No abstract

Reference strains for each of the 23 serogroups of Leptospira interrogans yielded different pulsed-field gel electrophoresis patterns of NotI digestion products. This was also the case for the 14 serovars belonging to serogroup Icterohaemorrhagiae (with one exception). The NotI restriction patterns of 45 clinical leptospiral isolates belonging to serovar icterohaemorrhagiae were analyzed and compared with those of type strains. No differences were observed between isolates from countries of different continents, namely, France, French Guiana, New Caledonia, and Tahiti. The pattern was indistinguishable from that of the reference strain of serovar icterohaemorrhagiae.

Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.

Metabolites such as diaminopimelate and some aromatic derivatives, not synthesized in mammalian cells, are essential for growth of bacteria. As a first step towards the design of a new human live vaccine that uses attenuated strains of Leptospira interrogans, the md, uroD and h p D genes, encoding aspartate P-semialdehyde dehydrogenase, 3-dehydroquinase and tetrahydrodipicolinate N-succinyltransferase, respectively, were cloned by complementation of Escherichia coli mutants. The complete nucleotide sequence of the md gene was determined and found to contain an open reading frame capable of encoding a protein of 349 amino acids with a calculated M, of 38007. Comparison of this deduced L. interrogans aspartate P-semialdehyde dehydrogenase amino acid sequence with those of the same enzyme from Succharomyces cereuisiue and Corynekterium glutmicum revealed 46% and 36% identity, respectively. By contrast, the identity between the L. interrogans enzyme and the Streptococcus mutans or E coli enzymes was less than 31%. Highly conserved sequences within aspartate semialdehyde dehydrogenase from the five organisms were observed at the amino and carboxyl termini, and around the cysteine of the active site.

Pulsed‐field gel electrophoresis allowed the determination of the size of the genome of Leptospira, a bacterium of the spirochete family. The three restriction enzymes, Not I (5′ GC/GGCCGC), Nhe I (5′‐G/CTAGC), Apa I (5′‐GGGCC/C) generated DNA fragments of suitable size. The results are compatible with a size of 5000 kb for the chromosome of both the pathogenic and the saprophytic species of Leptospira.