Paratuberculosis, also called Johne's disease, is a se- vere and incurable gastroenteritis of ruminants caused by Mycobacterium avium subspecies (ssp.) paratuberculosis (M. paratuberculosis ; Kreeger, J. M. 1991. Ruminant paratuberculosis-a century of progress and frustration.

J. Vet. Diagn. Invest 3: 373-382). In cattle, infection most commonly occurs in newborn calves through the fecal-oral route. After a long incubation period, clinical symptoms including persistent diarrhea and weight loss predomi- nantly develop in animals three to five years of age (Chiodini, R. J. , H. J. Van Kruiningen, and R. S. Merkal.

1984. Ruminant paratuberculosis (Johne's disease): the current status and future prospects. Cornell Vet. 74: 218- 262). The disease is prevalent in domestic animals and wildlife worldwide and has a considerable economic impact on the livestock industry (Harris, N. B. and R. G. Bar- letta. 2001. Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine. Clin. Microbiol. Rev. 14: 489-512).

M. paratuberculosis is a member of the Myobycterium avium complex (MAC) additionally comprising M. avium ssp. avium (M. avium) and M. avium ssp. silvaticum (M. silvaticum).

At the subspecies level, M. paratuberculosis can be dif- ferentiated phenotypically from M. avium and M. silvati- cum by its dependence on the iron chelator mycobactin for growth in culture (De Voss, J. J. , K. Rutter, B. G.

Schroeder, and C. E. Barry, III. 1999. Iron acquisition and metabolism by mycobacteria. J. Bacteriol. 181: 4443-

4451; Thorel, M. F. , M. Krichevsky, and V. V. Levy- Frebault. 1990. Numerical taxonomy of mycobactin- dependent mycobacteria, emended description of Mycobacte- rium avium, and description of Mycobacterium avium subsp. avium subsp. nov. , Mycobacterium avium subsp. paratuber- culosis subsp. nov. , and Mycobacterium avium subsp. sil- vaticum subsp. nov. Int. J. Syst. Bacteriol. 40: 254-260) and genotypically by the presence of multiple copies of the insertion elements IS900 (Green, E. P. , M. L. Tizard, M.

T. Moss, J. Thompson, D. J. Winterbourne, J. J. McFadden, and J. Hermon-Taylor. 1989. Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculo- sis. Nucleic Acids Res. 17: 9063-9073) and ISMav2 (Strom- menger, B. , K. Stevenson, and G. F. Gerlach. 2001. Isola- tion and diagnostic potential of ISMav2, a novel inser- tion sequence-like element from Mycobacterium avium ssp. paratuberculosis. FEMS Microbiol. Lett. 196: 31-37). The three closely related M. avium subspecies show distinct host preferences with M. paratuberculosis being patho- genic mainly for ruminants and M. avium and M. silvaticum being primarily pathogenic in birds (Thorel, M. F. , M.

Krichevsky, and V. V. Levy-Frebault. 1990). Despite its host preference, M. paratuberculosis has also been found in other lifestock (Grange, J. M. , M. D. Yates, and E.

Boughton. 1990. The avian tubercle bacillus and its rela- tives. J. Appl. Bacteriol. 68: 411-431.; Thorel, M. F. , H.

Huchzermeyer, R. Weiss, and J. J. Fontaine. 1997. Myco- bacterium avium infections in animals. Literature review.

Vet. Res. 28: 439-447), in non-ruminant wildlife (Beard, P.

M. , M. J. Daniels, D. Henderson, A. Pirie, K. Rudge, D.

Buxton, S. Rhind, A. Greig, M. R. Hutchings, I. McKen- drick, K. Stevenson, and J. M. Sharp. 2001. Paratubercu- losis infection of nonruminant wildlife in Scotland.

J. Clin. Microbiol. 39: 1517-1521; Beard, P. M. , S. M.

Rhind, D. Buxton, M. J. Daniels, D. Henderson, A. Pirie, K. Rudge, A. Greig, M. R. Hutchings, K. Stevenson, and J.

M. Sharp. 2001. Natural paratuberculosis infection in rabbits in Scotland. J. Comp Pathol. 124: 290-299; Greig, A. , K. Stevenson, V. Perez, A. A. Pirie, J. M. Grant, and J. M. Sharp. 1997. Paratuberculosis in wild rabbits (Oryctolagus cuniculus). Vet. Rec. 140: 141-143), and in intestinal tissue of Crohn's disease patients (Chiodini, R. J. 1989. Crohn's disease and the mycobacterioses: a review and comparison of two disease entities.

Clin. Microbiol. Rev. 2: 90-117; Hulten, K. , H. M. El Zi- maity, T. J. Karttunen, A. Almashhrawi, M. R. Schwartz, D. Y. Graham, and F. A. El Zaatari. 2001. Detection of Mycobacterium avium subspecies paratuberculosis in Crohn's diseased tissues by in situ hybridization.

Am. J. Gastroenterol. 96: 1529-1535).

This as well as the finding that M. paratuberculosis is shed with milk from infected cows, and, in contrast to other mycobacteria, survives pasteurization temperatures and cheese production (Sung, N. and M. T. Collins. 1998.

Thermal tolerance of Mycobacterium paratuberculosis.

Appl. Environ. Microbiol. 64: 999-1005; Sung, N. and M. T.

Collins. 2000. Effect of three factors in cheese produc- tion (pH, salt, and heat) on Mycobacterium avium subsp. paratuberculosis viability. Appl. Environ. Microbiol. 66: 1334-1339) led to the concern that the bacterium may present a potential risk for human health. Although the relationship between M. paratuberculosis and Crohn's dis- ease is still controversially discussed, evidence has ac- cumulated that M. paratuberculosis is very likely a cause of Crohn's disease (Cook, L. S. 2000. A causal role for Mycobacterium avium subspecies paratuberculosis in Crohn's disease? Can. J. Gastroenterol. 14: 479-480; Hermon- Taylor, J. , T. J. Bull, J. M. Sheridan, J. Cheng, M. L.

Stellakis, and N. Sumar. 2000. Causation of Crohn's dis- ease by Mycobacterium avium subspecies paratuberculosis.

Can. J. Gastroenterol. 14: 521-539).

The problem to be solved by the present invention is to provide a vaccine and antibodies as well as therapeutic and diagnostic peptides that are specific to M. paratu- berculosis.

The present invention provides a M. paratuberculosis spe- cific ABC-transporter operon which encodes several pro- teins useful for the induction of an immune response in a living body. The invention furthermore provides antibod- ies as well as therapeutic and diagnostic peptides spe- cific to M. paratuberculosis.

The present invention accordingly provides a M. paratu- berculosis specific ABC-transporter operon with the se- quence according to SEQ ID NO 1.

According to the invention, a M. paratuberculosis spe- cific ABC-transporter operon is preferred, that comprises six open reading frames mptA, mptB, mptC, mptD, mptE and mptF.

Furthermore, a M. paratuberculosis specific ABC- transporter operon that encodes five proteins is pre- ferred.

Preferred is a M. paratuberculosis specific ABC- transporter operon, that encodes the proteins MptAB, MptC, MptD, MptE and MptF.

Another preferred embodiment is the protein MptAB with the sequence according to SEQ ID NO 2.

It is further preferred that MptAB is an ABC-transporter protein.

Another preferred embodiment is the protein MptC with the sequence according to SEQ ID NO 3.

It is preferred that the protein MptC has a molecular mass of 64 kilodalton.

It is particularly preferred that the MptC protein is an ABC-transporter protein.

A preferred embodiment of the invention is the protein MptD with the sequence according to SEQ ID NO 4.

It is preferred that protein MptD is a transmembrane pro- tein.

Also preferred is the protein MptE with the sequence ac- cording to SEQ ID NO 5.

Furthermore preferred is the protein MptF with the se- quence according to SEQ ID NO 6.

Preferred are transformants of M. smegmatis mc2155 con- taining the M. paratuberculosis specific ABC-transporter operon, characterized in that the integrative and kanamy- cin-selectable shuttle vector pMV361 was used.

Also preferred is the protein MptC, characterized in that it was expressed in M. smegmatis.

The use of the proteins MptAB and/or MptC and/or MptD and/or MptE and/or MptF for the isolation of peptides that specifically bind to said proteins is preferred.

Preferred is an antibody against the M. paratuberculosis specific ABC-transporter encoded by the sequence accord- ing to SEQ ID NO 1.

Also preferred are antibodies against the proteins MptAB and/or MptC and/or MptD and/or MptE and/or MptF.

Furthermore preferred is the use of the proteins MptAB and/or MptC and/or MptD and/or MptE and/or MptF for manu- facturing an antiserum.

Also preferred is a method for the preparation of anti- sera against at least one of the proteins MptAB or MptC or MptD or MptE or MptF, characterized in that it is raised in an animal. It is furthermore preferred to raise the antibody in a rabbit. In another preferred em- bodiment, these antisera are obtained by an initial in- jection of protein MptC, that is followed by two booster injections of recombinant fusion protein, emulsified in adjuvant.

A preferred embodiment of the invention is a therapeuti- cal composition against paratuberculosis and/or Crohn's disease, that contains protein MptAB or protein MptC or protein MptD or protein MptE or protein MptF or at least one fragment of at least one of said proteins or a mix- ture thereof. In a particularly preferred embodiment, this therapeutical composition is a vaccine, which pref- erably further contains pharmaceutically acceptable agents, carriers, solvents and/or adjuvants.

According to the invention, a method for inducing an im- munological response in an animal or a human is pre- ferred, in which the M. paratuberculosis specific ABC- transporter operon or at least one of the proteins MptAB or MptC or MptD or MptE or MptF or at least one fragment of at least one of said proteins or a mixture thereof are used to produce antibodies to protect said animal or hu- man from paratuberculosis and/or Crohn's disease.

It was surprisingly found that unique membrane proteins and transport functions are involved in the host prefer- ence, the high tenacity, and the iron uptake mechanisms of M. paratuberculosis. Since a direct approach target- ing the identification of M. paratuberculosis-specific membrane proteins by comparative 2-D-electorphoresis was unlikely to succeed due to the high mechanic stability of mycobacterial membranes, a reverse genetics approach was chosen. A representational difference analysis (RDA; Lisitsyn, N. , N. Lisitsyn, and M. Wigler. 1993. Cloning the differences between two complex genomes. Science 259: 946-951) was performed followed by sequencing and specific analyses for the presence of open reading frames containing putative membrane-spanning regions. Using this approach we identified and characterized the first M. paratuberculosis specific membrane protein and ex- pressed it in M. smegmatis mc2155.

According to the present invention, we cloned and charac- terized the first M. paratuberculosis specific membrane protein designated as MptC. The data indicates that this protein is part of an M. paratuberculosis specific ABC- transporter. The cloned sequence of this transporter en- codes at least three proteins, namely MptAB, MptC, and MptD. Both MptAB and MptC contain an amino-terminal transmembrane domain and a carboxy-terminal ATP-binding

domain. A similar secondary structure has been described for the putative ABC-transporter proteins YbtP and YbtQ of Y. pestis (Fetherston, J. D. , V. J. Bertolino, and R.

D. Perry. 1999) and could also be demonstrated for the putative ATP-binding proteins Rv1348 and Rv1349 of M. tu- berculosis (Braibant, M. , P. Gilot, and J. Content.

2000). These two putative ABC-transporter protein encod- ing genes are followed by a gene encoding the transmem- brane protein MptD. Although the MptD protein has no se- quence homologies to known proteins, its secondary struc- ture and position correspond to the YbtX protein encoded within the ybt operon immediately downstream from the ybtP and ybtQ genes (Fetherston, J. D. , V. J. Bertolino, and R. D. Perry. 1999).

The start codon of the putative MptE protein does not overlap the stop codon of the MptD protein, but is lo- cated 78 base pairs (bp) downstream. The protein has only a limited degree of homology to an ABC transporter permease protein of Lactococus lactis. The following MptF protein has a significant homology to a structurally different putative ATP-binding protein. As similar ar- rangements are unknown for other ABC transporters, a functional association of the putative MptE and MptF pro- teins with the proteins mptAB, mptC, and mptD remains un- clear.

The function of the mpt-operon cannot be predicted based on sequence homologies. One of the highly homologous transporters is predicted to have an export function (Rv1348/1349) whereas the other one is known to be re- quired for iron uptake (ybtPQX). Therefore, in order to obtain an initial clue on possible functions, the puta- tive ABC transporter genes were transformed into M. smeg- matis mc2155, and a strongly reduced lag phase in the

presence of kanamycin was observed. The initial idea that the MptC protein might be involved in a process of efflux-mediated drug resistance, as it has been described for a M. smegmatis transport system (Banerjee, S. K. , K.

Bhatt, P. Misra, and P. K. Chakraborti. 2000. Involvement of a natural transport system in the process of efflux- mediated drug resistance in Mycobacterium smegmatis.

Mol. Gen. Genet. 262: 949-956) is unlikely, since M. paratu- berculosis strain 6783 has been shown to be kanamycin sensitive. A second hypothesis predicting an involvement in the excretion of regulatory molecules as it has been shown for gram positive and gram negative bacteria (Young, J. and I. B. Holland. 1999. ABC transporters: bacterial exporters-revisited five years on. Bio- chim. Biophys. Acta 1461: 177-200) could also not be con- firmed. Thus, preliminary experiments showed no influ- ence of supernatants from M. smegmatis pRDIII320 trans- formants on the lag phase of pMV361 transformants (data not shown). Another possibility for the function of the Mpt-operon could be an involvement in iron uptake, as suggested for the ybtPQX operon (Fetherston, J. D. , V. J.

Bertolino, and R. D. Perry. 1999). Such a function has previously been reported for a mycobacterial ABC- transporter involved in exochelin uptake in M. smegmatis (Zhu, W. , J. E. Arceneaux, M. L. Beggs, B. R. Byers, K.

D. Eisenach, and M. D. Lundrigan. 1998. Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes. Mol. Microbiol. 29: 629-639). However, for trans- porters mediating solute uptake the existence of an addi- tional substrate-binding protein located outside the cy- toplasmatic membrane is usually required (Fath, M. J. and R. Kolter. 1993. ABC Transporters: Bacterial Exporters.

Microbiol. Rev. 57: 995-1017), and such a protein is not encoded for on the DNA analysed so far. Since the DNA

fragment analyzed, even at the ends, has no significant homology to the M. avium genome, it might be part of a M. paratuberculosis specific pathogenicity island, possibly involved in iron uptake, as recently reported for Strep- tococcus (S.) pneumoniae (Brown, J. S. , S. M. Gilliland, and D. W. Holden. 2001. A Streptococcus pneumoniae patho- genicity island encoding an ABC transporter involved in iron uptake and virulence. Mol. Microbiol. 40: 572-585). This would be particularly interesting as it has been shown that the components of this system can protect mice against systemic S. pneumoniae infection (Brown, J. S., A. D. Ogunniyi, M. C. Woodrow, D. W. Holden, and J. C.

Paton. 2001. Immunization with Components of Two Iron Up- take ABC Transporters Protects Mice against Systemic Streptococcus pneumoniae Infection. Infect. Immun.

69: 6702-6706).

According to the invention we have cloned and character- ized the first M. paratuberculosis specific membrane pro- tein MptC. By showing that it most likely is part of an ABC-transporter operon which again might be part of a pathogenicity island, we have made a first step towards investigating the cause for the distinct features of M. paratuberculosis on a molecular basis.

Examples The following examples describe the present invention further.

The following materials and methods were used: Materials Bacterial strains, plasmids, primers and growth condi- tions. The bacterial strains used in this study are

listed in Table 1, plasmids and primers are noted in Ta- ble 2. Mycobacteria were grown on Middlebrook 7H10 agar (MB-agar) or in Middlebrook 7H9 medium (MB-medium; both DIFCO Laboratories, Detroit, MI) supplemented with OADC enrichment (DIFCO), Tween 80@ (0.05 %) and the appropri- ate antibiotics (kanamycin, 40 Hg ml-1) for M. smegmatis transformants; for M. paratuberculosis mycobactin (2 Hg mi-1 ; Synbiotics, Lyon, France) was added. Escherichia (E.) coli strains were grown in Luria Bertani (LB) medium supplemented with the appropriate antibiotics (ampicil- lin, 100 pg ml~1 ; kanamycin, 40 Hg ml-1).

Representational difference analysis (RDA) and isolation of RDA fragments Mycobacterial chromosomal DNA was extracted according to the method described by Bose et al. (Bose, M. , A. Chan- der, and R. H. Das. 1993. A rapid and gentle method for the isolation of genomic DNA from mycobacteria. Nucleic Acids Res. 21: 2529-2530). Then the RDA-technique (Lis- itsyn, N. , N. Lisitsyn, and M. Wigler. 1993) recently ap- plied to isolate a M. paratuberculosis-specific low G+C content island (Tizard, M. , T. Bull, D. Millar, T. Doran, H. Martin, N. Sumar, J. Ford, and J. Hermon-Taylor. 1998.

A low G+C content genetic island in Mycobacterium avium subsp. paratuberculosis and M. avium subsp. silvaticum with homologous genes in Mycobacterium tuberculosis. Mi- crobiology 144: 3413-3423. ) and the novel M. paratubercul- sosis-specific insertion sequence like element ISMav2 (Strommenger, B. , K. Stevenson, and G. F. Gerlach. 2001), was used as previously described (Strommenger, B. , K.

Stevenson, and G. F. Gerlach. 2001).

Manipulation and analysis of DNA Gel electrophoresis, Southern blot, plasmid preparation, PCR, DNA cloning, and transformation of E. coli were done

following standard procedures (Sambrook, J. , E. F.

Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Sping Harbor Laboratory Press, Cold Spring Harbor Laboratory, N. Y. ). DNA modify- ing enzymes were puchased from New England Biolabs (Karlsruhe, Germany). Nucleotide sequencing was done by "primer walking" ; primers were purchased from MWG Biotech AG (Ebersberg, Germany), and sequencing reactions were done by SeqLab (Göttingen, Germany). Sequencing data analyses were performed using the HUSAR 5.0 programme (dkfz, Heidelberg, Germany). The DNA sequence as well as the putative open reading frames were investigated for homologies in GenBank/EMBL-as well as in the TIGR and Sanger Center data bases.

Construction of recombinant mycobacterial shuttle plas- mids and deletion derivatives and their expression in M. smegmatis mc2155 The 5367 bp SacI-fragment RDIII300 (Fig. 1), comprising the complete ORFs for mptC, mptD and mptE and the incom- plete ORFs for mptB and mptF, was cloned into the myco- bacterial shuttle vector pMV361 (Tab. 2) under control of the vector-based hsp60 promoter. Based on this plasmid, designated pRDIII320, deletion derivatives were gener- ated. Deletion of the 3201 bp FseI fragment (position no. 1208 to 4409 in Fig. 1), comprising the ORFs mptB, mptC and mptD resulted in plasmid pRDIII320D1. Deletion of the DNA downstream of the SmaI restriction site at po- sition no. 4009 (Fig. 1) resulted in plasmid pRDIII320D2 lacking the ORFs mptE, mptF, and part of ORF mptD (Table 2). These plasmids were used to transform M. smegmatis mc2155 by electroporation as described by Sander and Boettger (Sander, P. and E. C. Boettger. 1998. Gene Re- placement in Mycobacterium smegmatis Using a Dominant Negative Selectable Marker, p. 207-216. In T. Parish and

N. G. Stoker (eds. ), Mycobacteria Protocols. Humana Press Inc. , Totowa, New Jersey, USA. ). Briefly, 250 ml cultures of M. smegmatis mc2155 were incubated in a roller bottle at 37 °C until an optical density at 660 nm (OD660) of 0.4 was reached. Cultures were placed on ice for 90 min, and bacteria were harvested by centrifugation (2.600 x g) for 15 min at 4 °C. Pellets were washed twice with glycerol (10%) and finally resuspended in 2.5 ml of glycerol (10%). For transformation, 100 1 of cells were mixed with 0.5 Ag DNA and incubated on ice for 10 min before transferring into an electroporation cuvette. Electropo- ration was performed with a single pulse using the fol- lowing settings: 2.5 kV, 1000 W and 25 yF. Bacterial cells were resuspended in MB-media immediately after electroporation and incubated on a rotating shaker at 37 °C for 2 hours before plating on agar plates containing kanamycin (40 Hg ml~1) for positive selection. The suc- cess of the transformations was controlled by PCR with primers specific to mptC (IPIII30 and IPIII31, Table 2, Fig. 1) and for the kanamycin resistence determinant (Kan3 and Kan4, Table 2), as well as by Southern blot analyses.

Preparation of antiserum A DNA fragment obtained by PCR using the primers ABC3 and ABC4 was cut with BamHI and EcoRI and cloned into pGEX5x- 3 resulting in plasmid pRDIII350 (Table 2). Upon induc- tion with isopropyl-thiogalactoside (IPTG; 1 mM final concentration) inclusion bodies were formed and purified as described previously (Gerlach, G. F. , C. Anderson, A.

A. Potter, S. Klashinsky, and P. J. Willson. 1992. Clon- ing and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae. Infect. Immun. 60: 892- 898). Serum against the MptC protein was raised in rab- bits by an initial intra-cutaneous injection and two

booster injections using 100 Ag of recombinant fusion protein emulsified in adjuvant (Emulsigen-Plus MVP Inc. , Ralston, Nebr.).

Protein preparations, electrophoresis and Western Blot- ting Mycobacterial whole cell lysates were prepared from 100 mg of bacterial pellet. Bacteria were resuspended in 500 Al H20, and cell disruption was achieved by mechanical treatment for 190 sec with zirconium beads in a mini-bead beater (Bio-Spec Products, Inc. , Bartlesville, Okla.) followed by sonication. Cell debris was removed by cen- trifugation (2 500 x g) for 5 min. Mycobacterial mem- branes were prepared according to the protocol of Hancock and Nikaido (Hancock, R. E. and H. Nikaido. 1978. Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J. Bacteriol.

136: 381-390). Briefly, 100 mg of bacterial pellet was re- suspended in 2 ml Tris-HC1 (30 mM, pH 8.0) containing 20% saccharose. Cell disruption was achieved as described above. Residual cellular debris was removed by centrifu- gation (17 000 x g) for 10 min; total mycobacterial mem- branes were obtained by ultracentrifugation (175 000 x g) for 2 h at 4 °C and dissolved in 500 Al TE buffer. Pro- tein aggregates were prepared as peviously described (Gerlach, G. F. , C. Anderson, A. A. Potter, S. Klashin- sky, and P. J. Willson. 1992). All protein preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot analysis as described earlier (Homuth, M. , P. Valentin-Weigand, M.

Rohde, and G. F. Gerlach. 1998. Identification and char- acterization of a novel extracellular ferric reductase from Mycobacterium paratuberculosis. Infect. Immun.

66: 710-716. ). For the detection of the MptC protein in M.

paratuberculosis the ECL Western blotting detection sys- tems (Amersham Pharmacia Biotech, Freiburg) was used ac- cording to the manufacturer's instructions; for other Western blots an alkaline-phosphatase-labelled goat anti- rabbit conjugate and a substrate containing Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate was used (Gerlach, G. F. , C. Anderson, A. A. Potter, S.

Klashinsky, and P. J. Willson. 1992).

Determination of growth kinetics Material from cultures of M. smegmatis mc2155 transfor- mants grown on MB-agar containing kanamycin was resus- pended in MB-medium and adjusted to an optical densitiy (OD660) of 0.5. Liquid cultures (2.5 ml) with and without kanamycin were inoculated with 5 iLl of this suspension and incubated on a rolling incubator at 37 °C. The OD660 was determined at various time points.

The present invention is further illustrated by the fig- ures 1 to 5, together with the detailed description of the invention.

Figure 1 shows the physical map of the mptC gene and ad- jacent sequences. The open arrows above indicate the size of the ORFs mptA-F; the numbers in parentheses indi- cate the position of start and stop codon. The grey bars underneath indicate the size and relative location of the DNA fragments originally cloned. The open bars indicate the fragments cloned into the vector pMV361 and trans- formed into M. smegmatis mc2155. The small arrows indi- cate the primers used in the examples of the present in- vention study; the position of the 5'-end is given in pa- rentheses.

Figure 2 shows PCR-analyses of M. paratuberculosis and M. avium isolates using (a) primers IPIII30 and IPIII31, (b) primers MK05 and MK06, (c) primers MK07 and MK08. The template DNA used is derived from 14 bovine clinical M. paratuberculosis isolates (lanes A to N), M. paratubercu- losis strain 6783 (tester; lane O), M. avium strain ATCC 25291 (driver; lane P), M. avium strain DSM 44157 (lane Q), and M. avium strain DSM 44158 (lane R).

In Figure 3, Coomassie blue-stained gels (left) and West- ern blots using serum raised against the recombinant MptC fusion protein and developed with (a) alkaline- phosphatase-labelled goat anti-rabbit conjugate and a substrate containing Nitro Blue Tetrazolium and 5-bromo- 4-chloro-3-indolyl phosphate or (b) the ECL-system are shown. The serum used is absorbed with whole cell lys- ates of Escherichia coli transformants containing pGEX5x- 3 (left hand blot) or pRDIII350 (right hand blot). In lane A, membranes prepared from M. smegmatis mc2155 were loaded, in lane B, membranes from M. paratuberculosis strain 6783 were loaded. The numbers on the right indi- cate the relative positions of size markers in kilodal- tons. The arrowheads indicate the expected position of the MptC protein (64 kilodaltons (kDa)).

Figure 4 depicts the analysis of the Mpt proteins. (A) Sequence identities as determined by BLAST search and (B) prediction of transmembrane helices in the proteins MptC and MptD (Sonnhammer, E. L. , G. von Heijne, and A. Krogh.

1998).

Figure 5 shows analysis of M. smegmatis mc2155 transfor- mants. (A) Coomassie blue-stained gel (left) and Western blot (right) of membrane preparations (A-D) and whole cell lysates (A'-D') using serum raised against the re-

combinant MptC fusion protein. Proteins are prepared from cells transformed with pMV361 (A), pRDIII320 (B), pRDIII320A2 (C), pRDIII320A1 (D). The numbers in the middle indicate the relative positions of size markers in kilodaltons. The arrowhead to the right indicates the expected position of the MptC protein (64 kDa). (B) Growth curves of M. smegmatis mc2155 transformants in MB medium without (top) and with the addition of kanamycin (40 Ag ml-1 ; bottom).

RDA-based isolation of the M. paratuberculosis specific DNA fragment RDIII30 Among several fragments cloned by an RDA approach, a 403 bp BclI-fragment designated as RDIII30 was identified to encode putative transmembrane domains by DNA sequencing and analysis of the predicted amino acid sequence with the programme TMHMM (Sonnhammer, E. L. , G. von Heijne, and A. Krogh. 1998. A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. Int.

Conf. Intell. Syst. Mol. Biol. 6: 175-182). Data base com- parison did not show a significant degree of DNA sequence homology to other mycobacterial species. Subspecies specificity of the sequence was further investigated by PCR analyses using the internal primers IPIII30 and IPIII31 (Table 2). A single amplification product of the predicted size was obtained for all seventy-nine M. para- tuberculosis strains tested; for the nine IS901-positive M. avium strains and for the reference strains of nine other mycobacterial species tested no amplification product was obtained (Fig. 2). These results supported the M. paratuberculosis specificity of fragment RDIII30.

Isolation of large M. paratuberculosis specific DNA frag- ments

Using RDIII30 as a probe, two overlapping fragments were cloned from M. paratuberculosis strain 6783. One of these fragments was a 5367 bp SacI fragment designated as RDIII300, the other a 4681 bp KpnI fragment designated as RDIII301 (Fig. 1). The two fragments together span 6419 bp of DNA; sequence comparison with the known genome se- quence of M. avium in a BLAST search using the TIGR data- base (www. tigr. org) showed an overall identity of the segment (including both ends) with the M. avium genome of only 60%. Analysis of the DNA sequence revealed the presence of six ORFs, designated mptA to mptF, with mptA and mptF being present only partially on the DNA fragment analyzed. The ORFs mptB, mptC and mptD as well as mptE and mptF directly follow each other with start and stop codons overlapping. The original RDA fragment RDIII30 is part of mptC coding for a predicted protein (MptC) of 593 amino acids in length and a calculated molecular mass of 64 kDa (Fig. 1).

Expression of the MptC protein in M. paratuberculosis In order to investigate the expression of mptC by M. paratuberculosis, a serum raised against a GST-MptC fu- sion protein was used to determine expression of MptC in M. paratuberculosis and in M. smegmatis (negative con- trol). Using a highly sensitive chemiluminescence detec- tion system, the expression of a 64 kDa protein was de- tected in M. paratuberculosis membrane preparations only (Fig. 3), demonstrating that the MptC protein is ex- pressed by M. paratuberculosis grown in standard culture conditions. The absence of a specific serological reac- tion with M. smegmatis membranes further supports the subspecies specificity of the Mpt protein.

Sequence analysis

A data base search at the protein level (Fig. 4A) showed that the MptC protein had obvious similarities to the pu- tative ATP-binding proteins Rv1349 from M. tuberculosis (34.4% ; GenBank accession no. Q11019 (Braibant, M. , P.

Gilot, and J. Content. 2000. The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis.

FEMS Microbiol. Rev. 24: 449-467) and YbtQ from Yersinia (Y.) pestis (38.2% GenBank accession no. T17436, (Fether- ston, J. D. , V. J. Bertolino, and R. D. Perry. 1999. YbtP and YbtQ: two ABC transporters required for iron uptake in Yersinia pestis. Mol. Microbiol. 32: 289-299). The pro- tein encoded by the ORF mptB, located upstream from mptC (Fig. 2), showed 41.4% similarity to another putative ATP-binding protein (Rv1348) of M. tuberculosis (GenBank accession no. Q11018; Braibant, M. , P. Gilot, and J. Con- tent. 2000) and 38.8% similarity to another ABC- transporter protein YbtP from Y. pestis (GenBank acces- sion no. T17437; Fetherston, J. D. , V. J. Bertolino, and R. D. Perry. 1999); both genes are located upstream of the respective mptC homologues. The similarity between the protein encoded by ORF mptB and Rv1348 is limited to the carboxy-terminal part of the putative ATP-binding protein Rv1348, whereas the protein encoded by ORF mptA shows 38% identity over 117 amino acids to the amino- terminal part of Rv1348. Based on these observations it can be hypothesized that the ORFs mptA and mptB encode a single protein MptAB via a programmed translational fra- meshift event occuring at a"slippery site" (CCC CCA; Snyder, L. and W. Champness. 1997. Exceptions to the code, p. 55. In Molecular Genetics of Bacteria. ASM Press, Washington, D. C. ) present 30 bp upstream of the start codon of ORF mptB. A similar translational frameshift event resulting in the expression of a single transporter protein has recently described by Barsom and Hatfull (Barsom, E. K. and G. F. Hatfull. 1997. A puta-

tive ABC-transport operon of Mycobacterium smegmatis.

Gene 185: 127-132) for a permease protein of an ABC- transporter operon of M. smegmatis.

While the protein encoded by ORF mptF shows 32.4% simi- larity over 430 amino acids to an ATP-binding protein of Methanobacterium thermoautotrophicum, the proteins en- coded by the ORFs mptD and mptE exhibit only weak or no similarities to known proteins (Fig. 4A). Analyses of the amino acid sequences of the putative Mpt proteins for the presence of conserved sequence motifs of ABC- transporter proteins revealed that the putative proteins MptAB and MptC comprised all typical sequence motifs, i. e. the Walker A-and Walker B-motif, the ABC-signature and the Linton and Higgins-motif (Linton, K. J. and C. F.

Higgins. 1998. The Escherichia coli ATP-binding cassette (ABC) proteins. Mol. Microbiol. 28: 5-13), whereas in the incomplete putative protein MptF only the Walker A-motif and the ABC-signature were found. However, the lacking motifs might be located on the unknown part of the ORF.

Using the programme TMHMM (Sonnhammer, E. L. , G. von Hei- jne, and A. Krogh. 1998. ) to identify putative transmem- brane domains, membrane spanning domains consisting of 5 (MptAB) or 6 to 7 (MptC) transmembrane helices were pre- dicted for the proteins MptAB (data not shown) and MptC (Fig. 4B). The putative protein MptD was predicted to be a transmembrane protein comprising six transmembrane helices (Fig. 4B). Based on these findings the 6419 bp DNA fragment described was considered to be part of a pu- tative ABC-transporter system. The cloned part of the system was assigned the GenBank accession no. AF419325.

It encompasses two putative ABC-transporter proteins, each of them consisting of a carboxyterminal ATP-binding domain fused to an N-terminal transmembrane domain (MptAB and MptC), followed by a transmembrane protein (MptD) and

two additional putative proteins (MptE and MptF) with un- certain function.

Expression of the MptC protein in M. smegmatis The MptC protein was not expressed in E. coli transfor- mants. In order to confirm the sequence and to investi- gate a possible function of the MptC protein, fragment RDIII300 (Fig. 1) was cloned into the mycobacterial shut- tle vector pMV361, yielding plasmid pRDIII320. M. smeg- matis mc2155 was transformed with this construct as well as with two deletion derivatives (pRDIII320D1 and pRDIII320D2 ; Table 2; Fig. 1). Using the serum raised against the recombinant GST-MptC fusion protein, the ex- pression of MptC was investigated in whole cell lysates and membrane preparations of the M. smegmatis transfor- mants. In membrane preparations of pRDIII320 and pRDI- II320D2 transformants (both containing ORF mptC) the ex- pression of a 64 kDa protein was demonstrated (Fig. 5A).

In order to investigate possible influences of MptC expression on the phenotype of M. smegmatis transfor- mants, growth kinetics were determined in medium with and without kanamycin (Fig. 5B). In medium without kanamycin cultures of all M. smegmatis transformants showed similar growth kinetics with visible growth after 20 to 24 hours, reaching the maximum OD660 4 to 6 hours later. In medium containing kanamycin only, M. smegmatis transformants carrying the plasmids pRDIII320 and pRDIII320D2 showed these growth kinetics; cultures of the pMV361 and pRDIII320D1 transformants had a clearly prolonged lag- phase with visible growth not occurring until 60 hours after inoculation and then also reaching the maximum OD660 within 4 to 6 hours.

Table 1 Strains Characteristics and source M. avium ssp. paratuberculosis American Type Culture Collection (ATCC, ATCC 19698 Rockville, Maryland, USA) laboratory ref erence strain Strain 6783 (DSM 44135) Strain collection, Institut für Mikro- biologie und Tierseuchen 14 bovine isolates 8 cervine, 8 caprine, 8 ovine, Strain collection, 8 leporine, 16 bovine and 16 Moredun Research Institute wildlife clinical isolates M. avium ssp. avium M. avium ssp. avium Deutsche Gesellschaft fuer ATCC 25291 (DSM 44156) DSM 44157 Mikroorgansismen und Zellkulturen DSM 44158 (DSMZ, Braunschweig, Germany) NCTC 8559 National Collection of Type Culture ! NCTC 8562 (NCTC, Central Public Health Labora NCTC 8562 1' NCTC 8551 tory, Colindale, UK NCTC 8552 NCTC 8553 JD88/118 Clinical isolate, strain collection, Moredun Research Institute, Edinburgh, U Other mycobacteria M. smegmatis mc2155 Snapper, S. B., R. E. Melton, S. MustafE T. Kieser, and W. R. Jacobs, Jr. 1990. Isolation and characterization of effi- cient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4 : 1911-1919 M. smegmatis NCTC 8159 M. scrofulaceum NCTC 10803 M. phlei NCTC 8151 National Collection of Type Cul- M. bovis BCG NCTC 5692 tures M. bovis NCTC 10772 (NCTC, Central Public Health M. kansasii NCTC 10268 Laboratory, M. microti NCTC 8710 Colindale, UK) M. gordonae NCTC 10267 M. fortuitum NCTC 10394 E. coli DHSaF F'/endAl hsdRl7 (rK-mK+) supE44 thi-1 recAl gyrA (NalR) relAl A (lacZYA-argF) U169 deoR [+80dlacA (lacZ) M15] (Raleigh, F. A., K. Lech, and R. Brent. 1989. Se- lected topics from classical bacterial genetics, p. 1. 4. 1-1. 4. 14. In : F. M. Ausubel, et al. (ed.), Current protocols in molecular biology. Publishing Associ- ates and Wiley Interscience, New York, N Y.) Topr) i n TOPO 10 F-mcrA A (mrr-hsdRMS-mcrBC) (D801acZAM15 AlacX74 rec Al deoR araD139 A (araleu) 769 galU galK rpsL (StrR) endAl nupG, TOPO TA Cloning (Invitrogen, Groningen, NL) Table 2 <BR> <BR> <BR> <BR> <BR> <BR> <BR> Plasmids and Characteristics, reference or source<BR> <BR> primers Plasmids <BR> <BR> <BR> UC19 E. coli cloning vector carrying an ampicillin resistance<BR> <BR> <BR> determinant, Pharmacia<BR> <BR> <BR> <BR> <BR> <BR> E. coli cloning vector carrying an amplicillin and kanaymcin<BR> pCR#II-TOPO resistance determinant, Invitrogen <BR> <BR> <BR> <BR> E. coli expression vector for the construction of glu-<BR> pGEX5xc-3 tathion-S-transferase (GST) fusion proteins, Pharmacia Integrative mycobacterial shuttlevector carrying a kanamycin resistance determinant (Burlein, J. E. , C. K. Stover, S. Of- futt, and M. S. Hanson. 1994. Exression of Foreign Genes in pMV361 Mycobacteria, p. 239-252. In B. R. Bloom (ed.), Tuberculo- sis: Pathogenesis, Protection and Control. ASM Press, Wash- ington, D. C.) pRDIII300 fragment RDIII300 in pUC19 pRDIII301 fragment RDIII301 in pUC19 pRDIII320 fragment RDIII300 in pMV361 deletion derivative of pRDIII320 with a 3201 bp FseI frag- ment deleted (Fig. 1) deletion derivative of pRDIII320 with a 2410 bp SmaI/SacI fragment deleted (Fig. 1) <BR> <BR> <BR> PCR-fragment obtained with primers ABC3 and ABC4, cut with<BR> pDRIII350 BamHI and EcoRI and ligated into pEX5x-3.

Primers GAT CCT CGG TGA (Lisitsyn, N. , N. Lisitsyn, and M. Wigler.

RBaml2 1993. Cloning the differences between two complex genomes.

Science 259: 946-951) AGC ACT CTC CAG CCT CTC ACC GAG (Lisitsyn, N. , N. Lisitsyn, RBam24 and M. Wigler. 1993. Cloning the differences between two complex genomes. Science 259: 946-951) TTC TTG AAG GGT GTT CGG GGC C (Doran, T. J. , J. K. Davies, <BR> <BR> <BR> <BR> A.J. Radford, and A. L. Hodgson. 1994. Putative functional<BR> MK5 domain within ORF2 on the Mycobacterium insertion sequences IS900 and IS902. Immunol. Cell Biol. 72: 427-434) GCG ATG ATC GCA GCG TCT TTG G (Doran, T. J. , J. K. Davies, <BR> <BR> <BR> <BR> A. J. Radford, and A. L. Hodgson. 1994. Putative functional<BR> MN6 domain within ORF2 on the Mycobacterium insertion sequences IS900 and IS902. Immunol. Cell Biol. 72: 427-434) GTC TGG GAT TGG ATG TCC TG (Kunze, Z. M. , S. Wall, R. Appel- berg, M. T. Silva, F. Portaels, and J. J. McFadden. 1991.

MK7 IS901, a new member of a widespread class of atypical inser- tion sequences, is associated with pathogenicity in Mycobac- terium avium. Mol. Microbiol. 5: 2265-2272) CAC CAC GTG GTT AGC AAT CC (Kunze, Z. M. , S. Wall, R. Appel- berg, M. T. Silva, F. Portaels, and J. J. McFadden. 1991.

MK8 IS901, a new member of a widespread class of atypical inser- tion sequences, is associated with pathogenicity in Mycobac- terium avium. Mol. Microbiol. 5: 2265-2272) ABC3 CCG CGG ATC CGC TTA CGA CGG AGG TCA A ABC4 5GCC GGA ATT CGA TGT TGA TGA GAAT CCC T IPIII30 CTA TGC GCA CTG ACG CTT C IPIII31 TTC CGA AGA ATC CGA TGA G Kan3 CTC ATC GAG CAT CAA ATG AAA CTG C Kan4 ATA TTC AAC GGG AAA CGT CTT GCT C