The Helicobacter pylori Anti-Sigma Factor FlgM Is ...MATERIALS AND METHODS Bacterial strains and...

JOURNAL OF BACTERIOLOGY, Aug. 2009, p. 4824–4834 Vol. 191, No. 150021-9193/09/$08.00�0 doi:10.1128/JB.00018-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Helicobacter pylori Anti-Sigma Factor FlgM Is PredominantlyCytoplasmic and Cooperates with the Flagellar

Basal Body Protein FlhA�†Melanie Rust,1 Sophie Borchert,1 Eike Niehus,1‡ Sarah A. Kuehne,1§ Eugenia Gripp,1

Afrodita Bajceta,1 Jonathan L. McMurry,3 Sebastian Suerbaum,1Kelly T. Hughes,2 and Christine Josenhans1*

Department for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Carl-Neuberg-Strasse 1,30625 Hannover, Germany1; Department of Biology, University of Utah, Salt Lake City, Utah2; and

Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia3

Received 7 January 2009/Accepted 5 May 2009

Helicobacter pylori requires flagellar motility and orientation to persist actively in its habitat. A particularfeature of flagella in most Helicobacter species including H. pylori is a membraneous flagellar sheath. Theanti-sigma factor FlgM of H. pylori is unusual, since it lacks an N-terminal domain present in other FlgMhomologs, e.g., FlgM of Salmonella spp., whose regulatory function is intimately coupled to its secretion throughthe flagellar type III secretion system. The aim of the present study was to characterize the localization andsecretion of the short H. pylori FlgM in the presence of a flagellar sheath and to elucidate its interaction withother flagellar proteins, such as the basal body protein FlhA, which was previously shown to cooperate withFlgM for regulation. H. pylori FlgM was only released into the medium in minor amounts in wild-type bacteria,where the bulk amount of the protein was retained in the cytoplasm. Some FlgM was detected in the flagellarfraction. FlgM was expressed in flhA mutants and was less soluble and differentially localized in bacterialfractions of the flhA mutant in comparison to wild-type bacteria. FlgM-green fluorescent protein and FlgM-V5translational fusions were generated and expressed in H. pylori. FlgM displayed a predominantly polardistribution and interacted with the C-terminal domain of FlhA (FlhAC). We suggest that, in H. pylori, FlgMsecretion may not be paramount for its regulatory function and that protein interactions at the flagellar basalbody may determine the turnover and localization of functional FlgM.

Helicobacter pylori causes chronic infection of the humanstomach mucosa in about 50% of the world population. H.pylori requires flagellar motility and taxis for persistent survivalin the gastric mucus of humans (11, 23, 27, 40). The flagellarapparatus of H. pylori shows some specific properties. Thebacterium may have acquired those properties as an adapta-tion to an inhospitable environment of low pH and high pro-teolytic activity (e.g., pepsin), which may be damaging to flagel-lar components and function. In particular, a flagellar sheathcovers each flagellum and is continuous with the bacterialouter membrane and of similar lipid composition (14).

Many H. pylori flagellar components are similar to the well-characterized flagellar apparatus of Salmonella spp. (3, 30).The complex hierarchical regulation of flagellar genes in H.pylori (24, 33, 51) suggests that flagellar components in thisspecies are also assembled in a highly ordered fashion. Theanti-sigma factor FlgM and the sigma factor FliA jointly con-trol the expression of late flagellar genes in H. pylori, in par-

ticular of the major filament protein FlaA (6, 24). Interestingly,H. pylori FlgM is truncated at its extreme N terminus in com-parison to its counterpart in Salmonella. Still, H. pylori FlgM isfunctional as an anti-sigma factor in Salmonella (24). FlgM inH. pylori is a central component in the regulation of flagellargenes. It is important at all regulatory levels and not only forthe regulation of late flagellar genes (33). In H. pylori andnumerous other Helicobacter spp., the presence of a flagellarsheath is a common and conspicuous trait and raises the ques-tion of whether FlgM secretion through the flagellar centralchannel into the environment is possible or whether it might behampered under these circumstances. In Salmonella, FlgM se-cretion is crucial for its function as an anti-sigma factor, asFlgM secretion from the bacterial cytoplasm through theflagellar type III secretion system itself is intimately coupled toits regulatory function (15, 20). An unusually short FlgM isfound not only in H. pylori but also in other Campylobacteralesspecies whose whole genome sequences have been decoded(e.g., Helicobacter mustelae, Helicobacter hepaticus, Campy-lobacter coli, Campylobacter jejuni, Wolinella succinogenes [12,35, 44; P. W. O’Toole et al., Sanger Center, unpublisheddata]). It is assumed that the type III secretion signal of FlgMis encoded in its N-terminal amino acid sequence as in othertype III secreted substrate proteins (43). Proposed evolution-ary degeneration at the N terminus of FlgM in all of theEpsilonproteobacteria may therefore suggest that the type IIIsecretion signal responsible for secretion of FlgM is notpresent in those bacterial species. This raises the question of

* Corresponding author. Mailing address: Hannover MedicalSchool, Institute for Medical Microbiology, Carl-Neuberg-Strasse 1,30625 Hannover, Germany. Phone: 49 511 532 4348. Fax: 49 511 5324355. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

‡ Present address: RMC Consulting, Rheinfelden, Germany.§ Present address: Institute for Infection, Immunity and Inflamma-

tion, University of Nottingham, Nottingham, United Kingdom.� Published ahead of print on 22 May 2009.

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whether FlgM cannot be secreted in those bacteria. Underthose circumstances, secretion may not be essential for FlgMfunction.

Our previous data on flagellar transcriptional regulation inH. pylori flhA flgM double knockout mutants (33) indicatedthat inactivation of flgM resulted in a partial suppressor muta-tion over a previous inactivation of flhA. It is of interest thatthe transcriptional regulatory phenotype of wild-type bacteriawas reconstituted in the double mutants. Despite the regula-tory suppressor phenotype, the morphological flagellar pheno-type was not reconstituted back to wild type (33). This wasprobably due to the total lack of FlhA protein in those mu-tants. Thus, the hypothesis was generated that FlhA and FlgMcooperate closely or may physically interact in order to coor-dinate flagellar gene regulation.

The goal of this study was therefore to establish whetherFlgM is secreted from the H. pylori cell and to determine FlgMexpression and localization in wild-type H. pylori and flagellarmutants with defined defects in flagellar regulation and flagel-lar secretion. We also hypothesized that protein-protein inter-actions at the flagellar basal body, in particular with FlhA, maybe required for the function and localization of H. pylori FlgM.

In this study we found that FlgM is only secreted in smallquantities from H. pylori wild-type cells. Furthermore, FlhAhad an influence on the localization of FlgM, and interactionbetween FlgM and the C-terminal domain of FlhA (FlhAC)was demonstrated by several methodologies.

MATERIALS AND METHODS

Bacterial strains and culture conditions. H. pylori strains N6 (13) and 88-3887(motile variant of the fully sequenced strain 26695 [21]) were used. The bacterialstrains and H. pylori mutants used are listed in Table 1. Bacteria were routinelycultured on blood agar plates (Oxoid blood agar base and 5% horse blood) or inbrain heart infusion broth supplemented with 2.5% yeast extract and 10% horseserum and the following antibiotics: vancomycin (10 mg/liter), polymyxin B(2,500 U/liter), trimethoprim (5 mg/liter), and amphotericin B (4 mg/liter). Theselective antibiotics included ampicillin (300 mg/liter), kanamycin (20 mg/liter),and chloramphenicol (10 mg/liter) and were added to the media as required.Escherichia coli strains used for cloning were DH5� and MC1061 (38), and foroverexpression of proteins, the strains ER2566 (New England Biolabs Inc.) andTG1 (38) were employed.

Techniques of molecular cloning and protein analysis. DNA purification,DNA manipulation, and cloning procedures were performed according to stan-dard protocols (38). PCRs were run in Perkin-Elmer or Biometra thermocyclersusing Amersham Taq polymerase. The Expand High-Fidelity kit (Roche) wasused for longer fragments (�2 kb) or if high amplification accuracy for proteinexpression was required. Plasmids are listed in Table 2. Protein analysis wasperformed using denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gelscontaining between 11% and 16% acrylamide, depending on the molecular massof the proteins to be detected (29), and applying Western immunoblot detectionaccording to the methods described by Towbin (46). Antibodies for immunola-beling were used as indicated in the results or in the figure legends. Proteinconcentrations were determined using the bichinchoninic acid assay. H. pyloriwas transformed with plasmids by natural transformation or electroporation. H.pylori crude flagellar preparations and bacterial lysates were obtained as previ-ously described (23, 41).

Crude fractionation of bacteria. Bacteria, grown either on plates or in liquidculture to defined optical densities, were fractionated into supernatant, insoluble(membrane-enriched), and soluble (cytoplasmic) fractions. Also, the surfaceappendages containing flagella and other surface-associated material were iso-

TABLE 1. Strains used in this study

Strain Genotypea Reference

H. pylori strains26695 Wild-type strain 4588-3887 Motile variant of 26695 21N6 Wild-type stain 13N6flhA N6 flhA::aphA3�-III 39N6flgM N6 flgM::aphA3�-III 24N6fliA N6 fliA::aphA3�-III 24N6fliI N6 fliI::aphA3�-III This studyN6rpoN N6 rpoN::aphA3�-III 3388-3887flgM 88-3887 flgM::aphA3�-III 2488-3887flhA 88-3887 flhA::aphA3�-III 3388-3887fliP 88-3887 fliP::aphA3�-III 2188-3887fliI 88-3887 fliI::aphA3�-III This study88-3887fliF 88-3887 fliF::aphA3�-III This studyN6flhF N6 flhF::aphA3�-III 33N6(pCJ604) N6 � plasmid pCJ604 This studyN6flgM(pCJ604) N6 flgM::aphA3�-III � plasmid pCJ604 (FlgM-GFP) This studyN6fliA(pCJ604) N6 fliA::aphA3�-III � plasmid pCJ604 (FlgM-GFP) This studyN6flhA(pCJ604) N6 flhA::aphA3�-III � plasmid pCJ604 (FlgM-GFP) This studyN6fliI(pCJ604) N6 fliI::aphA3�-III � plasmid pCJ604 (FlgM-GFP) This studyN6flgM(pCJ607) N6 flgM::aphA3�-III � plasmid pCJ607 (FlgM-V5) This study

E. coli strainsDH5� F� endA1 recA1 hsdR17 �(lacZYA-argF)U169 thi1 supE44 gyrA96 relA1 17ER2566 F� �� fhuA2[Ilon], ompT lacZ::T7 gene I gal sulA11�(mcrC-mrr)11::IS10 R

(mcr-73::miniTn10)2 R(zgb-210)::Tn10)1 (Tets) endA1 [dcm]New England

BiolabsMC1061 F� araD139 �(ara-leu)7696 galE15 galK16 �(lac)X74 rpsL (Strr) hsdR2 (rK

� mK�)

mcrA mcrB15

TG1 Expression of FlhAC 38BTH101 Cya-BACTH expression strain; F� cya-99 araD139 galE15 galK16 rpsL1 (Strr)

hsdR2 mcrA1 mcrB125

a Kmr (aphA3�-III), kanamycin resistance cassette (28).

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lated as previously described (21). Briefly, liquid culture bacteria were harvestedby centrifugation (4,500 � g for 20 min at 4°C). The cell culture supernatant wasremoved and further purified according to a method originally developed forSalmonella by Hughes and Karlinsey (26). The supernatant was first sterilefiltered through a 0.22-m-pore-size MillexR-GV polyvinylidene difluoridemembrane. The cell-free supernatant was then filtered through a Protran nitro-cellulose immobilization membrane (Schleicher & Schuell, Germany), whichbinds proteins present in the cell-free suspension. The membrane was thenbathed in 20 to 25 l of SDS-polyacrylamide gel electrophoresis (SDS-PAGE)sample buffer to elute the concentrated proteins from the membrane, which wereloaded onto SDS-PAGE gels. For isolation of flagellar fractions, the bacterialpellets were resuspended in 0.9% NaCl and subjected to mild shearing forces byrepeated (30 times) pushing of the suspension through 26-gauge syringe needlesto shear off appendages from the bacterial surface. Intact bacterial cells and thesheared-off material were then separated by differential centrifugation steps (firstat 9,000 � g for 30 min followed by 40,000 � g for 1 h in a Beckman Optima100ultracentrifuge). The quality of the surface preparations was confirmed by elec-tron microscopy and by Western immunoblotting using flagellin-specific antisera.These controls confirmed that the protocol successfully isolated surface-associ-ated material. The separation of bacteria into insoluble (mainly membrane-associated) and soluble (predominantly cytoplasmic) fractions followed after theseparation from the sheared-off material. The intact bacterial cells were resus-pended again in 0.9% NaCl and lysed by sonication (Branson sonifier). Aftercentrifugation (two times at 9,000 � g for 30 min at 4°C), the pellet was resus-pended in 0.9% NaCl and contained the crude membrane fraction, whereas thesupernatant represented the soluble fraction. The quality of the fractionationprocedure to isolate insoluble and soluble fractions was tested by electron mi-croscopy and by Western blot analysis for membrane proteins (using anti-HPFlhA antiserum).

RNA preparation and RT-PCR. RNA was prepared from H. pylori bacteriaharvested from liquid cultures in different growth phases using the QiagenRNeasy kit with slight modifications as described elsewhere (24). Semiquantita-tive reverse transcription-PCRs (RT-PCRs) were performed on 2 g of DNaseI-treated RNA samples. Reverse transcription was performed using a randomhexamer primer mix and SuperScript III reverse transcriptase (Invitrogen) at42°C for 2 h. The cDNA was adjusted to 50 l with double-distilled H2O.One-microliter aliquots of cDNA sample were amplified in different PCRs (in-cluding negative controls) with primers specific for the corresponding genes. H.pylori 16S rRNA-specific primers were used in each experiment to verify thatcomparable amounts of cDNA were used.

Construction of H. pylori flagellar mutants. Plasmid constructs for three ad-ditional flagellar mutants (fliI, gene for flagellar ATPase [32, 36]; fliF, flagellarMS ring protein gene [30]; and flhB1 and flhB2, genes for flagellar basal body

proteins cooperating with FlhA [30]) were generated in E. coli. In brief, PCR-amplified fragments of the genes were cloned into pUC18 or pILL570. Antibioticresistance cassettes were inserted into the 3� parts of the genes either by inversePCR amplification of the plasmids or using natural restriction sites. Insertion ofthe cassettes was in the same orientation with respect to the target gene, in orderto avoid polar effects. Resulting suicide plasmids (Table 2) were then introducedinto H. pylori strains N6 and the motile isogenic clone 88-3887 of 26695 (21) bynatural transformation as described previously (16). Transformants or allelicexchange mutants were selected on kanamycin- or chloramphenicol-containingplates. The genotypes of the mutants were verified by PCR using differentcombinations of oligonucleotide primers. All mutant clones were verified byPCR to carry the genomic disruptions as a result of a double-crossover and nota single-crossover event. Details of the mutant constructions and oligonucleotideprimers are provided in the supplemental material. In addition, we used H. pylorimutants in flhA (39), fliP (21), flgM and fliA (24), and rpoN and flhF (33) that hadbeen previously generated and characterized.

Construction of FlgM-GFP and FlgM-V5 transcriptional fusion plasmids forcomplementation in H. pylori in trans and for localization studies. (i) FlgM-GFP.For the expression of a FlgM-green fluorescent protein (GFP) fusion in H. pylori,a plasmid was constructed starting with pCJ105 (FlgM-FLAG [C. Josenhans,unpublished data]), which contains the H. pylori flgM gene followed by a FLAGtag sequence. This tag was removed via inverse amplification (primers HPFlgM_GFP_1and HPFlgM_GFP_2; both BglII sites). Using oligonucleotides GFP-FlgM_1and GFP-FlgM_2 (BglII sites) the gene encoding a red-shifted and enhancedGFP variant, GFPmut2 (7), was amplified. Both amplification products were cutand ligated to yield plasmid pCJ604 (FlgM-GFP), from which a FlgM proteinwith a C-terminally fused GFP protein could be expressed in H. pylori. Theshuttle plasmid pCJ604 was transformed into H. pylori N6 wild type and also forcomparison into N6flgM, N6flhA, N6fliI, and N6fliA mutant strains by naturaltransformation (50), yielding chloramphenicol-resistant colonies. The rationalefor using a fliI mutant was that it is expected to be impaired for separation ofFlgM from its chaperone FliA (36). The transformants were characterized byPCR, RT-PCR, Western blot analysis, transmission electron microscopy, andimmune fluorescence.

(ii) FlgM-V5. The flgM gene, including its own untranslated 5� region contain-ing putative promoter sequences, was also C-terminally fused to a V5 tag se-quence (GKPIPNPLLGLDST) with a 7-amino-acid linker (GGSSAAG [4]) andcloned into the E. coli/H. pylori shuttle plasmid pHel2 (18). Initially, the vectorpEF6-V5 (Invitrogen) was amplified by inverse PCR using primers (OLpEF6_Rand OLV5_F) and including the sequence encoding the 7-amino-acid linker(GGSSAAG) fused to the 5� end of the encoded V5 tag (from plasmid pCJ521[41]). The PCR product was digested with BglII and ligated with the BamHI-digested PCR product of the flgM gene (pCJ606). The flgM gene had been

TABLE 2. Plasmids constructed or used in this study

Plasmid Vector Descriptiona Reference orsource

pHel2 Cmr RepEc RepHp; multicopy shuttle vector for E. coli and H. pylori 18pUC18 Apr RepEc, high-copy-number cloning vector 48

pCJ105 pHel2 RepEc RepHp flgM-FLAG tag fusion; flgM from H. pylori 26695 C. Josenhans,unpublished

pCJ521 pEF6-V5 RepEc RepHp Ampr Blastr; source of V5 sequence 41pCJ601 pUC18 Apr Kmr RepEc fliI::aphA3�-III; fliI from H. pylori 88-3887 This studypCJ604 pHel2 RepEc RepHp Cmr flgM-gfpmut2 fusion; flgM from H. pylori 88-3887 This studypCJ607 pHel2 RepEc RepHp Cmr flgM-V5 tag fusion; flgM from H. pylori 88-3887 This studypILL600 pBR322 Apr Kmr RepEc; source of Kmr cassette (aphA3�-III) 28pSUS1608 pSUS2/pUC18 fliF-aphA3�-III This studypSUS1614 pUC18 flhB2-aphA3�-III This studypSUS1620 pUC18 flhB1-aphA3�-III This studypCJ1001 pKNT25 (BACTH) RepEc Kmr flhAC-t25 gene fusion flhAC from strain N6 This studypCJ1004 pUT18 (BACTH) RepEc Apr flgM-t18 gene fusion flgM from strain 26695 This study

pUT18 (BACTH) RepEc Apr 25pKNT25 (BACTH) RepEc Kmr 25

pUT18C-ZIP pUT18C (BACTH) RepEc Apr, leucin zipper gene 25pKT25-ZIP pKT25 (BACTH) RepEc Kmr, leucin zipper gene 25pCJ309 pTWIN1 (NEB) RepEc Apr, H. pylori FlgM expression plasmid This studypSUS225 pQE30 (Qiagen) RepEc Apr, H. pylori His6-FlhAC expression plasmid 39

a Apr, ampicillin resistance; Kmr, kanamycin resistance; Cmr, chloramphenicol resistance (50); Blastr, blasticidin resistance; RepEc and RepHp, replication origin forE. coli and H. pylori, respectively.

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amplified before from H. pylori 26695 with primers (HPFM5 and FlgM_rev_2).By sequencing pCJ606, the in-frame fusion of the flgM gene with the V5 tag waschecked. For cloning into pHel2, which was required for expression in trans in H.pylori, the FlgM-V5 gene fusion was amplified from the plasmid pCJ606 usingprimers (HPFM5 and OLV5_R, BamHI sites), which introduced a stop codon atthe 3� end. The amplified gene was cloned into the BamHI site of pHel2(resulting shuttle plasmid, pCJ607; FlgM-V5). pCJ607 was then introduced intothe N6flgM mutant using natural transformation. Plasmid-containing transfor-mant clones were selected on kanamycin-chloramphenicol-containing mediumand characterized. The expression of the FlgM-V5 fusion protein was assessed byWestern blotting. Primer sequences used for amplification and cloning are avail-able in the supplemental material.

Motility testing (motility plates). Motility testing was performed in semisolidplates consisting of brucella broth supplemented with 0.3% agar and 3% fetalcalf serum (FCS), both as stab tests and as single colony motility tests as de-scribed previously (23).

Recombinant expression and purification of H. pylori FlhA and FlgM proteinsin E. coli. A partial C-terminal protein domain of approximately 46.5 kDa of H.pylori FlhA (amino acids 351 to 732) was expressed as described previously usingplasmid pSUS225 (39). For technical reasons it was not feasible to overexpressthe complete H. pylori FlhA including its N-terminal membrane domain (39). H.pylori FlgM was expressed in the pTWIN system (New England Biolabs, Cam-bridge, MA) as a C-terminal FlgM-intein-chitin binding domain fusion protein.FlgM was cloned into the pTWIN1 C-term vector (according to the New EnglandBiolabs information manual) by PCR and ligation. Plasmid pCJ309 obtained inE. coli DH5� was characterized and verified by nucleotide sequencing of thecomplete insert. The expression of the fusion construct in E. coli ER2566 (NewEngland Biolabs) was induced at early exponential phase by administering 0.5mM isopropyl--D-thiogalactopyranoside (IPTG) for 4 h at 37°C. Purification ofthe fusion protein was performed by affinity chromatography using a chitincolumn as recommended by the manufacturer (New England Biolabs informa-tion manual). Binding to the column was followed by an overnight purificationstep involving an intein autocleavage reaction in the presence of the reducingagent dithiothreitol. Dithiothreitol causes the autocatalytic cleavage of the inteindomain from the fusion protein and enables the subsequent elution of an un-tagged version of the overexpressed protein (H. pylori FlgM). The eluted FlgMwas further purified by gel elution and repeated dialysis against phosphate-buffered saline.

Generation of antisera. An anti-HPFlhA antiserum raised against the recom-binantly expressed C-terminal domain (FlhAc) was generated in rabbits as pre-viously described (39). An antiserum directed against the recombinantly overex-pressed and gel-purified untagged FlgM (described above) was raised in rabbits(Eurogentec, Seraing, Belgium) by repeated intradermal injection.

Electron microscopy. Copper electron microscropy (EM) grids coated withFormvar and carbon were directly placed on plate-grown bacteria to let themstick and to negatively stain them with 1% phosphotungstate (pH 7.0). EM gridswere viewed in a Zeiss EM10 microscope at an acceleration voltage of 80 kV.

Immunofluorescence labeling and microscopy for intracellular detection oftagged FlgM. Coverslips were coated with 0.3% gelatin and were placed directlyon plate-grown bacteria to let them attach. Liquid grown bacteria were also usedbut did not become attached to the coverslips as reproducibly as plate-grownbacteria. The bacteria were fixed with 2% paraformaldehyde in 100 mM phos-phate buffer for 3 h and for another hour after exchanging the fixative with freshfixative. Nonadhering bacteria were removed by washing the coverslips fourtimes with 0.1% glycine in phosphate-buffered saline (PBS). To permeabilize thefixed bacteria, they were incubated for 30 min with 0.1% Triton X-100. Afterwashing three times with PBS, the coverslips were incubated for 30 min inblocking buffer (1% bovine serum albumin [BSA], 1% FCS in PBS). Due to aweak GFP signal in H. pylori cells transformed with the FlgM-GFP fusion con-struct, the bacteria had to be immunostained with polyclonal anti-GFP antibod-ies (R970-01; Invitrogen). The bacteria were incubated overnight with the anti-GFP antibodies diluted in blocking buffer (1:2,000). After washing four timeswith 0.1% BSA in PBS, the fixed bacteria were incubated with secondary anti-body (goat anti-rabbit Alexa488; 1:5,000; Invitrogen) for 1 h. The bacteria werewashed four times with 0.1% BSA in PBS and also three times with PBS. Thecoverslips were mounted with the cell side down on a microscope slide withmounting medium (2.5% DABCO in Mowiol) and dried overnight protectedfrom light. The coverslips were sealed with nail polish. Fluorescence microscopywas performed using an Olympus BX40 microscope with a mercury light sourceand a narrow-band-pass filter combination optimized for GFP detection (exci-tation at 490 nm, emission at 515 nm), outfitted with a ColorviewIII digitalcamera (Olympus). Immunofluorescent labeling for the V5-tagged FlgM wasperformed according to the same experimental protocol using mouse monoclo-

nal anti-V5 antibody (dilution of 1:500; Invitrogen) and goat anti-mouse-Al-exa488 (1:5,000; Invitrogen) as a secondary antibody. Labeling with anti-V5monoclonal antibody, in contrast to anti-GFP polyclonal serum, did not detect-ably stain all bacteria for FlgM-V5, and this was reproduced several times. Weassume that this is due to the single epitope being recognized by the antibody,which may be masked in some states of FlgM, e.g., when it is in a stable complexwith FliA.

Protein coprecipitations. Coprecipitations (pull-down assays) were performedeither with highly purified H. pylori FlgM or His6-FlhAC proteins or with bacte-rial lysates which were sonicated in modified radioimmunoprecipitation assay(RIPA) buffer (1% Nonidet P-40, 100 mM NaCl, 25 mM Tris-HCl, pH 7.5, 10%glycerol, 20 mM imidazole, 0.1% Tween 20) and cleared by centrifugation tocontain only soluble proteins. In the case of purified recombinant proteins (seeabove), His6-FlhAC (5 g) and FlgM (2 g) were mixed and modified RIPAbuffer was added to a total volume of 500 l. The samples were sonicated for 2min at 4°C and incubated with the buffer for 1 h at 4°C with rotation. They werefurther incubated for 2 h at 4°C with 25 l of TALON metal affinity resin(Clontech), which had been prewashed three times with ice-cold PBS. Thesamples were centrifuged at 1,006 � g for 1 min at 4°C, and the TALON resinpellets were washed three times with modified RIPA buffer (without NonidetP-40) and two times with ice-cold PBS. The proteins were eluted from theTALON beads by incubating at 100°C for 10 min in 30 l of 2� SDS-PAGEsample buffer. Alternatively, for pull-down from bacterial lysates, the amount ofFlhAC or FlgM-V5 in the respective lysates was determined in Western blotassay and compared to purified proteins loaded in adjacent lanes. Equivalentamounts of cleared lysates, containing approximately 5 g of His6-FlhAC and 2g of FlgM, were then used for the experiments. Purified FlgM (2 g) was mixedwith cleared lysate of E. coli expressing recombinant H. pylori His6-FlhAC. In thiscase, control experiments using the same amount of His6-FlhAc-expressing lysateonly were always performed in parallel. The mixtures were brought to a volumeof 500 l with modified RIPA buffer and further handled for the pull-down assayas described above, including five subsequent wash steps before the elution.

Bacterial two-hybrid system to investigate protein-protein interactions. Thebacterial two-hybrid system BACTH, which was originally developed by Ladant,Ullmann, and Karimova (25) and is now commercially available from EuroME-dEx Inc., was used to assess the potential interactions between H. pylori FlgMand FlhAC in a bacterial native system (in E. coli). The BACTH plasmids pUT18and pKNT25 were engineered to express FlgM and FlhAC (see Fig. S3 in thesupplemental material; primer sequences are also provided in the supplementalmaterial) as C-terminal fusion proteins to the T18 and T25 domains of Bordetellapertussis adenylate cyclase (cya). Again, only the C-terminal soluble part of FlhAwas cloned into pKNT25 (FlhAC; cloning primers HP_FlhApKNT_fw andHP_FlhApKNT_rvEco; resulting plasmid pCJ1001). FlgM was inserted intopUT18, starting from the first possible start codon of the open reading frame(primers HPFM24_fw and HPFM25_rvEco; resulting plasmid, pCJ1004).pKT25-zip and pUT18C-zip plasmids (25) were used as a positive control forprotein-protein interactions. All possible single and double transformants of theBACTH plasmids with and without inserts were prepared in E. coli BTH101(Table 2). The plasmid content of the transformants was screened using Qiagenplasmid spin columns, and protein expression of the transformants was detectedusing polyclonal antisera against FlgM and FlhA in Western immunoblot assays.Metabolic screening of the clones after transformation was performed on Luria-Bertani plates supplemented with IPTG (0.2 mM) and 5-bromo-4-chloro-3-indolyl--D-galacatopyranoside (X-Gal; 40 g/ml) at 37°C as described in theBACTH manual (EuroMedEx). Quantitative measurements of -galactosidaseactivities in the transformants were performed according to the method devel-oped by Miller as described previously (24).

RESULTS

H. pylori FlgM is a predominantly cytoplasmic protein andis only secreted in minor amounts into H. pylori flagella. In H.pylori, a flagellar sheath envelopes the whole filament and iscontinuous with the bacterial outer membrane. Under thesecircumstances, it is not easy to envisage how FlgM could workas a secreted regulator. Our hypothesis was that H. pylori FlgMmight undergo gain and loss of its regulatory anti-sigma func-tion without the absolute requirement of secretion or mightnot be secreted at all.

The potential for secretion of FlgM was first assayed by filter



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concentrating supernatants of H. pylori wild-type bacteriagrown in liquid medium to different optical densities. In con-centrated bacterial supernatants of wild-type H. pylori har-vested from liquid culture at different time points from lagphase to late exponential phase, almost no FlgM was detected(Fig. 1). At very late time points of growth (an optical densityof 600 nm [OD600] of approximately 2.5), minor amounts ofFlgM were detected in the cell supernatants (Fig. 1). We thenattempted to determine the subcellular localization of FlgM inH. pylori wild type further by crude fractionation, separatingthe sheared-off fraction (containing flagellar material includingflagellar sheaths), soluble (expected to contain predominantlycytoplasmic and periplasmic materials), and insoluble (crudemembranes) bacterial components (see Materials and Meth-ods). In these experiments, we detected the bulk amount ofFlgM in the soluble fraction at all time points during growth(Fig. 1). Osmolytic treatment of whole bacteria according tothe methods described in reference 1 (before and after shear-ing of flagella) (data not presented), intended to releaseperiplasmic proteins, did release some FlgM from the bacterialbodies, but only in the presence of flagella. This finding sug-gested that the soluble part of FlgM is predominantly cytoplas-

mic and to some extent flagellum bound. In concordance, mi-nor amounts of FlgM were also detected in the isolatedsheared-off flagellar preparations of wild-type bacteria, with anincrease of FlgM in this fraction at the onset of stationarygrowth (Fig. 1).

We therefore concluded that FlgM is predominantly a cell-bound and soluble cytoplasmic protein in H. pylori wild-typebacteria, while small amounts of FlgM can be released into andretained within the flagellar fraction, which includes the flagel-lar sheath.

FlgM is variably expressed in various H. pylori flagellarmutants. Previous results indicated that FlgM cooperatesclosely with FlhA to ensure the timely regulation of flagellarclass II and III regulons in H. pylori (33). We therefore hy-pothesized that flagellar basal body proteins, in particularFlhA, are involved in the function and cytosolic localization ofH. pylori FlgM. Thus, in order to determine the requirementsfor FlgM expression in H. pylori with regard to the presence ofdifferent flagellar proteins and regulons, we tested a panel offlagellar basal body and regulatory mutants in two different H.pylori strains, N6 and 88-3887 (flhA, flhB1, flhB2, flhF, fliA, fliF,fliI, fliP, and rpoN, which were generated and characterized inour laboratory) (21, 24, 33, 39).

Analysis of whole-cell lysates (Fig. 2) by SDS-PAGE andWestern blotting against FlgM demonstrated that FlgM ex-pression was low in flhB1, flhF, fliP, and rpoN allelic disruptionmutants and almost undetectable in isogenic flhB2, fliA, andfliF disruption mutants. These results indicated that eithertranscript abundance, translation, or stability of H. pylori FlgMdepended crucially on the flagellar basal body proteins FlhB1,FlhB2, FlhF, FliF, and FliP, as well as on the flagellar sigmafactors RpoN and FliA. The latter result also corresponds withearlier work performed in Salmonella, which determined thatFliA acts as a stabilizing type III secretion chaperone on FlgM(2). In contrast, the fliI mutants expressed FlgM. Likewise, inflhA mutants, which lack FlhA as a possible interaction partnerfor FlgM (33), FlgM expression in whole-cell lysates appearedto be similar to the wild type (Fig. 2).

FlgM displays a different subcellular distribution in H. py-lori flhA mutants compared to wild-type bacteria. Since theflagellar basal body protein FlhA was assumed to be an im-portant cooperative or interactive partner of FlgM in H. pylori,the next step was to investigate whether the subcellular local-ization of FlgM in H. pylori flhA mutant bacteria is altered incomparison to the wild type. Therefore, we cultured H. pyloriflhA mutants in liquid media to different cell densities andcrudely fractionated them, as with the wild-type bacteriaabove. We then analyzed these four fractions using Westernblotting and compared the fractions to those of the wild-typebacteria.

In several experiments, less overall FlgM was detected inflhA mutants after separation of the soluble and insolublefractions than in the crude lysate before fractionation of thesame flhA mutants (data not shown). This suggested that FlhAmight be involved directly or indirectly in stabilizing FlgM. Incontrast to the wild-type strain, where FlgM was predomi-nantly detected in the soluble fraction, we recovered the high-est amount of FlgM within the insoluble fraction of the flhAmutant (Fig. 3). Very little FlgM was detected at all time pointsin the soluble fraction. Thus, apparently, the soluble and cyto-

FIG. 1. Localization of native FlgM in cellular fractions of H. pyloriwild-type bacteria. Bacteria (wild-type HP 88-3887) grown in liquidculture were harvested in different growth phases (OD600 of 0.5 to 2.5)and fractionated. Western blots after SDS-PAGE (16% acrylamide)were incubated with anti-HPFlgM antiserum (1:200), and secondaryantibodies were goat anti-rabbit, peroxidase coupled (1:10,000). Ten-microliter aliquots of the respective fractions were loaded in each lane.The molecular mass of FlgM is designated by the arrows. IF, insolublefraction; CF, soluble fraction; EF, culture supernatant (external frac-tion); SF, sheared fraction (flagella-containing fractions).



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plasmic localization of FlgM was decreased in the absence ofFlhA. Some FlgM was also recovered in the secreted superna-tant of flhA mutants in later phases of growth. No FlgM wasdetected in the sheared-off fraction. This was consistent withexpectations, since the H. pylori flhA mutants have been re-ported to lack flagellar formation and also the formation offlagellar sheath structures (39).

Expression of tagged FlgM fusions and predominantly polarlocalization of FlgM-V5 in H. pylori. Subsequently, in order toinvestigate the localization of FlgM in situ with alternativemethods, two different expression plasmids were constructedwhich produce tagged fusion proteins of FlgM at its C-terminalend (FlgM-GFP and FlgM-V5). These shuttle plasmids codingfor FlgM fusions were transformed into H. pylori (strain N6)flgM mutants for the FlgM-GFP plasmid and also into wild-type N6, N6 flhA, and fliI mutants (Fig. 4). Owing to gene copyeffects, genes encoded by the pHel2 derivative plasmids areapproximately fivefold overexpressed in H. pylori (data notshown).

The tagged versions of FlgM were expressed in H. pyloriN6flgM(FlgM-GFP) and in the other FlgM-GFP transformants(data not shown) and in N6flgM(FlgM-V5) (Fig. 5A). Bothfusion proteins were functional as inhibitory anti-sigma factors,since they were able to diminish the transcript abundance formost FliA-dependent class III genes (flaA; HP0472) and oneintermediate class flagellar gene (fliD) tested in the trans-formed strains to at least the level of wild type for FlgM-V5(Fig. 4B) and to even lower levels for FlgM-GFP (Fig. 4A).This was in contrast to flgM mutants, which showed highertranscript levels than the wild type for class III and someintermediate genes, as previously reported (24). FlgM-GFPexpression enhanced class II gene transcripts in comparison towild type, which was not observed for FlgM-V5 (Fig. 4A).Bacteria expressing FlgM-GFP showed increased repressionabilities for class III genes in comparison to FlgM-V5, suggest-

FIG. 2. FlgM expression in various H. pylori flagellar basal body mutants. FlgM expression in H. pylori wild-type strains (HPN6 and HP88) anddefined isogenic mutants in flagellar regulatory and basal body genes (whole-cell lysates; see Materials and Methods for descriptions of themutants) and flgM mutant (negative control). M, molecular mass marker. Approximately 10 g of total cell lysate protein for the upper panel or30 g for the lower panel was loaded in each lane. Immunodetection was performed using anti-H. pylori FlgM antiserum (1:300).

FIG. 3. Localization of native FlgM in fractions of H. pylori flhAmutant bacteria. Bacteria (HP88-3887 flhA) grown in liquid culturewere harvested in different growth phases (OD600 of 0.5 to 2.5) andfractionated. Western blots after SDS-PAGE (16% acrylamide) wereincubated with anti-FlgM antiserum (1:200), and secondary antibodieswere goat anti-rabbit, peroxidase coupled (1:10,000). Ten-microliteraliquots of the respective fractions were loaded in each lane. Themolecular mass of FlgM is designated by the arrows. IF, insolublefraction; CF, soluble fraction; EF, culture supernatant (external frac-tion); SF, sheared fraction (surface associated, containing flagella).



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ing that the anti-sigma factor effect of the GFP fusion is stron-ger than that of wild-type FlgM and FlgM-V5 (Fig. 4) or thatrepression by FlgM-GFP does not become appropriately re-lieved.

GFP is a 27-kDa protein tag and, to a large extent, getsfolded into its barrel-like three-dimensional structure in thebacterial cytoplasm in the presence of oxygen (49). Therefore,the secretion of proteins through the narrow channel of theflagellar secretion system is largely abolished by GFP tagging(19) (unpublished data). V5 is a short epitope tag (14 aminoacids) which can be used as a specific tag for fluorescenceimmunodetection and is not predicted to hamper type III se-cretion. The morphology of the transformants expressingFlgM-GFP was characterized by the lack of flagella, whereas

FIG. 5. Localization of FlgM-V5 in subcellular fractions of H. pyloriand in intact bacteria. (A) FlgM-V5 was detected in fractions of H.pylori N6flgM(pCJ607) using anti-V5 monoclonal antibody (1:3,000;Invitrogen; secondary antibody was goat anti-mouse peroxidase cou-pled, at 1:15,000). Bacteria were cultured on plates for 2 days. SF,sheared-off surface-localized fraction; IF, insoluble fraction; CF, solu-ble fraction. (B) FlgM was localized by immunofluorescence on fixed,permeabilized intact H. pylori N6flgM(pCJ607) bacteria expressingFlgM-V5, using anti-V5 antibody (1:500; secondary antibody was goatanti-mouse Alexa 488 at 1:5,000). (C) Fluorescence micrograph aftercontrol labeling of intact, nontransformed H. pylori N6 wild type usingthe same antibodies.

FIG. 4. Flagellum-related transcripts in H. pylori transformants ex-pressing tagged FlgM. Shown is the detection of selected flagellartranscripts in H. pylori (HP) N6(FlgM-GFP), HPN6flhA(FlgM-GFP),HPN6flgM(FlgM-GFP), HPN6fliI(FlgM-GFP), and HPN6flgM(FlgM-V5)transformants in comparison to HPN6 wild type. (A) FlgM-GFP-ex-pressing strains; (B) FlgM-V5-expressing flgM(FlgM-V5). flhA and fliIflagellar basal body protein mutants with defined defects in the flagel-lar assembly process (described in Materials and Methods) trans-formed with FlgM-GFP-expressing plasmid were used as controls fortranscript-level comparison with transformed flgM and wild-type bac-teria. Relative transcript amounts of selected regulatory and flagellargenes of different hierarchical classes were determined using semi-quantitative PCR on equivalent amounts of cDNA (RNA preparationperformed from liquid cultures, all bacteria harvested at an equivalentOD600 of approximately 1.6). fcr, flagellar regulatory class; fc1, flagellarclass 1; fc2, flagellar class 2; fc3, flagellar class 3; fci, flagellar intermediateclass (see reference 33). In flgM mutants which are not depicted here,transcripts of class 3 genes HP0472 and flaA are highly upregulated (24).The respective gene names are shown on the right side. �, negativecontrol. H. pylori 16S rRNA-specific primers were used to control forequivalent amounts of cDNA.



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the FlgM-V5-expressing bacteria possessed flagella (data notshown). These results suggested that FlgM-V5 permits thechronology of events that lead to flagellar assembly to takeplace, whereas FlgM-GFP does not.

Fractionation of bacteria expressing FlgM-GFP andFlgM-V5 was performed. Neither tagged FlgM version wassecreted into the culture supernatants (data not shown), asshown for wild-type FlgM. Western blot analysis revealed thatFlgM-V5 was localized both in the soluble fraction and in theinsoluble fraction (Fig. 5A), while FlgM-GFP localized pre-dominantly to the insoluble fraction (data not shown). SomeFlgM-V5 was also detected in the sheared off-fraction (Fig.5A), similar to the nontagged native FlgM (Fig. 1). Further-more, fluorescent immunolabeling was performed for both fu-sion constructs in order to localize the tagged FlgM versions inintact bacteria. In fluorescence microscopy of live and fixedbacteria, GFP fluorescence of the FlgM-GFP fusion was quitelow and did not allow protein localization in microscopy (22).We therefore used very specific anti-GFP and anti-V5 antibod-ies for immunofluorescent labeling of both fusion proteins inintact bacteria (see Materials and Methods). Control strainsnot expressing the fusion proteins were negative in fluores-cence microscopy, whereas a clear signal could be observedwith both FlgM fusions. As the regulatory and morphologicalphenotype of the transformed FlgM-V5 bacteria resembledwild-type bacteria, we continued the immunolocalization ap-proach using only the FlgM-V5 fusion. For FlgM-GFP, severechanges in morphology and regulatory phenotype indicatedclogging of the flagellar secretion channel and possibly otherside effects, for which reason we did not continue localizationstudies with this strain. The FlgM-V5-specific signal in immu-nofluorescence microscopy was predominantly polar (Fig. 5B).Despite repeated attempts, it was not possible to obtain trans-formants with the FlgM-V5 plasmid in a flhA mutant. There-fore, FlgM-V5 localization in situ in this mutant could not bedetermined.

Interaction between H. pylori FlgM and FlhAC. In order togain some insight into the molecular basis of the function andsubcellular localization of H. pylori FlgM in cooperation withFlhA as hypothesized earlier, we investigated the potential forinteraction between FlgM and FlhA. For this purpose, thesoluble C-terminal domain of H. pylori FlhA, which is supposedreach into the bacterial cytoplasm (37, 47), was overexpressedas a His6-tagged fusion protein in E. coli and then purified aspreviously described (39). H. pylori FlgM was also expressedand purified from E. coli (see Materials and Methods). Usingboth H. pylori FlgM and FlhAC as highly purified proteins afteroverexpression in E. coli, we observed a nonreproducible in-teraction of the two proteins in coprecipitation experiments(data not shown). Surface plasmon resonance analysis, usingthe same purified proteins in standard buffers, posed persistenttechnical problems and yielded no clear interaction profile. Ina second biosensor setup using a Biolayer Interferometry plat-form, immobilized purified FlgM was screened against bacte-rial lysate containing H. pylori FlhA, but this method also failedto detect binding (see the supplemental material). Neither didwe observe an interaction between purified denatured FlhAC

and FlgM in overlay blot assays (data not shown). Thus, wehypothesized that the folding of one or both interaction part-ners might be hampered during or after the purification pro-

cess, which requires alternative strategies. A subsequent ap-proach simulated more-physiological conditions and includedthe possibility that FlgM and FlhA might cooperate indirectly,aided by other proteins. This approach included only one pu-rified partner, nontagged FlgM, which was combined in solu-tion with whole-cell lysate of His6-FlhAC-expressing E. coli.This combination finally yielded a reproducible interaction be-tween FlgM and FlhAC in pull-down assays (Fig. 6). Hence,FlgM was suggested to be able to interact with FlhAC, eitherdirectly or indirectly.

As a further strategy to support our interaction results, weused a bacterial two-hybrid system (BACTH) in order to assessthe interaction potential of those two molecules in a nativephysiological environment inside the bacterial cell. Clones ex-pressing C-terminal fusion proteins between FlhAC (pCJ1001)or FlgM (pCJ1004) and the T25 or T18 domains of Bordetellapertussis adenylate cyclase and appropriate positive and nega-tive controls (see Materials and Methods; see also Fig. S3 inthe supplemental material) were cotransformed into theBACTH expression strain E. coli BTH101. Only bacteria co-transformed with both FlgM and FlhAC plasmids and the pos-itive control consistently gave rise to blue colonies on IPTG–X-Gal–containing plates (see Fig. S3 in the supplementalmaterial). We could detect expression of both fusion proteinsin Western blot assays by using anti-HPFlhA and anti-HPFlgMantisera (data not shown). In -galactosidase assays, negativecontrols and single FlgM-T18 transformants (in combinationwith empty pKNT25) did not produce activities above back-ground. The doubly flhAC-t25- and flgM-t18-transformed bac-teria consistently yielded values of at least 7% of the -galac-tosidase activities obtained from a positive control (Table 3).Taken together, these findings indicate that H. pylori FlgM andthe cytoplasmic domain of the flagellar basal body proteinFlhA (FlhAC) are able to interact with each other.

DISCUSSION

Although only a few eubacterial species possess flagellarsheaths (e.g., Vibrio cholerae, Bdellovibrio bacteriovorus, Rose-

FIG. 6. Recombinantly expressed H. pylori FlhAC pulls down puri-fied H. pylori FlgM. Results of a Western immunoblot analysis afterpull down of overexpressed H. pylori His6-FlhAC contained in E. colilysate, which was mixed with purified H. pylori FlgM are shown. West-ern blots were immunolabeled using anti-HPFlhA (1:2,000) and anti-HPFlgM (1:300) polyclonal antisera. Various samples of the coprecipi-tation procedure are depicted: lanes 1, 2, and 3 are from a controlexperiment containing FlhAC lysate only (“without FlgM”); lanes 4, 5,and 6 are from one representative pull-down experiment (out of threesimilar experiments) combining both FlhAC-containing lysate and pu-rified FlgM (“with FlgM”). Lanes 1 and 4, starting material of FlhAC-containing lysate, either combined (lane 4) or not (control; lane 1) withpurified FlgM; lanes 2 and 5, the eluted material from TALON resinafter pull down. Lanes 3 and 6, the last wash steps of the TALON resinprior to elution.



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buria cecicola), most Helicobacter species including H. pylori dopossess a flagellar sheath. This probably serves to protect theflagellar apparatus and filament from inactivation in the harshenvironment within the intestinal mucus of various animals,which is rich in proteolytic activity and mucosal antibodies andhas a low pH. Possession of a flagellar sheath is likely tohamper FlgM secretion, which was one initial motivation tostudy FlgM secretion and function in H. pylori.

The present investigations revealed that in H. pylori, FlgM isa predominantly soluble cytoplasmic protein. It is released intothe flagellar compartment in only low amounts and is notdetectable in cell supernatants during exponential growth. Thisresult could be a consequence of the shortened FlgM N ter-minus, which may have a lower propensity of being secreted.Detection of FlgM in cell supernatants in late exponentialphase may be due to bacterial lysis. It cannot be excluded atpresent that FlgM in H. pylori is released in larger amounts intothe periplasm, although several observations suggest this is notthe case. First, FlgM was found in the sheared-off flagellarfraction, presumably inside the flagellar channel or collectedwithin the sheath surrounding the filament. Mild osmolytictreatment of bacteria posterior to flagellar shearing did notrelease FlgM from the bacterial cells and did not reduce theamount of FlgM found in the bacteria-bound soluble fraction.This argues against FlgM accumulating in the periplasm. InSalmonella basal body and flk mutants, but notably not in thewild type, FlgM was present in the periplasm, where it washighly unstable (1). The possibility that FlgM is released intothe periplasm in H. pylori wild type and then quickly degradedmerits further investigation. Still, the fact that some FlgMcould be reproducibly detected in flagellar fractions in wild-type bacteria indicates that FlgM can stay stable upon its re-lease. In a control experiment where H. pylori supernatantswere coincubated with purified H. pylori FlgM, FlgM remainedstable at room temperature for more than 2 h (M. Rust and C.Josenhans, unpublished data). In contrast to our results in H.pylori, FlgM in Vibrio cholerae was reported to be secreted intothe medium, although the bacteria possess sheathed flagella(8). For that study, it was not discussed how FlgM secretionoutside of the sheath might take place.

Summarizing from our present data, FlgM is a predomi-nantly cell-bound protein in H. pylori and very likely also inother species of the Campylobacterales, which possess a shortFlgM. Our data imply that the H. pylori FlgM may exert its

regulatory functions predominantly without being secreted.This is in contrast to Salmonella flagella, where the function ofFlgM is tightly connected to its secretion into the environmentthrough the flagellar type III secretion system (20). Cytoplas-mic localization of the anti-sigma factor FlgM in H. pylori canbe partially explained by its interaction with the sigma factorFliA, when FliA is in its inhibited state, before late flagellargene transcription is initiated.

However, as soon as FliA is released and becomes func-tional, which has to occur at later stages of flagellar assembly,a different mechanistic explanation is required for the predom-inantly cytoplasmic localization of FlgM in H. pylori. We hy-pothesized that the recycling or shuttling of FlgM between anactive, FliA-bound state and an inactive noninhibitory state inH. pylori is mediated by a mechanism which differs from theloss of activity by abundant secretion. Several possibilities canbe envisaged, including FlgM being degraded by proteolysis orinteractions with different proteins when FlgM is not bound toFliA. Using assays to test for FlgM stability in lysed or nativebacteria when protein synthesis was inhibited, we did not findany evidence for FlgM being rapidly degraded (Rust and Jo-senhans, unpublished). For this reason, we focused on thepossibility that FlgM interacts with cytoplasmic proteins otherthan FliA for being functionally inactivated, and one putativecandidate was FlhAC. Previously, data on flagellar regulationin H. pylori wild type and various flagellar mutants (33) sug-gested that FlgM may act as an intergenic suppressor of theflagellar basal body protein gene flhA and exerts its function inclose cooperation with FlhA. In the present study, the intra-bacterial localization of FlgM was altered in the absence ofFlhA, providing further evidence for a close cooperation be-tween these proteins. Furthermore, we gathered evidence thatFlgM interacts directly or indirectly with FlhAC.

We therefore propose a model for H. pylori and closelyrelated species in which the anti-sigma factor FlgM is predom-inantly retained in the cytoplasm and can be shuttled betweenan active FliA-bound state and an inactive state. We proposethat FlgM may be bound to a different protein complex, afterbeing transferred from FliA to the flagellar basal body. Onebinding partner is suggested to be the flagellar basal proteinFlhA, specifically, FlhAC. Interestingly, axial export substratesof different substrate classes, including the late flagellar exportsubstrate flagellin (FliC), interacted directly with denaturedSalmonella FlhAC in a blot overlay system (31). So far, these

TABLE 3. -Galactosidase assays of transformants for BACTH system suggest interaction of FlgM and FlhACa

Sample (doubly transformed E. coli BH101)

-Galactosidase activity (% of control) ina:

Expt 1 Expt 2 Expt 3

MU % MU % MU %

pUT18/pKNT25 (negative control) ND ND 0 0 0 0pKNT25/pCJ1004 (FlgM-only control) �20 0 �1 0 ND NDpKT25-Zip/pUT18C-Zip (I) (positive control) 7,622 100 4,147 100 1,188 100pKT25-Zip/pUT18C-Zip(II) 7,178 94.2 3,881 93.6 ND NDpCJ1001/pCJ1004 (I) (FlhA� FlgM) 508 6.7 293 7.1 547 46pCJ1001/pCJ1004 (II) (FlhA � FlgM) 324 4.3 172 4.1 329 27.6pUT18/pCJ1001 (FlhA-only control) ND ND ND ND 48 4

a Activities in Miller units (MU) and percentage of positive control for BACTH cotransformants in E. coli BTH101 from three separate induction experiments aredepicted. One or two separate cultures of each transformation (induced from mid-log growth with 0.2 mM IPTG, 37°C for 3 h) were tested for each experiment in the-galactosidase activity assay. All presented values are mean values of duplicate measurements. ND, not determined.



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interactions have not been confirmed in a native system. In blotoverlays, we were not able to detect an interaction using over-expressed H. pylori FlhAC and purified FlgM. Optical biosen-sor assays, using one purified interaction partner randomlyfixed on a solid support, did not reveal interaction either,further hinting at specific structural requirements for the in-teraction. In particular the ability of FlgM to interact may beimpeded when FlgM is immobilized, since FlgM has a dynamicor disordered structure in solution which is required for itsfunction (9, 10). Three-dimensional structural modeling of H.pylori FlhAC (data not shown) and the substantial overall sec-ondary structure and amino acid similarity between H. pyloriFliA and FlhAC (11% identity, 30% similarity) (see Fig. S2 inthe supplemental material) suggest that FlgM could bind to�-helically folded FlhAC in a similar conformational arrange-ment as FliA (34, 42). This mode of binding would requireFlhAC to be in a native conformation.

FlgM in Salmonella is released from FliA (acting as its chap-erone) with the help of the flagellar ATPase FliI (32, 36), aprocess which would occur concomitantly with FlgM beingtransferred to the flagellar basal body complex containingFlhAC. While bound to FlhAC, H. pylori FlgM might be stabi-lized and retained in the bacterial cytoplasm, which couldenable a recycling and renewed binding to FliA at an appro-priate later time point. Direct (31) and indirect (32, 36) bindingof axial flagellar export substrates other than FlgM to FlhA hasbeen reported previously. It is unclear at this point whether thenature of FlgM binding to FlhAC in H. pylori is similar to thebinding of other flagellar export substrates to FlhAC, which hasbeen demonstrated using partially denatured proteins (31).Further investigations will also have to elucidate how the trans-fer of FlgM from FliA to the FlhA protein complex is occurringand if other binding partners are involved. It also remains to bedetermined whether the cytoplasmic shuttling process postu-lated here is sufficient for full FlgM function or whether FlgMlocalization within the flagellar compartment is required.

ACKNOWLEDGMENTS

We thank Shin Ichi Aizawa and Ikuro Kawagishi for helpful com-ments. We are very grateful to Daniela Fischer, Verena Ryan, andDaniela Goeppel for expert technical assistance.

Work in the laboratory of C.J. was funded by the German ResearchCouncil, grant JO344/2-2, and the Pathogenomics/ERAnet HELDIV-Net research networks by the German Ministry of Education andResearch. J.L.M. acknowledges support by PHS grant GM080701 fromthe National Institutes of Health and Cottrell College Science AwardCC6900 from the Research Corporation. The K.T.H. laboratory wassupported by PHS grant GM056141 from the NIH.

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