Determination of the minimal acid-inducible promoter region of the lipF gene from Mycobacterium tuberculosis

The Mycobacterium tuberculosis gene lipF, Rv3487c, is transcriptionally upregulated by exposure to acidic growth media. We previously identified a 477 base pair (bp) region of DNA 147 bp upstream of lipF that is transcriptionally upregulated by exposure to growth media at pH 4.5 [Saviola, B., Woolwine, S., Bishai, W. R., 2003. Isolation of acid-inducible genes of Mycobacterium tuberculosis with the use of recombinase-based in vivo expression technology. Infect. Immun. 71, 1379–1388]. In this study we truncate the lipF promoter region first from the 3′ DNA end and then from the 5′ DNA end. The truncated promoter regions were placed upstream of the gene for the green fluorescent protein (gfp) and each promoter region was analyzed in Mycobacterium smegmatis for its ability to undergo transcriptional upregulation in response to acid stress. A minimal acid-inducible promoter region was identified and is located between − 515 bp and − 573 bp with respect to the start site of translation of lipF. The 59 bp minimal promoter region is a defined DNA sequence that confers full promoter activity that is transcriptionally upregulated in response to acid stress. Primer extension analysis was performed on acid-induced M. smegmatis bearing the minimal promoter region fused to gfp and revealed a start site of transcription specifically upregulated by acid stress corresponding to − 511 bp upstream of lipF with respect to the start of translation.