagellin subunits with apparent molecularweights (Mr) of 61,500 and 59,500, respectively. Impor-tantly, passage of VC167 Ti through nonimmune animalshas been shown to select for a transition to VC167 T2 cells(23). Amino-terminal sequence analysis of the flagellinsisolated from these antigenically different flagellar filamentsfurther showed that both Ti and T2 flagellins had identicalsequences to residue 30. Subsequent genetic analysesshowed that VC167 Ti and VC167 T2 both contained twoadjacent, tandemly oriented flagellin genes, termed flaA andflaB (12, 26). Sequence analysis showed that the flaA genecorresponded to the N-terminal sequence of the isolatedflagellins, suggesting that the Campylobacter filament was ahomopolymer of the flaA gene product. However, primerextension experiments indicated that both genes were ex-pressed concomitantly in both variants, and automatedEdman degradation of peptides produced by CNBr andproteolytic cleavage of the isolated proteins revealed the

* Corresponding author.

presence of trace amounts of peptides encoded by flaB,implying that the C. coli flagellum is a complex filamentcontaining two different flageilin species (12). However,neither the presence of theflaB gene product in the flagellarfilament nor the ability of the flaA andflaB gene products toform flagellar filaments by themselves has been establishedunequivocally. Indeed, in a preliminary genetic analysis of asingle flaA mutant, the flaB gene product was shown to befound intracellularly and was not assembled into a filament(12).

In this study we examined flagellar structure and functionin C. coli VC167 T2. We determined the nucleotide sequenceof theflaA andflaB genes and flanking DNA and deduced theprimary and secondary structures of their respective geneproducts. Further, utilizing gene replacement mutagenesistechniques we generated additional mutations in both flag-ellin genes of VC167 T2. Here we report for the first timethat bothflaA andflaB gene products are capable of formingfunctional flageilar filaments. However, the filament formedexclusively by the flaB product is truncated and confersgreatly reduced motility. The filament formed by the flaAgene product is full length but is slightly reduced in function.Thus, both gene products, while not essential for motility,are required for a fully active flagellum.

MATERIALS AND METHODS

Bacterial strains and culture conditions. C. coli VC167,serogroup L108, is a clinical isolate obtained from H. Lior,National Enteric Reference Centre, Ottawa, Ontario, Can-ada. Isolation of variants VC167 Ti and VC167 T2, whichproduce antigenically different flagella, has been previouslydescribed (14). Escherichia coli DH5 or DH5a was used asthe host for cloning experiments. Growth conditions were aspreviously described (14).DNA methods. Total DNA extractions from C. coli were

done by the method of Hull et al. (17). Plasmid DNAs werepurified as previously described (13). Restriction enzymeswere purchased from Boehringer Mannheim Biochemicals(Indianapolis, Ind.) or Pharmacia (Uppsala, Sweden) andwere used under the conditions recommended by the sup-pliers. Double-stranded dideoxy sequencing was performed

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after alkaline denaturing by using [35S]dATP (Dupont NENResearch Products, Boston, Mass.) and Sequenase (UnitedStates Biochemical Corp., Cleveland, Ohio) as specified bythe manufacturers. Primers were commercially availablepBR322 primers, and custom primers were synthesized on aBiosearch 8700 synthesizer (Milligen Biosearch, Burlington,Mass.) or purchased from Synthecell Corp., Gaithersburg,Md. Custom primers were synthesized at approximately 250-to 300-bp intervals.

Hybridizations. DNAs were nick translated with [a-32P]dCTP by using a commercial kit (Dupont, NEN). Hybrid-ization conditions have been previously described (13).

Genetic procedures. Gene replacement mutagenesis proce-dures for campylobacters have been previously described(12, 20). A new suicide vector, pGK2003, was constructedby cloning the origin of transfer from an IncP plasmid (10)into the unique EcoRI site of pUC18. This vector containsrestriction enzyme sites not available on other Campylobac-ter suicide vectors (20). Conjugation experiments betweenE. coli DH5 cells harboring plasmid RK212.2 and the deriv-ative of the suicide vector described in Results and C. coliwere done as previously described (12). Campylobactertransconjugants were selected on Mueller-Hinton mediumsupplemented with 100 ,ug of kanamycin sulfate per ml and10 ,ug of trimethoprim per ml.

Motility testing. Campylobacters were tested for motilityby spotting cultures onto plates of Mueller-Hinton mediumwith 0.4% agar or the same medium supplemented with 100,ug of kanamycin per ml. Zones of motility were examinedfollowing incubation at 37°C for 48 h.

Antibodies. Polyclonal antiserum SML2 to cross-reactive,non-surface-exposed Campylobacter flagellin epitopes andantiserum LAH2 specific to filaments on C. coli VC167 T2flagella were prepared as previously described (14). Whenrequired, antiserum LAH2 was absorbed with live cells ofthe flaA+ flaB mutant KX5, as previously described (14).Antisera were stored at -20°C. Prebleed serum sampleswere taken and used as control sera.

Electrophoresis and Western blotting (immunoblotting).SDS-PAGE was performed with a minislab gel apparatus(Hoeffer Scientific Instruments, San Francisco, Calif.) bythe method of Laemmli (21). Protein samples solubilized insample buffer were stacked in 4.5% acrylamide (100 V,constant voltage), separated in 12.5% acrylamide (200 V,constant voltage), and stained with Coomassie blue or trans-ferred to nitrocellulose for immunological detection as pre-viously described (25).

Flagellin purification. Flagella were isolated from 24-hcultures as follows. Bacterial cells were harvested in 20 mMTris-HCI, pH 7.5, and collected by centrifugation (30 min,3,000 x g, 4°C). Cells were resuspended in 25% sucrose-50mM Tris-HCl, pH 8.0, and incubated on ice for 60 min. Permilliliter of solution, 1 mg of lysozyme, 0.01 g of sarcosyl, 2mg of DNase, and 2 mg of RNase (Boehringer MannheimBiochemicals) were added and incubated at room tempera-ture for 20 min. After centrifugation at a low speed (500 x g,10 min), the supematant was subjected to ultracentrifugation(80 min, 120,000 x g, 40C). The pellet was resuspended in asolution of 20 mM Tris-HCl, pH 7.5, sarcosyl was added toa final concentration of 1%, and the mixture was incubated atroom temperature for 15 min. The sample was ultracentri-fuged as described before, and the pellet was subjected tothe sequential acid dissociation and ultracentrifugation pro-cedure previously described (24).

N-terminal amino acid sequencing. Flagellin-containingsamples were subjected to SDS-PAGE and electrophoreti-

cally transferred to an Immobilon transfer membrane (Milli-pore Corp., Burlington, Mass.) in 10 mM 3-cyclohexy-lamino-l-propanesulfonic acid, pH 11.0 (Aldrich ChemicalCo., Milwaukee, Wis.)-10% methanol for 2.5 h (22). Theflagellin product was detected by staining with Coomassiebrilliant blue R. The flagellin bands were excised and storedat -20°C. Amino acid sequencing of purified polypeptideswas performed on an Applied Biosystems 470A gas phasesequencer.

Electron microscopy. A grid covered with a Formvar filmwas floated on a 50-pJl drop of bacterial cells or purifiedflagella for 5 min. The grids were stained by floating on adrop of 1% (wt/vol) ammonium molybdate containing 0.1%(wt/vol) glycerol, pH 7.0, and were examined on either aPhillips EM300 or a JEOL JEM-1200EX electron micro-scope operated at an accelerating voltage of 60 kV. Imageswere recorded on 70-mm Fine Grain Release film (Kodak,Rochester, N.Y.).For immunoelectron microscopy the grid was floated on

the sample and then removed and floated on a drop of 10mMTris containing 150 mM NaCl-0.1% (wt/vol) bovine serumalbumin (BSA) in 0.05% (vol/vol) Tween 20 (Tris-NaCl-BSA-Tween) for approximately 30 min. The grid was thenincubated on polyclonal serum diluted in Tris-NaCl for 1 h.After incubation, the grid was removed and nonspecificallybound immunoglobulin G was removed by floating the gridon three drops of Tris-NaCl-Tween. The grid was thenfloated on a drop of Tris-NaCl-Tween containing a 1:50dilution of 15-nm colloidal gold particles coated with proteinA (Jannsen Biotech, Olen, Belgium). After incubation for 1h, the nonspecifically bound colloidal gold particles wereremoved by floating the grid on two drops of Tris-NaCl-Tween and one drop of distilled water. The grids werenegatively stained and examined as described above.

Nucleotide sequence accession number. The DNA se-quences have been deposited with GenBank under accessionnumbers M64670 (T1) and M64671 (T2).

RESULTS

Molecular cloning and sequence analysis of the VC167 T2flageilin genes. VC167 Ti, which produces a highly relatedbut antigenically distinguishable flagellar filament fromVC167 T2, contains two tandemly oriented flagellin genes,termed flaA and flaB (12, 26). Since previous data hadindicated that VC167 Ti and VC167 T2 DNAs displayed anidentical HindIII pattern when probed with a plasmid,pGK201, containing both VC167 Ti flagellin genes (12), thisplasmid was used as a probe to clone the flagellin genes fromVC167 T2. The VC167 T2 flagellin genes were cloned asthree HindIII fragments into pBR322, as shown in Fig. 1,and these three clones, pGK212, -213, and -214, were useddirectly for double-stranded dideoxy sequence analysis. TheDNA sequences of VC167 T2flaA and flaB genes and theirflanking regions as well as a comparison to the flaA andflaBgenes of Ti (12, 26) are shown in Fig. 2. The two flagellingenes from VC167 T2 are 91.9% identical to each other andare also highly homologous to the corresponding genes inVC167 Ti (12, 26). Both open reading frames are 1,709 baseslong, both contain an overall G+C content of 38%, and bothdisplay a strong bias (58.9%) for codons with T in the thirdposition. The codon adaptation indices are 0.227 and 0.230,respectively (36). The genes are separated by a 163-bpintergenic region which is 74.8% A+T.Upstream from both fla genes there is a well-positioned

ribosome binding site (AGGA). However, as shown in Fig.

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=R =Rc -L-F En

flaAI I

flaBpGK21 2

a i pGK21 3

pGK214

pGK21 0a i

1 kb

FIG. 1. Map of VC167 flagellin genes. Plasmids pGK212, pGK213, and pGK214 contain VC167 T2 DNA cloned into the Hindlll site ofpBR322. pGK210 contains the VC167 Ti flaA gene subcloned from pGK201 (26) as a 2.3-kb SspI fragment into pUC19.

2, the promoters of the two genes, as deduced by mRNAstart sites (12), are quite distinct from one another. Thepromoter of the flaA gene is typical of a .28 promoter, witha -35, -10 consensus sequence of CTAAAn17CGATAT, aspreviously shown for the flaA gene of VC167 Ti (12). TheflaB promoter, which is identical to the flaB promoter ofVC167 Ti, is a typical as" promoter with a -13, -26consensus sequence of TTGGn1OGC. The 3' region of theflaA gene contains a potential terminator structure beginning45 bases after the termination codon with a stem-loopstructure (free energy value of -14.8 kcal [-61.2 kJ/mol])followed by a cluster of thymine residues. No thermodynam-ically stable stem-loop structure has been located down-stream of the flaB gene.

Protein structure. Both the flaA and flaB genes of VC167T2 encode proteins of 572 amino acid residues (after removalof the N-terminal methionine), with predicted unmodifiedMrs of 58,884 and 59,124, respectively. The primary se-quences of the two flagellins are 93.9% identical, with only35 residue changes, 7 of which are conservative, while anadditional 7 involve substitution with the same class ofresidue. As seen in Fig. 3, 11 of the substitutions are locatedin the N-terminal 71 residues, 11 are in the central region ofthe sequence between residues 201 and 301, and the remain-ing 13 substitutions are in the C-terminal 68 residues. Thepredicted overall pl of the unmodifiedflaA flagellin was 5.52compared with the predicted pl of 6.0 for the flaB flagellin.Figure 3 also shows the protein sequences of the flaA andflaB gene products from VC167 Ti for comparison.

Generation of flagellar mutants. All mutants were gener-ated by gene replacement methods, using the campylobacterkanamycin resistance cassette of Labigne-Roussel et al. (20).The entire flaA gene of VC167 Ti was subcloned frompGK201 (26) as a 2.3-kb SspI fragment into the SmaI site ofpUC19 to generate plasmid pGK210, as seen in Fig. 1. Thekanamycin resistance gene was inserted into the uniqueEcoRV site located 1.0 kb into theflaA gene (26) to generatepGK211. The entire flaA gene disrupted by the kanamycinresistance cassette was subcloned as a KpnI-XbaI fragmentfrom pGK211 into the suicide vector pGK2003, described inMaterials and Methods, to generate pGK2004. pGK2004 wastransformed into DH5 cells harboring the conjugative plas-mid RK212.2. These transformants were used as donors inconjugation experiments with C. coli VC167 T2 cells, asdescribed in Materials and Methods. Fifteen kanamycin-resistant campylobacter transconjugants were obtained, and

all displayed reduced motility compared with that of VC167T2 in 0.4% agar plates (data not shown), although thechanges in motility fell into two phenotypic classes (seebelow). Since flaA and flaB are so highly homologous,pGK2004 should be capable of crossing over into eithergene. The mutants were further characterized by Southernblot analysis to determine which flagellin gene was affected.DNAs from individual mutants were digested with SspI,electrophoresed on 0.7% agarose gels, transferred to nitro-cellulose, and probed with 32P-labeled pGK213. This probehybridizes to a 2.3-kb fragment from VC167 DNA thatcontains all of the flaA gene and to a 1.6-kb fragmentcontaining most of the flaB gene, as shown schematically inFig. 1. A Southern analysis is shown in Fig. 4 for the wildtype (lane a) and for each class of mutant. In flaA' flaBmutants, as shown in Fig. 4, lane b, the flaA gene is intactand the mutated flaB gene is disrupted into one larger andone smaller fragment because of the presence of an asym-metric SspI site within the kanamycin resistance cassette. InflaAflaB+ mutants, theflaB gene is intact, and theflaA geneis disrupted, as seen in Fig. 4, lane c. We obtained eightflaAflaB + mutants and seven flaA + flaB mutants.

Motility testing. The relative degree of motility of VC167T2 wild-type cells and representatives of each mutant class isshown in Fig. 5. All mutants which were genotypicallycharacterized asflaA+ flaB showed a slight but reproducibledecrease in motility (Fig. 5B) compared with that of wild-type cells (Fig. SA). Mutants which produced only the flaBgene product were very slightly motile after 48 h of incuba-tion (Fig. 5C). One flaA+ flaB mutant (KX5) and one flaAflaB+ mutant (KX15), which are those shown in Fig. 5, werefurther characterized.

Physical characterization of mutant flagella. Examinationof the flaA+ flaB mutant KX5 by electron microscopyrevealed the presence of a flagellar filament at both poles ofall of the cells in the population (Fig. 6B). This flagellarfilament is indistinguishable in length from the wild-typefilament, as seen in Fig. 6A. The average length of 79wild-type flagellar filaments was 2.73 + 0.39 p.m, and theaverage length of 77 KX5 filaments was 2.83 + 0.46 ,um.Electron microscopic examination of KX15, the flaA flaB+mutant, revealed the presence of a truncated flagellar fila-ment with an average length of 0.47 + 0.24 pum (average of 61filaments) on the surface of only some cells in the popula-tion, as shown in Fig. 6C. The number of cells in thepopulation expressing the truncated filament ranged from 20

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TTTTAGTGATGATAACTTTATACAGGTGGTATTTTAAAAGGGGTATTGTATTATCGTGGGAGTTTTTTATCTGAAGCTATAAaAAAAAGATTTGAAAGATGAGGCGTATTATCTTTTAAATTTTATTAGCGCTTATCAAAAATGGCTTGAAAAGTTTATTGAGAGGCTAGAAATAAAAAATGCCATCATAAAAGATACAGTGTTAAAATATGGAAATTTGATATAAAATTTTATAAAACTC

-35 -10 MRNJa rho flaAjaCTATTTTTCCTTTT A AS AGTTTATAXCAAGTTCATGGATGGACTTGAATTTATTTAAA OTTTAaAA WGGATTTCGTATTAACACAAATGTTGCAGCATTAAATGCT

AAAGCAAATTCGGATCTAAACAGCAGAGCATTAGATCAATCACTTTCAAGACTCAGTTCAGGTCTTAGAATCAACTCCGCAGCAGATGATGCTTCAGGGATGGCGATAGCAGATAGTTTAAGATCTCAGGCAAATACTTTGGGTCAGGCTATATCTAATGGTAATGATGCTTTAGGTATCTTGCAAACTGCAGATAAGGCTATGGATGAGCAACTTAAAATCTTAGATACCATCAAGACTAAAGCGACTCAAGCTGCTCAAGATGGTCAAAGCTTAAAAACAAGAACTATGCTTCAAGCAGACATCAACCGTTTGATGGAAGAACTTGATAATATCGCAAATACCACTTCATTTAATGGC

GAAACAGGTTCACAAAGTTTTTCTTCAGGCACTGTAGGACTTACTATTAAAAACTACAACGGTATCGAAGATTTTAAATTTCAAAATGTAGTGATTTCTACTTCTGTAGGAACAGGTCTTG T G A

GGAGCTTTGGCTGAAGAGATCAACAGAAATGCAGATAAAACAGGAATTCGTGCAACTTTTGATGTAAAATCTGTAGGAGCCTATGCAATAAAAGCAGGAAATACTTCTCAGGATTTTGCTATCAATGGGGTTGTTATAGGTAAGGTTGATTATTCAGATGGTGATGAGAATGGTTCTTTAATTTCAGCTATCAATGCTGTAAAAGATACAACTGGTGTTCAAGCCTCTAAAGATGAAAATGGTAAACTTGTTCTTACTTCGGCCGATGGTAGAGGGATTAaAAATCACAGGTAGCATAGGTGTAGGAGCTGGTATATTGCACACTGAAAATTATGGAAGGTTATCTTTAGTTAAAAATGATGGTAGAGATATCAATATAAGTGGAACAGGTCTTTCAGCTATAGGTATGGGTGCTACAGACATGATTTCTCAATCTTCAGTATCTCTAAGAGAGTCAAAAGGGCAAATTTCAGCAGCCAATGCTGATGCTATGGGCTTTAATGCTTATAATGGCGGCGGCGCTAAGCAAATTATTTTCGCTTCTAGTATTGCAGGATTTATGTCTCAGGCTGGTTCAGGCTTCTCTGCTGGTTCGGGATTTTCAGTAGGTAGTGGTAAAAATTATTCAGCCATTTTATCAGCTTCTATACAGATAGTATCTAGCGCACGTTCTATCAGTAGCACCTATGTTGTTTCTACTGGTTCAGGTTTCTCTGCTGGTTCAGGTAATTCTCAATTTGCAGCTTTAAGAATAAGTACAGTAAGTGCTCATGATGAAACTGCAGG;TGTAACTACACTTAAGGGTGCAATGGCTGTGATGGATATAGCAGAAACTGCTATTACCAATCTTGATCAAATCAGAGCGGATATAGGTTCTGTGCAAAATCA.AATCACATCGACTATAAACAACATTACTGTAACCCAGGTAAATGTTAAATCAGCAGAATCACAAATCAGAGAT

stopGTAGATTTTGCAAGCGAGAGTGCAAATTACTCTAAAGCAAATATATTGGCTCAAAGTGGTTCTTATGCTATGGCTCAAGCAAATTCAAGCCAGCAAAATGTTTTAAGATTACTACAGAaa

-62 -26

TATATAAAACATTCTTTAGCGTTTACSTGAATTTATACAAATCCMOGZAWT v TTTTATTTCTAAATAAAATTTCAATTTGAATCAAAACTT aAACACTTCTT

-13 UREA rbs flaRGcTTTAATCTTTTCaATGCAATATTTTGAaA5aTTTAAAasGGGTTTTAGAATAAACACCAACATCGGTGCATTGAACGCACATGCAAATTCAGTTGTTAATGCTAGGGAGCTTGACAA

CCAAATCGGTTCAAGTTCAAATCAAACTATTAAAGCAAGTATAGGAGCAACTCAGTCTTCTAAAATCGGTGTAACAAGATTTGAAACAGGTTCACAAAGTTTTTCTTCAGGCACTGTAGGACTTACTATTAAAAACTACAACGGTATCGAAGATTTTAAATTTGATAGTGTAGTGATTTCTACTT,CAGTAGGAACAGGTCTTGGAGCTTTGGCTGAAGAGATCAACAGAAATGCAGATAAAACAGGAATTCGTGCAACTTTTGATGTAAAATCTGTAGGAGCCTATGCAATAAAAGCAGGAAATACTTCTCAGGATTTTGCTATCAATGGGGTTGTTATCGGACAAATAAATTATAATGACGGTGATAACAATGGTCAACTTATCTCAGCTATCAATGCTGTAAAAGATACAACTGGTATTGAAGCCTCTAAAGATGAAAATGGTAAACTTGTTCTTACTTCAAGAGATGGTAGAGGGAT

G C GGCC

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TAAAATCACAGGTAGCATAGGTGTAGGAGCTGGTATATTGCACACTGAAAATTATGGAAGGTTATCTTTAGTTAAAAATGATGGTAGAGATATCAATATAAGTGGAACAGGTCTTTCAGC 3240

TATAGGTATGGGTGCTACAGACATGATTTCTCAATCTTCAGTATCTCTAAGAGAGTCAAAAGGGCAAATTTCAGCAGCCAATGCTGATGCTATGGGCTTTAATGCTTATAATGGCGGCGG 3360

CGCTA,AGCAAATTATTTTCGCTTCTAGTATTGCAGGATTTATGTCTCAGGCTGGTTCAGGCTTCTCTGCTGGTTCGGGATTTTCAGTAGGTAGTGGTAAAAATTATTCAGCCATTTTATC 3480

AGCTTCTATACAGATAGTATCTAGCGCACGTTCTATCAGTAGCACCTATGTTGTTTCTACTGGTTCAGGTTTCTCTGCTGGTTCAGGTAATTCTCAATTTGCAGCTTTAAGAATAAGTAC 3600

AGTAAGTGCTCATGATGAAACTGCAGGTGTAACTACACTTAAGGGTGCAATGGCTGTGATGGATATAGCAGAAACTGCTATTACCAATCTTGATCAAATCAGAGCAGATATAGGTGCTGT 3720

GCAAAATCAGCTCCAAGTAACGATAAATAATATTACCGT,AACCCAGGTAAATGTTAAAGCAGCAGAATCAACCATAAGAGATGTGGATTTCGCTGCAGAAAGTGCAAATTTTTCTAAGTA 3840stop

CAATATCCTTGCGCAGTCGGGTTCATATGCTATGAGCCAACGTAACGCTGTGCAACAAAATGTCTTAAAACTTTTACAASaAUTTTTTCTA,AAGAGCAAATTTTTGCTCTTTAGATTAAAT 3960

TTCTTTTTTCTAATTCATCTTGTAA,AGGGATAATGGCTTTTTTAATTCTTATGTTTTtGACTTTCTATGAGATCAATTATAGTTTCAATTAAAGATTCGTTAGCATAAATCCAAGATAGAA 4080TTTTATTTTGTCTTTGGCTTTCATTGTTAACACTTTGGATATAAATAGGGGCAATTAAACTTTCTTCATGATATAGACTAGGTCCTAAAATTTCATGTAAAAAAAAGTATCTTTTCTTGC 4200

FIG. 2. DNA sequence of VC167 T2 flaA and flaB genes. The translational start and stop of each gene are indicated in boldface, andputative ribosome binding sites (rbs') are designated by double underlining. The mRNA start sites, which were determined previously (12),

28~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~34

are indicate'd. Onthe basis of the mRNA start sites, aputative C2 promoter at -10 and -35 is indicated forflaA, and aputativeCrS4 promoteris indicated at -26 and -13 for flaB. A potential transcriptional terminator forflaA is shown in boldface with underlining. Bases which differin the VC167 Tl sequence (12, 26) are shown below the T2'sequence. The sequence shown for VC167 Tl flaA includes corrections to thepreviously published (26) sequence.

to 50%, and the remaining cells in the population were

nonflagellated. There was no difference in the percentage ofthe cells expressing the truncated filament from 18- or 60-hcultures.The flagella from VC167 T2, KX5, and KX15 cells were

purified by glycine extraction and analyzed by Western blotanalysis using a flagellin-specific antiserum, SML2 (14). Theresults, shown in Fig. 7, indicate that flagellin from all three

filaments run at an Mr of 59,500 and react with SML2antiserum. N-terminal amino acid sequencing of flagellinpurified from KX15 confirmed that this filament is composedof the flaB gene product, since the sequence obtainedcorresponded to the first 28 amino acid residues predictedfrom the DNA sequence of the flaB gene, and not that offlaA, as seen in Fig. 3.

Immunogold electron microscopy was employed to exam-

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AAACAACTTTTAAGTGGTGGTTTTACCAATCAAGAATTCCAAATCGGTTCAAGTTCAAATCAAACTATTAAAGCAAGTATAGGAGCAACTCAGTCTTCTAAAATCGGTGTAACAAGATTT 840

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mnr_mxxmcxmnrm&ThMCTATrTTnC"ACTGCXGkTAAGGCTATGGA7GAGCAACTTAAAATCTTAGATACCATCAAGACTAAAGCGAC7CAAGC7GC7CAAGATGGTCAAAGCTTAAA

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65GFRINTNVAALNAKANSDLNSRALDQSLSRLSSGLRINSAADDASGMAIADSLRSQANTLGQAIS T2 FLA A:::::: :IG::::H::. :W:A:E: :K:::::::::::::::::::::::::::::: :A::::: :N T2 FLA B

T1FLA A.:::::::IG::: :H:: :W:A:E: :K:::::::::::::::::::::::::::::::A::::::N Tl FLA B

130NGNDALGILQTADKAMDEQLKILDTIKTKATQAAQDGQSLKTRTMLQADINRLMEELDNIANTTS T2 FLA A.::::.I:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::.T2 FLA B

T1 FLA ATl FLA B

195FNGKQLLSGGFTNQEFQIGSSSNQTIKASIGATQSSKIGVTRFETGSQSFSSGTVGLTIKNYNGI T2 FLA A

T2 FLA BT1 FLA ATl FLA B

260EDFKFQNVVISTSVGTGLGALAEEINRNADKTGIRATFDVKSVGAYAIKAGNTSQDFAINGVVIG T2 FLA A

T2 FLA B:::::DS::::::::::::::::::::::::::::::::::::::::::::::::::::::::::.T1FLAA:::::S::::::::::::::::::::::::::::::::::::::::::::::: Tl FLA B

325KVDYSDGDENGSLISAINAVKDTTGVQASKDENGKLVLTSADGRGIKITGSIGVGAGILHTENYG T2 FLA A

T2 FLA B::::::::::::::::::::::::::::::::::::::::::::::::::::: Tl FLA AQIN::::::Q:::::::::::::::::::::::::::::::::::::::::: Tl FLA B

390RLSLVKNDGRDINISGTGLSAIGMGATDMISQSSVSLRESKGQISAANADAMGFNAYNGGGAKQI T2 FLA A

T2 FLA B

T1 FLA A

455IFASSIAGFMSQAGSGFSAGSGFSVGSGKNYSAILSASIQIVSSARSISSTYVVSTGSGFSAGSG T2 FLA A

T2 FLA BT1FLAATI FLAB

520NSQFAALRISTVSAHDETAGVTTLKGAMAVMDIAETAITNLDQIRADIGSVQNQITSTINNITVT T2 FLA A

. .: :: A::: :LQv: :::: ~~~~~~~~T2FLABTl FLAA

.:::::::::::::::::::::::::::::::::::::::::::::::: A::::LQV:::::::: Tl FLA B

572QVNVKSAESQIRDVDFASESANYSKANILAQSGSYAMAQANSSQQNVLRLLQ T2 FLA A:::::A::AT:::::::A::: :F: :Y:::::::::::: :R:AV:::: :K::: T2FLA B

Tl FLAATl FLAB

FIG. 3. Comparison of the gene products of the VC167 T2 flaAandflaB genes and the VC167 Ti and T2 genes (12, 26). The T2flaAprotein sequence is shown on the top line, and the other flagellinsare compared with it. Colons indicate identical residues; changes areso indicated. Numbers at the right indicate amino acid residuenumbers. Amino acids are designated by the single-letter code.

ine the distribution of the flaB flagellin in the wild-typefilament. Antiserum LAH2 prepared to VC167 T2 flagellawas shown to be reactive with the flaA and flaB flagellins ofVC167 T2 in Western blot analysis. The antiserum alsoreacted with epitopes on the surface of wild-type flagellumfilaments, the flaA' flaB flagellar filaments produced byKX5, and the truncated flaA flaB+ filaments produced byKX15 (data not shown). Antiserum LAH2 which was pre-absorbed with KX5 cells to remove antibodies to surface-exposed epitopes of theflaA gene product also reacted withepitopes along the length of the wild-type filament (Fig. 8A)and the truncated flaA flaB+ filament of KX15 (Fig. 8C).While the labeling of the truncated filament with the ab-sorbed antiserum was strong, the wild-type filament waslabeled more sparsely than when reacted with unabsorbedantiserum, suggesting that only a small number of flaBepitopes were exposed on the surface of the wild-typefilament. As expected, the absorbed antiserum failed to reactwith the flaA+ flaB filament produced by KX5 (Fig. 8B).

DISCUSSION

The flagella of the majority of free-swimming bacteria arecomposed of a single species of flagellin, although there area number of species in which the flagellar filament is com-posed of two or more species of flagellin (11, 18, 28, 34). Our

a b c

2.3

Iwo

so

t., .....

FIG. 4. Southern blot analysis of flagellin mutants. DNAs weredigested with SspI, electrophoresed on 0.7% agarose, transferred tonitrocellulose, and probed with pGK213. Lane a, VC167 T2 wildtype; lane b, flaA+ flaB mutant; lane c, flaA flaB+ mutant. Thenumbers on the left indicate the positions of the fragments in theVC167 T2 wild type encoding all offlaA (2.3 kb) and most offlaB(1.6 kb), as diagrammed in Fig. 1.

previous studies on flagella production by C. coli VC167suggested that this organism fell into this latter category. Forexample, primer extension experiments indicated that boththe flaA and flaB genes are transcribed concurrently inVC167 cells (12). The genetic analyses presented here showthat both gene products are capable of assembly into aflagellar filament and indicate that the C. coli VC167 flagellarfilament is composed of two distinct flagellin species. Thefact that the filament formed exclusively by the flaB geneproduct in the KX15 mutant is truncated is consistent withour previous observation that the level of transcription of theflaB gene is less than that of theflaA gene (12). In this regardit is interesting to note that the sheathed flagellum ofHelicobacter pylori also contains two species of flagellinwhich are present in significantly different copy numbers(18). In this organism, the minor species makes up the hookproximal region of the filament, while the predominantflagellin species makes up the remainder of the flagellarfilament. This appears not to be the case in C. coli. Immu-nogold electron microscopy with polyclonal antiserum pre-pared against the native VC167 T2 flagellum filaments andabsorbed with the KX5 mutant showed that theflaA andfiaBflagellins, despite their high degrees of sequence similarity,could be distinguished on the basis of surface-exposedepitopes. The data further suggested that both flagellin

FIG. 5. Motility of C. coli VC167 T2 wild type (A) comparedwith those of the flaA+ flaB KX5 (B) and flaA flaB+ KX15 (C)mutants. Motility of the wild type was tested in 0.4% Mueller-Hinton agar, while those of the mutants were tested in this mediumsupplemented with 100 p.g of kanamycin per ml.

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A

.B

C

.,',,''s'>,2,.' .,

... : :.:.:: :: :. .:: .:

..:

B ze,.: .+,

.. .,, ... . .:

.: ::. : .:: :: ::. :.:: : .: .: .:

....: :. :.. :.. ..:^ . . . ..*|G :fP : : .._ .. . , .FIG. 6. Electron micrographs of VC167 T2 wild type (A), flaA+

flaB KX5 (B), and flaA flaB+ KX15 (C) negatively stained with 1%ammonium molybdate. The truncated filaments composed of theflaB gene product are readily seen on four of the cells in panel C. Bar= 1 p.m.

species were distributed along the length of the assembledcomplex Campylobacter flagellar filament, albeit with theflaB gene product in significantly lower copy number andhaving fewer exposed epitopes.The presence of two subunits in the wild-type filament

results in greater motility than occurs with either the flaA orflaB gene product alone, although a filament composed ofeither subunit is functional. This is similar to the situationfound in Caulobacter crescentus, which also has a complexflagellum filament. However, in this species, the filamentcontains three flagellin species incorporated into separatespatial regions of the flagellar filament. Mutation in any onesubunit species results in decreased motility, but all three areneeded for a fully active flagellum (28). In C. coli the

1 2 3

* 106 kDa

* 80.0 kDa

~~ *~- 49.5 kDa

FIG. 7. Western blot analysis of glycine extracts of flaA flaB+KX15 (lane 1), flaA+ flaB KX5 (lane 2), and VC167 T2 wild type(lane 3) reacted with a 1:2,000 dilution of antiserum SML2. Thesmall quantity of flaB gene product produced by KX15 can beclearly seen in lane 1. Molecular mass standards are indicated on theright.

FIG. 8. Immunogold electron microscopy labeling of wild-typeflagellum filaments (A), flaA flagellum filament from KX5 (B), andthe truncated flaB flagellum filament of KX15 (C) reacted withantiserum LAH2 absorbed againstflaA+flaB KX5 cells. Bars = 0.5pum.

wild-type flagellar filament containing both subunits resultsin greater motility than a filament of equivalent lengthcontaining only the flaA protein (KX5).The difference in the amounts of flaA and flaB gene

products incorporated into the filament is most likely ex-plained by the fact that the two genes are under the controlof different promoters. Regulation of flagellin genes byspecialized sigma factors is not unusual (15, 16). Flagellingenes in members of the family Enterobacteriaceae and inBacillus spp. are controlled by C28 promoters (5, 15, 29), andin C. crescentus, genes in the lower level of the regulatoryhierarchy of flagellum assembly, including flagellin genes,are temporally controlled by U5M promoters (27, 31, 32). InPseudomonas aeruginosa PAK the flagellin structural geneis transcribed from a U28 promoter, although the product ofthe rpoN gene (a54) is necessary for flagellin synthesis (40),either for expression of positive regulators of the flagellingene or for expression of the &28 factor in a cascade of sigmafactors (38). C. coli is unusual in having two flagellin geneswhose expression involves two alternate sigma factors. Theobservation that only a minority of cells in the populationproduce theflaB filament may be a reflection of metabolic orchemotactic controls on the U54 promoter. Comparativelength measurements of the wild-type filament to that pro-duced by flaA flaB+ mutants suggest that the flaB geneproduct constitutes less than 20% of the wild-type filamentunder the growth conditions used. However, the two distinctpromoters may offer a mechanism by which the organismcan vary the amounts of each gene product in response toglobal regulatory signals. One of the unique characteristicsof U(4 promoters is the presence of upstream activator sites,which, in the systems studied to date in other organisms, aregenetically and physiologically distinct (19, 35). The pres-ence of such an upstream activator for theflaB gene remainsto be determined. However, there are several regions up-

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ROLE OF TWO FLAGELLIN GENES IN CAMPYLOBACTER MOTILITY 4763

stream offlaB which are strikingly similar to the consensusactivator site found for o54-controlled genes which areregulated by nitrogen and oxygen (TGT-N10-ACA) (20). Themost comparable site is TGT-N7-ACA, found 82 bp up-stream of the u54 promoter (Fig. 2).

Previous data obtained from a singleflaA mutant of VC167Ti, VC167-B2, suggested that theflaB gene product was notassembled into a filament but accumulated intracellularly(12). The data presented here shown that the flaB geneproduct of VC167 T2 is assembled into a filament. Resultsobtained with additionalflaA mutants of VC167 Ti indicatethat this flaB gene product can also be assembled into afilament (4) and would suggest that VC167-B2 contains somesecondary defect in transport and/or assembly. Hybridiza-tion studies suggest that many strains of both C. jejuni andC. coli contain two copies of flagellin (26, 39), and it has beendemonstrated that C. jejuni 81116 also contains two tan-demly oriented flagellin genes, one of which (flaA) has a o.28promoter and the other of which (flaB) has a C54 promoter(33). However, no expression of the strain 81116 flaB genehas been detected by primer extension or Northern blotanalyses (33). Nonetheless, mutational analysis of severalother strains of C. jejuni suggest that complex flagellarfilaments are not unusual among campylobacters (3). Sinceflagella are virulence determinants for campylobacters, itwill also be interesting to assess the relative virulence andimmunogenicity of various flagellin mutants in in vitro and invivo models.

ACKNOWLEDGMENTS

This work was supported in part by U.S. Navy Research andDevelopment Command Research Work Unit no. 61102A3M161102BS13 AK.111 and by a grant from the Medical ResearchCouncil of Canada to T.J.T.We thank Denny Cautivar and Steve Martin for expert technical

assistance.

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