Repetitive sequence of Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No. 1: a sensitive probe for demonstration of Leptospira interrogans strains.
A 4.8-kilobase (kb) repetitive sequence element generated with KpnI digestion was cloned from the Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No. 1. The sequence, repeated in tandem, was located on the 280-kb fragment between the FseI and AscI sites on the chromosome by hybridization using the 4.8-kb fragment as a probe. We cloned the fragment containing the element for the Ictero No. 1 strain in a lambda EMBL3 bacteriophage DNA, and one out of 5 clones was sequenced. Within the sequenced 9-kb segment that partially repeated, 9 putative open-reading frames and 2 transfer RNA genes, for alanine and isoleucine, were identified. A similarity search for the products deduced from the sequenced data revealed that the repeated sequence includes both beta-oxidation enzymes, acyl-CoA dehydrogenase and enoyl-CoA hydratase, and hydroxythiazole kinase protein homologues. Hybridization experiments against different leptospiral strains using the element as a probe showed a similar sequence in the strains of L. interrogans and L. kirschneri, but not in any strains of L. borgpetersenii, L. weillii, L. meyeri or L. biflexa. Results indicated that the highly repeated element in the Ictero No. 1 strain exists as a well conserved sequence, though at a moderate level of repetition, in certain strains of L. interrogans and L. kirschneri. PCR amplification targeting the repetitive element was successful and indicated that the procedure provides a sensitive and specific probe to detect leptospires.
Takahashi
Y
Y
Laboratory of Molecular Microbiology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Hiroshima, Japan.
Kishida
M
M
Yamamoto
S
S
Fukunaga
M
M
D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific; D007921:Leptospira interrogans / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific; C029312:GGTACC-specific type II deoxyribonucleases