Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae

Abstract

Serpulina hyodysenteriae B204 cells treated with mitomycin (20 g of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsCl density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm 3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa. The purified bacteriophage has been designated VSH-1 (VSH for virus of S. hyodysenteriae). VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species. VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy. Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S. hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S. hyodysenteriae chromosomal genes nox and flaA1. When purified bacteriophages induced from cultures of S. hyodysenteriae A203 (⌬flaA1 593-762::cat) were added to growing cells of strain A216 (⌬nox 438-760::kan), transductants (Cm r Km r ) were obtained at a frequency of 1.5 ؋ 10 ؊6 per phage particle (enumerated by electron microscopy). These findings indicate that induced VSH-1 virions package DNA of S. hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete.Natural gene transfer mechanisms (conjugative plasmids and transducing bacteriophages) have not been demonstrated for spirochetes. Indirect evidence of lateral gene transfer has been obtained for Borrelia spp. (16). The existence of DNAfilled membrane vesicles on the surfaces of Borrelia burgdorferi cells has been reported, although their possible role in gene transfer is unknown (7). In addition, several extrachromosomal elements have been identified in B. burgdorferi, and an extrachromosomal plasmid of 2.6 kb has been isolated from Treponema denticola and characterized (10,25). In cultures of various spirochetes, bacteriophages have been observed free, attached to cells, or within cells as noted previously (9). Three lytic phages of Leptospira biflexa have been isolated and characterized (24). However, to our knowledge, there is no evidence that these three phages play a role in gene transfer.The spirochete Serpulina hyodysenteriae causes swine dysentery, an enteric disease producing a severe mucohemorrhagic diarrhea in infected swine (8). Potential genetic transfer elements for S. hyodysenteriae have been identified. Ritchie and colleagues observed bacteriophages with the same morphology in 18 different cultures of S. hyodysente...

Gene transfer agents: phage-like elements of genetic exchange

“…2c), and contain 7.5 kb of linear double-stranded DNA 50 . The element is named VSH-1, and the packaged DNA is a random collection of fragments from the genome of the producing cell 50 .…”

“…2c), and contain 7.5 kb of linear double-stranded DNA 50 . The element is named VSH-1, and the packaged DNA is a random collection of fragments from the genome of the producing cell 50 . VSH-1 mediates the transfer of many markers between B. hyodysenteriae cells [50][51][52][53] .…”

“…The element is named VSH-1, and the packaged DNA is a random collection of fragments from the genome of the producing cell 50 . VSH-1 mediates the transfer of many markers between B. hyodysenteriae cells [50][51][52][53] . Although mitomycin C is required to observe the particles 49,50 , DNA fragments matching those packaged by VSH-1 have been found in B. hyodysenteriae that has not been treated with mitomycin C 54,55 , and co-culture experiments have confirmed that VSH-1 can be produced in the absence of an exogenous inducer 52,53 .…”

“…VSH-1 mediates the transfer of many markers between B. hyodysenteriae cells [50][51][52][53] . Although mitomycin C is required to observe the particles 49,50 , DNA fragments matching those packaged by VSH-1 have been found in B. hyodysenteriae that has not been treated with mitomycin C 54,55 , and co-culture experiments have confirmed that VSH-1 can be produced in the absence of an exogenous inducer 52,53 . Two important differences between VSH-1 and other GTAs are the fact that VSH-1 production can be induced with mitomycin C and the fact that detectable cell lysis is associated with particle release 49 (discussed further below).…”

“…It has been suggested that VSH-1 plays a part in the genomic and population structures and the pathogenic properties of Brachyspira spp. 63,64 , and VSH-1 does transfer virulence and antibiotic resistance genes between cells [50][51][52][53] . VSH-1 is induced by treatment of Brachyspira spp.…”