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Journal of Clinical Microbiology, April 1999, p. 1018-1023, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Identification and Characterization of IS2404 and IS2606: Two Distinct Repeated Sequences for Detection of Mycobacterium ulcerans by PCR

Timothy Stinear,1,* Bruce C. Ross,2 John K. Davies,1 Lui Marino,3 Roy M. Robins-Browne,3 Frances Oppedisano,3 Aina Sievers,4 and Paul D. R. Johnson1,3,5

Department of Microbiology, Monash University, Clayton,1 Research and Development, CSL Limited,2 Department of Microbiology and Infectious Diseases, Royal Children's Hospital,3 Victorian Infectious Diseases Reference Laboratory,4 and Department of Infectious Diseases and Clinical Epidemiology, Monash Medical Centre,5 Victoria, Australia

Received 9 September 1998/Returned for modification 22 November 1998/Accepted 21 December 1998

Molecular analysis of Mycobacterium ulcerans has revealed two new insertion sequences (ISs), IS2404 and IS2606. IS2404 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 (AsIs1), Escherichia coli (H repeat element), Vibrio cholerae (VcIS1), and Porphyromonas gingivalis (PGIS2). The second IS, IS2606, was discovered by sequence analysis of a HaeIII 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 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), Corynebacterium diphtheriae, and Yersinia pestis. PCR screening of DNA from 45 other species of mycobacteria with primers for IS2404 confirm that this element is found only in M. ulcerans. However, by PCR, IS2606 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 IS2404 and IS2606 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.

* Corresponding author. Mailing address: Department of Microbiology, Monash University, Wellington Rd., Clayton 3168, Australia. Phone: 61 3 9905 4809. Fax: 61 3 9905 4811. E-mail: tim.stinear{at}med.monash.edu.au.

Journal of Clinical Microbiology, April 1999, p. 1018-1023, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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