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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.

A lambda gt11 library constructed with Leptospira borgpetersenii DNA was screened with monoclonal antibodies (mAb) recognizing a periplasmic flagella-associated protein. A plaque expressing a fusion protein (lambda F15) which reacted with the mAb was isolated and the nucleotide sequence analyzed. The deduced amino-acid (aa) sequence indicates that the pfaP gene belongs to a group of bacterial genes whose products share aa sequence and possibly functional homologies with sppA, an Escherichia coli signal peptidase-encoding gene.

A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes.

The Leptospira biflexa rpsL and rpsG genes were sequenced. Although similar in many respects, proteins encoded by these L. biflexa genes had several unusual features when compared with homologous proteins of other organisms. Unlike the rpsL genes of other eubacteria, the L. biflexa rpsL gene is adjacent to a rpoC-like gene.

The genus Leptospira, as a member of the order Spirochaetales, forms one of the most ancient evolutionary branches of the eubacteria. These spirochetes are morphologically and physiologically different from most eubacteria, and little is known about Leptospira genetics. In this communication, we report the first nucleotide sequence of a Leptospira gene. A gene which complements an argE mutation in Escherichia coli was isolated from a plasmid-based genomic library composed of Leptospira biflexa serovar patoc DNA. The functional region for the complementing activity was localized by transposon mutagenesis and restriction enzyme mapping and by subcloning. Nucleotide sequence analysis indicated a single open reading frame within the region containing argE complementing activity. The size of the predicted protein, 31,071 daltons, was in excellent agreement with data obtained from coupled transcription-translation reactions primed with cloned L. biflexa DNA. One surprising result was that the predicted amino acid sequence of this protein closely resembles portions of the beta' subunits of RNA polymerases from bacteria and chloroplasts.