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United States Department of Agriculture

Agricultural Research Service

Title: Vsh-1, a Bacteriophage-Like Gene Vector for Serpulina Hyodysenteriae

Authors
item Humphrey, Samuel
item Stanton, Thaddeus
item Jensen, Neil
item Zuerner, Richard

Submitted to: Gordon Research Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: January 26, 1996
Publication Date: N/A

Technical Abstract: When S. hyodysenteriae B204 cultures are treated with mitomycin C (8 ug/ml broth), the cells lyse and release bacteriophages, designated "VSH-1". Purified VSH-1 virions had a buoyant density of 1.375 g/cm**3 and consisted of a head (45 nm diameter) and a noncontractile tail (64 nm x 9 nm). The virus was incapable of lytic growth on any of 5 intestinal spirochete strains, representing 3 Serpulina spp. VSH-1 nucleic acid was determined to be 7.5 kB, linear, double-stranded DNA. Viral DNA digested by different restriction enzymes gave electrophoretic banding patterns nearly identical to those of digested S. hyo chromosomal DNA. Additionally, VSH-1 DNA hybridized with probes complementary to S. hyo chromosomal genes (nox and flaA). Thus, restriction endonuclease analysis and Southern hybridization findings indicated that VSH-1 virions package host DNA. To evaluate whether or not VSH-1 is a gene vector, purified bacteriophages induced from cultures of S. hyo strain A201 (delta-flaA::Cm**R) were added to growing cells of recipient strain A216 (delta-nox::Km**R) for 8 hr and plated onto Trypticase soy blood agar containing both chloramphenicol (10 ug/ml) and kanamycin (200 ug/ml). Transductants (Cm**R, Km**R) were presented at a level of 1 per 2 x 10**5 viable cells in A216 cultures receiving virus and were not detected in control cultures to which no virus had been added. Homologous recombination incorporating the delta-flaA::Cm**R locus from donor strain A203 was confirmed by PCR and Southern blot analysis. These results suggest that VSH-1, a phage apparently incapable of lytic growth, packages the DNA of its bacterial host, S. hyodysenteriae, and is capable of transferring genes between cells of that spirochete.

Last Modified: 10/30/2014
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