Identification and characterization of IS2404 and IS2606: Two distinct repeated sequences for detection of Mycobacterium ulcerans by PCR

Molecular analysis of Mycobacterium ulcerans has revealed two new insertion sequences (ISs), IS2404 and IS2606, IS2404 was identified by complete sequ encing of a previously described repetitive DNA segment from M. ulcerans, T his element is 1,274 bp long, contains 12-bp inverted repeats and a single open reading frame (ORF) potentially encoding a protein of 327 amino acids (aa), and apparently generates 7-bp direct repeats upon transposition. Amin o acid similarity was found between the putative transposase and those enco ded by ISs in other bacterial sequences from Aeromonas salmonicida (AsIs1), Escherichia coli (N repeat element), Vibrio cholerae (VcIS1), and Porphyro monas gingivalis (PGIS2). The second IS, IS2606, was discovered by sequence analysis of a HaeIII fragment of M. ulcerans genomic DNA containing a repe titive sequence. This element is 1,404 bp long, with 12-bp, inverted repeat s and a single ORF potentially encoding a protein of 445 aa, Database searc hes revealed a high degree of amino acid identity (70%) with the putative t ransposase of IS1554 from M. tuberculosis, Significant amino acid identity (40%) was also observed with transposases from several other microorganisms , including Rhizobium meliloti (ISRm3), Burkholderia cepacia (IS1356), Cory nebacterium diphtheriae, and Yersinia pestis. PCR screening of DNA from 35 other species of mycobacteria with primers fur IS2404 confirm that this ele ment is found only in M. ulcerans, However, by PCR, IS2606 was also found i n Mycobacterium lentiflavum, another slow-growing member of the genus Mycob acterium that is apparently genetically distinct from M. ulcerans. Testing the sensitivity of PCR based on IS2404 and IS2606 primers demonstrated the ability to detect 0.1 and 1 M. ulcerans genome equivalents, respectively, T he ability to detect small numbers of cells by using two gene targets will be particularly useful fur analyzing environmental samples, where there may be law concentrations of M. ulcerans among large numbers of other environm ental mycobacteria.