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Negative transcriptional regulation of the mce3 operon in Mycobacterium tuberculosis

Institute of Biotechnology¹, Institute of Pathobiology², Institute of Virology³, CICVyA/INTA, 1712 Castelar, Argentina

Author for correspondence: Angel A. Cataldi. Tel: +54 11 4621 1447 or 0199. Fax: +54 11 4481 2975. e-mail: acataldi{at}correo.inta.gov.ar

mce3 is one of the four mce operons in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence of this bacterium. Upstream of mce3 there is a putative regulatory gene (Rv1963) that harbours a double tetR-family signature. To study the role of this putative regulatory gene in the transcriptional regulation of the mce3 operon, Mycobacterium smegmatis mc²155 and M. tuberculosis H37Rv strains that harboured gene fusions between the mce3 promoter region and the Escherichia coli lacZ gene, either containing or not containing the Rv1963 gene, were used. The presence of the Rv1963 gene in the strains greatly reduced ß-galactosidase activity, suggesting that the Rv1963-encoded protein is a transcriptional repressor of the mce3 operon. Expression of mce3 by recombinant M. tuberculosis was increased when it was grown in a macrophage-like cell line (J774), compared to the level of expression seen when the recombinant bacterium was grown under in vitro conditions. However, no lifting of repression was induced. The mce3 promoter was defined by deletion and cloning of the Rv1963–Rv1964 intergenic region in a 200 bp DNA fragment harbouring the region upstream of the Rv1964 start codon. Gel-shift experiments determined that the Rv1963-binding site was located in this region. These results indicate that the mce3 operon is transcriptionally regulated and that under certain, unknown, conditions repression of gene expression could be lifted.

Keywords: repressor, tetR, reporter

Abbreviations: mce3; Rv1963; TSP, transcription start point; BAC, bacterial artificial chromosome

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