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Deciphering kas operon locus in Mycobacterium aurum and genesis of a recombinant strain for rational‐based drug screening
Author(s) -
Gupta N.,
Singh B.N.
Publication year - 2008
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2008.03888.x
Subject(s) - operon , biology , antimycobacterial , reporter gene , recombinant dna , gene , mycobacterium tuberculosis , microbiology and biotechnology , mycobacterium , gene expression , genetics , escherichia coli , tuberculosis , bacteria , medicine , pathology
Aims: To generate a recombinant Mycobacterium aurum strain for screening of antimycobacterial compounds affecting fatty acid synthase type II (FAS‐II) elongation pathway. Methods and Results: kas operon locus was delineated in M . aurum , a fast growing nonpathogenic strain. Cloning and sequencing all the genes of the operon showed similar organization and sequence similarities with Mycobacterium tuberculosis (H37Rv) orthologues. Further, we cloned the upstream region of M. tuberculosis kas operon in fusion with lacZ reporter gene and put it in M. aurum . Recombinant M. aurum strain showed continued expression of reporter gene throughout the growth while an increased expression of the reporter gene was noticed only after treatment with FAS‐II pathway inhibitors. Swapping of the regulatory sequence aborts the increased reporter gene expression after same antibiotic treatments. Conclusions: kas operon genes are similarly organized in M. tuberculosis and M. aurum . H37Rv kas operon promoter upregulates the reporter gene expression in M. aurum only upon treatment with drugs inhibiting FAS‐II pathway. Significance and Impact of the Study: It would serve as a good second‐line screen for characterization of compounds showing antimycobacterial activity in a first‐line screen. With the simplicity of β‐galactosidase enzyme assay the system can be easily adapted in high‐throughput mode.