I00004Identification of genes encoding virion-associated proteins of VSH-1, a bacteriophage of Serpulina hyodysenteriae
M. GREG THOMPSON (1), THAD B. STANTON (2), SAM B. HUMPHREY(2), NEIL S. JENSEN(2).
(1) Dept. of Microbiology, Immunology, and Preventive Medicine, Iowa State University, Ames,IA. (2) National Animal Disease Center, USDA-ARS, Ames, IA.
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 (J Bacteriol. 179:323). To further investigate the nature of VSH-1 and its potential application in S. hyo gene transfer, we have begun a search for viral genes. Proteins from purified virions were separated by SDS-PAGE and electroblotted to PVDF membranes. Of the thirteen distinct bands detected by Coomassie blue staining, five (migrating at rates equivalent to 13, 19, 38, 55, and 100 kDa) were harvested from PVDF membranes, and the sequences of their N-terminal twenty to forty amino acids were determined. Degenerate primers were designed based on the 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 two open reading frames. One contained a region encoding the forty N-terminal amino acids of the 19 kDa protein, and the other contained a region encoding the thirty 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.
Comments:
Address questions and comments about this abstract to Thad B. Stanton ( tstanton@iastate.edu).
Previously presented at Conf. Res. Workers in Animal Diseases on Nov 1996.