Journal of Bacteriology, March 2004, p . 1537-1545, Vol . 186, No . 5

Identification of a 349-Kilodalton Protein [Gli349] Responsible for Cytadherence and Glass Binding during Gliding of Mycoplasma mobile

Atsuko Uenoyama,¹ Akiko Kusumoto,^1, and Makoto Miyata^1,2*

Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585,¹ PRESTO, JST, Osaka, Japan²

Received 30 July 2003/ Accepted 5 November 2003

 
Several mycoplasma species are known to glide in the directionof the membrane protrusion [head-like structure], but the mechanism underlying this movement is entirely unknown . To identify proteins involved in the gliding mechanism, protein fractions of Mycoplasma mobile were analyzed for 10 gliding mutants isolated previously. One large protein [Gli349] was observed to be missing in a mutantm13 deficient in hemadsorption and glass binding . The predictedamino acid sequence indicated a 348,758-Da protein that wastruncated at amino acid residue 1257 in the mutant . Immunofluorescencemicroscopy with a monoclonal antibody showed that Gli349 islocalized at the head-like protrusion's base, which we designatedthe cell neck, and immunoelectron microscopy established thatthe Gli349 molecules are distributed all around this neck . Thenumber of Gli349 molecules on a cell was estimated by immunoblotanalysis to be 450 ± 200 . The antibody inhibited boththe hemadsorption and glass binding of M . mobile . When the antibodywas used to treat gliding mycoplasmas, the gliding speed andthe extent of glass binding were inhibited to similar extentsdepending on the concentration of the antibody . This suggestedthat the Gli349 molecule is involved not only in glass bindingfor gliding but also in movement . To explain the present results,a model for the mechanical cycle of gliding is discussed.

 
Mycoplasmas are parasitic, small-genome bacteria that lack a peptidoglycan layer [28] . Several mycoplasma species, includingMycoplasma pneumoniae, M . genitalium, M . pulmonis, M . gallisepticum,and M . mobile, have distinct cell polarity and exhibit glidingmotility, a smooth translocation of cells across solid surfacesin the direction of the tapered end [17, 22, 36] . The glidingmotility of mycoplasmas is believed to be involved in pathogenicity,but the mechanisms underlying gliding motility have not beeninvestigated well [19] . Mycoplasmas have no surface structures,such as flagella or pili, or any homologs of genes that encodesuch structures . Neither do they have genes related to otherbacterial motility systems or to eukaryotic motor proteins [5, 11, 13, 21] . These facts suggest that mycoplasmas glide by anentirely unknown mechanism.

M . mobile, isolated in the early 1980s from the gills of a freshwater fish, is the fastest-gliding mycoplasma [18] . M . mobile glideson glass in the direction of its tapered end, where its so-calledhead-like structure is . Its average speed is 2.0 to 4.5 µm/s,about 3 to 7 times its cell length per second [32], and itsmaximum force can reach as high as 27 pN [23] . It binds easilyto glass and glides smoothly without pausing regardless of itsgrowth stage . These distinct characteristics have allowed fordetailed analyses of its gliding [9, 23-25, 32, 33] as wellas for the isolation of gliding mutants, which are characterizedby reduced or deficient gliding or by enhanced speed [26] . However,no proteins related to gliding have been identified . In thisstudy, we identified a huge protein that is truncated in a nonadhesivemutant and that is responsible for hemadsorption and glass-bindingduring gliding.

 
Strains and culture conditions. M . mobile strain 163K [ATCC 43663] and its mutants [26] weregrown at 25°C in Aluotto medium, consisting of 2.1% heartinfusion broth, 0.56% yeast extract, 10% horse serum, 0.025%thallium acetate, and 0.005% ampicillin [1], to an optical densityat 600 nm of around 0.07, which corresponds to 7 x 10⁸ CFU/ml.

Triton X-100 extraction. The cultured cells were centrifuged at 12,000 x g for 10 min at 4°C and washed three times with phosphate-buffered saline[PBS] . The cells were suspended in 20 mM Tris-HCl [pH 7.5],0.15 M NaCl, and 0.1 mM phenylmethylsulfonyl fluoride and extractedwith 1% [vol/vol] Triton X-100 . After incubation at 37°Cfor 20 min, the suspension was centrifuged at 25,000 x g for 15 min at 4°C, and the Triton-insoluble fraction was recovered as a pellet . The insoluble fraction was analyzed by sodium dodecyl sulfate [SDS]-5, 10, and 15% polyacrylamide gel electrophoresis [PAGE] and stained with silver . The molecular mass was estimatedby a broad-range protein marker [New England BioLabs, Inc.,Beverly, Mass.].

Cloning and sequencing. The Triton-insoluble fraction obtained from a 150-ml culturewas subjected to SDS-5% PAGE and stained with Coomassie brilliantblue . The Gli349 protein bands were excised and equilibratedwith Tris-SDS buffer, consisting of 125 mM Tris-HCl [pH 6.8]and 10% SDS . The bands were put into three wells, each 7 mmwide, 1 mm thick, and 15 mm deep . The bands in each well wereoverlaid with 5 µl of a solution containing 2 µgof V8 protease . Gli349 was partially digested and separatedon a gel according to the Cleveland method [6], transferred to an Immobilon-PSQ membrane [Millipore, Inc., Billerica, Mass.],and stained with amido black . Edman degradation of 18- and 20-kDaprotein bands revealed N-terminal amino acid sequences of eightresidues, EITNLVQG and EVSDQNII, respectively . Four degenerateDNA sequences were designed from the amino acid sequences asoverlapping nested primers: M1-18-5F [5'-GANATHACNAAYYTNGTNC-3'],M1-18-5S [5'-HACNAAYYTNGTNCARGG-3'], M1-20-3F [5'-DATDATRTTYTGRTCNSWNA-3'], and M1-20-3S [5'-RTTYTGRTCNSWNACNTC-3'] . The primary PCR was performed with the primers M1-18-5F and M1-20-3F with chromosomalDNA obtained by the Genomic-tip system [Qiagen, Hilden, Germany]as a template . A 5-kb DNA fragment was amplified by the secondaryPCR with the primers M1-18-5S and M1-20-3S and sequenced byusing ABI PRISM 310 [Applied Biosystems, Foster City, Calif.].The regions outside the 5-kb fragment were sequenced directly,starting with primers inside the 5-kb fragment, by using thedirect genomic DNA sequencing protocol [Qiagen] . A region ofabout 30 kb was sequenced . The gli349 gene of mutant m13 wasamplified by PCR as described above and sequenced.

Immunoblotting analysis and estimation of the number of Gli349 molecules. Whole-cell lysate was fractionated by SDS-10% PAGE and subjectedto immunoblotting analysis . This analysis used a hybridoma mediumcontaining a mouse monoclonal antibody obtained by immunizing a BALB/c mouse with intact M . mobile cells [unpublished data]. The concentration of monoclonal antibody in the ascitic fluid was titrated by enzyme-linked immunosorbent assay [30] with a mouse immunoglobulin G1 [IgG1] antibody [Ancell, Bayport, Minn.] as the standard . The antibody bound to the target proteinon the membrane was detected by using anti-mouse IgG horseradish peroxidase conjugate [Promega, Madison, Wis.] and a chemiluminescence reaction [Western lightning; PerkinElmer Life Sciences Inc., Wellesley, Mass.].

The number of Gli349 molecules on a cell surface was estimated based on the amount of antibody needed to saturate its bindingsites on a cell . Various concentrations [0, 0.0008, 0.002, 0.008,0.02, 0.08, 0.2, 0.8, 2, 8, and 20 µg/ml] of the anti-Gli349antibody and of a mock mouse IgG1 antibody [Ancell] were incubatedwith mycoplasma cells at 5 x 10⁸ CFU/ml in 1 ml of Aluotto mediumat 4°C for 3 h . The cells bound with antibodies were collectedby centrifugation at 12,000 x g for 4 min at 4°C, washed three times with PBS, and subjected to SDS-PAGE with the bound antibodies . The antibody bound to cells was detected by immunoblotting on an X-ray film with the anti-mouse IgG antibody conjugated with horseradish peroxidase . The band intensity was measuredby NIH Image [version 1.61] from band images scanned by a transmission scanner [GT9800F; Epson, Nagano, Japan] . The amount of antibodywas calculated by using various amounts of the anti-Gli349 andthe mock antibodies applied to immunoblots as standards . Thelinear correlation was confirmed between the amount of standardantibody and its band intensity . Four measurements were takenfrom each independent culture, and the average was obtained.

Immunofluorescence microscopy. Cultured cells were collected by centrifugation at 12,000 x g for 4 min at 20°C and suspended in fresh Aluotto mediumat a 10-fold-higher concentration . Then, 200-µl suspensionsamples were put on coverslips and incubated at 25°C for1 h . After the medium was removed, the attached cells were fixedwith 3.0% [wt/vol] paraformaldehyde and 0.1% [vol/vol] glutaraldehydein PBS . The following procedure was done as described previously[34, 35], except that the cells were not treated with Triton X-100 or dried . The fixed cells were stained with 0.2 µgof monoclonal antibody/ml . The secondary antibody was a goatantibody labeled with Cy3 . The cells were observed with OlympusBX50 and IX71 microscopes and photographed with an ORCA-ER charge-coupleddevice [CCD] camera [Hamamatsu Photonics, Hamamatsu, Japan]attached to the IX71 microscope.

Immunoelectron microscopy. The cultured cells were collected by centrifugation and suspendedin fresh Aluotto medium at a 50-fold-higher concentration . Thecell suspension was placed on a collodion-coated nickel gridfor 10 min at room temperature to let mycoplasmas bind to thegrid . After removal of the medium, the cells were fixed with3.0% [wt/vol] paraformaldehyde and 0.1% [vol/vol] glutaraldehydein PBS for 10 min at room temperature and subsequently fixedfor 30 min at 4°C . The cells were washed three times with PBS and incubated for 1 h at room temperature with hybridoma medium diluted 1:10 with PBS and containing 0.2 µg ofanti-Gli349 antibody/ml . After three washes with PBS, labelingfor 1 h at room temperature was performed with an electron microscopygoat anti-mouse IgG [heavy plus light chains] conjugated to10-nm-diameter gold beads [BB International, Cardiff, UnitedKingdom] diluted 1:20 with PBS . The cells were washed threetimes with PBS, negatively stained with 2% ammonium molybdate[7], and observed with a transmission electron microscope ata tilt angle of 10°.

Hemadsorption assay. Colonies grown on 0.7% agar medium for 6 days were overlaidwith sheep red blood cells [RBCs] suspended in PBS, as previouslydescribed [26, 37], and observed under an inverted microscope.The antibody's effect on hemadsorption was tested by addingit to the RBC suspension before the suspension was overlaidon colonies . A monoclonal antibody against a 57.5-kDa cell surfaceprotein of M . mobile was used as a mock antibody . The colonieswere photographed with an Olympus CK2 inverted microscope equippedwith a Photometrics CoolSNAP CCD camera [Roper, Atlanta, Ga.].

Inhibition of gliding. Cultured cells were collected by centrifugation and suspendedin Aluotto medium at a 30-fold-higher concentration . The cellsuspension was poured into a tunnel [8-mm interior width, 26-mmlength, and 60-µm wall thickness] constructed by tapinga coverslip to a glass side . The glasses were cleaned with saturatedethanolic KOH [3] . The suspension was then incubated for 5 minat room temperature . The nonbinding cells were removed by aflow of 200 µl of Aluotto medium . The effects of the antibodyon the gliding M . mobile cells were observed after the Aluottomedium was replaced with medium containing various concentrationsof anti-Gli349 monoclonal antibody . The antibody against a 57.5-kDacell surface protein was used as a mock antibody . The cell movementswere observed under an Olympus BX50 microscope . The images wererecorded by a WV-BP510 CCD camera [Panasonic, Osaka, Japan]onto mini-DV tapes and were transferred to a personal computer via DV Raptor [Canopus Co., Kobe, Japan] . To make still images, the video footage was converted into sets of 20 frames for 2s by using Video Editor, version 2.0, editing software [UleadSystems, Inc., Taipei, Taiwan] . An average image was producedfrom each set of frames inverted and the last inverted framesubtracted with Scion Image PC, beta version 4.0.2, software[Scion Corp., Frederick, Md.] . To measure the number of boundcells and their gliding speeds, the video was converted intosets of 6 frames per 2 s by using Video Editor, version 6.0[Ulead Systems] . The number of cells bound to a glass surface65.3 µm wide by 48.9 µm long was counted . The initial number of cells was more than 150 . To measure gliding speeds, the mass center of cells gliding in the square was determinedby Scion Image, and their speeds were calculated from their displacements, as described by Miyata et al . [23] . Speed as reported here is the average speed of 10 gliding cells [or lower at the points where the log₁₀ bound cell ratio was less than -1.5 because there were fewer than 10 gliding cells in the field].

Nucleotide sequence accession numbers. The nucleotide sequences reported in this paper were depositedin the DDBJ, EMBL, and GenBank nucleotide sequence databasesunder accession no. AB074422 [wild-type] and AB074421 [m13].

 
Identification of protein missing in nongliding mutant. Ten gliding mutants were obtained previously by UV irradiation[26] . To identify the mutated genes, we examined the proteinprofiles of Triton X-100-insoluble fractions for the wild-typestrain and these gliding mutants . As the adhesion protein, P1,of M . pneumoniae is found in Triton-insoluble fractions [29],we focused on the Triton-insoluble fraction of M . mobile . M.mobile cells were treated with 1% Triton X-100 at 37°C for20 min followed by washing with PBS and were then divided intoTriton-insoluble and Triton-soluble fractions . The former wereanalyzed by SDS-PAGE at three different concentrations of polyacrylamidefor all strains, resulting in the detection of about 40 majorprotein bands . Only one band was determined to be missing ina mutant; the position of this band was extrapolated to an apparentmass of 240 kDa in the wild-type strain [Fig . 1A] . The mutantm13 is characterized by an inability to bind to glass and consequentlyby an inability to glide.

 
Cloning and sequencing of gli349 gene. Focused protein bands were collected from the Triton X-100-insolublefraction following separation by 5% gel electrophoresis . Thebands were then partially digested with V8 protease and fractionated electrophoretically according to a method previously reported[6] . The 8 N-terminal amino acid residues of two peptide digestswere sequenced by Edman degradation . DNA primers were designedfrom the amino acid sequences, and the DNA sequence of the geneencoding the missing protein was determined [Fig . 1B] . A long open reading frame [ORF] was found to encode a polypeptide of3,183 amino acids . The predicted molecular mass and isoelectricpoint of the protein, which we refer to as Gli349, were 349kDa and 4.9, respectively . Edman degradation analysis of theN terminus of the whole protein resulted in a sequence correspondingto the predicted N-terminal sequence of the ORF . The amino acidsequences deduced by the partial digests corresponded to thesequences of amino acids 1251 to 1258 and 2847 to 2854, respectively.These results shows that the ORF is at least translated fromamino acids 1 to 2854 and not processed in this region . Sequenceanalysis of the gene in mutant m13 revealed a nonsense mutationat amino acid residue 1257 [Fig. 1B] . A CAA codon in the wild-typestrain is mutated to a TAA nonsense codon in mutant m13 . Theprimary structure of the gene was significantly similar to thatof the ORF MYPU_2110 from the gliding mycoplasma M . pulmonis,which is a mouse pathogen [5] . The amino acid sequences weresimilar in all regions; 22% identity and 40% similarity werefound especially in the region expanding from amino acid residues623 to 2929 of the gli349 gene of M . mobile . Neither proteinsequence has any cysteine residue . Using SOSUI, an algorithmto predict a membrane-spanning segment, a transmembrane segmentpreceded by a positively charged region can be predicted nearthe N terminus for both sequences [14] . No significant similarityto other protein sequences was detected, and no motifs, suchas a P-loop involved in the motor proteins, were found.

Truncated gli349 gene product in mutant m13. The anti-Gli349 monoclonal antibody was obtained by screeninga hybridoma established after immunizing a BALB/c mouse withintact M . mobile cells . Cell lysates from the wild-type strainand mutant m13 were examined by immunoblot analysis with a hybridomamedium containing the anti-Gli349 monoclonal antibody [Fig.2] . The antibody recognized only the Gli349 protein band inthe whole-cell lysate of the wild-type strain . In mutant m13,a band was detected with an approximate mass of 137 kDa, whichcorresponds to the size of the truncated polypeptide predictedby the DNA sequence.

 
Subcellular localization and number of Gli349 molecules. The localization of Gli349 in the wild-type cells was examinedby immunofluorescence microscopy [Fig . 3, upper panel] . The results clearly showed that Gli349 is located at the head-like structure's basal position, which we call the neck . The localization of the truncated product in mutant m13 was examined by immunofluorescence microscopy [Fig . 3, lower panel], but no signal was found withthe same exposure time as that for the wild-type cells . A 10-times-longerexposure revealed the signal on the cell surface, but its positionwas not uniform, unlike the case for the wild-type cells . Necklocalization of wild-type Gli349 was confirmed by using electronmicroscopy to examine subcellular localization at a higher resolution[Fig . 4] . Three-dimensional immunoelectron microscopic imagesrevealed gold particles on all sides of a cell.

 
The number of Gli349 molecules on the cell surface was examinedbased on the amount of antibody needed to saturate its bindingsites on a cell . Various concentrations [0 to 20 µg/ml]of the antibody were incubated with mycoplasma cells at 5 x 10⁸ CFU/ml, and the amount of antibody bound to the cell surfacewas titrated by centrifugation and immunoblotting . The amount increased linearly with the antibody concentration used, and saturation occurred at the concentration of 0.8 µg/ml.The number of Gli349 molecules on the cell surface was estimatedto be 445 ± 197 per cell . The mock antibody used as acontrol did not bind to the cells.

To learn Gli349's association with the structures included inthe Triton-insoluble fraction, we examined the existence ofthis protein in the extract and in the insoluble fraction . Sixtypercent of the Gli349 was found in the soluble fraction, andthe remainder was found in the insoluble fraction, which wasslightly solubilized after two additional extractions.

Inhibition of hemadsorption by antibody. The mutant m13 is deficient in both the glass binding and staticbinding activities of animal cells, as estimated by the adsorptionof RBCs to colonies formed on an agar plate [hemadsorption activity][26] . Therefore, Gli349 protein may be involved in both activities.To examine the possible involvement of this protein in staticbinding, the effect of the antibody on hemadsorption activitywas examined [Fig . 5] . RBC suspension with the addition of 0, 0.1, or 1 µg of anti-Gli349 antibody/ml was overlaid ontomycoplasma colonies . When no antibody was added, the coloniesbound to RBCs across the entire area . The addition of 1 µgof anti-Gli349 antibody/ml inhibited the binding, whereas thesame concentration of antibody against another surface proteinshowed no effect . The addition of 0.1 µg of anti-Gli349antibody/ml resulted in no significant reduction in hemadsorptionactivity.

 
Inhibition of gliding by antibody. The effects of the antibody on gliding were examined [Fig . 6].Mycoplasma cells suspended in a fresh medium without antibodywere inserted into a tunnel slide, and the medium was replacedby that containing various concentrations of the antibody, rangingfrom 0 to 40 µg/ml . Gliding cells were then observed continuously.When the medium had a 40-µg/ml concentration of antibody,all mycoplasmas lost their binding to glass, resulting in Brownianmotion, whereas the same concentration of an antibody againstanother surface protein showed no effects [Fig . 6A] . Detailedanalyses showed that, with time, the anti-Gli349 antibody reducedboth glass binding and gliding speed, although both were affectedslightly by the addition of a mock antibody [Fig . 6B and C].The inhibition rates were estimated from the time required toreach 0.1 and 0.5 of the start values for glass binding andgliding speed, respectively . The rates were similar betweenglass binding and gliding speed at a wide range of antibodyconcentrations [Fig . 6D].

 
We fractionated M . mobile proteins by Triton X-100 extraction and looked for differences in protein contents between the wild-type strain and the mutants . Only in the Triton-insoluble fractionwas Gli349 obviously missing from the mutant m13, which lacksboth hemadsorption and glass binding activities [Fig . 1] [26]. This suggested that Gli349 is responsible for hemadsorptionand glass binding in gliding . This inference was proven by thespecific inhibitory effects of an anti-Gli349 antibody [Fig.5 and 6A].

The ORF for the protein truncated in the mutant m13 encodeda 349-kDa protein . The ORF contained some TGA triplets generallyused as stop codons, suggesting that the TGA codon of M . mobile encodes tryptophan, as observed with other mycoplasmas [39]. A homologous gene was found in M . pulmonis but not in the recently determined genome of Mycoplasma hyopneumoniae, [C . Minion, personal communication], although this species is believed to be more closely related to M . mobile [38] . This might be consistentwith the fact that neither gliding motility nor a tapered cellpole has been reported for M . hyopneumoniae thus far . Severalcytadherence proteins have been identified from M . pneumoniae[2, 8, 16] and some other mycoplasma species [4, 10, 12, 15, 40], but none have shown any similarities to Gli349 in size or in primary structure . This suggests that adhesion proteins are diversified among mycoplasma species.

In immunoblotting, the monoclonal antibody recognized the Gli349 protein band for the wild-type strain and a 137-kDa band formutant m13 [Fig . 2] . The 137-kDa band is presumably a peptide of the N-terminal region and is truncated at the mutated site because the molecular mass estimated from the electrophoresis corresponded to the predicted molecular mass obtained from the sequence . Evidently, the epitope for the monoclonal antibodyresides in this fragment . The existence of Gli349 in the othernine gliding mutants, including six strains with deficient orreduced glass binding activity [26], was examined by immunoblotting, but none showed significant differences from the wild-type strain. This suggests that additional proteins are involved in glass binding [data not shown].

Immunofluorescence and immunoelectron microscopy revealed that Gli349 molecules are localized to the neck of a cell [Fig . 3 and 4] . Based on the results of previous studies, we expectedthat the gliding proteins would be located at the distal end of the head-like structure because gliding cells that each carried a bead attached to its tail were reoriented to glide upstream by the flow of fluid to the tail [23] and because an elongatedhead-like structure found in a very old culture moved forward,sometimes leaving the cell body in one position, resulting ina stretched head-like structure [25] . The localization of Gli349at the cell neck is consistent with these previous observations.The three-dimensional immunoelectron microscopic images revealedthat Gli349 molecules are distributed around the cell neck,indicating that Gli349 molecules on the cell surface exist alsoon sides other than that facing the glass surface.

The truncated protein in m13 mutant cells appeared to be present in reduced amounts and was randomly localized on the cell surface [Fig . 3, lower panel] . As permeabilization of cells in the stainingprocedure by a previously described method [34, 35] did notincrease the signal intensity [data not shown], it is unlikelythat the major part of the truncated protein exists inside thecell . We failed to detect the truncated molecule by immunoblotanalysis in the supernatant of m13 culture [data not shown].These facts suggest that the truncated molecules were exportedfrom inside the cell and anchored to the cell membrane but werenot localized normally . Possibly, the physical interaction withother structures involving the truncated region is essential for the normal localization of the Gli349 molecule on a cell.

The maximum force generated in gliding was previously measuredas 27 pN [23] . Considering this rather large force and the possibleparticipation of this protein in the gliding mechanism as explainedbelow, the Gli349 molecule should be supported by a robust structure.Such a structure may be included in the Triton-insoluble fraction,in which 40% of Gli349 molecules were detected . One transmembranestretch preceded by a cluster of positively charged residueswas predicted at the N terminus of Gli349 [14] . A monoclonalantibody raised against the cell surface affected hemadsorptionand glass binding from the outside [Fig . 5 and 6] . These observationssuggest that Gli349 is a membrane-anchored protein, a largepart of which is outside the cell, and also that the moleculeis supported by the robust structure . Gli349 shares this featurewith the cytadherence-related proteins of M . pneumoniae, linkedto a Triton-insoluble structure, called the Triton shell [27, 29], which is thought to involve cytoskeletal structures, i.e.,a filamentous network and a rod structure [20].

The anti-Gli349 antibody inhibited hemadsorption [Fig . 5], andit removed the gliding mycoplasmas from the glass in a concentration-dependentmanner [Fig . 6B and D] . The monoclonal antibody against a 57.5-kDaprotein had no effect on either activity, although it also recognizedthe protein from the outside . Two other antibodies that recognizedthe surface proteins showed effects on neither gliding nor glassbinding [data not shown] . These observations show that Gli349is responsible for both static binding of animal cells and glassbinding essential for gliding . This is consistent with the previousobservations that the gliding mutants showed similar extentsof reduction in both activities, suggesting that these two activitiesshare the same machinery, in M . mobile [26].

The addition of antibody against Gli349 reduced gliding speedwith time in a concentration-dependent manner, in a way similarto that for glass binding [Fig . 6B, C, and D] . As the antibody ultimately removed the gliding mycoplasmas from the glass surface, the binding of the antibody should release Gli349 molecules from the glass surface . Then how did the antibody reduce thegliding speed? This can be explained by an assumption that thegliding mechanism involves the movement of the Gli349 molecule,which can be inhibited by the antibody . One possible model ispresented in Fig. 7, where the cell propels itself by repeatinga cycle of binding the Gli349 protein as follows: [i] bindingof Gli349 to glass, [ii] transfer of generated force to Gli349,[iii] stroke of Gli349, [iv] release of Gli349 from glass, and[v] return of Gli349 to its original form . Here, we know neitherthe actual molecular shape nor the manner of movement . If weassume that the step i is inhibited by the antibody, the reductionof speed should be caused by the lack of force needed to thrusta cell, which resulted from a decrease in the number of workingmolecules . However, the actual maximal force of gliding, 27pN, is 1,800 times larger than 15 fN, the force required tothrust an unbound M . mobile cell in a medium [23, 24, 31] . Therefore,it is unlikely that step i is inhibited by the antibody . Ifwe assume that step ii is inhibited by the antibody, the Gli349 molecule would generate drag force, thereby reducing speed.In this case, the ultimate release of cells from glass can occurthrough a backward transition in step i.

 
We thank Howard C . Berg of Harvard University for comments onthe manuscript and Shintaro Seto of Osaka City University forhelpful discussions.

This work was supported in part by grants-in-aid for scientific research [C] and for science research on priority areas [motor proteins, genome science, and infection and host response] fromthe Ministry of Education, Science, Sports, Culture, and Technologyof Japan to M.M.

 
* Corresponding author . Mailing address: Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan . Phone: 81 [6] 6605 3157 . Fax: 81 [6] 6605 3158 . E-mail: miyata@sci.osaka-cu.ac.jp.

Present address: Division of Biological Science, Graduate Schoolof Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602,Japan.

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