Species differentiation of mycoplasmas by EF-Tu specific monoclonal antibodies

Journal of Immunological Methods, 147 (1992) 73-81 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00

73

JIM 06192

Species differentiation of mycoplasmas by EF-Tu specific monoclonal antibodies Volker Kamla, Birgit Henrich and Ulrich Hadding Institute for Medical Microbiology and Virology, Heinrich-Heme-University, 4000 DUsseldorf, Germany

(Received 22 July 1991, revised received 10 September 1991, accepted 17 October 1991)

Ten mouse hybridoma cell lines producing IgG monoclonal antibodies to mycoplasmal elongation factor Tu (EF-Tu) were established. These mAbs showed different degrees of cross-reactivity between mollicutes and even other bacteria. This finding, indicating protein structure diversities of pan bacterial EF-Tu should permit species differentiation of mycoplasmas by epitope pattern analysis of a single protein. Epitope patterns of 23 mollicute type strains and of 40 M hominis isolates were determined by ELISA. All M hominis patterns were found to be closely related whereas intrageneric patterns differed in a species specific manner. KEy words: Mycoplasma; Elongation factor Tu; Monoclonal antibody; Species differentiation of mycoplasmas

Introduction Mycoplasmas or mollicutes are the smallest living bacteria. Some species are known as plant and insect pathogens, others cause veterinary and human diseases or live as apathogenic commensals in animal and man. Beside this they are well known tissue culture contaminants, capable of changing several properties of eUkaryotic host cells (McGarrity et aI., 1984). The diagnosis of

Correspondence to: V. Kamla, Institut fUr Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universitiit, Moorenstrasse 5. 4000 Dusseldorf, Germany Tel.: 0211-311 2460; Fax: 0211-342229). Abbreviations: EC. enzyme commission; IEC, ion exchange chromatography; kOa. kilodalton; SDS-PAGE. sodium dodecyl sulphate polyacrylamide gel electrophoresis; PBS. phosphate-buffered saline (10 mM Na2HP04 /NaH 2 P04 , 140 mM NaO, pH 7.4); PPLO, pleuropneumoniae like organisms; RT, room temperature; TBS. Tris-buffered saline (200 mM NaQ, SO mM Tris-Ho, pH 7.5).

mycoplasmas includes testing of biochemical and metabolic properties, culture examination, evaluation of colony morphology, detection of serum antibodies and in some cases detection with specific gene probes using the polymerase chain reaction (Bernet et aI., 1989; Jensen et aI., 1989; Kunita et aI., 1990). Recently we described the detection of tissue culture mycoplasmas by an immunofluorescence assay using a single monoclonal antibody (mAb). This mAb CCM-2 recognizes a common antigen shared by most mycoplasmas (Blazek et aI., 1990). Nucleic acid sequencing of the encoding gene from M. hominis, cloned and expressed in E. coli showed this mycoplasma common antigen to be elongation factor Tu (EF-Tu) (Liineberg et aI., 1991). This protein, essential for all prokaryotes, mediates the transport of aminoacyl-tRNA to the codon recognition site of ribosomes (Kaziro, 1978). EF-Tu is in part responsible for the fidelity of translation (Tapio and Kurland, 1986). It has been shown by hybridization experiments (Filer

74

et aI., 1981) and by sequence comparisons that EF-Tu encoding tuf genes are highly conserved within Gram-positive and Gram-negative prokaryotes, cyanobacteria and even in plastids and mitochondria (Seidler et aI., 1987; Yogev et aI., 1990). In this study we report the generation and application of a set of monoclonal antibodies (mAb) directed against the EF-Tu of mycoplasmas. These antibodies exhibited specificity differences reflecting sequence diversities at the epitope level, which could be measured by ELISA. Such species-specific epitope patterns of EF-Tu should permit Mycoplasma differentiation.

Materials and methods

Mycoplasma strains and culture media The following type strains were obtained from the National Collection of Type Cultures (NCTC, London, UK):

Acholeplasma axonthum NCTC 10138,

A. /aidlaw;i

NCTC 10116,

Mycoplasma arginini NCTC 10129,

M. bods NCTC 10131

M. buccale NCTC 10136

M. capricolum NCTC 10154

M. fermentans

NCTC 10117

M. gallisepticum NCTC 10115 M. hyorhinis NCTC 10130 M. /ipophilum NCTC 10173 M. pneumoniae NCTC 10119 M. pulmonis NCTC 10139

A. granularum NCTC 10128,

A. modicum

NCTC 10134,

M. arthritidis

NCTC 10162,

M.bovoculi NCTC 10141

M. canis NCTC 10146

M·faucium NCTC 10174

M. gallina rum

NCTC 10120 M. hominis PG21 NCTC 10111

M. neurolyticum NCTC 10166

M. orale

NCTCI0112

M. prima tum

NCTC 10163

M. salivarium NCTC 10113.

Clinical isolates of M. hominis and all nonmollicute bacteria were from our departmental stocks.

Antigen preparation Lyophilized mycoplasma species were inoculated in 10 mt broth medium recommended for each species by the American Type Culture Collection (ATCC). Precultures of mycoplasmas were diluted 1/100 in a conventional mycoplasma broth medium consisting of PPLO broth base (Difco, Detroit, MI), 20% heat-inactivated mycoplasmafree horse serum (Gibco, Grand Island, NY), 10% fresh yeast extract (Flow, Meckenheim, Germany), 1% glucose or arginine (Merck, Darmstadt, Germany), 0.002% phenol red (Merck). The final pH was adjusted to 6.5 (arginine medium) or 7.5 (glucose medium). Mycoplasma cells were centrifuged 05,000 x g, 30 min, 4 0 C). Pellets were resuspended in phosphate-buffered saline pH 7.4 (PBS), followed by centrifugation. After a second wash cells were lysed osmotically in distilled water by repeatedly freezing and thawing. Protein amounts were determined by the method of Bradford (976).

Purification of M. hominis proteins For the sequencing of N terminal amino acids the proteins of M. hominis were purified by ion exchange chromatography (I EC). Sonicated cell extract was applied to Mono Q 5/5 (Pharmacia LKB, Freiburg, Germany) in 20 mM Tris-HO, pH 7.6. Bound protein was eluted by a linear sodium chloride gradient.

N terminal amino acid sequencing IEC-purified protein of M. hominis was fractionated by SOS-PAGE and electroblotted to Immobilon membrane (Millipore, Bedford, MA, USA). After staining with amido black lOB (Merck) the desired protein band was cut out and analysed on a 477A Protein Sequencer (Applied Biosystems) to determine the N terminal amino acid sequence.

Affinity chromatography of M. hominis EF- Th The mAb CCM-2 was purified from ascites fluid by ammonium sulphate precipitation. Antibody (5 mg) was coupled to O.S g CNBr-activatcd

75

Sepharose 4B (Pharmacia LKB) following the instructions of the manufacturer. This affinity column was charged with 30 mg protein equivalents of M. hominis cell extract diluted in 10 ml PBS. Bound protein was eluted with 2.5 M MgCI 2 , chromatographically transferred in PBS and yielded about 500 ILg purified EF-Tu. Production of hybridoma cells and monoclonal antibodies After immunization of 3-4 months old female BALB/c mice hybridoma cells were generated by PEG-fusion of spleen cells with the murine myeloma cell line X63Ag8 653. The generation of hybridomas, tissue culture techniques and production of mAb have been described earlier (Fazekas de St. Groth and Scheidegger, 1980; Blazek et aI., 1990). Microtitre plate enzyme immunoassay-ELISA For antibody capture ELISA Maxisorb microtitre plates (Nunc, Wiesbaden, Germany) were used. Incubation steps were performed at room temperature (RT) with agitation, followed by washing the wells three times with PBS. Sonicated cell extracts were diluted to final concentrations of 20 ILg/ml protein in coating buffer (50 mM Na 4 B40 7 , 150 mM NaCI, pH 9.0). Each well was coated with 1ILg (50 ILl) antigen for 2 h, then blocked with 100 ILl of 1% skim milk (Oxoid, Basingstoke, Hampshire, England) in PBS for 1 h. Hybridoma culture supernatants (50 ILl/well) diluted if required with 1% skim milk were added and incubated for 1 h. After incubating for 1 h with 50 ILl/well rabbit anti-mouse peroxidase conjugated antibodies (Dianova, Hamburg, Germany) diluted 1/5000 with 1% skim milk, enzyme reaction was performed by incubation in the dark with 100 ILl/well of freshly prepared peroxidase substrate (50 rnI 0.1 M citrate, pH 5.1,50 JLI 30% H 2 0 2 , 50 mg o-phenylene-diamine). Mter 30 min the reaction was stopped by adding 100 ILl/well 2 M HCI. Absorbance was measured with an NJ2000 ELISA Reader (Nunc) at 490 nm wavelength. Determination of mAb isotypes Isotypes were determined in a variant of the above described antibody capture ELISA MAb

bound to solid phase antigen of M hominis PG21 were labelled by consecutive incubations with biotinylated class-specific and subclass-specific goat antimouse antibodies (Dianova, Hamburg, Germany) and peroxidase conjugated streptavidin prior to the enzyme reaction. SDS-PAGE and immunoblot assay Denaturation of protein samples and electrophoresis were carried out according to the method of Laemmli (1970). Proteins were transferred to nitrocellulose or Immobilon filters using a semi dry blot apparatus (Phase, Molin, Germany). For immunostaining the filters were blocked with 5% skim milk in PBS, and then incubated with mAb hybridoma supernatant and finally with rabbit anti-mouse peroxidase conjugated antibody (Dianova) diluted 1/5000 in blocking solution. Each incubation step was performed for 1 h at RT with gentle agitation. Mter washing two times with PBS the filters were developed by incubating for 1 h in freshly prepared substrate (50 mg 4-cliloro-1-naphthol in 10 ml methanol, 50 ml TBS and 50 ILl 30% H 2 0 2 ). The reaction was stopped by washing the filters in water. Sequence alignment and software Protein sequence data were taken from the EMBL database (EMBL, Heidelberg, Germany) except for the sequences of M hominis from our paper (LUneberg et aI., 1991) and M. pneumoniae (Yogev et aI., 1990), M. genitalium (Loechel et aI., 1989), M. gallisepticum Onamine et aI., 1989). Sequence alignment was done using the computer program package CLUSTAL (Higgins and Sharp, 1989). Evaluation of ELISA data and generation of epitope pattern diagrams were performed with programs developed by one of us (VK).

Results Identification of mycoplasma common antigen From cell extracts of M hominis the 43 kDa antigen defined by monoclonal antibody CCM-2 (Blazek et aI., 1990) was purified by ion exchange chromatography. P43-antigen eluted at 230 mM

76

NaCl and was subjected to SOS-PAGE prior to microsequencing. The N terminal amino acid sequence shows strong homology to sequences of prokaryotic elongation factor Tu deduced from the nucleic acid sequence data of several species (Table I). The determination of the nucleic acid sequence of the corresponding gene (Liineberg et aI., 1991) confirmed that the mycoplasma common antigen is identical to the elongation factor Tu (EF-Tu). Generation of monoclonal antibodies against EFTu We produced five murine mAbs directed against EF-Tu by immunization with purified EFTu from M. hominis PG21. Five additional antiEF-Tu mAbs were generated by immunizing with cell extracts of M. hyorhinis. M. orale. A. laidlawii and M. hominis 7447 (clinical isolate). The designation, isotype and immunizing antigen of all mAbs are listed in Table II. Specificity of monoclonal a~tibodies The specificities of all mAbs for EF-Tu were tested by immunoblot analysis. All of them detected EF-Tu from M. hominis purified byaffinity chromatography (Fig. O. Several proteins of lower molecular weight were detected by nine mAbs. These mAb-reactive proteins were be-

TABLE II DESIGNATION, ISOTYPE AND IMMUNIZING ANTIGEN OF TEN MONOCLONAL ANTIBODIES DIRECTED AGAINST MYCOPLASMAL EF-Tu Designation

Isolype

Immunizing antigen

BAS CA6 GCI ME3 MF9 HE6 FE3 KD2(CCM-2) GBB RB3

IgGIK IgGlA IgGIK IgGlK IgGlK IgGIK IgGIK IgGIK IgGIK IgGIK

M. hominis purified EF-Tu M. hominis purified EF-Tu M. homims purified EF-Tu M. hominis purified EF-Tu M. hominis purified EF-Tu M. hominis 7447 clinical isolate M. orale cell extract M. orale cell extract M. hyorhinis cell extract A. la;dlaw;; cell extract

Iieved to be EF-Tu fragments since their amounts increased after incubation of purified EF-Tu in acid buffer (pH 3.7 for 2 h) suggesting that they arose as a result of protein degradation. Peptide mapping (Cleveland et aI., 1977) of EF-Tu and the large immunoreactive fragment of 37 kOa produced by endoprotease V-8 (E.C. 3.4.21.19) cleavage revealed several proteolytical fragments of identical length when partially digested, suggesting that the 37 kOa protein was part of EF-Tu (data not shown). Cross reactivity of the mAbs with other mycoplasmal species was tested by immunoblot anal-

TABLE I COMPARISON OF THE N TERMINAL AMINO ACID SEQUENCE OF MYCOPLASMA COMMON ANTIGEN WITH EF-Tu SEQUENCES OF SEVERAL SPECIES The amino acids indicated by question marks could not be determined. Minus signs indicate gaps which were inserted in order to reach optimal alignment. Abbreviations: M., Mycoplasma; S., Saccharomyces; Eugl., Euglena. N terminal 43 kDa antigen

M. homims PG21 M. gallisepticum M. genitalium M. pneumonille Escherichia coli Micrococcus IUleus Melhanococcus L'anielli Thermus thermophl/US Thermus aquolicus and Thermothoga maritima Spirulina pialensis S. cereL'isille (mitochondria) Eugl. gracilis (plastids)

? "AIeL "AKE "ARE "ARE -SKE "AKA "AKT "AK6

?FDRSKPHVN DFDRSKPHVN RFDRSKPHVN KFDRSKPHVN KFDRSKPQLN KFERTKPHVN KFERTKAHVN K-----PILN EFVRTKPHVN

IGTIGHVDDG IGTIGHVDHG IGTIGHIDHG VGTIGHIDHG VGTIGHIDHG VGTIGHVDHG IGTIGHVDHG VAF IGHVDAG V6TIGHVDHG

K-T K-TT-----L K-TT-----L K-TT-----L K-TT-----L K-TT-----L K-TT-----L KSTTVGRLLL K-TT-----L

TUIATVL-TAAICTVL TAAICTVL-TAAICTVL-TAAIT----TAAISKVLYO DGGAIDPQLI TAU TV----

"AKE "ARA SYAA "AU

KFVRTKPHVN KFERNKPHVN AFDRSKPHVN KFERTKPHIN

VGTIGHIDHG IGTIGHVDHG IGTIGHVDHG IGTIGHVDHG

K-ST-----L K-TT-----L K-TT-----L K-TT-----L

TAAlTKYLSL TAAIT----TAAlTKTLU TUlT-----

77

ysis. Fig . .2 shows two typical hlots immunostained with two anti EF-Tu mAhs: GCI, which reacts with only a few species (top) and ME3, which detects most mycoplasma species (hottom). The molecular weights of the immunoreactive EF-Tu hand differed hetween species. All mAhs showed different degrees of cross reactivity except for mAhs BAS and GCI which recognized EF-Tu of the same suhset of species. MAhs CA6, GBS, KD.2. ME3 and RB3 reacted with antigen from non-mollicutes and the latter three also detected EF-Tu of Acholeplasma. No reaction was ohserved with M. fallcium antigen, and no reaction occurred with any mAh to cell extracts of the murine myeloma cell line X63AgS 653 and to several human cell lines. In order to confirm and quantify these cross reactivity data an antibody capture ELISA was estahlished using cell extract antigens in excess as

BAS

CA6

FE3

GBa

GC1

HE6

the solid phase. MAhs were prediluted to reach an ahsorhance of 2.0-2.5 with M. hominis PG21 antigens. In each test plate M. 110minis PG21 antigen served as a mAh-specific positive control. Using these conditions we ohtained reproducihle quantitative differences in the reactivities of antigen from 23 type strain mycoplasmas with all tcn anti-EF-Tu mAbs. Fig. 3 (left) shows epitope pattern diagrams of type strains derived by ELISA data. These reactivity patterns differed in some cases from the immunohlot data: mAb HE6 reacted only with antigen from M. hominis in ELISA, but detected EF-Tu from 9 of 24 species hy immunoblot. Similar differences were found using mAb MF9, which detected three spccies by ELISA and seven species by immunoblot. In hoth cases the epitopes were presumahly only accessible when the protein was denaturated. On the other hand M. faucium antigen reacted only with

KD2

ME3

RB3

MF9

A B A B A B A BA B A B AB AB A B A B

Coomassie A B M

· 97.4 • 66.2

~I

· 45

• 31

· 21.5 · 14.4 Fig. I. Immunoblot analysis of mAb specificity for EF-Tu. A: M. hominis EF-Tu. purified by affinity chromatography. 2 j.Lg EF-Tu/lane: B: M. homillis cell extract. 10 j.Lg protein/lane. M: molecular weight marker (Bio-Rad, Munich. Germany).

78

mAb GBS in ELISA and with no mAb in the immunoblot, suggesting a native structure for this epitope. In order to determine reactivity pattern differences within one species 40 clinical isolates of M. hominis were tested in the same manner. All isolates reacted qualitatively with all ten mAbs as did type strain M. hominis PG21, but seven mAbs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

(BAS, CA6, GB8, GCI, HE6, ME3, RB3) exhibited considerable quantitative differences in absorbance between several strains. A selectio~ of reaction patterns of 23 M. hominis isolates is illustrated in Fig. 3 (right). In Fig. 4 the epitope pattern diagrams are represented in one graph for one group at a time in order to visualize the frequency of mAb-bind19 20 21 22 23 24 2526 27 28 29 30 31 32 • 116.5

80.0 49.5

- 32.5 - 27.5 • 18.5

1 2

3 4

5 6 7

8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 2425 28 27 28 29 30 31 32 • 116.5

- 80.0 - 49.5

• 32.5 • 27.5

- 18.5

Fi,. 2. Immunoblot analysis of cross reactivity of mAbs GCI (top) and ME3 (bottom) with different mycoplasma species (8 1"

protein per lane): Acholep/4smo tutlIIIhum (1), A. gnuwlGrum (2), A. lIIidlIIwjj (3), A. modicum (4), Mycoplama IU'fiItbti (5), M.

arthritidis (6). M. boois (7). M. bovocu/i (8). M. buccale (9). M. ca";' (10), M. capricoium (10. M. laucium (12), M. formmtflllS (13). M. ga/Jinanun (14). M. gG/Ji.repticum (1S). M. homini.r (16), M; hyorhini.r (17), M. IIpophiJum (18), M. ()I'tI/e (19), M. ptIftIIIIOIIiM (20), M. primIItum (21), M. pu/monis (22), M. IGlivarium (23), prestalned molecular welabt marker (Siama,

Deisenhofen Germany) (24), murine myeloma cellliue X63A18 653 (25), EICMrichlG coli (26), Enttrobackr clotlcflt (27) ~ ptttU1IIOIIiM (28), NtUItriII ~ (29), N. mtningitidi.r (30), ProttIU mirabilis (30, PltudontOftlll atf'UlinolG (32).

Mollicute type strain species

21124

ru

0

4980

~

[L

D

21260

~

-D

r=1

24280

[L..r

0

0

7447

ru

1636

~

894

~

18891

~J

2760

~

10938

~

1014

~

19781

~

13932

~

&278

~

9597

C

M ••rginini

rt----J

M .•rthritidla

~ jL,

M.bovla M.bovoculi M.buecal. M.eanla

D

.r--, 0 £::J

M.f.ueium

M.g.llin.rum M.galilaepticum M.hyorhinla

n

n 0

M.e.prlcolum

M.fermentanl

I

n 0

0 0 0 0 n

...D

~

IL.n

D

c=-

ILJl IL-, 0

D-

IL ...Sl

0

[L [b 0 n M.neurolytlcum 0 --11 ru- ] [L n M.or", M.lipophilum

0

-

D

M.pneumonlae M.primltum

n n

M.pulmonla

CJ

M ....Iv.rium

0

0

L.rL,

--r=uL, ~

~

r--r-

L.JL-r-J

0

7089

~

~

....,

11417

[

0

1189

0

071

0 .....fL

0

0

A.granul.rum

D

r=1

A.I.icIl.wi

0

0

.D

12

0 0 £::J

1 2 34& 878 910

019

79

~

[b

A.axanthum

A.modlcum

M.hominis clinical isolates

~

lJI--rI

~:

0117

~

034

~ , 2 3 4 5 6 7 8 9 10

Fig. 3. EUSA reactivity patterns of mycoplasma type strain species (left) and M. hominis isolates sorted by pattern similarities (right). The bars represent the ratio between absorbance reached with cell extract antigen from the test species and absorbance with M. hominis PGll antigens. All measurements were performed in duplicate. Bars symbolize absorbance ratios of mAbs in the following order: (I) BAS, (2) CA6, (3) FE3, (4) GB8, (5) GCI, (6) HE6, (7) KD2, (8) ME3, (9) MF9, (10): RB3.

80

Mollicute type strain species

BAS CA6 FE3 GBS GC1 HE6 KD2 ME3 MF9 RB3

M.hominis clinical isolates

BAS CAS FE3 GBS GC1 HES KD2 ME3 MF9 RI3

Fig. 4. Heterogeneity in epitope patterns of 23 different mycoplasma type strain species (top) and 40 M. hominis isolates (bottom) with anti·EF-Tu mAbs. Epitope pattern diagrams of both groups in Fig. 3 are represented in one graph for each. Bars symbolize overlayed absorbance ratios between absorbance of cell extract antigens from the test organism and from M. hominis P021.

ing to any type strain (top) or any M hominis isolate (bottom), respectively. The bar-heights of type strains were scattered over the total range whereas they were clustered close to the means in the case of M. hominis isolates.

Discussion We have identified a mycoplasma common protein antigen with an apparent molecular weight ranging from 42 to 48 kDa in different species. Nucleic acid sequence analysis revealed that this protein was elongation factor Tu. Recently, Sasaki (1991) reported the production of two monospecific mouse antisera raised against a 45 kDa protein antigen derived from M. hominis

and M. termenlans respectively. The reactivities of both antisera suggest that this antigen, which we believe is the EF-Tu protein, is shared by mycoplasmas, Gram-negative bacteria and certain Gram-positive bacteria. In this study we describe ten monoclonal antibodies to mycoplasmal elongation factor Tu and their use in differentiating several mycoplasma species by analysing epitope patterns within this defined antigen molecule. EF-Tu as a test antigen in serological diagnostics offers certain attractive advantages: (1) it is present in every prokaryotic organism/ organelle independent of growth conditions and number of passages; (2) it is expressed in high amounts and therefore can be easily detected. We estimate that about 2% of total cellular protein is EF-Tu; (3) its amino acid sequence contains regions which are nearly identical even in organisms/ organelles which, in evolutionary terms are regarded as distinct such as mollicutes, Gram-positive and Gram-negative bacteria, cyanobacteria, archaebacteria, mitochondria and plastids. This is why mAbs raised against EF-Tu of one species cross react with the homologous protein of other species; (4) nevertheless, there exist amino acid sequence differences between members of a genus and these differences are detectable with mAbs. These properties make EF-Tu an appropriate candidate for determining immunological distances. Epitope 'fingerprints' of EF-Tu from 23 mollicute type strain species as determined in this study showed sufficient differences to identify these species. For example in order to evaluate intraspecies variability we determined epitope patterns of several M. hominis isolates. Although pattern differences occurred within this species, these patterns were more similar to each other than to other species. This finding suggests that the immunological relationship between EF-Tu proteins from different species parallels their taxonomic relationship. Analysis of EF-Tu epitope gene differences as well as comparisons of sequences may provide a very suitable approach to confirming or correcting taxonomic data in the same manner as do differences in ribosomal RNA sequences (Woese, 1987) or restriction fragment

tut

81

length polymorphism (RFLP) analysis using a tuf-gene probe (Yogev et aI., 1988). In addition to its applicability in species differentiation of mycoplasmas, this kind of 'fingerprinting' can be an effective tool in epidemiological studies since minor but still detectable differences occur within members of a species as illustrated in the case of M. hominis isolates. In the case of higher taxa we observed cross reactivity with several but not with all tested non-mollicutes. EF-Tu epitope patterns as determined with our ten antibodies against mycoplasma-EF-Tu do not permit detailed differentiation between these species. However, we believe that the generation of suitable mAbs following immunization with EF-Tu from Gram-positive and Gram-negative bacteria would lead to similar results. We are presently investigating epitope patterns of more mollicute species and genera and more strains of species other than Mycoplasma hominis.

Fazekas de St. Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: Strategy and tactics. J. Immunol. Methods 35, 1. Filer, D., Dhar, R. and Furano, A.V. (1981) The conservation of DNA sequences over very long periods of evolutionary time. Eur. J. Biochem. 120,69. Higgins, D.G. and Sharp, P.M. (1989) Fast and sensitive multiple sequence alignments on a microcomputer. CABIOS 5, 151. Inamine, I.M., Loechel, S. and Hu, P.e. (1989) Nucleotide sequence of the tuf gene from Mycoplasma gallisepticum. Nucleic Acids Res. 17, 10126. Jensen, I.S., Sondergard Andersen, I., Uldum, S.A. and Lind, K. (1989) Detection of Mycoplasma pneumoniae in simulated clinical samples by polymerase chain reaction. APMIS 97,1046. Kaziro, Y. (1978) The role of guanoside 5'-triphosphate in polypeptide chain elongation. Biochim. Biophys. Acta 505, 95. Kunita, S., Terada, E., Goto, K. and Kagiyama, N. (1990) Sensitive detection of Mycoplasma pulmonis by using the polymerase chain reaction. Iikken Dobutsu 39, 103. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,

680.

Acknowledgements This work was supported by a grant from the Bundesministerium fUr Forschung und Technologie (01 KI 8813/14). We acknowledge Dr. U. Jahnke (KFA Jiilich AG, Germany) for sequencing the N terminal amino acids and P. Gehrmann for supports at DNA database recherches.

References Bernet, c., Garret, M., De Barbeyrac, B., Bebear, C. and Bonnet, 1. (1989) Detection of Mycoplasma pneumoniae by using the polymerase chain reaction. 1. Clin. Microbiol. 27, 2492. Blazek, R., Schmitt, K., Krafft, U. and Hadding, U. (1990) Fast and simple procedure for the detection of cell culture mycoplasmas using a single monoclonal antibody. 1. Immunol. Methods 131, 203. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72, 248. Cleveland, D.W., Fischer S.G., Kirschner, M.W. and Laemmli, UK (1977) Peptide mapping by limited proteolysis in sodium dodecylsulfate and analysis by gel electrophoresis. 1. BioI. Chern. 252, 1102.

Loechel, S., Inamine, J.M. and Hu, P.C. (1989) Nucleotide sequence of the tuf gene from Mycoplasma genitalium. Nucleic Acids Res. 17, 10127. Liineberg, E., Kamla, V., Hadding, U. and Frosch, M. (1991) Sequence and expression in Escherichia coli of a Mycoplasma hominis gene encoding elongation factor Tu. Gene 102, 123. McGarrity, G.J., Vanamann, V. and Sarama, J. (1984) Cytogenic effects of mycoplasmal infection of cell cultures: A review. In Vitro 20, 1. Sasaki, T. (1991) Evidence that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen. 1. Bacteriol. 173,2398. Seidler, L., Peter, M., Meissner, F. and Sprinzl, M. (1987) Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8. Nucleic Acids Res. 15, 9263. Tapio, S. and Kurland, C.G. (1986) Mutant EF-Tu increases missense error in vitro. Mol. Gen. Genet. 205, 186. Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221. Yogev, D., Sela, S., Bercovier, H. and Razin, S. (1988) Elongation factor (EF-Tu) gene probe detects polymorphism in Mycoplasma strains. FEMS Microbiol. Lett. 50, 145. Yogev, D., Sela, S., Bercovier, H. and Razin, S. (1990) Nucleotide sequence and codon usage of the elongation factor Tu (EF-Tu) gene from Mycoplasma pneumoniae. Mol. Microbiol. 4, 1303.