Molecular Structure of Isolated MvspI, a Variable Surface Protein ofthe Fish Pathogen Mycoplasma mobile

Jun Adan-Kubo,a Shu-hei Yoshii,a Hidetoshi Kono,b and Makoto Miyataa

Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka, Japan,a and Molecular Modeling and Simulation, Quantum Beam Science,Japan Atomic Energy Agency, Kizugawa, Kyoto, Japanb

Mycoplasma mobile is a parasitic bacterium that causes necrosis in the gills of freshwater fishes. This study examines the molec-ular structure of its variable surface protein, MvspI, whose open reading frame encodes 2,002 amino acids. MvspI was isolatedfrom mycoplasma cells by a biochemical procedure to 92% homogeneity. Gel filtration and analytical ultracentrifugation sug-gested that this protein is a cylinder-shaped monomer with axes of 66 and 2.7 nm. Rotary shadowing transmission electron mi-croscopy of MvspI showed that the molecule is composed of two rods 30 and 45 nm long; the latter rod occasionally features abulge. Immuno-electron microscopy and epitope mapping showed that the bulge end of the molecular image corresponds to theC terminus of the amino acid sequence. Partial digestion by various proteases suggested that the N-terminal part, comprised of697 amino acids, is flexible. Analysis of the predicted amino acid sequence showed that the molecule features a lipoprotein and16 repeats of about 90 residues; 15 positions exist between residues 88 and 1479, and the other position is between residues 1725and 1807. The amino acid sequence of MvspI was mapped onto a molecular image obtained by electron microscopy. The presentstudy is the first to elucidate the molecular shape of a variable surface protein of mycoplasma.

Mycoplasmas are commensal and occasionally parasitic bac-teria with small genomes and no peptidoglycan layer (30).

They bind to host tissues via adhesion proteins. Some species at-tach to solid surfaces through a membrane protrusion and glide bya unique mechanism which is thought to be involved in parasitism(11, 12, 19–22, 30). In addition to this adhesion and gliding activ-ity, mycoplasmas have various systems for surface variation toevade host immune systems, allowing for the frequent modifica-tion of the expression and structures of surface proteins (7,39–42).

Mycoplasma mobile, which causes necrosis in the gill organ offreshwater fishes, glides on solid surfaces at a rate of 2.0 to 4.5 �mper second, making it the fastest-moving mycoplasma species re-ported so far (23, 24, 31). M. mobile expresses mobile variablesurface proteins (Mvsps), which are encoded by 16 genes, mvspAto mvspP, on its genome (10, 13). Eleven genes, mvspB to mvspL,are clustered tightly on the genome, from nucleotide (nt) 398037to 430685 nt, with few intervening genes. Another small cluster,mvspM to mvspP, is located from nt 746365 to 777079. mvspA islocated by itself from nt 128047 to 129,525.

These proteins have been suggested to be involved in surfacevariation represented by phase and antigenic variations, for threereasons: (i) the sequences of all Mvsps except MvspG are sug-gested to have transmembrane segments or a lipid anchor at the Nterminus; (ii) when mice were immunized by intact M. mobilecells, antibodies against Mvsps were produced preferentially; (iii)Mvsps other than MvspG contain repeat sequences. These prop-erties are common to proteins for surface variation of mycoplas-mas: Vsa of Mycoplasma pulmonis, Vlp of Mycoplasma hyorhinis,Vsp of Mycoplasma bovis, and so on (5–7). Recently, Wu et al.showed that MvspI on cells drastically decreases in a reversibleway, responding to addition of an anti-MvspI antibody, and theysuggested a novel mechanism of surface variation, designated“mycoplasmal antigen modulation” (41). The flask-shaped M.mobile cell can be divided into three parts—the head, neck, andbody from the pole of membrane protrusion— based on the loca-

tions of surface proteins (13, 36). Interestingly, the localizations ofat least four Mvsps are restricted to those parts on the cell surfacewhere MvspI, MvspN and MvspO, and MvspK are localized at thehead and body, head, and body, respectively (13).

The surface variations of mycoplasmas have been analyzedmainly for expression dynamism, antibody reactivity, and caus-ative DNA changes, including deletion, insertion, and inversion,altering on/off switching. However, although changes in the anti-genicity of variable surface proteins should depend on theseshapes, the molecular shapes of mycoplasmas have not beenstudied.

Here, we focused on the molecular shape of MvspI, which witha mature form of 218 kDa is the largest Mvsp. In our previousstudies, isolated Gli349 (349 kDa) and Gli521 (521 kDa) proteinswere visualized by rotary-shadowing electron microscopy (EM),which is suitable for visualizing protein molecules whose molec-ular masses are larger than 100 kDa (1, 18, 29). In the presentstudy, we isolated MvspI protein and analyzed its molecular shapeby hydrodynamics and rotary-shadowing EM and by determiningthe domain structure and amino acid sequence.

MATERIALS AND METHODS

Strains and culture conditions. M. mobile strain 163K (ATCC 43663) wasgrown at 25°C in Aluotto medium, consisting of 2.1% heart infusionbroth, 0.56% yeast extract, 10% horse serum, 0.0025% thallium acetate,and 0.005% ampicillin, to an optical density of around 0.1 at 600 nm(3, 25).

Received 11 February 2012 Accepted 12 March 2012

Published ahead of print 23 March 2012

Address correspondence to Makoto Miyata, miyata@sci.osaka-cu.ac.jp.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.00208-12

3050 jb.asm.org Journal of Bacteriology p. 3050–3057 June 2012 Volume 194 Number 12

Purification of MvspI. We modified the Gli349 purification proce-dure to fit MvspI isolation (1, 29). All procedures were done on ice exceptthe gel filtration, which was performed at room temperature (RT). Cellsfrom 1 liter of culture were collected by centrifugation at 14,000 � g for 10min and washed twice with phosphate-buffered saline (PBS) consisting of75 mM Na-phosphate (pH 7.3) and 68 mM NaCl. The cells were sus-pended to an optical density of 20 at 600 nm in 10 mM Tris-HCl (pH8.0)– 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and then weremixed with Triton X-100 to 0.5% (vol/vol). After gentle shaking for 1 h,the suspension was centrifuged at 450,000 � g for 30 min (step i). Thesupernatant was fractionated by stepwise salting out with ammoniumsulfate of 35% and 40% saturations. The insoluble fractions of 40% satu-ration were recovered by centrifugation at 22,000 � g for 15 min (step ii).The recovered fraction was dissolved and dialyzed overnight by 10 mM2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.9). The insolublefraction caused by this pH shift was removed by centrifugation at22,000 � g for 15 min (step iii). The soluble fraction was applied to aHiLoad 16/60 Superdex 200 prep grade column set on an AKTA prime orAKTA purifier (GE Healthcare, Milwaukee, WI) and eluted with a bufferconsisting of 0.2 M NaCl, 0.1% Triton X-100, and 10 mM Tris-HCl, pH8.0, with a flow rate of 1 ml/min at RT. The sample elution was monitoredby absorbance at 280 nm and analyzed by sodium dodecyl sulfate-polyac-rylamide gel electrophoresis (SDS-PAGE) (step iv). The homogeneity ofprotein fractions was estimated by densitometry of Coomassie brilliantblue (CBB)-stained SDS-PAGE gels with the use of a GT-9800F scanner(Epson, Nagano, Japan) and analysis software (ImageJ, version 1.44p;NIH). MvspI fractions were pooled and concentrated using a Biomax-10instrument (Millipore, Bedford, MA) to 1 to 3 mg/ml, followed by re-moval of Triton X-100 by Carbiosorb adsorbent (Calbiochem, Darm-stadt, Germany).

Gel filtration and analytical centrifugation. To analyze the associat-ing properties, MvspI at a concentration of 1 mg/ml was applied to a gelfiltration column (TSK G5000PWXL gel set; Tosoh, Tokyo, Japan),equipped with a series 1200 high-performance liquid chromatograph(Agilent Technologies, Palo Alto, CA) and was eluted with 0.2 M NaCl,0.1% Triton X-100, and 10 mM Tris-HCl, pH 8.0, at a flow rate of 0.5ml/min at 20°C, with the absorbance monitored at 280 nm (29). Thyro-globulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), and albumin(68 kDa) (with each mass shown in parentheses) were used as the stan-dards for Stoke’s radii of 8.5, 6.1, 4.8, and 3.6 nm, respectively (9). Weexamined the effect of Triton X-100 on the result of gel filtration andfound that Triton X-100 does not influence the behavior of isolatedMvspI. The analytical ultracentrifugation was performed by an XL-I an-alytical ultracentrifuge equipped with an An-60Ti rotor (Beckman-Coulter Inc., Fullerton, CA). MvspI at 0.75 �M (0.17 mg/ml) in the PBSwas centrifuged at 42,000 rpm at 20°C for 360 min and scanned for theabsorbance at 220 nm with 210-s intervals. The sediment coefficient[C(s)] distribution was analyzed by SEDFIT, version 12.2 (33), and thedimensions of the molecule were calculated by sednterp, version 1.09,assuming that the hydration of the molecule is 0.3 g/g.

Rotary-shadowing electron microscopy. MvspI at 20 to 200 �g/ml in33% (vol/vol) glycerol and 0.3 M ammonium acetate was sprayed to afreshly cleaved mica surface. The following procedure was performed asdescribed previously (1, 4, 29). For the analysis of MvspI decorated by ananti-MvspI monoclonal antibody (MAb14), the antibody was purifiedfrom ascites fluid by using a HiTrap Protein G HP column (GE Health-care, Milwaukee, WI) (13). MvspI and MAb14 were mixed to 0.2 and 0.6mg/ml, respectively, incubated for 1 h at 4°C, and sprayed onto a micasurface. Each particle image was picked up by EMAN, version 1.6 (http://ncmi.bcm.tmc.edu/�stevel/EMAN/doc/) (16). The lengths, angles, andprofiles of molecular images were analyzed by ImageJ, and image averag-ing was performed using Adobe Photoshop, version 7.0.1 (Adobe, SanJose, CA).

Digestion and mapping of MvspI. For epitope mapping of MAb14,purified MvspI was digested with trypsin or V8 protease at a 1/25 (wt/wt)

ratio to MvspI at 37°C for 5 h. For the domain analysis of MvspI, purifiedMvspI was digested with proteinase K (ratio of 1/100, 1/50, 1/10, and 1/5[wt/wt] to MvspI), chymotrypsin (1/100, 1/50, 1/25, and 1/10 [wt/wt] toMvspI), or trypsin (1/25 [wt/wt] to MvspI) at 37°C for 30 min. The digestswere analyzed by SDS-PAGE, Western blotting (WB), and peptide massfingerprinting (PMF), as reported previously (27–29, 43). Briefly, in PMF,protein bands in SDS-PAGE are digested by a residue-specific protease,for example, trypsin. The resulting peptide mixture is extracted and sub-jected to mass spectrometry. The combination of mass measurements ofmany peptides and the genome database allows us to identify the corre-sponding open reading frame (ORF) for each protein band. In the casethat the peptide band of SDS-PAGE is part of the protein, we can identifythe corresponding position in the whole sequence from the covering re-gions of peptides detected by the mass spectrometry.

RESULTSIsolation of MvspI. The MvspI protein was isolated using thefour-step procedure as presented in Fig. 1. In step i, MvspI wasextracted with 0.5% Triton X-100 treatment, and most of it wasrecovered as a soluble fraction, as shown in lanes 1 to 3 in Fig. 1. Instep ii, the MvspI fraction was applied to stepwise ammoniumsulfate fractionation. The MvspI protein was recovered between35% and 40% saturations as shown in lane 4 in Fig. 1. In step iii,the ammonium sulfate precipitate was dissolved and dialyzedagainst the gel filtration buffer. No obvious precipitation wasfound in this dialysis. In step iv, the MvspI protein was purifiedthrough gel filtration column chromatography in the presence of0.1% Triton X-100 and used in the following experiments. TheMvspI sample did not show any other major bands in the gelimage of CBB-stained SDS-PAGE, as shown in lane 6, with 92.0%homogeneity, based on densitometry. The major protein bandwas confirmed to be MvspI by WB and PMF. Finally, about 100 �gof MvspI was obtained from 1 liter of mycoplasma culture.

Molecular mass and dimensions shown by hydrodynamics.The isolated MvspI protein was applied to a gel filtration assay

FIG 1 Protein profile of each fraction in MvspI isolation procedure. MvspIprotein was purified through four steps as described in the text. Lane 1, whole-cell lysate; lane 2, Triton-insoluble fraction of step i; lane 3, Triton-solublefraction of step i; lane 4, precipitate of 40% saturated ammonium sulfate instep ii; lane 5, supernatant of step iii; lane 6, fraction obtained after step iv. Eachfraction was subjected to SDS–10% PAGE with a 5-mm lane width and stainedby the CBB staining method. Protein fractions derived from 1- and 3-ml cul-tures were applied to lanes 1 to 5 and 6, respectively. Molecular masses areindicated on the left. The solid triangle indicates the protein band of MvspI.

Structure of a Variable Surface Protein of Mycoplasma

June 2012 Volume 194 Number 12 jb.asm.org 3051

(Fig. 2A). The elution pattern showed a single peak with an esti-mated Stoke’s radius of 8.6 nm. Next, the protein was analyzed byanalytical ultracentrifugation (Fig. 2B). The 94.6% protein sedi-mented with a sedimentation constant of 5.2 S20.w (sedimentationcoefficient standardized to 20°C in water) with a Stoke’s radius of9.7 nm, which is consistent with the result from the gel filtration.Based on the results of centrifugation, the mass of MvspI wascalculated as 200 kDa, corresponding to the 218 kDa which waspredicted from the mature amino acid sequence. Therefore, weconcluded that MvspI behaves as a monomer in water. The mo-lecular dimensions were estimated as a length of 66 nm and adiameter of 2.7 nm, assuming that the molecule is a cylinder ratherthan an ellipsoid.

Shape and dimensions of MvspI revealed by electron micros-copy. The purified MvspI protein was rotary shadowed and ob-served by EM (Fig. 3A). Gooseneck particle images were foundbasically in all fields of a grid, and the frequency of their appear-ance changed in correspondence with the MvspI concentrationsin the specimen. These facts suggest that the gooseneck particlesare MvspI molecules. Some of the images featured a bulge at oneend and a bend around the center. We picked 845 isolated imagesand examined their features. The frequencies of images with andwithout a bulge were 15.4% and 84.6%, respectively, and the fre-quencies of those with and without a bend were 45.5% and 54.5%,respectively. The images were classified into four types, bulge andbend (bulge-bend), no bend (bulge-none), no bulge (none-bend),and no bend or bulge (none-none), with frequencies of 5.0%,10.4%, 40.5%, and 44.1%, respectively (Fig. 3). Next, we mea-sured the total length, bend position, and bulge size as shown inFig. 3B. The averages of total length were 66.4 � 6.2, 68.8 � 7.8,76.4 � 8.7, and 77.0 � 9.5 nm for bulge-bend, bulge-none, none-bend, and none-none images, respectively. The total lengthsshown in the images of the bulge-bend and bulge-none types wereshorter than those of none-bend and none-none types by 10.0 and8.2 nm, respectively, suggesting that these differences are causedby the alignment of the bulge part relative to the rod, as shownschematically in Fig. 3B (see also Fig. 7A). These molecular lengthsare consistent with the estimation of a cylinder 66 nm long and 2.7nm thick, based on the analytical centrifugation (Fig. 2B).

The average width of the bulge was 25.0 nm, as shown in Fig.3B. The bend positions measured from the closer end were 28.8nm and 31.5 nm, on average, for the bulge-bend and none-bend

types, respectively. These observations suggest that the moleculeshave a bend at the corresponding positions in both the bulge-bendand none-bend types.

Binding positions of MAb14 antibody on MvspI amino acidsequence and molecular image. To determine the orientation ofthe amino acid sequence on the molecular shape, we examined thebinding positions of a previously isolated anti-MvspI monoclonalantibody, MAb14, on the amino acid sequence and EM images(13). Purified MvspI was partially digested by trypsin and V8 pro-tease, applied to SDS-PAGE gels, and analyzed by CBB stainingand WB (Fig. 4A). Protein bands ii and iii (Fig. 4A), with apparentmasses of 31 kDa and 43 kDa, were reactive to MAb14, whilebands i and iv of 43 kDa and 30 kDa were not. PMF showed thatthe reactive bands, ii and iii, cover the regions of amino acids 1679to 1985 and 1635 to 1985, respectively, while the nonreactivebands, i and iv, cover the region of amino acids 62 to 429 and bothamino acids 62 to 303 and 1724 to 1985, respectively, showing thatband iv was a mixture of two peptides (Fig. 4A, right). The pep-tides detected in the PMF covered 19.1%, 38.2%, 28.9%, and28.0% lengths of each region for bands i to iv, respectively. Theoverlapping region among the reactive bands, except the regionsof nonreactive protein bands, was the region of amino acids 1679to 1723, located at the 0.85 position to the whole sequence lengthfrom the N terminus. Therefore, we concluded that the epitope ofMAb14 is at this position.

Next, MAb14 was mixed with MvspI at twice the molar ratio,rotary shadowed, and observed by EM (Fig. 4B). MvspI moleculeswere found to be decorated by a globular particle of about 17.5 �2.5 nm (n � 35) in diameter, presumably MAb14, at one end ofthe molecular image, with a frequency of one per five particleimages. The molecules bound by the antibody did not appear tohave a bulge. This observation can be explained by either of thefollowing possibilities: the binding of the antibody inhibits thebulge formation or the antibody hides the bulge, with its bindingto the bulge end. Considering that the epitope of MAb14 lies at0.85 from the N terminus of the total amino acid sequence, weconcluded that the bulge end should be the C terminus.

Regions sensitive to protease in the MvspI molecule. To char-acterize the domain structures, the purified MvspI was partiallydigested with various amounts of proteinase K, chymotrypsin,and trypsin and then analyzed by WB using MAb14 (Fig. 5). Mostof the major products were reactive to MAb14 (marked as bands v

FIG 2 Hydrodynamic analyses of MvspI. (A) Gel filtration assay. The isolated MvspI was applied to gel filtration at a flow rate of 0.5 ml/min and monitored byabsorbance. Standard proteins were analyzed in the same way, and their peak positions were plotted against their known Stoke’s radii, 8.5, 6.1, 4.8, and 3.6 nm.The Stoke’s radius of MvspI was estimated to be 8.6 nm, as indicated by an open circle. (B) Analytical ultracentrifugation. The isolated MvspI (left) was appliedto the centrifugation and scanned for absorbance with 3.5-min intervals. Nine representative traces with 36-min intervals are shown. The x axis shows theposition from the rotational center. The right panel shows the C(s) distribution of S20.w.

Adan-Kubo et al.

3052 jb.asm.org Journal of Bacteriology

to ix). The masses of these bands were 144, 149, 141, 144, and 135kDa, respectively, for bands v to ix. These protein bands weresubjected to PMF and shown to cover at least the regions of aminoacids 614 to 1985, 422 to 1985, 614 to 1985, 614 to 1985, 614 to1985, and 698 to 1985, respectively, with length coverages of23.3%, 14.7%, 20.9%, 24.7%, 25.7%, and 51.7% (Fig. 5B). Majorpeptide bands smaller than 135 kDa were not produced during thedigestion of the N-terminal region. This shows that the N-termi-nal region of amino acids 28 to 613, including repeats a to g (Fig.5B), was digested into small pieces, suggesting that this region issensitive to proteases, probably because of its flexibility.

Sequence analysis of MvspI. We analyzed the amino acid se-quence of Mvsps to infer the topology of the molecule on the cellmembrane using SMART (15) and DOLOP (17) (Fig. 6). MvspIwas predicted to be cleaved at the C-terminal side of the 27thalanine with the modification of the following cysteine by diacyl-glycerol for lipid anchoring. MvspA, -H, -J, -K, -L, and -P werealso predicted to be processed and modified in similar ways. OtherMvsps except MvspG were predicted to have a transmembranesegment at the N terminus. Mvsp proteins are also known to befeatured with sequence repeats of about 90 amino acids, which arecommon in most Mvsp proteins (10). To list all repeats in Mvsp

FIG 3 Rotary-shadowed EM images of MvspI molecules. (A) At left is an image of a 775-nm-square field. Scale bar, 200 nm. At right four images of bulge-bendand bulge-none types and nine images of none-bend and none-none types are presented in a circle of 130-nm diameter with a schematic shown in the left-mostpanel. (B) The total length (T) and bend positions (B). The averages of bend positions from the closer rod end were 28.8 and 31.5 nm for images of the bulge-bendand none-bend types, respectively, as indicated by open triangles. The averages of total length for each type of molecule were 66.4 � 6.2, 68.8 � 7.8, 76.4 � 8.7,and 77.0 � 9.5 nm for bulge-bend, bulge-none, none-bend, and none-none types, respectively, as indicated by filled triangles. The right-most panel shows thedistribution of bulge width in the bulge-bend and bulge-none types. The average bulge length was 25.0 nm, as indicated by a gray triangle.

Structure of a Variable Surface Protein of Mycoplasma

June 2012 Volume 194 Number 12 jb.asm.org 3053

proteins, we analyzed the sequences of MvspA to MvspP, using theHMMER package (http://hmmer. janelia.org/) based on a hiddenMarkov model (8) in the same way that we did for Gli349 sequenceanalysis (18), and identified 52 repeats of about 90 amino acids,ranging from 72 to 105 amino acids whose E-values were less than0.001 (Table 1 and Fig. 6). The sequence of MvspI had 16 repeats,which were clustered mostly on the N-terminal side, as shown inTable 1 and Fig. 5 and 6. The N-terminal ends of protease-resistantregions exist in repeats d, f, and g (Fig. 5B).

DISCUSSIONIntegrated molecular structure of MvspI. We produced an imageof an MvspI molecule (Fig. 7A) by integrating the EM images andthe sequence analysis. The molecule is a 77-nm rod with a bend atthe position two-fifths from the N terminus and the occasionalbulge at the C terminus. A plausible lipid anchor was suggested atthe N terminus. The 16 repeat sequences, a to p, of about 90 aminoacids exist mainly at the N-terminal side, and the region of repeatsa to g is more flexible than the other parts.

The amino acid sequence can be mapped onto the molecular

image, based on their features and the orientation of amino acidsequence, as shown in Fig. 7. The flexible repeats, a to g, are as-signed to the 30-nm rod at the other side of bulge, and the solidrepeats, h to p, are assigned to the 38-nm rod at the bulge side. Thesegments of 246 and 195 amino acid residues on each side ofrepeat p are assigned to the bulge. This assumption is consistentwith the fact that the bulge appears to be composed of two smallglobules (Fig. 3A).

We carried out a PSI-BLAST search (2) to find a known three-dimensional (3D) structure whose sequence is homologous toMvsps and to obtain more concrete images of repeats. We re-peated the PSI-BLAST search six times until we found no new hitswith the default parameters and thus obtained the position-spe-cific site matrix for Mvsps. Unfortunately, with the use of thematrix, there was no hit against the sequences in the Protein DataBank, where known 3D structures of proteins and nucleic acidshave been deposited.

The region including repeats a to g was sensitive to protease,while the other repeats were not (Fig. 5B). To find out the possiblecauses of this sensitivity in the amino acid sequence, we carried

FIG 4 Binding positions of MAb14 antibody on MvspI amino acid sequence and rotary-shadowed molecular image. (A) Trypsin and V8 protease were addedto purified MvspI at the mass ratio of 1 to 25 and incubated at 37°C for 2 and 5 h for trypsin and V8 protease, respectively (left). Each product was subjected toSDS–12.5% PAGE followed by WB or CBB staining. Filled and open triangles indicate the peptides reactive and nonreactive to MAb14, respectively. Bands i toiv were analyzed by PMF. At right bands i to iv were mapped on the amino acid sequence of MvspI. The positions of peptides detected in PMF are marked as filledand open dots for nonreactive and reactive fragments, respectively. Peptide bands i to iv are mapped on the whole amino acid sequence from the residues ofdetected amino acid fragments. The evident residue numbers of the ends are indicated with an open triangle. The region of amino acid residues 1679 to 1723,shown by hash marks, should contain the epitope of MAb14. (B) At left is an image of a field 775 nm wide and 523 nm high. The MAb14 molecules attached toMvspI are marked by a white triangle. Scale bar, 200 nm. At right, isolated images of MvspI molecules attached by MAb14 were picked up in a circle 130 nm indiameter. Upper and lower panels represent images of molecules with and without bends, respectively.

Adan-Kubo et al.

3054 jb.asm.org Journal of Bacteriology

out multiple sequence alignments of the repeats. Although therepeat regions did not show any features specific to the protease-sensitive regions, the lengths of the amino acid sequences connect-ing the repeats were different between the sensitive (repeats a to g)and resistant (repeats h to o) regions. That is, in the sensitiveregion, the lengths were 4 amino acids for a to b, 1 for b to c, 4 forc to d, 4 for d to e, 8 for e to f, and 4 for f to g, while in the resistantregion, all the lengths were 1 or 2 amino acids. The longer con-

necting sequences that are unlikely to form a specific structure willcause flexibility, resulting in structures sensitive to proteases.

In a previous EM study of M. mobile cells, we found filamen-tous structures on the cell surface (28, 32, 35), consistent with themolecular image of MvspI obtained in the present study. How-ever, we could not identify the MvspI molecule on the cell surfacebecause at least MvspN, -O, and -K exist at the overlapping re-gions on the cell (10, 13).

Cell surface proteins of M. mobile. The flask-shaped cell of M.mobile can be divided into three parts: the head, neck, and bodyfrom the pole of the membrane protrusion (13, 36). MvspI local-izes at the head and body, and at least three proteins involved ingliding motility exist at the neck (34, 36, 37). In previous studies,the molecular shapes of Gli349 and Gli521, responsible for bind-ing and force transmission, respectively, have been clarified (1,29). The present study showed that the MvspI protein moleculeshares some features with these molecules although they do notshare the amino acid sequences. The molecules have a rod-shapedmorphology about 100 nm long, an oval structure at one termi-nus, and a transmembrane segment at the other terminus. Lowisoelectric points are also common features of 12 Mvsps and glid-ing proteins, i.e., pI values of 5.26, 5.05, and 5.38 for MvspI,Gli349, and Gli521, respectively. Moreover, sequence repeats ofabout 100 amino acids exist also in Gli349 (Fig. 7B) (1, 18, 38).Although these proteins play different roles in cellular activities,some aspects of protein functions are common; i.e., they stick outfrom the cell membrane and have some interactions at their distalends with other molecules, antibodies for MvspI, sialyllactose forGli349, and Gli349 for Gli521 (13, 14, 26, 29, 38). The commonfeatures in the structures and functions of these proteins may sug-gest that they evolved from a common ancestor. Alternatively,these features are essential for the functions and localization of the

FIG 5 Domain analyses of MvspI by partial digestion. (A) MvspI protein wastreated with various amounts of proteinase K, chymotrypsin, or trypsin, sub-jected to SDS-PAGE, and analyzed by WB and CBB staining. The MvspI pro-tein and the peptides resulting from protease treatment were analyzed by SDS-PAGE with 5.5%, 7.5%, 5.5%, and 7.5% PAGE for untreated, proteinase K,chymotrypsin, and trypsin products, respectively. Peptide bands, marked v toix and reactive to MAb14, were analyzed by PMF. (B) Mapping of peptidefragments v to ix on the amino acid sequence of MvspI. The positions ofpeptides detected in PMF are marked as open dots. The features of amino acidsequence are shown at the bottom. White and gray boxes present sequencerepeats a to g (protease sensitive) and repeats h to p (protease resistant), re-spectively. This protein was predicted to be processed between the 27th and the28th amino acid residues and attached to a lipid at the 28th residue, cysteine.The epitope of MAb14 is positioned in the region of amino acid (aa) residues1679 to 1723, as indicated by the bracket.

FIG 6 Amino acid sequences of Mvsps. Transmembrane segment, lipid at-tachment site, and repeat sequences are shown. The number indicated at theright of each sequence is the number of amino acids of each protein. Thenumbers in brackets show amino acid numbers after processing. The se-quences are aligned as the initiation codons are at the same horizontal posi-tions represented by dashed lines.

Structure of a Variable Surface Protein of Mycoplasma

June 2012 Volume 194 Number 12 jb.asm.org 3055

surface-exposed proteins of M. mobile and became commonamong unrelated proteins as a result of convergent evolution.

Molecular shape of protein for surface variation. The struc-tures of proteins responsible for surface variation represented byphase and antigenic variations may be critical to understandingthe strategies of parasitic bacteria because surface variation is oneof the determinants of their survival. However, these molecularshapes have not been examined experimentally. The present studymay be the first example of such studies. Vlps, the antigenic vari-ation proteins of M. hyorhinis, have been illustrated based on thefeatures in amino acid sequences, where a filamentous molecule isanchored to the membrane at its N terminus while the other partsfloat outside (40). These predicted features of Vlps are commonwith those of MvspI although the total amino acid numbers afterprocessing, ranging from 75 to 344, are much smaller than 1975,the number of amino acids in MvspI. We showed that the MvspImolecule is flexible, i.e., that it is a flexible rod with an occasionalcentral bend and the occasional C-terminal bulge. Generally, theproteins for surface variations are known to be modified fre-quently through the modifications of the genome sequence (7).The flexibilities of the rod and the switch between the forms of theMvspI molecule with and without the bulge may produce addi-tional structural variations on the molecules without genomemodifications. Recently, MvspI has been shown to be involved ina novel mechanism of surface variation, designated mycoplasmalantigen modulation, where MvspI decreases in a reversible way,responding to binding of MAb14 (41). The molecular structure ofMvspI clarified in the present study would be essential informa-tion to elucidate this novel mechanism of surface variation.

ACKNOWLEDGMENTS

We are grateful to Daisuke Nakane and Heng Ning Wu for helpful discus-sions.

This work was supported by Grants-in-Aid for Scientific Research in

TABLE 1 Positions of the predicted repeats and their lengths amongopen reading frames of Mvsp genes

Protein (accession no.)

Repeat position (aa)a

Length(aa)Start End

MvspA (MMOB0980) 39 116 77123 212 89238 338 100343 438 95

MvspB (MMOB3220) 10 80 7087 172 85179 263 84270 350 80

MvspC (MMOB3230) 83 172 89179 259 80264 345 81

MvspD (MMOB3280) 6 83 7790 179 89185 274 89281 361 80

MvspE (MMOB3290) 85 177 92184 273 89281 372 91379 470 91477 558 81563 637 74

MvspF (MMOB3300) 85 177 92184 273 89281 372 91379 470 91477 558 81

MvspG (MMOB3320) None 211MvspH (MMOB3330) 87 174 87

177 263 86

MvspI (MMOB3340)b

a 88 172 84b 177 272 95c 274 379 105d 384 468 84e 473 558 85f 567 660 93g 665 757 92h 760 848 88i 851 938 87j 940 1032 92k 1034 1122 88l 1124 1209 85m 1211 1297 86n 1299 1392 93o 1395 1479 84p 1725 1807 82

MvspJ (MMOB3340) 81 171 90MvspK (MMOB3370) 89 179 90

185 277 92283 380 97383 467 84

MvspL (MMOB3380) 179 266 87272 354 82367 451 84459 539 80

MvspM (MMOB6070) 4 91 8798 193 95

MvspN (MMOB6080) 110 204 94211 308 97

MvspO (MMOB6090) 112 207 95212 307 95314 411 97

MvspP (MMOB6330) 7 79 72

a aa, amino acid.b Sixteen repeats of MvspI were named a to p.

FIG 7 Schematics of MvspI and Gli349 molecules. (A) Molecules of the bulge-bend (upper) and none-bend (lower) types. At left is a schematic of EM imageswith approximate dimensions in nanometers. Two rods, a bend, and a bulgeare shown. At right amino acid sequences are mapped onto EM images. Thebright and dark repeats a to g and h to o show the repeats sensitive and resistantto proteases, respectively. (B) Schematic of the Gli349 molecule. The capitalletters in the right model present the repeat sequences composed of about 100amino acids specific to this protein (1, 18).

Adan-Kubo et al.

3056 jb.asm.org Journal of Bacteriology

Priority Areas (Structures of Biological Macromolecular Assemblies andSystem Cell Engineering by Multi-Scale Manipulation to M.M.), by aGrant-in-Aid for Scientific Research (A) (to M.M.) from the Ministry ofEducation, Culture, Sports, Science, and Technology of Japan, and by agrant from the Institution for Fermentation Osaka (to M.M.).

REFERENCES1. Adan-Kubo J, Uenoyama A, Arata T, Miyata M. 2006. Morphology of

isolated Gli349, a leg protein responsible for glass binding of Mycoplasmamobile gliding revealed by rotary-shadowing electron microscopy. J. Bac-teriol. 188:2821–2828.

2. Altschul SF, et al. 1997. Gapped BLAST and PSI-BLAST: a new genera-tion of protein database search programs. Nucleic Acids Res. 25:3389 –3402.

3. Aluotto BB, Wittler RG, Williams CO, Faber JE. 1970. Standardizedbacteriologic techniques for the characterization of mycoplasma species.Int. J. Syst. Bacteriol. 20:35–58.

4. Arata T. 1998. Electron microscopic observation of monomeric actinattached to a myosin head. J. Struct. Biol. 123:8 –16.

5. Behrens A, Heller M, Kirchhoff H, Yogev D, Rosengarten R. 1994. Afamily of phase- and size-variant membrane surface lipoprotein antigens(Vsps) of Mycoplasma bovis. Infect. Immun. 62:5075–5084.

6. Bhugra B, Voelker LL, Zou N, Yu H, Dybvig K. 1995. Mechanism ofantigenic variation in Mycoplasma pulmonis: interwoven, site-specificDNA inversions. Mol. Microbiol. 18:703–714.

7. Citti C, Nouvel LX, Baranowski E. 2010. Phase and antigenic variation inmycoplasmas. Future Microbiol. 5:1073–1085.

8. Eddy SR. 1998. Profile hidden Markov models. Bioinformatics14:755–763.

9. Erickson HP. 2009. Size and shape of protein molecules at the nanometerlevel determined by sedimentation, gel filtration, and electron micro-scopy. Biol. Proced. Online 11:32–51.

10. Jaffe JD, et al. 2004. The complete genome and proteome of Mycoplasmamobile. Genome Res. 14:1447–1461.

11. Kirchhoff H. 1992. Motility, p 289 –306. In Maniloff J, McElhaney RN,Finch LR, Baseman JB (ed), Mycoplasmas: molecular biology and patho-genesis. American Society for Microbiology, Washington, DC.

12. Krunkosky TM, Jordan JL, Chambers E, Krause DC. 2007. Mycoplasmapneumoniae host-pathogen studies in an air-liquid culture of differenti-ated human airway epithelial cells. Microb. Pathog. 42:98 –103.

13. Kusumoto A, Seto S, Jaffe JD, Miyata M. 2004. Cell surface differenti-ation of Mycoplasma mobile visualized by surface protein localization. Mi-crobiology 150:4001– 4008.

14. Lesoil C, et al. 2010. Molecular shape and binding force of Mycoplasmamobile’s leg protein Gli349 revealed by an AFM study. Biochem. Biophys.Res. Commun. 391:1312–1317.

15. Letunic I, Doerks T, Bork P. 2009. SMART 6: recent updates and newdevelopments. Nucleic Acids Res. 37:D229 –232.

16. Ludtke SJ, Baldwin PR, Chiu W. 1999. EMAN: semiautomated softwarefor high-resolution single-particle reconstructions. J. Struct. Biol.128:82–97.

17. Madan Babu M, Sankaran K. 2002. DOLOP: database of bacterial lipo-proteins. Bioinformatics 18:641– 643.

18. Metsugi S, et al. 2005. Sequence analysis of the gliding protein Gli349 inMycoplasma mobile. Biophysics 1:33– 43.

19. Miyata M. 2008. Centipede and inchworm models to explain Mycoplasmagliding. Trends Microbiol. 16:6 –12.

20. Miyata M. 2005. Gliding motility of mycoplasmas: the mechanism cannotbe explained by current biology, p 137–163. In Blanchard A, Browning G(ed), Mycoplasmas: pathogenesis, molecular biology, and emerging strat-egies for control. Horizon Bioscience, Norfolk, United Kingdom.

21. Miyata M. 2007. Molecular mechanism of mycoplasma gliding: a novelcell motility system, p 137–175. In Lenz P (ed), Cell motility. Springer,New York, NY.

22. Miyata M. 2010. Unique centipede mechanism of mycoplasma gliding.Annu. Rev. Microbiol. 64:519 –537.

23. Miyata M, Ryu WS, Berg HC. 2002. Force and velocity of Mycoplasmamobile gliding. J. Bacteriol. 184:1827–1831.

24. Miyata M, Uenoyama A. 2002. Movement on the cell surface of glidingbacterium, Mycoplasma mobile, is limited to its head-like structure. FEMSMicrobiol. Lett. 215:285–289.

25. Miyata M, et al. 2000. Gliding mutants of Mycoplasma mobile: relation-ships between motility and cell morphology, cell adhesion and micro-colony formation. Microbiology 146:1311–1320.

26. Nagai R, Miyata M. 2006. Gliding motility of Mycoplasma mobile canoccur by repeated binding to N-acetylneuraminyllactose (sialyllactose)fixed on solid surfaces. J. Bacteriol. 188:6469 – 6475.

27. Nakane D, Adan-Kubo J, Kenri T, Miyata M. 2011. Isolation andcharacterization of P1 adhesin, a leg protein of the gliding bacterium My-coplasma pneumoniae. J. Bacteriol. 193:715–722.

28. Nakane D, Miyata M. 2007. Cytoskeletal “jellyfish” structure of Myco-plasma mobile. Proc. Natl. Acad. Sci. U. S. A. 104:19518 –19523.

29. Nonaka T, Adan-Kubo J, Miyata M. 2010. Triskelion structure of theGli521 protein, involved in the gliding mechanism of Mycoplasma mobile.J. Bacteriol. 192:636 – 642.

30. Razin S, Yogev D, Naot Y. 1998. Molecular biology and pathogenicity ofmycoplasmas. Microbiol. Mol. Biol. Rev. 62:1094 –1156.

31. Rosengarten R, Kirchhoff H. 1987. Gliding motility of Mycoplasma sp.nov. strain 163K. J. Bacteriol. 169:1891–1898.

32. Rosengarten R, Kirchhoff H, Kerlen G, Seack K-H. 1988. The surfacelayer of Mycoplasma mobile 163K and its possible relevance to cell cohe-sion and group motility. J. Gen. Microbiol. 134:275–281.

33. Schuck P, Perugini MA, Gonzales NR, Howlett GJ, Schubert D. 2002.Size-distribution analysis of proteins by analytical ultracentrifugation:strategies and application to model systems. Biophys. J. 82:1096 –1111.

34. Seto S, Uenoyama A, Miyata M. 2005. Identification of 521-kilodaltonprotein (Gli521) involved in force generation or force transmission forMycoplasma mobile gliding. J. Bacteriol. 187:3502–3510.

35. Shimizu T, Miyata M. 2002. Electron microscopic studies of three glidingmycoplasmas, Mycoplasma mobile, M. pneumoniae, and M. gallisepticum,by using the freeze-substitution technique. Curr. Microbiol. 44:431– 434.

36. Uenoyama A, Kusumoto A, Miyata M. 2004. Identification of a 349-kilodalton protein (Gli349) responsible for cytadherence and glass bind-ing during gliding of Mycoplasma mobile. J. Bacteriol. 186:1537–1545.

37. Uenoyama A, Miyata M. 2005. Identification of a 123-kilodalton protein(Gli123) involved in machinery for gliding motility of Mycoplasma mobile.J. Bacteriol. 187:5578 –5584.

38. Uenoyama A, Seto S, Nakane D, Miyata M. 2009. Regions on Gli349 andGli521 protein molecules directly involved in movements of Mycoplasmamobile gliding machinery, suggested by use of inhibitory antibodies andmutants. J. Bacteriol. 191:1982–1985.

39. Wise KS. 1993. Adaptive surface variation in mycoplasmas. Trends Mi-crobiol. 1:59 – 63.

40. Wise KS, Yogev D, Rosengarten R. 1992. Antigenic variation, p 473– 490.In Maniloff J, McElhaney RN, Finch LR, Baseman JB (ed), Mycoplasmas:molecular biology and pathogenesis. American Society for Microbiology,Washington, DC.

41. Wu H, Kawaguchi C, Nakane D, Miyata M. 2012. “Mycoplasmal antigenmodulation,” a novel surface variation suggested for a lipoprotein specif-ically localized on Mycoplasma mobile. Curr. Microbiol. 64:433– 440.

42. Yogev D, Browning GF, Wise KS. 2002. Genetic mechanisms of surfacevariation, p 417– 443. In Herrmann R, Razin S (ed), Molecular biology andpathogenicity of mycoplasmas. Kluver Academic, London, UnitedKingdom.

43. Zhang W, Chait BT. 2000. ProFound: an expert system for protein iden-tification using mass spectrometric peptide mapping information. Anal.Chem. 72:2482–2489.

Structure of a Variable Surface Protein of Mycoplasma

June 2012 Volume 194 Number 12 jb.asm.org 3057