EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator

Jean Engohang-Ndong, David Baillat, Marc Aumercier, Flore Bellefontaine, Gurdyal S. Besra, Camille Locht, Alain R. Baulard

Research output: Contribution to journalArticle

108 Citations (Scopus)

Abstract

Ethionamide (ETH) is an important second-line anti-tubercular 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 mycobacterlal 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.

Original languageEnglish (US)
Pages (from-to)175-188
Number of pages14
JournalMolecular Microbiology
Volume51
Issue number1
DOIs
StatePublished - Jan 2004
Externally publishedYes

Fingerprint

Ethionamide
Mycobacterium
Isoniazid
dimethylaniline monooxygenase (N-oxide forming)
Mycobacterium tuberculosis
Genetic Promoter Regions
DNA Footprinting
Multidrug-Resistant Tuberculosis
Lac Operon
Surface Plasmon Resonance
Prodrugs
Dimerization
Electrophoretic Mobility Shift Assay
Reporter Genes
Pharmaceutical Preparations
Gel Chromatography
Culture Media
Enzymes
Genes

ASJC Scopus subject areas

  • Molecular Biology
  • Microbiology

Cite this

EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator. / Engohang-Ndong, Jean; Baillat, David; Aumercier, Marc; Bellefontaine, Flore; Besra, Gurdyal S.; Locht, Camille; Baulard, Alain R.

In: Molecular Microbiology, Vol. 51, No. 1, 01.2004, p. 175-188.

Research output: Contribution to journalArticle

Engohang-Ndong, J, Baillat, D, Aumercier, M, Bellefontaine, F, Besra, GS, Locht, C & Baulard, AR 2004, 'EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator', Molecular Microbiology, vol. 51, no. 1, pp. 175-188. https://doi.org/10.1046/j.1365-2958.2003.03809.x
Engohang-Ndong, Jean ; Baillat, David ; Aumercier, Marc ; Bellefontaine, Flore ; Besra, Gurdyal S. ; Locht, Camille ; Baulard, Alain R. / EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator. In: Molecular Microbiology. 2004 ; Vol. 51, No. 1. pp. 175-188.
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abstract = "Ethionamide (ETH) is an important second-line anti-tubercular 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 mycobacterlal 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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AU - Engohang-Ndong, Jean

AU - Baillat, David

AU - Aumercier, Marc

AU - Bellefontaine, Flore

AU - Besra, Gurdyal S.

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AB - Ethionamide (ETH) is an important second-line anti-tubercular 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 mycobacterlal 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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