Identification and Characterization of IS 2404 and IS 2606 : Two Distinct Repeated Sequences for Detection of Mycobacterium ulcerans by PCR

Abstract

ABSTRACT Molecular analysis of Mycobacterium ulcerans has revealed two new insertion sequences (ISs), IS 2404 and IS 2606 . IS 2404 was identified by complete sequencing of a previously described repetitive DNA segment from M. ulcerans . This 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. Amino acid similarity was found between the putative transposase and those encoded by ISs in other bacterial sequences from Aeromonas salmonicida ( As Is 1 ), Escherichia coli (H repeat element), Vibrio cholerae ( Vc IS 1 ), and Porphyromonas gingivalis ( PG IS 2 ). The second IS, IS 2606 , was discovered by sequence analysis of a Hae III fragment of M. ulcerans genomic DNA containing a repetitive sequence. This element is 1,404 bp long, with 12-bp inverted repeats and a single ORF potentially encoding a protein of 445 aa. Database searches revealed a high degree of amino acid identity (70%) with the putative transposase of IS 1554 from M. tuberculosis . Significant amino acid identity (40%) was also observed with transposases from several other microorganisms, including Rhizobium meliloti (IS Rm3 ), Burkholderia cepacia (IS 1356 ), Corynebacterium diphtheriae , and Yersinia pestis . PCR screening of DNA from 45 other species of mycobacteria with primers for IS 2404 confirm that this element is found only in M. ulcerans . However, by PCR, IS 2606 was also found in Mycobacterium lentiflavum , another slow-growing member of the genus Mycobacterium that is apparently genetically distinct from M. ulcerans . Testing the sensitivity of PCR based on IS 2404 and IS 2606 primers demonstrated the ability to detect 0.1 and 1 M. ulcerans genome equivalents, respectively. The ability to detect small numbers of cells by using two gene targets will be particularly useful for analyzing environmental samples, where there may be low concentrations of M. ulcerans among large numbers of other environmental mycobacteria.