Stinear T, Ross B C, Davies J K, Marino L, Robins-Browne R M, Oppedisano F, Sievers A, Johnson P D
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
J Clin Microbiol. 1999 Apr;37(4):1018-23. doi: 10.1128/JCM.37.4.1018-1023.1999.
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.
溃疡分枝杆菌的分子分析揭示了两个新的插入序列（ISs），即IS2404和IS2606。IS2404是通过对先前描述的溃疡分枝杆菌重复DNA片段进行全序列分析而鉴定出来的。该元件长度为1274 bp，含有12 bp的反向重复序列和一个单一的开放阅读框（ORF），可能编码一个327个氨基酸（aa）的蛋白质，并且在转座时显然会产生7 bp的直接重复序列。在推测的转座酶与来自杀鲑气单胞菌（AsIs1）、大肠杆菌（H重复元件）、霍乱弧菌（VcIS1）和牙龈卟啉单胞菌（PGIS2）的其他细菌序列中的ISs所编码的转座酶之间发现了氨基酸相似性。第二个IS，即IS2606，是通过对溃疡分枝杆菌基因组DNA中一个含有重复序列的HaeIII片段进行序列分析而发现的。该元件长度为1404 bp，具有12 bp的反向重复序列和一个单一的ORF，可能编码一个445 aa的蛋白质。数据库搜索显示，与结核分枝杆菌的IS1554的推测转座酶具有高度的氨基酸同一性（70%）。还观察到与来自其他几种微生物的转座酶具有显著的氨基酸同一性（40%），这些微生物包括苜蓿根瘤菌（ISRm3）、洋葱伯克霍尔德菌（IS1356）、白喉棒状杆菌和鼠疫耶尔森菌。用IS2404的引物对45种其他分枝杆菌的DNA进行PCR筛选，证实该元件仅在溃疡分枝杆菌中发现。然而，通过PCR，在淡黄分枝杆菌中也发现了IS2606，淡黄分枝杆菌是分枝杆菌属中另一种生长缓慢的成员，在遗传上显然与溃疡分枝杆菌不同。基于IS2404和IS2606引物的PCR检测灵敏度分别显示能够检测到0.1和1个溃疡分枝杆菌基因组当量。通过使用两个基因靶点来检测少量细胞的能力对于分析环境样品将特别有用,在环境样品中，在大量其他环境分枝杆菌中可能存在低浓度的溃疡分枝杆菌。
