title: Induction and transcription of VSH-1, a prophage-like gene transfer agent of Brachyspira hyodysenteriae.
abstract: The anaerobic spirochete Brachyspira hyodysenteriae is host to a bacteriophage-like agent known as VSH-1. VSH-1 is a novel gene transfer mechanism which does not self-propagate and transfers random 7.5kb fragments of host DNA between B. hyodysenteriae cells. In these investigations early events during VSH-1 induction by mitomycin C were examined. Quantitative PCR analysis revealed that VSH-1 hvp38 and hvp53 genes did not detectably increase in copy numbers during induction. Based on Northern blot hybridization assays, transcription of VSH-1 genes hvp38, hvp53, hvp45, hvp101, and lys increased fivefold to tenfold between 2 and 4h after induction whereas mRNA levels for B. hyodysenteriae flaA1 declined over the same time period. Chloramphenicol prevented the mitomycin C-induced increases in VSH-1 gene transcription. Hydrogen peroxide (300muM) substituted for mitomycin C as an inducer of VSH-1 gene transcription and is a possible 'natural' inducer of VSH-1 production in vivo. Northern blot hybridization, RT PCR, and primer extension analyses showed that VSH-1 genes are co-transcribed at an initiation site upstream of the VSH-1 gene operon. Two direct heptanucleotide repeats (ACTTATA) were identified between the putative -35 and -10 positions of the VSH-1 gene operon and are likely to represent a binding site for transcription proteins. These findings indicate VSH-1 virion production does not require genome replication, consistent with the inability of VSH-1 to self-propagate. Early events in VSH-1 induction include de novo synthesis of protein(s) essential for transcription of VSH-1 genes as polycistronic mRNA initiating upstream of the hvp45 gene.
authors.affiliation: Department of Microbiology, Iowa State University, Ames, IA 50010, USA.
authors.forename: Eric G
authors.initials: EG
authors.lastname: Matson
authors.forename: Richard L
authors.initials: RL
authors.lastname: Zuerner
authors.forename: Thad B
authors.initials: TB
authors.lastname: Stanton
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D002701:Chloramphenicol / Q000494:pharmacology; D004279:DNA, Viral / Q000096:biosynthesis; D005814:Genes, Viral; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016685:Mitomycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009876:Operon; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D039002:Prophages / Q000235:genetics* / Q000254:growth & development*; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D013144:Spirochaetales / Q000821:virology*; D014775:Virus Activation
chemical_list: D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D016685:Mitomycin; D002701:Chloramphenicol; D006861:Hydrogen Peroxide