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Brucella spp. are gram-negative, facultative intracellular bacterial pathogens that cause brucellosis, an infectious disease affecting animals and humans. Brucella melitensis is the most important species involved in ovine and caprine brucellosis, which is characterized by abortion, low production, and infertility in infected animals. B. melitensis is also the most pathogenic species for humans.

One of the principal aims in brucellosis research is the identification of Brucella antigens eliciting humoral and/or cell-mediated responses, which might be of interest for the development of diagnostic tests or subcellular vaccines that avoid the drawbacks of those currently used. B. melitensis Rev 1, a live attenuated vaccine strain that is currently used in sheep and goats, has been successful in disease eradication and control programs in some countries (1). However, there have been significant problems associated with its use. The most important among them are the residual virulence of Rev1 for humans and the development of agglutinating antibodies in animals vaccinated as adults which are indistinguishable from those elicited by natural infection (8). The construction of new brucellosis vaccines and associated diagnostic tests lacking these indesirable properties would be of great interest to veterinary medicine.

A number of immunogenic proteins have been previously identified by immunoblotting, such as the BP26 protein, and are currently being considered for the development of new diagnostic tests for ovine brucellosis (3, 6, 7, 10). Recently, two-dimensional electrophoresis, immunoblotting, and N-terminal microsequencing have considerably facilitated the identification of immunogenic proteins in ovine brucellosis (15, 16). Among the proteins identified by these methods, one with an apparent molecular mass of 45 kDa was recognized by sera from Brucella-infected sheep, and its N-terminal sequence showed homology to a dihydrolipoamide succinyltransferase (SucB) described in many bacteria (2, 5, 9, 13, 19). A monoclonal antibody (MAb) was raised against this protein (16) to allow easy screening of genomic libraries to clone the corresponding gene. The present report describes the cloning and the nucleotide sequence of the gene termed sucB encoding dihydrolipoamide succinyltransferase (E2o), an enzyme of the -ketoglutarate dehydrogenase complex, and its expression in Escherichia coli.

Specificity of the anti-SucB MAb. The MAb raised against Brucella SucB did not cross-react with E. coli and other bacteria closely genetically related to Brucella, such as Ochrobactrum anthropi, Phyllobacterium rubiacearum, Rhizobium leguminosarum, and Agrobacterium tumefaciens (20; data not shown). Thus, the MAb appeared to be specific for Brucella and therefore particularly useful for screening genomic libraries constructed in E. coli.

Cloning of the B. melitensis sucB gene and its expression in E. coli. A B. melitensis 16M genomic library was constructed in lambdaGEM-12 XhoI half-site arms (Promega, Madison, Wis.) by following the instructions of the manufacturer. Briefly, B. melitensis 16M DNA, extracted and purified as described previously (17), was partially digested for 30 min at 37°C with Sau3 AI (Promega) at 0.014 U/µg of DNA, the enzyme concentration giving the highest percentage of fragments ranging from 15 to 23 kb. DNA fragments were partially filled in with dGTP and dATP, by using Klenow DNA polymerase (Promega), ligated with T4 DNA ligase (Promega) to lambda-GEM-12 digested with XhoI, and partially filled in with dTTP and dCTP. Recombinant phage DNA was packaged in vitro with the Packagene System (Promega), and the library was titrated by determination of the number of PFU that appeared after infection of E. coli KW251 cells (Promega). Recombinant phages were transferred to nitrocellulose filters, and phages expressing the sucB gene were identified by reactivity with the anti-SucB MAb. DNA of a positive phage was extracted from culture supernatants of E. coli KW251 cells infected with the phage and cultured until lysis was observed. Phage DNA was then cut with NotI, BamHI, EcoRI, or SacI, and restriction fragments were ligated into pGEM-5Zf+ (Promega) cut with NotI or into pGEM-7Zf+ (Promega) cut with BamHI, EcoRI, or SacI, respectively. Competent E. coli JM109 cells (Promega) were transformed with recombinant plasmid DNA as described previously, and bacteria were spread on Luria-Bertani (LB) broth-ampicillin (50 µg/ml) plates containing isopropyl-1-thio--D-galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal). E. coli JM109 colonies bearing recombinant plasmids were transferred to nitrocellulose, lysed with 10% sodium dodecyl sulfate (SDS), and screened with the anti-SucB MAb by a colony blotting technique. One positive colony was found bearing a plasmid with a large BamHI insert of about 15 kbp. This plasmid was named pMZ4501. A 6.5-kbp NotI-BamHI fragment from this insert was further subcloned into pCR2.1 (In Vitrogen, San Diego, Calif.), resulting in plasmid pMZ4503. Expression of sucB in E. coli bearing plasmid pMZ4503 was further confirmed by immunoblotting with the anti-SucB MAb (Fig 1, lane 1). The MAb detected one band with an apparent molecular mass of 45 kDa, which was overproduced in E. coli as demonstrated by Coomassie blue staining (data not shown). Control E. coli cells bearing nonrecombinant pGEM-7Zf showed no reaction at all with the anti-SucB MAb (Fig. 1, lane 2).

View larger version (111K): [in this window] [in a new window]   FIG. 1.   Immunoblotting after SDS-PAGE of E. coli (pMZ4503) cells expressing the sucB gene of B. melitensis 16M with anti-SucB MAb (lane 1), with sera from brucellosis-negative sheep (lanes 3 and 4), and with sera from naturally infected sheep (lanes 4 to 10), all adsorbed with E. coli JM109 cells carrying the control vector pGEM7Zf+. Lane 2, immunoblotting after SDS-PAGE of E. coli JM109 carrying the control vector pGEM7Zf+ with anti-SucB MAb.

DNA sequence analysis of the Brucella melitensis 16M sucB gene. Recombinant plasmid pMZ4503 bearing the B. melitensis 16M sucB gene was sequenced for both strands by the chain termination method of Sanger et al. (11). Computer analysis of the sequence data was performed by BLAST analysis through the National Center for Biotechnology Information. Nucleotide sequencing of the 6.5-kbp NotI-BamHI fragment revealed the presence of two partial and two complete open reading frames (ORFs). Comparison of the ORFs to those in the GenBank database by using BLAST showed that three of the ORFs encoded proteins homologous to SucA, SucB, and LpdA previously identified in E. coli (12). The first partial ORF contained 713 codons with 80.7% amino acid sequence identity to the sucA gene product of B. abortus S19 (GenBank accession no. AF07932) and 41.7% identity to the sucA-encoded E1o protein of E. coli (GenBank accession no. X00661). There was a complete ORF immediately downstream of the partial sucA gene that contains 409 codons with 88% amino acid sequence identity to the sucB gene product of B. abortus strain S19 and 51.2% identity to the sucB-encoded E2o protein of E. coli (GenBank accession no. X00664) (13). The N-terminal amino acid sequence of the protein deduced from the nucleotide sequence matched the first 14 amino acids of the protein identified by two-dimensional electrophoresis and N-terminal microsequencing (16). The differences between the B. abortus S19 and B. melitensis 16M sucB genes consisted of the following: single nucleotide substitutions; one, two, or three nucleotide deletions; one nucleotide addition; and, most importantly, a 42-bp deletion in B. abortus S19 sucB (Fig. 2). These nucleotide substitutions and additions in the B. abortus S19 sucB gene relative to the B. melitensis 16M sucB gene altered the predicted amino acid sequence for many amino acids. The 42-bp deletion (coding for 14 amino acids) could possibly have a greater importance and perhaps cause an antigenic shift, as previously described for other proteins (4).

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Alignment of the B. melitensis (B. mel) 16M sucB and B. abortus (B. ab) S19 sucB nucleotide sequences. Identical nucleotides are indicated by stars, and gaps are indicated by hyphens.

View larger version (6K): [in this window] [in a new window]   FIG. 3.   Organization of the B. melitensis 16M suc genes. The 6,246-bp NotI-BamHI fragment comprised, besides the sucB gene, part of the sucA gene, ORF1 coding for an amino acid efflux-like protein, and part of the lpdA gene. Arrows indicate the direction of transcription.

Serum activities. The antibody responses of naturally infected sheep against recombinant SucB protein were analyzed. pMZ4503-transformed E. coli cells were cultured in LB broth, and total cell protein extracts were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by Western blotting as described previously (22). All sera have been adsorbed with E. coli JM109 carrying plasmid pGEM7Zf+ to remove nonspecific antibodies reacting with proteins from E. coli. Figure 1 shows the positive reaction with infected sheep sera of the recombinant SucB protein showing an apparent molecular mass of 45 kDa (lanes 5 to 10) corresponding to the SucB protein migration identified by using a MAb against the recombinant protein (lane 1). Antibody responses against the recombinant SucB protein were detected in all naturally infected sheep and not in Brucella-free sheep (lanes 3 and 4).

Nucleotide sequence accession number. The nucleotide sequence of the B. melitensis 16M sucB gene and flanking regions has been deposited in GenBank under accession no. AF235020.