Gene expression in mycobacteria: transcriptional fusions based on xylE and analysis of the promoter region of the response regulator mtrA from Mycobacterium tuberculosis

Abstract

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 xyIE 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 xyIE 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 xyIE, 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 xyIE-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.

Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two‐component response regulator

“…The processes by which M. tuberculosis achieves this task is an intensively investigated area of research. While the reasons for sharp upregulation of MtrA in M. bovis BCG during intramacrophage growth (Curcic et al, 1994;Zahrt and Deretic, 2000; are unclear, our data suggest that levels of MtrA and ratios of MtrA∼P to MtrA are likely to be tightly regulated in virulent M. tuberculosis, and conditions that interfere with this regulation could compromise proliferation of the pathogen.…”

Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis

“…The gentamicin resistance gene is not a reliable marker in M. tuberculosis, so we reasoned that this reporter activity could facilitate the screening by discriminating possible allelic exchange mutants that have lost the xylE gene, from sacB mutants with the whole vector integrated into the chromosome and that are thus phenotypically XylE ϩ . Indeed, xylE expression in mycobacteria can easily be tested by spraying colonies on plates with a solution of catechol and by observing a bright yellow coloration (25). Plasmid pMJ103 was introduced in M. tuberculosis by electroporation, and transformants were selected at 32°C on 7H10-kanamycin.…”

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model

“…Recombinant expressions systems have been developed recently that can be used in fast-growing non-pathogenic mycobacteria. These systems have made use of modified mycobacterial plasmids that have been engineered to over-express protein, via the hsp60 promoter (Curcic et al, 1994;Delogu et al, 2004;DeMaio et al, 1997;Dziadek et al, 2002;O'Donnell et al, 1994). Inducible systems for mycobacteria expression have also been developed, using tetracycline induction, which provide controlled amounts of protein expression (Blokpoel et al, 2005;Carroll et al, 2005;Ehrt et al, 2005;Triccas et al, 1998).…”