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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.

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 genomic locus was discovered within Brucella abortus that contains a novel transposon-like element designated Tn2020. Tn2020 is bounded by a copy of an insertion sequence designated IS2020 and a truncated imperfect copy of IS2020 (tIS2020A). The truncated copy is immediately adjacent to a second copy of IS2020. These data are consistent with the locus having evolved by a complex rearrangement following the transposition of a second copy of Tn2020. Analysis of the organization, orientation, and open reading frames (ORFs) of IS2020 places it within the IS6 family. Four ORFs from Tn2020 were translated in vitro producing a potential transposase with an apparent molecular mass of 27.5 kDa, and three polypeptides with apparent molecular masses of 71 kDa, 22 kDa, and 14 kDa. The central region of Tn2020 encodes the 71 kDa and 14 kDa proteins, while the 22 kDa protein is likely an internal translation initiation product of the IS2020 transposase. The 71 kDa protein shares sequence similarity with several bacterial transcriptional regulatory proteins and may form a helix-turn-helix structure capable of binding DNA. No homologous protein sequences to the 14 kDa peptide were detected in available databases. The 27.5 kDa transposase and its 22 kDa internal translation product shared significant similarity to several transposases from diverse bacterial hosts. Immediately 5' of Tn2020 are genes encoding ribosomal proteins RplU and RpmA. This region also contains a 90 bp sequence that shares significant homology to a repetitive element with inverted repeats from the Sinorhizobium genome. Downstream from Tn2020 is an ORF encoding a protein having significant similarity to a hypothetical protein from Caulobacter. The locus is lower in G+C content from that of the genome. These data suggest that lateral transfer of genetic material has occurred.

Genetic variability among Leptospira interrogans (sensu stricto) serovars was assessed by Southern blot hybridization and PCR analyses. The experiments used probes directed to sequences in a recently described insertion element, IS1500. Hybridization analysis showed that IS1500 was present on polymorphic fragments and that differences in these patterns could be used to identify serovars. Hybridization analysis was also useful in discriminating between serovar pomona type kennewicki isolates, making possible the identification of 15 previously unrecognized genetic groups. A PCR assay was developed in which the primers are positioned near the terminal inverted repeats of the element and directed outward. This assay yielded characteristic amplification patterns from isolates, allowing them to be identified. We applied these assays to several new animal isolates of L. interrogans from Nicaragua, which recently had an outbreak of human leptospirosis. Three groups of isolates were identified: one strain of serovar pomona type kennewicki and two genetically distinct groups of isolates which may be genetic intermediates between serovars canicola and portlandvere. The IS-based typing assays described should be useful for epidemiological analysis of leptospirosis.

A PCR-based assay was developed for typing L. interrogans sensu lato serovars. The assay is designed to exploit the presence of many copies of the leptospiral insertion sequence IS1533 and IS1533-like sequences present in the genomes of most leptospiral serovars. The PCR primers were designed to amplify DNA of unknown sequence between closely placed IS1533 or IS1533-like sequences. Amplification reactions primed with IS1533-based primers generated products of different sizes. When few copies of IS1533 were present in the genome, amplification of a few products was still detected. These results suggest that IS1533 elements may be found close together. Analysis of DNA amplified from different serovars showed the presence of differently sized products, thus enabling the serovars to be identified. Genetic variation among isolates within the same serovar was also demonstrated with the IS1533-based primers. Amplification reactions using DNA extracted from the urine of infected animals generated specific products which were similar to the products generated from purified bacterial DNA. These results demonstrate that this assay is selective enough to be used for typing leptospiral serovars from clinical material and thus allows leptospiral typing without isolation of the bacteria in pure culture.

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.