Enzymes with phospholipase C activity in Mycobacterium tuberculosis have been recently described. The three genes encoding these proteins, plcA, plcB, and plcC, are located at position 2351 of the genomic map of M. tuberculosis H37Rv and are arranged in tandem. We have previously described the presence of variations in the restriction fragment length polymorphism patterns of the plcA and plcB genes in M. tuberculosis clinical isolates. In the present work we investigated the origin of this polymorphism by sequence analysis of the phospholipase-encoding regions of 11 polymorphic M. tuberculosis clinical isolates. To do so, a long-PCR assay was used to amplify a 5,131-bp fragment that contains the plcA and plcB genes and part of the plcC gene. In the M. tuberculosis strains studied the production of an amplicon ϳ1,400 bp larger than anticipated was observed. Sequence analysis of the PCR products indicated the presence of a foreign sequence that corresponded to an IS6110 element. We observed insertion elements in the plcA, plcB, and plcC genes. One site in plcB had the highest incidence of transposition (5 out of 11 strains). In two strains the insertion element was found in plcA in the same nucleotide position. In all the cases, IS6110 was transposed in the same direction. The high level of transposition in the phospholipase region can lead to the excision of fragments of genomic DNA by recombination of neighboring IS6110 elements, as demonstrated by finding the deletion, in two strains, of a 2,837-bp fragment that included plcA and most of plcB. This can explain the negative results obtained by some authors when detecting the mtp40 sequence (plcA) by PCR. Given the high polymorphism in this region, the use of the mtp40 sequence as a genetic marker for M. tuberculosis sensu stricto is very restricted.The mtp40 gene was first described as a 402-bp open reading frame (ORF) encoding a 13.8-kDa specific protein of Mycobacterium tuberculosis (21). This gene was cloned in a 3.1-kbp BamHI fragment, and, after sequencing the whole insert, Leão et al. noted the presence of an ORF of 1,170 bp and the beginning of another (15). Johansen et al. (13) completely sequenced the second ORF, and they also demonstrated in vitro that these genes encode phospholipase C activities. They named the ORFs mpcA and mpcB. The sequence called mtp40 actually constitutes only a part of the mpcA gene. After the whole M. tuberculosis H37Rv genome was sequenced, two more phospholipase genes were decribed: an ORF beside mpcB and another related sequence at position 1755 of the genome, located beside an IS6110 element (4). From this point on these genes were designated plc (phospholipase C) genes; the three ORFs arranged in tandem were called plcA, plcB, and plcC, and the fragment at position 1755 in M. tuberculosis H37Rv was called plcD. We use this nomenclature in the present work.Since there have been conflicting results concerning the presence of the mtp40 sequence in the M. tuberculosis complex, we previously studied its distribution within a c...
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