| Summary: | MbcTA is a type II toxin/antitoxin (TA) system of <i>Mycobacterium tuberculosis</i>. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD<sup>+</sup> and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the <i>mbcAT</i> operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in <i>M. tuberculosis</i> and in the fast growing model species <i>Mycobacterium smegmatis</i> to: (i) assess the autoregulation of <i>mbcAT</i>; (ii) perform a genetic dissection of the <i>mbcA</i> promoter/operator region; and (iii) explore the regulation of <i>mbcAT</i> transcription from the <i>mbcA</i> promoter (P<i><sub>mbcA</sub></i>) in a variety of stress conditions, including in vivo in mice and in macrophages.
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