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Leptospira interrogans is a diverse species in which individual serovars have distinctive restriction fragment length polymorphisms that are useful in strain identification. Many of these polymorphisms can be detected using hybridization probes derived from insertion sequences; an observation that suggests these IS elements are active and can transpose in L. interrogans. Two spontaneous mutants of L. interrogans serovar Pomona strain RZ11 were isolated by immune selection and characterized. Changes in the size and antigenicity of LPS from these mutants were detected. Genetic analysis showed that both mutants have additional copies of an IS3-like element, designated IS1501, that are not present in the parental strain. One mutant, GT211, has a single additional copy of IS1501, whereas the other mutant, GT210 has three additional copies of IS1501 relative to strain RZ11. IS1501 transposition generated 3-bp direct repeats from target sequences flanking the insertion site. RT-PCR analysis of transcripts at altered loci showed IS1501 transcripts extended into adjacent sequences. These data are the first to show spontaneous transposition of an endogenous Leptospira insertion sequence, and suggest that IS1501 may be capable of gene activation.

VSH-1 is a mitomycin C-inducible, non-lytic, phage-like agent that packages random 7.5-kb fragments of the Brachyspira hyodysenteriae genome. VSH-1 is the first recognized mechanism for gene transfer between B. hyodysenteriae cells. To analyze the distribution of VSH-1 among spirochetes, a 344-bp probe for gene svp38, encoding the VSH-1 major head protein, was amplified by polymerase chain reaction and used in Southern blot hybridizations with genomic DNA from various spirochete genera. The svp38 probe hybridized to a 40-kb SalI-SmaI fragment of the B. hyodysenteriae B78(T) chromosome, indicating VSH-1 DNA insertion into the chromosome at a unique site. Restriction endonuclease digested DNAs of 27 spirochete strains representing six Brachyspira species (B. hyodysenteriae, B. innocens, B. pilosicoli, B. murdochii, B. intermedia, B. alvinipulli) contained a single fragment hybridizing with the svp38 probe. DNAs from spirochete species of the genera Treponema, Spirochaeta, Borrelia, and Leptospira did not hybridize with the probe. VSH-1-like agents appear to be widely distributed among Brachyspira species and, as has been demonstrated for B. hyodysenteriae, may serve as useful gene transfer agents for those other species.

A region of the Leptospira interrogans serovar pomona genome encoding DNA replication genes was characterized. This region, designated the ppa-ntrC locus, includes 19 open reading frames and a new insertion sequence, IS1502. Although this locus resembles replication origins from many eubacteria, it lacks several genes common to homologous loci. Some replication-related genes were previously located near rrf, and may have been moved to that location by homologous recombination between short sequence elements common to both loci. Further analysis showed that the ppa-ntrC region has undergone substantial change during spirochete evolution. Transcription analysis using RT-PCR revealed uniquely organized polycistronic mRNAs in the ppa-ntrC locus. The dnaN and recF intergenic region of serovar pomona was different from the homologous sites of 41 L. interrogans serovars by the presence of IS1502. The distribution of IS1502 throughout pathogenic Leptospira species varies. This result suggests that IS1502 may have been recently introduced into Leptospira.

A ribosomal protein gene cluster from the spirochaete Leptospira interrogans was characterized. This locus is homologous to the Escherichia coli S10, spc, and alpha operons. Analysis of L. interrogans RNA showed that the ribosomal protein genes within this cluster are co-transcribed, thus forming an operon. Two transcription initiation sites were mapped by primer extension, upstream of fus, the first gene in this cluster, and sequences from this region provided promoter activity in E. coli. Transcription terminates near a predicted stem-loop structure following rplQ, the last gene in the cluster. These data suggest that two promoters upstream of fus direct transcription of this 17.5-kb ribosomal protein gene cluster. Comparison of the L. interrogans S10-spc-alpha cluster to homologous loci from Borrelia burgdorferi and Treponema pallidum provided evidence that this region of the genome underwent several rearrangements during spirochaete evolution.

The size and physical structure of the Leptospira interrogans genome was characterized using contour-clamped homogenous electric field (CHEF) gel electrophoresis. The L. interrogans genome is approximately 4750 kb in size and is composed of two molecular species of DNA: a 4400 kb chromosome; and a 350 kb plasmid, pLIN1. A physical map of the chromosome was constructed with the restriction enzymes NotI and SfiI. A physical map of pLIN1 was constructed with ApaI, NotI, Sse83871, SgrAI, and SmaI. Both the L. interrogans chromosome and pLIN1 are circular.