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1.
FEBS J ; 290(2): 521-532, 2023 01.
Article in English | MEDLINE | ID: mdl-36017630

ABSTRACT

TetR/AcrR-like transcription regulators enable bacteria to sense a wide variety of chemical compounds and to dynamically adapt the expression levels of specific genes in response to changing growth conditions. Here, we describe the structural characterisation of SCO3201, an atypical TetR/AcrR family member from Streptomyces coelicolor that strongly represses antibiotic production and morphological development under conditions of overexpression. We present crystal structures of SCO3201 in its ligand-free state as well as in complex with an unknown inducer, potentially a polyamine. In the ligand-free state, the DNA-binding domains of the SCO3201 dimer are held together in an unusually compact conformation and, as a result, the regulator cannot span the distance between the two half-sites of its operator. Interaction with the ligand coincides with a major structural rearrangement and partial conversion of the so-called hinge helix (α4) to a 310 -conformation, markedly increasing the distance between the DNA-binding domains. In sharp contrast to what was observed for other TetR/AcrR-like regulators, the increased interdomain distance might facilitate rather than abrogate interaction of the dimer with the operator. Such a 'reverse' induction mechanism could expand the regulatory repertoire of the TetR/AcrR family and may explain the dramatic impact of SCO3201 overexpression on the ability of S. coelicolor to generate antibiotics and sporulate.


Subject(s)
Repressor Proteins , Streptomyces coelicolor , Repressor Proteins/metabolism , Streptomyces coelicolor/genetics , Streptomyces coelicolor/chemistry , Streptomyces coelicolor/metabolism , Anti-Bacterial Agents/pharmacology , Protein Domains , DNA , Bacterial Proteins/metabolism , Crystallography, X-Ray , Gene Expression Regulation, Bacterial
2.
Biochem Biophys Res Commun ; 450(1): 513-8, 2014 Jul 18.
Article in English | MEDLINE | ID: mdl-24928397

ABSTRACT

SCO3201, a regulator of the TetR family, is a strong repressor of both morphological differentiation and antibiotic production when overexpressed in Streptomyces coelicolor. Here, we report the identification of 14 novel putative regulatory targets of this regulator using in vitro formaldehyde cross-linking. Direct binding of purified His6-SCO3201 was demonstrated for the promoter regions of 5 regulators (SCO1716, SCO1950, SCO3367, SCO4009 and SCO5046), a putative histidine phosphatase (SCO1809), an acetyltransferase (SCO0988) and the polyketide synthase RedX (SCO5878), using EMSA. Reverse transcriptional analysis demonstrated that the expression of the transcriptional regulators SCO1950, SCO4009, SCO5046, as well as of SCO0988 and RedX was down regulated, upon SCO3201 overexpression, whereas the expression of SCO1809 and SCO3367 was up regulated. A consensus binding motif was derived via alignment of the promoter regions of the genes negatively regulated. The positions of the predicted operator sites were consistent with a direct repressive effect of SCO3201 on 5 out of 7 of these promoters. Furthermore, the 2.1Å crystal structure of SCO3201 was solved, which provides a possible explanation for the high promiscuity of this regulator that might account for its dramatic effect on the differentiation process of S. coelicolor.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial/genetics , Promoter Regions, Genetic/genetics , Streptomyces coelicolor/genetics , Trans-Activators/chemistry , Trans-Activators/genetics , Bacterial Proteins/ultrastructure , Base Sequence , Computer Simulation , Gene Targeting/methods , Models, Chemical , Models, Genetic , Molecular Sequence Data , Repressor Proteins/genetics , Structure-Activity Relationship , Trans-Activators/ultrastructure
3.
Nucleic Acids Res ; 40(9): 4178-92, 2012 May.
Article in English | MEDLINE | ID: mdl-22238377

ABSTRACT

Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/chemistry , Gene Expression Regulation, Bacterial , Trans-Activators/chemistry , Transcriptional Activation , Bacillus subtilis/drug effects , Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Base Sequence , Cysteine/chemistry , DNA-Binding Proteins/metabolism , Diamide/pharmacology , Models, Molecular , Molecular Sequence Data , Nitroreductases/genetics , Operator Regions, Genetic , Oxidation-Reduction , Oxidoreductases/biosynthesis , Oxidoreductases/genetics , Sodium Hypochlorite/pharmacology , Stress, Physiological/genetics , Trans-Activators/metabolism
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