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GeneticandserologicalanalysisoflipoproteinLppAinMycoplasmamycoidessubsp.mycoidesLCandMycoplasmamycoidessubsp,capri

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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/99/$04.0010

Mar. 1999, p. 224–230 Vol. 6, No. 2

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Genetic and Serological Analysis of Lipoprotein LppA inMycoplasma mycoides subsp. mycoides LC and

Mycoplasma mycoides subsp. capriMARIE-PIERRE MONNERAT,1 FRANCOIS THIAUCOURT,2 JOSE B. POVEDA,3

JACQUES NICOLET,1 AND JOACHIM FREY1*

Institute for Veterinary Bacteriology, University of Berne, CH-3012 Berne, Switzerland1; CIRAD-EMVT,Campus International de Baillarguet, F-34032 Montpellier, France2; and Facultad de Veterinaria,

Universidad de Las Palmas, E-35071 Las Palmas, Spain3

Received 29 June 1998/Returned for modification 6 November 1998/Accepted 4 December 1998

The genes encoding the 62-kDa lipoproteins from the Mycoplasma mycoides subsp. mycoides large-colony type(LC) strain Y-goat and the M. mycoides subsp. capri strain PG3 were cloned and analyzed by sequencing. Thesetwo lipoproteins have been named LppA[MmymyLC] and LppA[Mmyca], and their corresponding genes havebeen named lppA[MmymyLC] and lppA[Mmyca], respectively. The nucleotide and deduced amino acid se-quences of these two lipoproteins showed a very high degree of similarity between these two mycoplasmas.Given the sequence data, LppA seems to fulfill the same structural functions as the previously described majorlipoproteins P72 of M. mycoides subsp. mycoides small-colony type and P67 of the Mycoplasma species bovinegroup 7. Based on lppA gene sequences of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri typestrains, a specific PCR assay was developed so that it amplified this gene in all field strains of the two speciesanalyzed in this study but not in the other members of the M. mycoides cluster. Analysis of the PCR-amplifiedlppA genes with frequently cutting restriction enzymes showed a certain degree of genetic variability which,however, did not cluster the two subspecies. This PCR therefore allows a rapid identification of M. mycoidessubsp. mycoides LC and M. mycoides subsp. capri but does not distinguish between these two closely relatedsubspecies. LppA was expressed in Escherichia coli K-12 and used for the production of polyclonal mouseantiserum. Antibodies against recombinant LppA[MmymyLC] reacted with a 62-kDa protein in all M. mycoidessubsp. mycoides LC and M. mycoides subsp. capri type strains and field strains tested but not with the othermembers of the M. mycoides cluster, thus showing the antigenic specificity of LppA and further supporting theconcept that a close relationship exists between these two mycoplasmas.

The Mycoplasma mycoides subsp. mycoides large-colony type(LC) and M. mycoides subsp. capri strains belong to the M. my-coides cluster, a group of six closely related mycoplasmas (13).M. mycoides subsp. mycoides LC causes mastitis, keratocon-junctivitis, polyarthritis, pneumonia, and septicemia in goats(13, 14, 34, 36). It has also been isolated, rarely, from cattle (20,27) and sheep (25). M. mycoides subsp. capri is reported tocause a pattern of diseases similar to those induced by M. my-coides subsp. mycoides LC specifically in goats, including mas-titis, arthritis, and pulmonary diseases (13, 21, 23, 28, 40, 41).From an epidemiological point of view, differential identifica-tion of the members of the M. mycoides cluster is of majorimportance. An accurate and rapid means of identification ofthe subspecies or subtypes of mycoplasmas of the M. mycoidescluster is prerequisite for the differentiation, since the differentmembers of this cluster show very strong differences in viru-lence and epidemiological impact. However, many methodsfail in specificity because they are hampered by strong sero-logical cross-reactions between the different members of theM. mycoides cluster (7, 12, 15, 17, 32, 39).

It has been reported that M. mycoides subsp. mycoides LCand M. mycoides subsp. capri are antigenically very similar asassessed by numerical analysis of one-dimensional sodium do-decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

protein patterns (11, 22). Identical results have been obtainedby two-dimensional PAGE protein patterns, which confirmthat M. mycoides subsp. mycoides LC strains are more closelyrelated to M. mycoides subsp. capri strains than to M. mycoidessubsp. mycoides small-colony type (SC) strains (26, 35).

A DNA probe based on a randomly chosen genomic frag-ment was developed for the differentiation of the differentmembers of the M. mycoides cluster into four groups. ThisDNA probe grouped M. mycoides subsp. mycoides LC strainstogether with M. mycoides subsp. capri and distinguished themfrom the other members of the M. mycoides cluster. However,it did not allow differentiation between these two mycoplasmas(38). DNA-DNA hybridization studies revealed variable valuesfor DNA homology between M. mycoides subsp. mycoides LCand M. mycoides subsp. capri (75 to 94%), between M. my-coides subsp. mycoides LC and M. mycoides subsp. mycoides SC(88 to 93%), and between M. mycoides subsp. capri and M. my-coides subsp. mycoides SC (75 and 93%) depending on exper-imental conditions (2, 9). Phylogenetic studies based on se-quence analysis of 16S rRNA genes (rrs) revealed 99.9%similarity between M. mycoides subsp. mycoides LC and M. my-coides subsp. capri. These results suggested that these twomycoplasmas could be grouped into a single subspecies, onedistinct from M. mycoides subsp. mycoides SC (29).

With the objective of getting a better insight into the anti-genic and genetic differences among the different members ofthe M. mycoides cluster, the major surface-located lipoproteinantigens of M. mycoides subsp. mycoides SC, P72, and of My-coplasma sp. bovine group 7, P67, had been characterized (8,

* Corresponding author. Mailing address: Institute for VeterinaryBacteriology, University of Berne, CH-3012 Berne, Switzerland.Phone: 41 31 631 2484. Fax: 41 31 631 2634. E-mail: joachim.frey@vbi.unibe.ch.

224

16). Immunoblot analysis of type and field strains of the differ-ent species from the M. mycoides cluster by using monospecificpolyclonal antibodies against each of these proteins revealedthat these two proteins were antigenically species specific.

Southern blot hybridization with a gene probe for P72 re-vealed that the M. mycoides subsp. capri strain PG3 and theM. mycoides subsp. mycoides LC strain Y-goat as well con-tained an analogous gene. The aim of the present study wasto clone, sequence, and analyze the genes encoding the majorlipoproteins from M. mycoides subsp. capri and M. mycoidessubsp. mycoides LC.

MATERIALS AND METHODS

Strains and growth conditions. Mycoplasma strains used in this study and theirorigins are listed in Table 1. The Mycoplasma species were cultured in standardmycoplasma medium at 37°C (5). Cells were pelleted by centrifugation at20,000 3 g for 20 min, washed in TES buffer (10 mM Tris, 1 mM EDTA, 0.8%NaCl [pH 8.0]) and resuspended in TES buffer to reach a cell concentration of109 cells ml21. Escherichia coli XL1-Blue MRF9 {D(mcrA)183 D(mcrCB-hsdS-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F9 proAB laclqZDM15 Tn10(Tetr)]} and XLOLR {D(mcrA)183 D(mcrCB-hsdS-mrr)173 endA1 thi-1 recA1gyrA96 relA1 lac [F9 proAB laclqZDM15 Tn10(Tetr)]lr, Su2} (Stratagene, La Jolla,Calif.) were grown on Luria-Bertani broth at 37°C (3). E. coli YN2980 (leuUGAlacZ659UGA trpA9605UAG his-29UAG ile thyA metB argH rpoB rpsL prfB3

[pISM3001]) was used as a suppressor strain for UGATrp and was grown as de-scribed (37). Expression vector pBK-CMV phagemid (Stratagene) was propa-gated in XLOLR strain.

DNA manipulation, construction and screening of gene library, and PCR. Ge-nomic DNA was extracted by the guanidinium thiocyanate method as indicated(31). DNA from the M. mycoides subsp. mycoides LC strain Y-goat and theM. mycoides subsp. capri strain PG3 was partially digested with SauIIIA and usedto construct l ZAP Express phage banks (Stratagene) as described (8). The genelibraries were screened with a digoxigenin-11-dUTP-labeled DNA probe, andpositive clones were transformed to phagemids as indicated (16). All standardtechniques of molecular biology have been described previously (3). PCR am-plifications were carried out as indicated (16), by using the oligonucleotide prim-ers listed in Table 2.

Nucleotide sequencing and sequence analysis. Plasmids pJFFMMLC2 (fromM. mycoides subsp. mycoides LC) and pJFFMMCA4 (from M. mycoides subsp.capri) containing 2.0-kb and 4.5-kb inserts, respectively, were sequenced on bothstrands. Sequencing was performed with an Applied Biosystems DNA Sequena-tor AB373 by using the Taq Dye Deoxy Terminator Cycle kit (Applied Biosys-tems/Perkin Elmer, Norwalk, Conn.), using oligonucleotide primers matching T3and T7 promoter sequences contained on the cloning vector (Table 2). Full-length sequencing of the insert was obtained by producing an ordered nested setof deletion mutations in the same cloning vector with a Nested Deletion kitaccording to the directions of the supplier (Pharmacia Biotech, Uppsala, Swe-den) and subsequent DNA sequence analysis of the plasmids with the T3 and T7primers.

The DNA sequences and their deduced amino acid sequences were analyzedwith the program PROSITE (4) of the DNA analysis software PC/Gene and withthe program PSORT (24) and were aligned with the software GCG (Genetics

TABLE 1. Strains of mycoplasmas used and signals of PCRs for the lppA gene

Strain Species Origin Host PCR (MMMLC2-L/MMMLC1-R)e

Y-goat M. mycoides subsp. mycoides LC Type strain Goat Pos.LC8065 M. mycoides subsp. mycoides LC France Goat Pos.D 2503 M. mycoides subsp. mycoides LC Bern, Switzerland Goat Pos.D 2482/91 M. mycoides subsp. mycoides LC Bern, Switzerland Goat Pos.D 2083/91 M. mycoides subsp. mycoides LC Bern, Switzerland Goat Pos.B671/93 M. mycoides subsp. mycoides LC Portugal Cattle Pos.266/94 M. mycoides subsp. mycoides LC Gran Canaria Goat Pos.6P M. mycoides subsp. mycoides LC Gran Canaria Goat Pos.2/93 M. mycoides subsp. mycoides LC Gran Canaria Goat Pos.152/93 M. mycoides subsp. mycoides LC Gran Canaria Goat Pos.153/93 M. mycoides subsp. mycoides LC Gran Canaria Goat Pos.80x3 M. mycoides subsp. mycoides LC Gran Canaria Goat Pos.83/93 M. mycoides subsp. mycoides LC Gran Canaria Goat Pos.CP271 M. mycoides subsp. mycoides LC Portugal Goat Pos.9096-C9415 M. mycoides subsp. mycoides LC Nigeria Goat Pos.8756-13 M. mycoides subsp. mycoides LC United States Goat Pos.8794-Inde M. mycoides subsp. mycoides LC India Goat Pos.PG3 M. mycoides subsp. capri Type strain Pos.Capri N108 M. mycoides subsp. capri Nigeria Pos.Capri L M. mycoides subsp. capri France Goat Pos.9139-11/91 M. mycoides subsp. capri Turkey Pos.WK354/80 M. mycoides subsp. capria Switzerland Cattle Neg.b

PG1 M. mycoides subsp. mycoides SC Type strain Cattle Neg.Afade M. mycoides subsp. mycoides SC Chad Cattle Neg.L2 M. mycoides subsp. mycoides SC Italy Cattle Neg.B17c M. mycoides subsp. mycoides SC Chad Zebu Neg.T1/44d M. mycoides subsp. mycoides SC Tanzania Cattle Neg.PG50 Mycoplasma sp. bovine group 7 Reference strain Cattle Neg.PAD3186 Mycoplasma sp. bovine group 7 India Goat Neg.CP291 Mycoplasma sp. bovine group 7 Portugal Goat Neg.California kid M. capricolum subsp. capricolum Type strain Goat Neg.173/87 M. capricolum subsp. capricolum Greece Sheep Neg.F38 M. capricolum subsp. capripneumoniae Type strain Goat Neg.Gabes M. capricolum subsp. capripneumoniae Tunisia Goat Neg.9081-487p M. capricolum subsp. capripneumoniae Oman Goat Neg.KS1 M. putrefaciens Type strain Neg.

a Taxonomic identification unclear; originally characterized as Mycoplasma sp. bovine group 7.b Fragment of smaller size amplified.c Complement fixation test antigen.d Vaccine strain (30), passage 44.e Pos., positive; Neg., negative.

VOL. 6, 1999 LppA IN MYCOPLASMA MYCOIDES SUBSPECIES 225

Computer Group, Madison, Wis.). Sequence comparisons with GenBank/EMBLand NBRF databases were performed with the BLAST programs (1).

Preparation of recombinant LppA[MmymyLC] antigen and production ofpolyclonal mouse anti-LppA[MmymyLC] serum. The lppA gene of M. mycoidessubsp. mycoides LC cannot be expressed in its full length in E. coli K-12, due tothe mycoplasma-specific UGATrp codons (42), which are read in E. coli as stopcodons. We have therefore used an expression system which has been developedfor efficient suppression of UGATrp for the expression of mycoplasmal proteinsin E. coli (37). Strain YN2980 (37) was transformed with plasmid pJFFMMLC2and grown to the mid-exponential growth phase. Expression of the clonedlppA[MmymyLC] gene from the vector’s plac promoter was induced by additionof 1 mM isopropyl-b-D-thiogalactopyranoside and growth for 4 h further. Cellswere then harvested, washed with TES buffer, and resuspended in a 0.1 volumeof TES buffer. The resuspended cells were sonicated on ice with a BransonSonifier 250 (Branson Ultrasonics, Danbury, Conn.) equipped with the Microtipat an output of 3 for 1 min. The protein concentration of the suspension obtainedwas measured by the method of Bradford (6) and reached 400 mg ml21.

In order to obtain polyclonal anti-LppA[MmymyLC] serum, 150 ml of thesuspension described above containing 60 mg of protein per administration wasmixed with an equal volume of Freund’s complete adjuvant (Difco Laboratories,Detroit, Mich.) as described (18) and used to immunize mice. After 3 weeks themice were booster immunized with the same amount of protein, but Freund’sincomplete adjuvant was used. Blood for serum was taken 2 weeks later. Theserum was adsorbed with E. coli YN2980 carrying the empty vector pBK-CMV.The antigen for adsorption was prepared as indicated (8). Immunoblot analysiswas done according to standard protocols (3, 8). The mouse serum was diluted1:2,000, and reaction products were visualized with affinity-purified goat phos-phatase-labeled anti-mouse immunoglobulin G (heavy and light chains) (Kirke-gaard & Perry Laboratories, Gaithersburg, Md.) diluted 1:2,000.

Nucleotide sequence accession numbers. The GenBank/EMBL sequence ac-cession numbers for the lppA[MmymyLC] gene from M. mycoides subsp. my-coides LC and the lppA[Mmyca] gene from M. mycoides subsp. capri areAF072714 and AF072715, respectively.

RESULTS

Cloning of the lppA genes of M. mycoides subsp. mycoidesLC and M. mycoides subsp. capri. Southern blot analysis ofHindIII-digested genomic DNA from the type and reference

strains of all members of the M. mycoides cluster was per-formed with a digoxigenin-labeled gene probe for the P72 (lipo-protein) gene of M. mycoides subsp. mycoides SC (8). Thesehybridization results showed the presence of genes analogousto the P72 gene in all members of the M. mycoides cluster.

Gene libraries of M. mycoides subsp. mycoides LC andM. mycoides subsp. capri containing about 106 phage clonesml21 were constructed with l phage ZAP Express vectorand screened. From both libraries, plasmids which wereshown to contain the entire genes of the lipoproteins namedlppA[MmymyLC] and lppA[Mmyca], as assessed from sequenc-ing data of the extremities of the inserts, were retained. Plas-mid pJFFMMLC2 contained a 2.0-kb insert from M. mycoidessubsp. mycoides LC, and plasmid pJFFMMCA4 contained a4.5-kb insert from M. mycoides subsp. capri (Fig. 1). The in-tegrity of the inserts of these plasmids was verified by PCR am-plification of the corresponding fragments from genomic DNAof M. mycoides subsp. mycoides LC and from M. mycoidessubsp. capri by using primers matching the sequenced extrem-ities of the inserts (Table 2).

DNA sequence analysis of the lipoprotein genes and offlanking genes. The inserts of plasmids pJFFMMLC2 andpJFFMMCA4 were sequenced in both directions. The nucle-otide sequence of the insert pJFFMMLC2 contained an openreading frame (ORF) encoding the lipoprotein LppA[Mmy-myLC] precursor of 526 amino acid (aa) residues with a pre-dicted molecular mass of 60.288 kDa (Fig. 2). It is preceded bya consensus sequence for a ribosome binding site (RBS) lo-cated five nucleotides (nt) upstream of the initiation codon,AUG. The analogous ORF in plasmid pJFFMMCA4 encodedthe lipoprotein LppA[Mmyca] precursor, which shows 523 aaresidues and a predicted molecular mass for LppA[Mmyca] of

FIG. 1. Locations of the lppA gene (black boxes) and surrounding genes on plasmids pJFFMMLC2 containing a 2.0-kb insert cloned from the DNA of theM. mycoides subsp. mycoides LC strain Y-goat (a) and pJFFMMCA4 containing a 4.5-kb insert cloned from the DNA of the M. mycoides subsp. capri strain PG3 (b).The ORFs are represented by white boxes, and arrowheads indicate the direction of transcription and translation. The locations of the oligonucleotide primers areshown by arrows. Filled triangles indicate the location of plac promoter of the cloning vector.

TABLE 2. Primer pairs used in PCR assays and for DNA sequencing

Primer paira Nucleotidepositionsb Sequence Annealing

temperature (°C)Fragmentsize (bp)

MMMLC2-L 668–689 59-CAATCCAGATCATAAAAAACCT-39 49 1,049MMMLC1-R 1716–1697 59-CTCCTCATATTCCCCTAGAA-39

JFFMMCA4-L 141–160 59-GATTTAACTTCTCATGCAAC-39 47 4,403JFFMMCA4-R 4543–4522 59-AAGTAAAAATGAAAGAATTTTA-39

T3 T3 promoter 59-GCGCGCAATTAACCCTCACTAAAG-39T7 T7 promoter 59-GTAATACGACTCACTATAGGGC-39

a The primer pairs were used as follows: MMMLC2-L and MMMLC1-R, for specific lppA[MmymyLC] and lppA[Mmyca] gene amplification; JFFMMCA4-L andJFFMMCA4-R, for amplification of genomic DNA for the verification of the integrity of the insert of pJFFMMCA4; and T3 and T7, for DNA sequencing.

b For MMMLC2-L and MMMLC1-R, the nucleotide positions correspond to those in the pJFFMMLC2 insert according to GenBank accession no. AF072714, andfor JFFMMCA4-L and JFFMMCA4-R, they correspond to those in the pJFFMMCA4 insert according to GenBank accession no. AF072715.

226 MONNERAT ET AL. CLIN. DIAGN. LAB. IMMUNOL.

59.822 kDa (Fig. 2). This ORF is preceded 6 bp upstream by acanonical RBS sequence. The two lppA genes contain myco-plasma-specific UGATrp codons corresponding to aa 147 and415 and to aa 144 and 412, respectively, and are terminated byUAA stop codons. Analysis of the amino acid sequences ofLppA indicated a consensus sequence for a potential recogni-tion site of a prokaryotic signal peptidase II (19) at aa 21 to 25and a lipid attachment site at the cysteine residue starting at aa25 (Fig. 2). A potential transmembrane region is located at aaresidues 7 through 23 of the leader sequence. The amino acidsequences of the mature LppA[MmymyLC] and LppA[Mmyca]are virtually the same. They have 94% identical and 95%positive (identical 1 similar) amino acids. Differences aremainly located at the N terminals of the proteins, where a fewdeletions and substitutions are located (Fig. 2). The matureLppA proteins of both M. mycoides subsp. mycoides LC andM. mycoides subsp. capri showed a similarity (42 to 44% iden-tity and 50 to 52% positivity for aa residues) to P72 of M. my-coides subsp. mycoides SC.

The sequence analysis of both plasmids pJFFMMLC2 andpJFFMMCA4 revealed a part of an ORF, ORF1, upstream oflppA. Its function is currently unknown. However, ORF1 inboth M. mycoides subsp. mycoides LC and M. mycoides subsp.capri showed a high similarity to the ORF upstream of the geneencoding P72 in M. mycoides subsp. mycoides SC (8). TheORF1 of M. mycoides subsp. mycoides LC showed 52% iden-tical amino acids and 69% identical nucleotides at the DNAlevel with the ORF1 of M. mycoides subsp. capri. In bothplasmid clones, the lppA gene is followed by a hairpin structurerepresenting a potential transcriptional termination signal(Fig. 1). In pJFFMMCA4, the hairpin structure was followedby two ORFs that sequenced in the opposite direction; one,ORFX, encodes a polypeptide with unknown function, and thesecond has 63% identical nucleotides with a DNA methylase inSpiroplasma species (accession no. X17195) (33) (Fig. 1). Up-stream of the potential DNA methylase gene we found a hair-pin structure that acts as a transcription stop signal and is partof an ORF showing 93% identical nucleotides to the mtlD(which encodes mannitol-1-phosphate dehydrogenase) geneanalogue found in M. mycoides subsp. mycoides SC (8) (Fig. 1).In pJFFMMLC2, an ORFX was found downstream of thetranscription stop signal following lppA. It showed 89% iden-tical nucleotides to the analogous location in M. mycoidessubsp. capri (Fig. 1).

Expression and serological specificity of LppA of M. my-coides subsp. mycoides LC and M. mycoides subsp. capri. Theexpression and the antigenic specificity of LppA in M. mycoidessubsp. mycoides LC and M. mycoides subsp. capri were ana-lyzed on immunoblots by using adsorbed polyclonal mouseantibodies directed against recombinant LppA[MmymyLC].Whole cells of the M. mycoides subsp. mycoides LC and M. my-coides subsp. capri type strains and field strains, as well as thetype and reference strains of the other members of the M. my-coides cluster (Table 1), were solubilized in SDS sample bufferand used as antigens on the immunoblots. The mouse anti-LppA[MmymyLC] serum strongly reacted with a 62-kDa bandor a 60- and 62-kDa doublet for all strains of M. mycoidessubsp. mycoides LC and M. mycoides subsp. capri tested exceptstrain WK354/80. The apparent molecular mass of LppA onSDS polyacrylamide gels (62 kDa) is slightly higher thanthe expected molecular mass as calculated from its DNA se-quence-deduced amino acid composition (60 kDa). StrainWK354/80, for which the taxonomic classification is not clear,showed weak reactions with a doublet band of 58 and 60 kDa.The other mycoplasmas of the M. mycoides cluster did notreact with anti-LppA[MmymyLC] antibodies, thus showingthe antigenic specificity of LppA[MmymyLC] to M. mycoidessubsp. mycoides LC and M. mycoides subsp. capri (Fig. 3).

Specificity of lppA to M. mycoides subsp. mycoides LC andM. mycoides subsp. capri. The presence of lppA[MmymyLC]and lppA[Mmyca] was studied in M. mycoides subsp. mycoidesLC and M. mycoides subsp. capri strains and in the othermembers of the M. mycoides cluster by PCR amplification withthe primer pair MMMLC2-L and MMMLC1-R (Table 2) byusing as the templates chromosomal DNA from all myco-plasma strains used in this study (Table 1). All strains of M. my-coides subsp. mycoides LC and M. mycoides subsp. capri showedthe expected DNA fragment of 1.05 kb with the exception ofstrain WK354/80, which amplified a smaller fragment (0.85kb). The PCR product obtained from WK354/80 was analyzedfurther by DNA sequencing. DNA sequencing results showedthat the edges of the PCR fragment but not the central part ofthe segment were identical to lppA[MmymyLC] and lppA[Mmyca]. In total, this 0.85-kb fragment showed 63 to 64%similarity to lppA[MmymyLC] and lppA[Mmyca], 95% similar-ity to the P67 gene of the Mycoplasma sp. bovine group 7,and 93% similarity to the P72 gene of M. mycoides subsp.mycoides SC. None of the other mycoplasmas of the M. my-

FIG. 2. Amino acid sequence comparison of LppA precursors from the M. mycoides subsp. mycoides LC strain Y-goat and from the M. mycoides subsp. capri strainPG3. Underlined amino acids correspond to the consensus sequence for the signal peptidase II recognition site. The arrow indicates the potential cleavage site for thispeptidase. Vertical bars show identical amino acids, and dots between sequences show similar amino acids.

VOL. 6, 1999 LppA IN MYCOPLASMA MYCOIDES SUBSPECIES 227

coides cluster analyzed showed any amplification product (Fig.4 and Table 1). These results confirmed that the primer pairMMMLC2-L and MMMLC1-R amplified a specific 1.05-kbfragment from M. mycoides subsp. mycoides LC and M. my-coides subsp. capri. For further analysis, the PCR amplifica-tion products of the lppA[MmymyLC] and lppA[Mmyca]genes were digested with the restriction enzyme AluI and ex-amined (Fig. 5). Profiles obtained from the different strainsshowed some variations, indicating a certain heterogeneity ofthe lppA gene within the different strains. The profiles showthat the same differences are found within strains of the samesubspecies as well as among strains of different subspecies andhence do not differentiate M. mycoides subsp. mycoides LCstrains from M. mycoides subsp. capri strains.

DISCUSSION

Sequence data show that the lipoproteins LppA[MmymyLC]and LppA[Mmyca] form a family together with the major li-poproteins of M. mycoides subsp. mycoides SC, P72, and of

Mycoplasma sp. bovine group 7, P67, and are hence suggestedto have a function which is analogous to the two latter lipopro-teins. Accordingly, we propose that P72 and P67 be renamedlipoproteins LppA[MmymySC] and LppA[Mbgr7], respectively.

Analysis of LppA from both M. mycoides subsp. mycoides LCand M. mycoides subsp. capri showed that the potential transmem-brane region located in the leader sequence of LppA[Mmyca] isidentical to that found in LppA[MmymySC] (P72) of M. mycoidessubsp. mycoides SC and in LppA[Mbgr7] (P67) of Mycoplasma sp.bovine group 7 (8, 16) and differs only in a single amino acid(residue 11) from the leader sequence of LppA[MmymyLC]. Thecorresponding gene fragment is also highly conserved and couldtherefore serve as a valuable probe for the cloning of other my-coplasmal lipoprotein genes.

DNA sequence analysis of the lppA genes of M. mycoidessubsp. mycoides LC and M. mycoides subsp. capri revealed avery high degree of similarity which is also reflected antigeni-cally, as shown on immunoblots. Minor variations, which canbe observed in the lppA genes of various field strains, are notspecific to the two subspecies. The differences seem to have

FIG. 3. Expression of lipoprotein LppA[MmymyLC] and LppA[Mmyca] in mycoplasmas of the M. mycoides cluster (a) and in the M. mycoides subsp. mycoides LCand M. mycoides subsp. capri field strains (b and c, respectively). Immunoblots containing total antigens of the different mycoplasmas (Table 1) were probed withanti-LppA[MmymyLC] serum from a mouse immunized with recombinant LppA[MmymyLC]. The positions of the lipoproteins LppA[MmymyLC] and LppA[Mmyca]and of molecular mass standards (broad range) (Bio-Rad Laboratories) are indicated. Certain strains showed a doublet band reacting with anti-LppA[MmymyLC]antibodies (b). We interpret these doublets to be due to posttranslational modification of these lipoproteins. In certain strains, no doublets are visible, which might bedue to the fact that the cells were taken in a late stage of cell growth. Secondary bands with molecular masses not within the 60 to 62 kDa range are interpreted asbackground reactions due to nonspecific serological reaction of the mouse sera. The faint doublet at 58 and 60 kDa seen with strain WK354/80 is supposed to be dueto a lipoprotein closely related to, but different from, LppA[MmymyLC] and LppA[Mmyca].

FIG. 4. PCR fragments obtained from amplification with the primer pairMMMLC2-L and MMMLC1-R and genomic template DNA from type andreference strains of the M. mycoides cluster and from the M. mycoides subsp.mycoides LC and M. mycoides subsp. capri field strains (Table 1). As size markers,HindIII-digested l DNA fragments were used. Their sizes are given in kilobaseson the left side.

FIG. 5. Restriction fragment analysis of PCR-amplified lppA genes from theM. mycoides subsp. mycoides LC and M. mycoides subsp. capri strains (Table 1).PCR products of DNA from type strains and field strains were digested with AluIand analyzed by electrophoresis on 8% polyacrylamide gels. The standards usedwere HinfI-digested pBR322 fragments. Their sizes are indicated in base pairs.

228 MONNERAT ET AL. CLIN. DIAGN. LAB. IMMUNOL.

only minor phenotypic impact since they are not reflected an-tigenically as revealed by immunoblots. Genetic variation amongdifferent field isolates from M. mycoides subsp. mycoides LCand M. mycoides subsp. capri has also been detected in othergene loci (40a). Hence, LppA seems to be a common andspecific antigen of the two closely related mycoplasmas M. my-coides subsp. mycoides LC and M. mycoides subsp. capri. Thechromosomal location of the lppA genes seems to be conservedin the vicinity of the mtlD gene (which encodes mannitol-1-phosphate dehydrogenase).

The specific PCR using the primer pair MMMLC2-L andMMMLC1-R amplified the lppA genes from M. mycoidessubsp. mycoides LC and M. mycoides subsp. capri field strainsof various geographic origins but not those from other mem-bers of the M. mycoides cluster. We therefore propose that thisPCR be used for the identification of the phylogenetic andantigenic entity M. mycoides subsp. mycoides LC/M. mycoidessubsp. capri.

Interestingly, for strain WK354/80, PCR amplification of thelppA gene resulted in a shorter-than-expected fragment whichwas shown to be most similar to the lppA[Mbgr7] (P67) gene ofMycoplasma sp. bovine group 7. WK354/80 strain was first de-scribed in the literature as Mycoplasma sp. bovine group 7 (10)and was later retyped by us as M. mycoides subsp. capri. Se-quence analysis of the rrs gene of strain WK354/80, on theother hand, showed its strong similarity to that of M. mycoidessubsp. mycoides LC. These results reflect the ambiguous taxo-nomic status of WK354/80, which must be located intermediatebetween those of Mycoplasma sp. bovine group 7 and M. my-coides subsp. mycoides LC/M. mycoides subsp. capri. It illus-trates further the good discriminatory potential of the lppAgenes, which encode the major lipoproteins, in the differenti-ation of the members of the M. mycoides cluster.

ACKNOWLEDGMENTS

We are grateful to Yvonne Schlatter for technical assistance withDNA sequence analysis and PCR and to Margrit Krawinkler for experthelp with identification and cultivation of mycoplasmas. We thankChris Minion, Ames, Iowa, for the kind gift of strain YN2980, whichproved to be most helpful for the expression of cloned mycoplasmalgenes, and Shmuel Razin, Jerusalem, Israel, Kevin Dybvig, Birming-ham, Alabama, and Karl-Erik Johansson, Uppsala, Sweden, for theirhelpful suggestions in naming lipoproteins.

This study is part of European COST action 826 on ruminants’mycoplasmoses and was supported by grant C96.0073 of the SwissMinistry of Education and Science and by the Swiss Federal VeterinaryOffice.

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