Mapping of IS6110 insertion sites in two epidemic strains of Mycobacterium tuberculosis

Abstract

A widely distributed strain designated 210 was identified in a study of the diversity of Mycobacterium tuberculosis DNA fingerprints from three geographically separate states in the United States. This strain is characterized by a 21-band fingerprint pattern when probed with IS6110, and the pattern is similar to that displayed by strains designated W. Intracellular growth of strain 210 isolates in human macrophages is significantly faster than that of isolates from other clusters or nonclustered isolates. The purpose of this study was to identify the sites of IS6110 insertions in strain 210 and compare these to IS6110 insertion sites in strain W. Our hypothesis is that an IS6110 insertion site(s) could possibly be responsible for a strain's increased capacity for transmission and/or replication. In this report, the insertion sites in strains 210 and W are described and referenced to their location in the M. tuberculosis H37Rv genome sequence. The W and 210 strains have 17 identical sites of IS6110 insertion and additional sequence not found in H37Rv but present in other clinical isolates. The IS6110 insertion site in the 36-bp direct repeat (DR) region of strains 210 and W has 15 spacers in the left flanking region. The DR region on the right side of IS6110 has been deleted. Five sites of insertion in strain 210 not found in strain W are described, as well as two unique sites in strain W. One copy of IS6110 was found to reside 55 bp in the ctpD gene. This gene is expressed, indicating that IS6110 can provide a promoter sequence for the transcription of genes.

FIG. 1

IS6110 fingerprint patterns of strains 210 (lane 1) and W (lane 2). Size markers are indicated on the right. Numbers 1 to 21 correspond to the flanking sequences listed in Table 1. These were numbered in the order in which they were isolated.

FIG. 2

Map of the DR region of strains 210 and W on the left side of IS6110. The sequence of IS6110 is underlined with a black arrow. Each 36-bp DR is underlined with a black bar. The three DR sequences that do not have the consensus sequence are marked with an asterisk. The spacer sequences are underlined with an open bar. The spacers are labeled with the new spoligotyping designation system with the old number in parentheses.

FIG. 3

RT-PCRs of the ctpD gene for strains 210 and H37Rv and two 21-band clinical isolates. Lane M, 1-kb ladder; sizes listed are in base pairs. Lane 1, H37Rv positive RT; lane 2, H37Rv negative RT; lane 3, 210 positive RT; lane 4, 210 negative RT; lane 5, H1110 positive RT; lane 6, H1110 negative RT; lane 7, TB545 positive RT; lane 8, TB545 negative RT; lane 9, negative PCR control; lane 10, positive PCR control, 10 pg of 210 DNA.