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

Abstract

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 Brachyspira hyodysenteriae is an anaerobic spirochete and the etiologic agent of swine dysentery, a severe, mucohemorrhagic intestinal disease afflicting animals in the postweaning period of growth (20). B. hyodysenteriae cells contain a defective prophage, VSH-1, which is induced when mitomycin C is added to cultures of growing cells (27). This prophage has been purified and characterized previously (28). VSH-1 particles resemble virions in morphology but, with a head diameter of 45 nm and a tail length of 64 nm, are substantially smaller.VSH-1 virions contain random 7.5-kb fragments of host genomic DNA (28), making it difficult to identify VSH-1 genes. Purified virions are "noninfectious," that is, they do not lyse spirochete cells when added to cultures of B. hyodysenteriae or of other Brachyspira species. VSH-1 transfers bacterial genes between B. hyodysenteriae cells (28, 52) and has likely contributed to the recombinant population structure of B. hyodysenteriae (55). This generalized transduction activity of VSH-1 is useful for constructing B. hyodysenteriae mutant strains (35).VSH-1 is the first natural gene transfer mechanism to be described for a spirochete.The ability to recognize VSH-1-specific genes would be a major advance for investigating intracellular and intercellular activities of VSH-1 and for evaluating its ecological significance to its host bacterium. To overcome the inability to identify distinct VSH-1 genes carried by virions, structural protein genes were identified from the sequences of VSH-1 head and tail proteins. Genes for endolysin and holin proteins, proteins enabling the release of bacteriophage from bacterial cells, were discovered during nucleotide sequence analyses. Recombinant endolysin was shown to degrade peptidoglycan. Based on its genome and biological properties, VSH-1 resembles prophagelike gene transfer agents (GTA) reported for other bacteria (3,32,37,46,56). MATERIALS AND METHODSSeparation of VSH-1 whole virions and tailless heads. VSH-1 particles were harvested from B. hyodysenteriae strain B204 cultures (400 ml) in NT broth 7 to 8 h after...

Gene transfer agents: phage-like elements of genetic exchange

“…3b) include a gene that is similar to a putative phage endolysin and is located next to the region encoding the VSH-1 structure. The protein encoded by this gene degrades B. hyodysenteriae peptidoglycan 56 , and a putative holin-encoding gene has also been identified close by 56 . One of the proteins associated with VSH-1 did not correspond to any of the genes that were originally mapped 56 , and subsequent work identified a separate cluster of three contiguous VSH-1 tail genes located a short distance away 57 (FIG.…”

“…VSH-1 gene distribution, using the current VSH-1-encoding region 56,57 (FIG. 3b), appears to be limited to members of the Brachyspira genus (FIG.…”

Identification of a Divided Genome for VSH-1, the Prophage-Like Gene Transfer Agent of Brachyspira hyodysenteriae

“…VSH-1 structural and escape (lysis) proteins are encoded by contiguous clusters of VSH-1 head, tail, and lysis genes ( Fig. 1; Table 1) occupying 16.3 kb of the B204 chromosome (15). In those studies, the gene encoding Hvp60, a tail-associated protein with the sequence NЈ-M_K_ MPYHFLRNKIYKLPPAPYINE was not found within the 16.3-kb VSH-1 genome cluster (15).…”

“…VSH-1 particles contain 7.5-kb fragments of B. hyodysenteriae DNA and transfer genes, including antibiotic resistance genes, between strains of the spirochete (11,15,20,21). VSH-1-like elements are widespread in Brachyspira species (3,4,22,26).…”

“…Previously, head (capsid) and tail proteins of VSH-1 particles were sequenced, and the sequences were used to establish the VSH-1 genome (15). VSH-1 structural and escape (lysis) proteins are encoded by contiguous clusters of VSH-1 head, tail, and lysis genes ( Fig.…”