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Identification of Genes of VSH-1, a Prophage-Like Gene Transfer Agent of Brachyspira hyodysenteriae

Enteric Diseases and Food Safety Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 50010,¹ Department of Microbiology, Iowa State University, Ames, Iowa 50010,² Zoonotic Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 50010³

Received 5 April 2005/ Accepted 10 June 2005

VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. Purified VSH-1 virions are noninfectious, contain random 7.5-kb fragments of the bacterial genome, and mediate generalized transduction of B. hyodysenteriae cells. In order to identify and sequence genes of this novel gene transfer agent (GTA), proteins associated either with VSH-1 capsids or with tails were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of 11 proteins were determined. Degenerate PCR primers were designed from the amino acid sequences and used to amplify several VSH-1 genes from B. hyodysenteriae strain B204 DNA. A clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence overlapping DNA inserts containing additional VSH-1 genes. VSH-1 genes spanned 16.3 kb of the B. hyodysenteriae chromosome and were flanked by bacterial genes. VSH-1 identified genes and unidentified, intervening open reading frames were consecutively organized in head (seven genes), tail (seven genes), and lysis (four genes) clusters in the same transcriptional direction. Putative lysis genes encoding endolysin (Lys) and holin proteins were identified from sequence and structural similarities of their translated protein products with GenBank bacteriophage proteins. Recombinant Lys protein hydrolyzed peptidoglycan purified from B. hyodysenteriae cells. The identified VSH-1 genes exceed the DNA capacity of VSH-1 virions and do not encode traditional bacteriophage early functions involved in DNA replication. These genome properties explain the noninfectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, GTAs of other bacterial species, such as the GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this GTA and should facilitate investigations of VSH-1-like prophages from other Brachyspira species.

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