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. 2001 Dec;183(23):6801-6.
doi: 10.1128/JB.183.23.6801-6806.2001.

Transcriptional Regulation of furA and katG Upon Oxidative Stress in Mycobacterium Smegmatis

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Free PMC article

Transcriptional Regulation of furA and katG Upon Oxidative Stress in Mycobacterium Smegmatis

A Milano et al. J Bacteriol. .
Free PMC article

Abstract

The DNA region upstream of katG in Mycobacterium smegmatis was cloned and sequenced. The furA gene, highly homologous to Mycobacterium tuberculosis furA, mapped in this region. The furA-katG organization appears to be conserved among several mycobacteria. The transcription pattern of furA and katG in M. smegmatis upon oxidative stress was analyzed by Northern blotting and primer extension. Although transcription of both furA and katG was induced upon oxidative stress, transcripts covering both genes could not be identified either by Northern blotting or by reverse transcriptase PCR. Specific transcripts and 5' ends were identified for furA and katG, respectively. By cloning M. smegmatis and M. tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of two promoters, pfurA, located immediately upstream of the furA gene, and pkatG, located within the terminal part of the furA coding sequence. Transcription from pfurA was induced upon oxidative stress. A 23-bp sequence overlapping the pfurA -35 region is highly conserved among mycobacteria and streptomycetes and might be involved in controlling pfurA activity. Transcription from a cloned pkatG, lacking the upstream pfurA region, was not induced upon oxidative stress, suggesting a cis-acting regulatory role of this region.

Figures

FIG. 1
FIG. 1
Transcription of the furA-katG region in M. smegmatis. (A) Map of the region. The furA-katG map of M. smegmatis is drawn to scale according to the sequence in the GenBank database with accession no. AF196484 (coordinate 1 corresponds to nt 245 of the deposited sequence). The genes are indicated by boxes. The potential Rho-independent termination site, downstream of katG, is shown. The transcripts are indicated by arrows, and the 5′ ends are indicated by vertical arrows below the map. The positions of the riboprobes and oligonucleotides used for Northern blotting, primer extension analysis, and RT-PCR are reported. (B to E) Northern blotting analysis of furA-katG transcription upon oxidative stress. RNAs were extracted from a culture of M. smegmatis mc2155 treated for 1 h with the millimolar concentration of hydrogen peroxide indicated on the top of the lanes, and equal amounts of each sample (20 μg) were separated by agarose (B, C, and D) or polyacrylamide (E) gel electrophoresis, blotted, and hybridized to the different probes. (B) Riboprobe katG (coordinates 724 to 1113); (C and E) riboprobe furA (coordinates 332 to 511); (D) the same filter used in panel C was hybridized successively to oligonucleotides 554 (coordinates 366 to 385), 553 (coordinates 715 to 742), and 244 (coordinates 803 to 822). Molecular size markers, run in the same gel, are indicated on the left. For panels C and D, probe 554 was exposed about three times longer. In agarose gels, two nonspecific bands (about 1.5 and 3 kb) were observed after prolonged exposure of the filters (particularly visible in panels C and D, oligonucleotide 554). These bands are likely to be furA-katG RNA entrapped by rRNA (5). (F) Primer extension of 5′ ends of furA and katG transcripts. The primer extension experiments were performed with the 554 (furA) and 244 (katG) oligonucleotides. Sequence reactions, performed with the same oligonucleotides on MS3 DNA (Table 1), are reported in the first four lanes of the two panels. The coordinates of the 5′ ends are reported on the left of each panel.

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