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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.

Brachyspira pilosicoli and B. hyodysenteriae are anaerobic pathogenic intestinal spirochetes differing in host range and disease manifestations. Little is known about the size, organization, or genetic content of the B. pilosicoli genome and only limited information is available regarding the genetic organization in B. hyodysenteriae. Both B. hyodysenteriae and B. pilosicoli exist as recombinant populations, and this may be due, in part, to an unusual phage-like gene transfer agent, VSH-1. To compare genetic organization in these two species, the number of mapped loci on an existing physical and genetic map of B. hyodysenteriae B78(T) was expanded, and a combined physical and genetic map of B. pilosicoli P43/6/78(T) was constructed. The B. pilosicoli genome size was about 2.5 Mb, nearly 750 kb smaller than the B. hyodysenteriae genome. Several chromosomal rearrangements have contributed to differences in the size, organization, and content of the two bacterial genomes, and such differences may influence the ability of these species to infect different hosts and cause disease. To evaluate these differences further, comparisons were focused on genes thought to contribute to host-parasite interactions. Four genetic loci (bit, fruBC, vspA, and vspH) were found in B. hyodysenteriae, but not in B. pilosicoli, while two genetic loci (clpX and mglB) were found in B. pilosicoli, but not in B. hyodysenteriae. Contrary to a previous study, an intact copy of the hlyA gene, encoding the B. hyodysenteriae beta-hemolysin gene was detected in B. pilosicoli. Although the hlyA genes of these two species were nearly identical, sequence variation was detected in the intergenic region upstream of hlyA that may alter transcription and translation efficiency of this gene in B. pilosicoli. In addition, divergence in genes flanking hlyA may affect the chemical composition of lipid attached to the mature B. pilosicoli HlyA protein resulting in reduced hemolytic activity.

The nucleotide sequence of IS1533, an insertion sequence-like element cloned from the spirochete Leptospira borgpetersenii, was determined. IS1533 contains imperfect terminal inverted repeats (IVR) of 31 bp flanking a 1402-bp internal sequence. A putative target sequence was identified, and insertion may result in duplication of 2 bp. The internal sequence has a single open reading frame (ORF). IS1533 encodes two proteins (43.5 and 41 kDa) initiating alternatively at either the first or the second AUG codons of the ORF. These proteins are related to a recently recognized family of IS-encoded transposases and bacterial recombinases, all which share a region of homology with the active site of the HIV reverse transcriptase. The IS1533-encoded proteins were expressed in Escherichia coli. Both the 43.5- and 41-kDa proteins bound IS1533 DNA probes in a Southwestern blot assay. These data suggest that one or both proteins function during transposition of IS1533.

Outer membrane and secreted proteins were isolated from Leptospira interrogans serovar pomona and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblot and radioimmunoprecipitation techniques. The L. interrogans outer membranes were extracted with Triton X-114 and contained several proteins. The major cellular protein with a molecular mass of 31 kDa was associated exclusively with the L. interrogans outer membrane. Using a whole cell immunoprecipitation method, five hydrophobic, Triton X-114 extractable proteins (22, 26, 31, 36 and 42 kDa) were exposed on the surface of L. interrogans. The 31 kDa protein was heat labile and was a potent antigen in animals experimentally infected with L. interrogans serovar pomona. Several proteins were secreted by L. interrogans including a 60 kDa protein tentatively identified as the L. interrogans hemolysin.