VETERINARY RESEARCHFirst identification of proteins involved in motilityof Mycoplasma gallisepticumIndikova et al.Indikova et al. Veterinary Research 2014, 1/99

Indikova et al. Veterinary Research 2014, 1/99RESEARCHVETERINARY RESEARCHOpen AccessFirst identification of proteins involved in motilityof Mycoplasma gallisepticumIvana Indikova, Martin Vronka and Michael P Szostak*AbstractMycoplasma gallisepticum, the most pathogenic mycoplasma in poultry, is able to glide over solid surfaces. Althoughthis gliding motility was first observed in 1968, no specific protein has yet been shown to be involved in gliding. Weexamined M. gallisepticum strains and clonal variants for motility and found that the cytadherence proteins GapA andCrmA were required for gliding. Loss of GapA or CrmA resulted in the loss of motility and hemadsorption and led todrastic changes in the characteristic flask-shape of the cells. To identify further genes involved in motility, a transposonmutant library of M. gallisepticum was generated and screened for motility-deficient mutants, using a screening assaybased on colony morphology. Motility-deficient mutants had transposon insertions in gapA and the neighbouringdownstream gene crmA. In addition, insertions were seen in gene mgc2, immediately upstream of gapA, in twomotility-deficient mutants. In contrast to the GapA/CrmA mutants, the mgc2 motility mutants still possessed theability to hemadsorb. Complementation of these mutants with a mgc2-hexahistidine fusion gene restored the motilephenotype. This is the first report assigning specific M. gallisepticum proteins to involvement in gliding motility.IntroductionMotility is regarded as a virulence factor in many pathogenic bacteria. The ability to move enables microorganisms to reach a specific niche or to leave hostileenvironments. Amongst motile bacteria, various mechanisms to create a momentum have evolved. In Bordetellabronchiseptica, Escherichia coli, and Salmonella entericaserovar Typhimurium flagellar motility has been shownto be crucial for the initial stages of infection, while inLegionella pneumophila motility is necessary to establishand maintain infection [1]. In contrast to these species,in which motility can be downregulated to favor aspecific life-style, some bacteria, such as Helicobacter,Campylobacter, and Pseudomonas aeruginosa, dependon constitutive flagellar motility for successful infection[1]. Experiments showing that only motile bacteria canbe reisolated after infection with a mixed population ofmotile and non-motile variants underline the importance of motility in the infection process [2].Mycoplasmas lack a cell wall and are considered to bethe smallest self-replicating microorganisms. They havelimited biosynthetic capabilities as they are highly adapted* Correspondence: [email protected] of Pathobiology, Institute of Bacteriology, Mycology andHygiene, University of Veterinary Medicine Vienna, Veterinaerplatz 1, A-1210Vienna, Austriato a parasitic life-style [3]. In spite of the many limitationsthat have resulted from their degenerative evolution, somemycoplasmas have the ability to travel over inert surfaces,like glass, plastic or over eukaryotic cells, even though theylack any obvious locomotory appendages such as flagellaor pili [4].Mycoplasma gallisepticum is an avian pathogen causing chronic respiratory disease in chickens and infectious sinusitis in turkeys, that is known to possessgliding motility. Like the majority of gliding mycoplasmas, M. gallisepticum belongs to the M. pneumoniaecluster [5], named after M. pneumoniae, the causativeagent of human bronchitis and atypical pneumonia [6].The mechanism that enables M. pneumoniae and othermycoplasmas to glide has been the subject of a numberof studies [7].The best studied gliding mechanism is that of M. mobile, isolated from the gills of a fresh-water fish [8],which is phylogenetically distant from the pneumoniaecluster. M. mobile can be cultivated at room temperatureand its average gliding speed is 2 to 4.5 μm/s [9], thusvisualization of the gliding process is not dependent onadditional microscope equipment such as a plate heateror a computer-connected CCD video camera. Severalproteins of M. mobile have been identified as motilityproteins [10]. Centered at the neck region of the jellyfish 2014 Indikova et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (, which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver ) applies to the data made available in this article,unless otherwise stated.

Indikova et al. Veterinary Research 2014, 1/99shape-like M. mobile, the Gli349 leg protein binds tosialylated oligosaccharides on glass or animal cells.Together with the Gli521 gear protein and the Gli123mount protein, a large number of legs may act in a continuous “bind, pull, and release” mode, thereby creatinga continuous pull in the forward direction. The multiplelegs involved suggested the term “centipede-like” locomotion [11].However, no homologs of these M. mobile motilitygenes have been found in M. pneumoniae or M. gallisepticum, indicating that different mycoplasmas may have developed different gliding machineries. The motile members ofthe M. pneumoniae cluster share a characteristic morphological feature, cellular polarity. These mycoplasmas have aflask-shaped appearance, strengthened by a cytoskeleton,and have a differentiated tip structure, often called theattachment tip or terminal organelle (TO). In M.pneumoniae, the TO mediates adherence to the hostrespiratory epithelium, a prerequisite for successfulcolonization [12]. In addition, the TO is the leadingend in gliding motility [13], as cells always glide in thedirection of the tip structure.Formation of the TO appears to be a complex processthat has to be well orchestrated, chronologically andspatially [14]. The TO of M. pneumoniae consists of anetwork of cytadherence proteins, including P1, P30, theaccessory proteins P65, B, C, and the structural proteinsHMW1, HMW2, and HMW3 [15]. Mutations affectingcytadherence or the correct assembly of the TO havedirect effects on gliding motility. Loss of proteins P1, P30,or P65 lead to a non-motile, as well as hemadsorptionnegative, phenotype [16]. Similarly, mutations in the TOproteins P41 and P24 have an impact on the velocityand frequency of gliding [17]. Although several elements of the gliding machinery have been identified, itis still unclear how these motility-associated proteinswork in concert to generate a propulsive force andmove the cell forward.Studies to elucidate the motility mechanisms of members of the pneumoniae cluster have also included M.genitalium, a close relative of M. pneumoniae. Theirproteins share a high degree of homology [18]. Many ofthe proteins involved in M. pneumoniae motility havecounterparts in M. genitalium [19]. Surprisingly, no protein involved in motility has yet been identified inM. gallisepticum, and although M. gallisepticum wasincluded in a recent study of mycoplasma gliding [20],little is known about the proteins involved. Therefore,we examined the gliding ability of M. gallisepticumstrain R and clonal variants of it, including a library oftransposon insertion mutants. The aim of this study wasto identify proteins that contribute to the motilityprocess of M. gallisepticum, to investigate the molecularproperties of such motility proteins, and to further refinePage 2 of 13the tools for screening and complementing motilitymutants.Materials and methodsStrains and growth conditionsM. gallisepticum strains Rlow, Rhigh [21], RCL1, RCL2,mHAD3 [22], motility mutants and complementedmotility mutants were cultured in modified Hayflickmedium [23] (HFLX) at 37 C. To grow tetracycline(TcR) or chloramphenicol- resistant (CmR) M. gallisepticum transformants, either Tc (4 μg mL 1; RocheDiagnostics, Penzberg, Germany) or Cm (17 μg mL 1;Carl Roth GmbH & Co KG, Karlsruhe, Germany) wereadded to HFLX medium. Escherichia coli DH10B(Invitrogen Corp., Carlsbad, CA, USA) was used forthe propagation of plasmids used in this study.Motility assaysTo detect satellite growth of M. gallisepticum, a freshlygrown culture was seeded in a 24-well microtiter plateat a concentration of 40 CFU per 400 μL of HFLXmedium per well. After 2 h of attachment, the mediumwas replaced by HFLX containing 2% gelatin and, iftransformants were to be analyzed, Tc was added to theHFLX-gelatin mixture. Colony morphology was examined after growth at 37 C for five to seven days using anSMZ-U stereomicroscope (Nikon Corp., Tokyo, Japan).Characterization of M. gallisepticum gliding motilitywas performed using a microcinematography motilityassay (MMA). For this purpose, 100 μL of a culturefreshly grown in HFLX medium was placed on a standard microscope glass slide (Thermo Fisher ScientificInc., Waltham, MA, USA). After 1 h of incubation at37 C, attached cells were overlaid with 100 μL of freshmedium containing 2% gelatin. After 1.5 h of incubation,cell movement was examined using an Olympus AX70microscope equipped with a heating plate set at 37 C,and phase-contrast images were captured at 1-s intervalsfor a total of 180 s with a Color View CCD digital camera controlled using CellPlus (Olympus Soft Imaging Solutions GmbH, Muenster, Germany).Computer-assisted qualitative analysis of motility wasperformed by overlaying 180 single frames of a 3 minmicroscope movie with the Z project tool of the Fijiimage processing package [24], choosing “MinimumIntensity” as the critical parameter. Bacterial pathswere highlighted by standard layer manipulations usingPhotoshop CS3 version 10.0 (Adobe Systems Inc., SanJose, CA, USA).For a quantitative analysis of motility, the ten mycoplasmas with the longest Z project paths in each field ofview were selected, and their movements were trackedusing the ImageJ/Fiji MTrackJ plugin [25]. Using theanalysis tool of MTrackJ, the distance travelled and the

Indikova et al. Veterinary Research 2014, 1/99overall speed of motility, including resting periods, weredetermined. The best three results of 5 independent experiments were chosen for graphical representation.Results of quantitative MMAs were analyzed for statistical significance by using a two-tailed Student’s T test[26]. P values of 0.05 were considered to indicate significant differences between groups.DNA isolation and sequencing reactionsGenomic DNA from mycoplasmas was isolated usingthe GenElute Mammalian Genomic DNA Miniprep Kit(Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany).Plasmid DNA from E. coli cultures was purified usingthe PureYield Plasmid System Kit (Promega, Mannheim,Germany). Oligonucleotide synthesis was performed byeither Microsynth (Microsynth AG, Balgach, Switzerland)or Invitrogen (Life Technologies GmbH, Darmstadt,Germany), and DNA sequencing was conducted byLGC Genomics (LGC Genomics GmbH, Berlin, Germany).If not otherwise mentioned, all enzymes used in this studywere purchased from Promega. For DNA/PCR purification,the Wizard SV Gel and PCR Clean-Up System (Promega)was used.Construction of plasmidsTransposon Tn4001camTo use transposon mutants in gentamicin-based cellinvasion assays, we first had to replace the gentamicinresistance gene of Tn4001. The chloramphenicol-resistancecassette CmR of plasmid pACYC184 (Invitrogen) was amplified using primers Xcat5 and Xcat3, introducing BamHIand NarI cleavage sites, respectively. The purified ampliconwas cloned into the corresponding sites of plasmidp5TlacZ [27], thereby placing the CmR cassette under thecontrol of tufPO in plasmid p5xCAT. Left (ISL) and right(ISR) IS256 elements of S. aureus transposon Tn4001modwere amplified from plasmid pISM2062 [28], using primersISR-f and ISR-r, which introduced MluI and KasI cleavagesites, and ISL-f and ISL-r, which contained SacII and SalIcleavage sites. The amplicons ISL and ISR were cloned intothe corresponding sites to the right and to the left end, respectively, of the CmR cassette on plasmid p5xCAT. Transformants of E. coli DH10B were selected on Luria-Bertaniagar containing Cm (30 μg/mL). Transposon mutants ofM. gallisepticum were stable for at least 20 passages without Cm and no re-transposition or excision of Tn4001camcould be detected (data not shown).Integration plasmid p5HmgcTn4001mod on plasmid pISM2062 [28] was modified byadding a 6xHis-tag and a multiple cloning site: a 51-bpDNA fragment, created by annealing oligonucleotidesHisC-f and HisC-r, was inserted between the BamHIand SmaI cleavage sites of pISM2062, resulting inPage 3 of 13plasmid pTnHis. The M. gallisepticum gene mgc2 wasthen amplified by PCR using genomic DNA of strainRlow as template and primers ISM-mgcF and ISMmgcR, and subcloned into pTnHis using the BamHI andSphI cleavage sites. The resulting plasmid, pTHmgc, waslinearized with NotI, treated with the Klenow fragmentof DNA polymerase I (New England Biolabs GmbH,Frankfurt/Main, Germany) to fill in the 5′ overhang, andsubsequently digested with BamHI. A 1093-bp fragmentwas gel-purified and ligated to a 3.5-kb fragment of plasmid pINT [27], obtained after digestion with BamHIand SfoI.Transformation of mycoplasmasM. gallisepticum transposon mutants were generated byelectroporation of strain RCL1 with 3–5 μg of pTnC, asdescribed previously [22]. For the transformation withintegration plasmid p5Hmgc, 20–30 μg of plasmid DNAwas used. Following electroporation, mycoplasma cellswere cultured on HFLX plates containing either 17.5 μgchloramphenicol mL 1 or 4 μg tetracycline mL 1.Ligation mediated PCR (LM-PCR)Transposon insertion sites were determined by LM-PCRusing the method of Sharma et al. [29], with modifications. Briefly, genomic DNA of M. gallisepticum transposon mutants was digested with BglII, and ligated tothe BglII-adaptors Ad1B and Ad2B. Prior to ligation, theadaptor oligo nucleotides were dissolved separately indouble distilled water at a concentration of 100 μM andequal volumes of both were mixed together. The mixture was incubated at 70 C for 10 min, allowed to coolgradually to 40 C, and then incubated at 40 C for10 min. The mixture was then cooled gradually to 25 Cand stored frozen in small aliquots until further usage.The ligation product was used as a template for PCRamplification using the adaptor-specific primer Bgl andprimer IS-I, specific for IS256 of Tn4001mod. The PCRproduct was then used as template for a semi-nestedPCR using primers Bgl and IS-N, and the gel-purifiedamplicons were sequenced (Microsynth).Production of antibodiesMGC2The full-length mgc2 gene was amplified using the LRPCR kit (Roche) and primers mgc2 3 and mgc2 4(Table 1), introducing EcoRI and HindIII cleavage sitesat either end. The TGA codon was mutagenized to TGGusing primer mgc tga and the Site-directed Mutagenesiskit (Stratagene) according to the manufacturer’s instructions. The mutated mgc2 gene was then cloned betweenthe EcoRI and HindIII cleavage sites in pRSET (Invitrogen)and introduced into E. coli BL21 (DE3)pLys Star (Invitrogen). Expression of MGC2 was induced by addition of

Indikova et al. Veterinary Research 2014, 1/99Page 4 of 13Table 1 Oligonucleotides used in this studyPrimerSequence (5′ to 3′)Product(length AggatccTGTTGAAAAGCGCTTAGCMG tsIS-NAAAGGACTGTTATATGGC(variable)mgc2 3ACGCAGgaattcATAACAATTATGMG mgc2mgc2 4TTTACAaagcttGTCTTATCTAGG(894)mgc tgaGAAAGATTACCTCCGAACCATGGTTTTATCCAGTAGTGGGTGA TGGXcat5TAGATGggatccATGGAGAAAAAAATCACTGpACYC184 (751)Xcat3ATAAATggcgccCGCTTATTATCACTTATTCCmPO CmRLaboratories Inc., Hercules, CA, USA) and electroelution inan Electro-Eluter Model 422 (BioRad). The immunizationof rabbits with the purified C-terminal part of GapAfollowed exactly the same procedures as for MGC2antibodies.MG gapA(911 bp)CrmAsynthetic6xHis-tagThe generation of CrmA-specific antibodies has beendescribed previously [30].Western blot analyses and tryptic digestionIS256L(1365 bp)IS256R(1342 bp)0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) andthe protein was purified using the MagneHis Protein Purification System (Promega) and immobilized metal affinitychromatography using ProBond nickel-chelating resin(Invitrogen) according to the manufacturers’ instructions.Purity of the MGC2 protein was confirmed by Westernblotting using Anti-Xpress antibodies (1:5000) (Invitrogen). After elution and dialysis against phosphate-bufferedsaline (PBS), the protein was used for immunization of rabbits as described elsewhere [30].Five millilitre aliquots of overnight cultures of mycoplasmas were centrifuged at 4200 g for 30 min, and the cellpellets washed once with PBS and resuspended in PBScontaining 0.05% of a commercial trypsin/EDTA solution (Life Technologies, #15400). Control samples weretreated in the same way but without trypsin. The samples were incubated at 37 C, 1-mL samples removedafter 10, 30, 40, 50, 70 and 90 min, and trypsin activitystopped by adding phenylmethylsulfonyl fluoride (PMSF)at a final concentration of 1 mM. Cells were then collected by centrifugation and the presence of MGC2 wasassessed by Western blot analysis. The solubilization andseparation of mycoplasma cell lysates using 10% SDSPAA gel electrophoresis, and Western blot analysis isdescribed elsewhere [31]. Membranes were probed withantibodies against MGC2 (1:500), GapA (1:6000), orCrmA (1:2000) using peroxidase-conjugated swine-antirabbit IgG (1:2000, Dako) as secondary antibody, or withanti-6xHis antibody (1:6000, Aviva Systems BiologyCorp., San Diego, CA, USA) in combination with theAffiniPure Goat Anti-Mouse IgG, Fc Fragment specific(1:10 000, Jackson ImmunoResearch Laboratories, Inc.,West Grove, PA, USA).Hemadsorption assayThe ability of mycoplasma colonies grown on HFLXagar plates to hemadsorb was tested as described previously [30].Scanning electron microscopy (SEM)C-GapAThe 3′-terminal part of the gapA gene was amplifiedusing the LR-PCR system (Roche) and primers C’gapA5and C’gapA3, introducing BamHI and HindIII cleavagesites, at either end. The gel-purified amplicon was ligated into plasmid pRSET-B (Invitrogen) and the resulting plasmid was introduced into E. coli BL21 (DE3)pLysStar (Invitrogen). The recombinant culture was grown at28 C, and gene expression was induced by addition of0.3 mM IPTG at the early logarithmic growth phase. Aprotein of 35 kDa was retrieved from a sodium dodecylsulfate polyacrylamide (SDS-PAA) gel after negativestaining with a zinc stain and destain kit (BioRadMycoplasma samples for SEM were prepared as described previously [32], except that mycoplasma cultureswere grown at 37 C on glass coverslips, precoated withpoly-L-lysine (Sigma-Aldrich) according to the manufacturer’s instructions.ResultsIdentification of non-motile M. gallisepticum strainsTo establish a method for screening for motility mutants ofM. gallisepticum, we first identified motile and non-motilestrains in our culture collection. Gliding of M. mobile andM. pneumoniae depends on cytadherence-associated components [16,33,34]. Therefore, we analyzed selected strains

Indikova et al. Veterinary Research 2014, 1/99Page 5 of 13of M. gallisepticum that differed in hemadsorption (HA)(Table 2) using a qualitative microcinematography motilityassay (MMA). The HA-positive (HA ) M. gallisepticumstrain Rlow and its clonal variant RCL1, expressing themajor cytadherence gene gapA as well as the cytadherencerelated gene crmA, were capable of gliding. At any time,60% of cells were moving, interrupted by short restingperiods. Mycoplasma gliding paths were visualized bycomputer-generated overlay of all frames of a 3 min videoand consisted mainly of circles and bends (Figure 1). Incontrast, no gliding could be visualized for strains Rhigh,RCL2, or mHAD3, which lack either GapA and/or CrmA.These HA strains appeared to have lost the ability to glide,as no moving cells were observed in MMAs, in spite of numerous trials under a variety of conditions.Colonies of motile M. gallisepticum form microsatellitesFormation of microsatellites around colonies grown onagar plates has been described for M. mobile [33] andM. pneumoniae [16], and appears to be an indicator ofgliding motility in mycoplasmas. M. gallisepticum colonies have been reported to grow without satellite formation under the conditions that allow M. mobile toform microsatellites [33]. Conditions for microsatelliteformation by M. gallisepticum were established usingstrains Rlow and Rhigh as prototypes of motile and nonmotile strains (Table 2). Diffuse colonies spreading in alldirections were observed for both strains when grownon HFLX medium solidified with a range of concentrations of agar (0.05 - 0.3%) (Figure 2). Higher concentrations of agarose have already been shown to allow onlythe formation of the typical fried-egg colonies and weretherefore not tested.When M. gallisepticum cells were first allowed to attach to the surface of a cell culture dish and then overlaid with HFLX medium containing gelatin, motilestrains could be differentiated from non-motile strains(Figure 2). At 0.5% gelatin, Rlow and Rhigh were notable to form colonies on the bottom of the dish, butcloudy regions in the overlay medium indicated thatmycoplasma cells had spread throughout the medium.Figure 1 Gliding paths of motile M. gallisepticum. A stack ofphase-contrast pictures of M. gallisepticum RCL1, captured at 1-sintervals, was manipulated with Fiji by applying a Z projectionmethod [24] to visualize the paths of gliding mycoplasmas.Differential colouring of mycoplasmas (red) and their paths(green) was done with Adobe Photoshop. This qualitativemicrocinematography motility assay allowed the rapid assessment ofthe motility of wild-type and mutant strains.With increasing gelatin concentrations, the number ofcolonies increased, while growth in the overlay mediumdecreased. At 2% gelatin, the motile strains Rlow andRCL1 formed round colonies with a smooth surface, surrounded by many satellites, while the non-motile strainsRhigh, RCL2, and mHAD3 formed colonies with a roughsurface, uneven edges, and without satellite colonies.Higher concentrations of gelatin resulted in partial detachment of colonies and formation of microcolonies inthe overlay medium. Therefore, for further experimentsto detect colonies of M. gallisepticum with a satellitegrowth altered (SGA) phenotype, HFLX medium solidified with 2% gelatin was used.Generation of motility-deficient mutants andcomplementationTo identify proteins involved in the gliding motility of M.gallisepticum, RCL1 was transformed with transposonTable 2 Protein content, motility and hemadsorption ability of MG strainStrainHem-adsorptionMotilityCell ShapePresence ofReferenceMGC2GapACrmARlow flask [21]Rhigh--round --[21]RCL1 flask [22]RCL2--round --[22]mHAD3--round ( )-[22]T932A -rounded flask- this studyT932C -distorted flasktrc1 this study1trc; truncated.

Indikova et al. Veterinary Research 2014, 1/99Page 6 of 13HFLX / agarHFLX / gelatine0.5%2%0.1%0.3%RCL1mHAD3Figure 2 Morphology of M. gallisepticum colonies. Cultures of RCL1 and mHAD3 were either grown in HFLX medium solidified with 0.5 or 2%gelatin or on low-agar plates containing 0.1 or 0.3% agar. In gelatin-containing medium, the non-motile strain mHAD3 formed compact colonies,in contrast to the motile RCL1 which formed colonies surrounded by microsatellites.Tn4001cam and transformed colonies were screened forthe SGA phenotype. In a proof-of-concept study, 4000colonies were screened and 38 mutants were found to exhibit defects in satellite colony formation. Their ability toglide was further examined individually using MMAs.Eight mutants had a low proportion of motile cells (20%),so they were stored for later analysis. Southern blot analyses of thirty motility mutants found that the majoritycarried multiple Tn4001cam insertions within their genome. Only ten mutants had contained only one or twotransposon insertions. They were subjected to LM-PCR toprecisely determine the transposon insertion sites. In fourmutants the transposon had integrated into the gapAgene, and in another four mutants Tn4001cam wasinserted into the crmA gene (Figure 3). These mutantswere not analyzed further, because the same genes wereaffected in the cytadherence-negative, non-motile strainsRhigh, RCL2, and mHAD3. However, in two motilitydeficient mutants the transposon had integrated intodifferent sites in the mgc2 gene (also known as theMGA 0932 coding region) and these mutant strains weredesignated T932A and T932C (Figure 3). After filter cloning, a LM-PCR analysis revealed that T932A containedthe Tn4001cam at position 222 517 (numbering accordingto GenBank Accession AE015450.2) of the genomic DNA,344 bp downstream of the initial coding nucleotide of themgc2 gene (Figure 3). The ORF therefore terminated 119amino acids (aa) after the start codon, whereby the last 5aa were encoded by the transposon. Even after repeatedfilter cloning mutant T932C still appeared to contain twotransposons, one within the mgc2 gene at position 222749, which would allow for translation of 193 aa of theMGC2 protein. The second transposon was found in theCRISPR region at position 930 903.To confirm that disruption of the mgc2 ORF by transposition was responsible for the loss of motility, mutantsT932A and T932C were complemented with an mgc26xHis fusion gene. For this purpose, the fusion gene wassubcloned into a derivative of plasmid pINT [27], whichintegrates into the oriC region of M. gallisepticumby homologous recombination. The resulting plasmidp5Hmgc was introduced into the mutants by electroporation, and the integration into the genomic oriC locuswas proven by Southern blot analyses.Characterization of mgc2 mutants and complementedmutantsExpression and surface localization of MGC2The effect of transposon integration on expression ofmgc2 and the gapA and crmA genes immediately downstream of it was investigated by Western blot analyses.For this purpose, polyvalent rabbit antisera againstMGC2, GapA and CrmA were produced. MGC2 wasequally well detected in HA and HA M. gallisepticumstrains (Table 2). In contrast, no MGC2 could be detected in T932A lysates (Figure 4A, lane 2), and only atruncated MGC2, with an apparent size of 19 kDa,was detected in T932C (Figure 4A, lane 3). Whencomplemented with p5Hmgc, both T932A and T932Cexpressed full-length MGC2 at concentrations comparable to RCL1 (Figure 4A, lanes 4–5). The Cterminal 6xHis-tag did not appear to influence thestability of the recombinant MGC2.As it has been suggested that the gapA transcript initiates in the 3′ region of mgc2 [35], we analyzed the expression of gapA and crmA. Western blot analysesrevealed that expression of these genes did not appear tobe affected by the Tn4001cam insertion into mgc2, asGapA and CrmA were detected at wild-type levels inboth the mgc2 mutants (Figures 4B and 4C).MGC2 has been identified by immunoelectron microscopy on the mycoplasma cell surface [36]. To confirm

Indikova et al. Veterinary Research 2014, 1/99Page 7 of 13T932A T932CgapAmgc21344 576crmA884Figure 3 Insertion sites of Tn4001cam transposon in motility mutants. Non-motile RCL1 mutants harbored transposons in the mgccytadherence locus, consisting of mgc2, gapA (formerly mgc1), and crmA (formerly mgc3). In mutants T932A and T932C Tn4001cam integrated344 and 576 bp after the translational initiation nucleotide of mgc2.this finding and to assess the size of the surface-exposedportion of MGC2, whole M. gallisepticum RCL1 cellswere incubated with trypsin for different time periodsand subjected to Western blot analyses with anti-MGC2antiserum. Tryptic digestion produced a shorter fragment of MGC2, migrating at an apparent molecularmass of 31 kDa (Figure 4D), while untreated MGC2 migrated at 33 kDa. The 31-kDa band was first detectedafter 10 min of trypsin treatment (not shown), and became more prominent with longer periods of digestion.After 90 min of trypsin digestion the 33-kDa proteinwas no longer detectable (Figure 4D), indicating the degree of surface accessibility of MGC2 to trypsin. The factAkDa1M2that trypsin digestion reduced the molecular weight ofMGC2 by only 2 kDa suggested that only a small regionof MGC2 was exposed on the surface. Trypsin digestionpatterns for the mgc2-complemented mutants T932A::p5Hmgc and T932C::p5Hmgc were comparable tothose seen with RCL1 (Figure 4D), indicating the samedegree of surface localization of the 6xHis-taggedMGC2 protein.Hemadsorption activityThe hemadsorptive activity of wild-type strains, andcytadherence and motility mutants was analyzed using astandard HA assay. In contrast to the non-motile strains345554033MGC224MGC2 17B 17013510072GapAC 170CrmA135100RCL1DMT0T1T932A::p5Hmgc T932C::p5HmgcT2T0T1T2T0T1T24033MGC224Figure 4 Absence of MGC2 protein in gliding mutants. Immunoblot analysis of RCL1 (lane 1), motility mutants T932A (lane 2) and T93

the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Plasmid DNA from E. coli cultures was purified using the PureYield Plasmid System Kit (Promega, Mannheim, Germany). Oligonucleotide synthesis was performed by either Microsynth (Microsynth AG, Balgach, Switzerland)