GENE-EXPRESSION IN MYCOBACTERIA - TRANSCRIPTIONAL FUSIONS BASED ON XYLE AND ANALYSIS OF THE PROMOTER REGION OF THE RESPONSE REGULATOR MTRA FROM MYCOBACTERIUM-TUBERCULOSIS

Understanding promoter regulation and signal-transduction systems in pathogenic mycobacteria is critical for uncovering the processes that govern interactions of these bacteria with the human host. In order to develop additional genetic tools for analysis of mycobacterial promoters, the xylE gene from Pseudomonas was tested as a transcriptional fusion reporter in fast- and slow-growing mycobacteria. initially, its utility was demonstrated by expression behind the hsp60 promoter in Mycobacterium smegmatis and Mycobacterium bovis BCG. The presence of an active promoter in front of the promoterless xylE cassette on a plasmid was scored by development of a bright yellow colour upon spraying of mycobacterial colonies on plates with a solution of catechol. The gene product of xylE, catechol 2,3 dioxygenase, was measurable in sonic extracts and whole cells, permitting quantitative determination of promoter activity in both fast- and slow-growing mycobacteria. The xylE-based mycobacterial transcriptional fusion plasmid pRCX3 was constructed and used to assess promoter activity within the sequences located upstream of the newly characterized Mycobacterium tuberculosis H37Rv response regulator mtrA, a member of the superfamily of bacterial signal-transduction systems.