Regulation of expression of mas and fadD28, two genes involved in production of dimycocerosyl phthiocerol, a virulence factor of Mycobacterium tuberculosis

Abstract

Transcriptional regulation of genes involved in the biosynthesis of cell wall lipids of Mycobacterium tuberculosis is poorly understood. The gene encoding mycocerosic acid synthase (mas) and fadD28, an adjoining acyl coenzyme A synthase gene, involved in the production of a virulence factor, dimycocerosyl phthiocerol, were cloned from Mycobacterium bovis BCG, and their promoters were analyzed. The putative promoters were fused to the xylE reporter gene, and its expression was measured in Escherichia coli, Mycobacterium smegmatis, and M. bovis BCG. In E. coli, the fadD28 promoter was not functional but the mas promoter was functional. Both fadD28 and mas promoters were functional in M. smegmatis, at approximately two- and sixfold-higher levels, respectively, than the BCG hsp60 promoter. In M. bovis BCG, the fadD28 and mas promoters were functional at three- and fivefold-higher levels, respectively, than the hsp60 promoter. Primer extension analyses identified transcriptional start points 60 and 182 bp upstream of the translational start codons of fadD28 and mas, respectively. Both promoters contain sequences similar to the canonical -10 and -35 hexamers recognized by the sigma(70) subunit of RNA polymerase. Deletions of the upstream regions of both genes indicated that 324 bp of the fadD28 and 228 bp of the mas were essential for promoter activity. Further analysis of the mas promoter showed that a 213-bp region 581 bp upstream of the mas promoter acted as a putative transcriptional enhancer, promoting high-level expression of the mas gene when present in either direction. This represents the identification of a rare example of an enhancer-like element in mycobacteria.

FIG. 1.

Mapping of the transcription initiation sites of the fadD28 (A) and mas (B) genes by primer extension analysis. Lanes G, A, T, and C, DNA sequence ladder of the fadD28 and mas promoter regions; lanes 1 and 2, primer extension with annealing temperatures of 45 and 65°C, respectively. The major extension products are indicated, and the transcription initiation sites of both genes are shown by arrows (details are in Materials and Methods).

FIG. 2.

Reporter gene expression driven by fadD28 and its derivatives fused to the xylE reporter gene. The locations of the ATG translation initiation codons of fadD28 and mas are marked. CDO activities in M. smegmatis and M. bovis BCG are expressed in milliunits per milligram of protein in extracts from cells at mid-exponential phase. +, promoter in the same direction as xylE; −, promoter in the opposite direction.

FIG. 3.

xylE gene expression driven by mas promoter and its derivatives fused to xylE gene. The translation initiation sites are marked and the enzymatic activity of the xylE gene product (CDO) is shown in milliunits per milligram of protein in the cell extracts. +, promoter in the same direction as xylE; −, promoter in the opposite direction.