Recombinase-based reporter system and antisense technology to study gene expression and essentiality in hypoxic nonreplicating mycobacteria.
The study of the mechanisms used by Mycobacterium tuberculosis to survive in the absence of growth is hampered by the absence of appropriate genetic tools. Here, we report two strategies, a recombinase-based reporter system and an antisense technology, to study gene expression and essentiality in hypoxic nonreplicating mycobacteria. The recombinase-based reporter system relies on the resolution of an antibiotic marker flanked by the gammadelta-res sites. This system was developed to identify M. tuberculosis promoters, which are specifically expressed under anaerobic conditions. The antisense strategy was designed to study the role of a gene candidate during anaerobic survival. To validate this approach, the dosR, narK2 and rv2466c promoters were selected to drive dosR antisense mRNA expression in quiescent mycobacteria. The conditional knockout strains were found to be attenuated to adapt and survive under anaerobic conditions, as observed for the dosR knockout strain. Together, our work demonstrates that the recombinase-based reporter system and antisense technology represent two genetic tools useful for the identification and characterization of genes essential for the survival of hypoxic nonreplicating M. tuberculosis.
Novartis Institute for Tropical Diseases, Chromos, Singapore.
Srinivasa P S
SP
Rao
Luis
L
Camacho
Bee
B
Huat Tan
Calvin
C
Boon
David G
DG
Russel
Thomas
T
Dick
Kevin
K
Pethe
D000693:Anaerobiosis; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015374:Biosensing Techniques / Q000379:methods*; D015169:Colony Count, Microbial; D020868:Gene Silencing; D017930:Genes, Reporter; D008967:Molecular Biology / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology; D013753:Tetracycline Resistance / Q000235:genetics
D000900:Anti-Bacterial Agents; D016372:RNA, Antisense; D045522:Recombinases; D013752:Tetracycline