Deciphering kas operon locus in Mycobacterium aurum and genesis of a recombinant strain for rational-based drug screening.
OBJECTIVE
To generate a recombinant Mycobacterium aurum strain for screening of antimycobacterial compounds affecting fatty acid synthase type II (FAS-II) elongation pathway.


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.


CONCLUSIONS
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 beta-galactosidase enzyme assay the system can be easily adapted in high-throughput mode.
Gupta
N
N
Division of Microbiology, Central Drug Research Institute, Lucknow, India.
Singh
B N
BN
D054889:Fatty Acid Synthase, Type II / Q000037:antagonists & inhibitors; D015964:Gene Expression Regulation, Bacterial; D007763:Lac Operon / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009161:Mycobacterium / Q000235:genetics*; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012335:RNA, Ribosomal / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
D012335:RNA, Ribosomal; D054889:Fatty Acid Synthase, Type II