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Serpulina (Treponema) hyodysenteriae, a gram-negative anaerobic spirochete, is the etiologic agent of swine dysentery (5, 19, 21). After ingestion the pathogen colonizes the colon of the infected animal, commonly a postweaning pig, leading to disease characterized by severe mucohemorrhagic diarrhea, dehydration, rapid weight loss, and, in some cases, death. In recent years several genes have been cloned from S. hyodysenteriae which may be involved in the colonization of the swine colon and the pathogenicity of the spirochete or in the generation of a protective immune response by the host. These include the flaA1, flaB1, and flaB2 genes, encoding components of the spirochete's periplasmic flagella (4, 8, 9, 15, 16); the smpA gene, encoding an outer membrane lipoprotein (22); and the hlyA, hlyB, and hlyC genes, encoding hemolysins (7, 14, 20).

We have previously described a rapid method for releasing extracytoplasmic proteins (ECP) from S. hyodysenteriae (B204) with the nonionic detergent Tween 20 and the subsequent subfractionation and purification of several of these proteins by centrifugation, differential urea solubility, and reverse-phase chromatography (4). One ECP, characterized by its partitioning during subfractionation, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and amino-terminal sequencing, was a sedimentable, urea-insoluble 39-kDa protein. In this paper we report the identification of two closely related and contiguous genes which have striking homology to each other and are predicted to have marked homology to the still-unidentified structural gene for the 39-kDa ECP we have purified.

S. hyodysenteriae B204, obtained from Joann Kinyon, Iowa State University, was grown anaerobically (80% nitrogen, 10% carbon dioxide, 10% hydrogen) from a 5 to 10% inoculum in a Braun Biostat M fermentor at 37°C, pH 6.8, in Difco brain heart infusion broth (37 g/liter) supplemented with 5% heat-inactivated fetal bovine serum, 0.5% dextrose, and 20 mg of spectinomycin (Sigma)/liter. Cells were harvested by centrifugation in late log phase (A₆₀₀ = 1.5 to 2) 18 to 24 h after inoculation, with a yield of approximately 10 g (wet weight) per liter.

Protein biochemistry. Enzymatic iodination of surface proteins of intact S. hyodysenteriae cells was performed essentially as described by Marchalonis et al. (12). Twenty milliliters of an exponentially growing culture of S. hyodysenteriae (approximately 5 × 10⁸ cells/ml) was centrifuged, washed with 0.5 volume of phosphate-buffered saline (PBS) (10 mM sodium phosphate [pH 7.2], 150 mM NaCl), and resuspended in a final volume of 2 ml of PBS. First, 25 µl of 0.5 M sodium phosphate (pH 7.3) was added, then 10 µl of lactoperoxidase (CalBiochem) (made up to 200 IU/ml in PBS), then 40 µl of 10 µM hydrogen peroxide, and finally 450 µCi of ¹²⁵I (20 mCi/ml in 0.1 N NaOH). The mixture was incubated at 30°C for 5 min, another 40 µl of hydrogen peroxide was added, and after an additional 10 min of incubation the reaction was stopped by the addition of 400 µg of tyrosine (40 µl of a 10 mg/ml solution in PBS). A whole-cell lysate sample of iodinated S. hyodysenteriae was electrophoresed on a 12% acrylamide gel, dried, and then exposed on X-ray film for 2 h before development. We observed one predominant iodinated component (Fig. 1), whose electrophoretic mobility matched that of the Coomassie-staining 39-kDa protein purified from S. hyodysenteriae (4). Subsequent extraction of these labeled cells with Tween 20 and urea showed that this same iodinated protein partitioned like the 39-kDa protein (specifically, it is released by Tween 20 from the cells and is insoluble following extraction with 6 M urea [data not shown]). Trace amounts of iodinated protein components of both higher and lower molecular masses were also noted.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Autoradiography of a whole-cell lysate sample of iodinated S. hyodysenteriae cells after gel electrophoresis with the buffer system of Laemmli (10). The numbers correspond to the MW (in thousands) of prestained protein markers (Diversified Biotech). DF, dye front.

View larger version (78K): [in this window] [in a new window]   FIG. 2.   Comparison of amino acid sequences for actual and predicted 39-kDa products. The LysC peptide fragments of the 39-kDa protein are presented on the top line. Question marks indicate sequence ambiguity at the specific cycle position. By the numbering system used in the figure, the peptides sequenced correspond to (i) Met1 to Arg41, (ii) Pro132 to Lys166, (iii) Thr174 to Ala220, (iv) Ala266 to Lys296, (v) His297 to Lys311, (vi) Val312 to Asn341, and (vii) Arg346 to Gln371. The sequences are aligned for best fit, with possible gaps or deletions between proteins indicated by dashed lines. Conserved sequences are shaded, the apparent variable regions (six are identified) are underlined (solid bar) and numbered, and apparent hydrophobic regions are underlined (broken bar).

Molecular cloning and analysis. S. hyodysenteriae B204 DNA was prepared from a 1-liter cell pellet of early-log-phase cells (optical density at 600 nm = 0.5) and purified in CsCl gradients according to the method of Maniatis et al. (11). Linkers containing an EcoRI restriction site were blunt end ligated to an AluI partial digest of the S. hyodysenteriae DNA, and a library was created by cloning these fragments into the EcoRI site of lambda phage gt11 (Promega). The Escherichia coli strain Y1090r (Promega) was used to screen the lambda gt11 phage library, and strains JM83 and DH5 were used for subcloning and DNA sequencing. The cloning of the vspA gene was done with the (degenerate) oligonucleotides COD555 [ATG-TA(T/C)-GG(T/C/A/G)-GA(T/C)-AG(T/C/ A/G)-GA], derived from amino acids Met¹ to Asp⁶, and COD553 [TGG-AT(T/C/A)-GA(T/C)-TT(T/C)-TT(T/C/A/ G)-AC], derived from amino acids Trp⁸ to Thr¹³ of the 39-kDa ECP. Oligonucleotide probes were synthesized with an automated DNA synthesizer (Biosearch 8700) and purified by acrylamide gel prior to labeling with [-³²P]dATP (New England Nuclear) and T4 polynucleotide kinase (Boehringer Mannheim). Degenerate oligonucleotides or plasmid subclone fragments were labeled by nick translation and used to probe Southern blots or to screen the phage library (11). A phage containing a 1.5-kb EcoRI fragment which hybridized to both oligonucleotide probes was identified, and its insert was subcloned into pUC19 (pTrep106) and sequenced by the dideoxy termination method of Sanger et al. (17). It was found to contain an extended open reading frame (ORF-1) which encoded 386 amino acids as shown in Fig. 3. The first 21 amino acids encoded by the ORF were typical of those corresponding to cleavable signal peptides directing transmembrane transport (23). The following 365 codons are predicted to encode a protein whose molecular weight (MW) is 41,000. Codon 22 of ORF-1 matches the N-terminal Met determined for the purified 39-kDa protein, consistent with a signal peptide-processing event between Gly²¹ and Met²² (23). Glycine or other small amino acids are typically found adjacent to the new N termini generated by signal peptidase processing. From Met²² onward the predicted polypeptide sequence matched 36 of the 40 amino acids obtained from the amino terminus of the 39-kDa ECP (Fig. 2). However, the four mismatches noted could not be reconciled with the nucleotide sequence of ORF-1 in pTrep106.

View larger version (64K): [in this window] [in a new window]   View larger version (65K): [in this window] [in a new window]   FIG. 3.   Nucleotide sequence of vspA and vspB genes from S. hyodysenteriae (length, 2,091 nucleotides). Insert sequences from two overlapping plasmids, pTrep106 and pTrep330, were combined, as indicated by arrows adjacent to Alu cloning sites (*). The underlined sequences (AGGT) upstream of the ORFs are apparent RBSs. The underlined sequences following ORF-1 form an apparent stem-loop structure. The first amino acid obtained from the isolated 39-kDa protein (Met, indicated with a #) is an apparent site of posttranslational signal peptide processing for both vsp gene products.

Concluding remarks. Surface iodination of S. hyodysenteriae identified a predominant component of the cell which had the same apparent MW and extraction properties as the 39-kDa protein released from whole cells by treatment with Tween 20 (4). It is likely that this 39-kDa protein is also one of the several surface proteins iodinated on cells incubated in neutral-pH buffer as reported by Wannemuehler et al. (24). We sought to obtain both its amino acid and nucleotide sequences in hopes of revealing a possible role this protein might play in either the pathogenesis of infection with S. hyodysenteriae or the stimulation of protective antibodies in animals which have recovered (and become immune to further infection). Such a role has already been suggested for a 16-kDa envelope antigen of S. hyodysenteriae (18). To our surprise, we found a tandem pair of closely related genes, neither of whose products precisely matches the amino acid sequence obtained from the 39-kDa ECP. We believe that this protein is the product of yet another closely related gene, an idea supported by the observation that some of the oligomer probes used in the cloning were found to hybridize to more than one region of a Southern blot of HindIII-digested genomic DNA. Since neither of the cloned fragments within pTrep106 or pTrep330 contains a HindIII site, the hybridizing regions of both of the cloned genes should reside on a single genomic HindIII fragment.

Nucleotide sequence accession number. The nucleotide sequence from the cloned S. hyodysenteriae DNA has been deposited in the GenBank library of DNA sequences under accession no. AF012102.