Identification and characterisation of endogenous inducible promoters in Mycobacterium tuberculosis
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Mycobacterium tuberculosis is one of the world’s most devastating
pathogens. Despite completion of the genome sequence in 1998, research
progress has been hampered by a lack of genetic tools and the difficulty of
working with the organism. Existing genetic systems are limited by their
lack of tight regulation or genetic instability. The aim of this study was to
characterise and utilise a range of promoters to express mycobacterial
genes in a controllable fashion by generating knockdown strains of a
number of target genes, using both sense and antisense approaches. This
would help to elucidate the function of a particular gene of interest and
identify or validate new drug targets. Sets of genes shown to be inducible
by certain stimuli such as tetracycline (Rv0277c, Rv0608, Rv0748,
Rv1015c, Rv2487c and Rv3898c), streptomycin (whiB7), sodium dodecyl
sulphate or ethanol (whiB6), hypoxia, nitric oxide and stationary phase
(Rv2625c, Rv2626c, Rv2627c and hspX), or salicylate (Rv0560c) were
selected from the literature. The upstream region of each gene was cloned
in front of a reporter gene and activity was tested in M. smegmatis and/or
M. tuberculosis. No inducible promoter activity was found for the upstream
regions of the tetracycline, streptomycin, sodium dodecyl sulphate or
ethanolresponsive
genes. Inducible promoter activity was found for some
of the hypoxiaresponsive
genes and was monitored in relation to growth
phase in M. tuberculosis wild type, a dosR deletion mutant and a Rv2625c
deletion mutant. The upstream region of Rv0560c was found to contain a
salicylateinducible
promoter. Promoter elements of this promoter were
identified and characterised in M. tuberculosis. Attempts to use the most
promising promoters in an antisense setting using the reporter gene lacZ or
the mycobacterial gene rpoB were unsuccessful.
Authors
Schuessler, Dorothée LauraCollections
- Theses [3919]