Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: November 11, 1996
Publication Date: N/A
Technical Abstract: VSH-1 is a mitomycin C-inducible bacteriophage of Serpulina hyodysenteriae (S. hyo). VSH-1 does not exhibit detectable lytic growth when incubated with S. hyo cultures. Purified virions contain 7.5 kb fragments of host DNA and can serve as generalized transducing phages of S. hyo. We began a search for viral genes to investigate VSH-1 and its potential application in S. hyo gene transfer. Proteins from purified virions were separated by SDS-PAGE and electroblotted to PVDF membranes. Of the 13 distinct bands detected by Coomassie blue staining, 5 (migrating at rates equivalent to 13, 19, 38, 55 and 100 kDa) were harvested from PVDF membranes, and the sequences of their N-terminal 20-40 amino acids were determined. Degenerate primers were designed based on amino acid sequences and used to PCR amplify the genes encoding these proteins from S. hyo strain B204 DNA. A forward primer (5'-AAA ATT/A ACT/A GAA AAA AAT/C AT) based on the 19 kDa protein and a reverse primer (5'-TGA ATA/T CCT/A GCT TTT/A ATT/A AT) based on the 13 kDa protein yielded a 1.1 kb product. The sequence of this product was used to design inverse PCR primers and amplify an additional 400 bp of flanking regions from B204 chromosomal DNA. Analysis of the resulting 1.5 kb nucleotide sequence revealed 2 open reading frames. One contained a region encoding the 40 N-terminal amino acids of the 19 kDa protein, and the other contained a region encoding the 30 N-terminal amino acids of the 13 kDa protein. BLAST and BLASTX searches of GenBank revealed no sequences with significant similarity to the 1.5 kb region. Knowledge of VSH-1 gene sequences will allow synthesis of probes to survey other S. hyo strains and Serpulina species for the virus and determine the location(s) of the prophage within the bacterial genome.