|Matson, Eric - IOWA STATE UNIVERSITY|
|Thompson, M Greg - UNIVERSITY OF WASHINGTON|
Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 10, 2005
Publication Date: September 20, 2005
Citation: Matson, E.G., Thompson, M., Humphrey, S.B., Zuerner, R.L., Stanton, T.B. 2005. Identification of genes of VSH-1, a prophage-like gene transfer agent of Brachyspira hyodysenteriae. Journal of Bacteriology. 187(17):5885-5892. Interpretive Summary: The prevalence and persistence of antibiotic resistant bacteria in food animals with and without antibiotic use are problems for veterinary practitioners, food animal producers, pharmaceutical representatives, and physicians. In this paper, we describe how we found and sequenced the genes of VSH-1. VSH-1 is a novel gene transfer mechanism in bacteria known as Brachyspira spirochetes. These spirochetes cause intestinal diseases in pigs, chickens, wild birds, and possibly in humans. VSH-1 looks like a bacterial virus or bacteriophage but it is different. VSH-1 is different because its small particles carry all the genes of its host bacterium Brachyspira hyodysenteriae. Some of the genes that VSH-1 carries are genes for antibiotic resistance. Thus, VSH-1 can spread genes for resistance to the antibiotic tylosin from one B. hyodysenteriae cell to another. Our identification of the genes for VSH-1 will now let us identify animal management practices that encourage (or prevent) the production of VSH-1 by B. hyodysenteriae cells. This breakthrough research will let us design strategies to reduce the spread of antimicrobial resistance genes carried by VSH-1. This research should be of interest for researchers in university and industry settings especially those interested in swine intestinal diseases and antimicrobial resistance.
Technical Abstract: VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. VSH-1 purified virions are non-infectious, 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, proteins associated either with VSH-1 capsids or with tails were purified by SDS-PAGE. The N-terminal amino acid sequences of eleven 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 lambda clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence over-lapping 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 (7 genes), tail (7 genes), and lysis (4 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 non-infectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, gene transfer agents of other bacterial species, such as GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this gene transfer agent and should facilitate investigations of VSH-1-like prophages in other Brachyspira species.