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Ethionamide (ETH) is an important second-line antitubercular drug used for the
treatment of patients infected with multidrug-resistant Mycobacterium
tuberculosis. Although ETH is a structural analogue of isoniazid, only little
cross-resistance to these two drugs is observed among clinical isolates. Both
isoniazid and ETH are pro-drugs that need to be activated by mycobacterial
enzymes to exert their antimicrobial activity. We have recently identified two
M. tuberculosis genes, Rv3854c (ethA) and Rv3855 (ethR), involved in resistance
to ETH. ethA encodes a protein that belongs to the Flavin-containing
monooxygenase family catalysing the activation of ETH. We show here that ethR,
which encodes a repressor belonging to the TetR/CamR family of transcriptional
regulators, negatively regulates the expression of ethA. By the insertion of the
ethA promoter region upstream of the lacZ reporter gene, overexpression of ethR
in trans was found to cause a strong inhibition of ethA expression,
independently of the presence of ETH in the culture media. Electrophoretic
mobility shift assays indicated that EthR interacts directly with the ethA
promoter region. This interaction was confirmed by DNA footprinting analysis,
which, in addition, identified the EthR-binding region. Unlike other TetR/CamR
members, which typically bind 15 bp operators, EthR recognises an unusually long
55 bp region suggesting multimerization of the repressor on its operator.
Identification by primer-extension of the ethA transcriptional start site
indicated that it is located within the EthR-binding region. Taken together,
bacterial two-hybrid experiments and gel filtration assays suggested a
dimerization of EthR in the absence of its operator. In contrast, surface
plasmon resonance analyses showed that eight EthR molecules bind cooperatively
to the 55 bp operator, which represents a novel repression mechanism for a
TetR/CamR member.
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