title: The PaaX-type repressor MeqR2 of Arthrobacter sp. strain Rue61a, involved in the regulation of quinaldine catabolism, binds to its own promoter and to catabolic promoters and specifically responds to anthraniloyl coenzyme A.
abstract: The genes coding for quinaldine catabolism in Arthrobacter sp. strain Rue61a are clustered on the linear plasmid pAL1 in two upper pathway operons (meqABC and meqDEF) coding for quinaldine conversion to anthranilate and a lower pathway operon encoding anthranilate degradation via coenzyme A (CoA) thioester intermediates. The meqR2 gene, located immediately downstream of the catabolic genes, codes for a PaaX-type transcriptional repressor. MeqR2, purified as recombinant fusion protein, forms a dimer in solution and shows specific and cooperative binding to promoter DNA in vitro. DNA fragments recognized by MeqR2 contained a highly conserved palindromic motif, 5'-TGACGNNCGTcA-3', which is located at positions -35 to -24 of the two promoters that control the upper pathway operons, at positions +4 to +15 of the promoter of the lower pathway genes and at positions +53 to +64 of the meqR2 promoter. Disruption of the palindrome abolished MeqR2 binding. The dissociation constants (K(D)) of MeqR2-DNA complexes as deduced from electrophoretic mobility shift assays were very similar for the four promoters tested (23 nM to 28 nM). Anthraniloyl-CoA was identified as the specific effector of MeqR2, which impairs MeqR2-DNA complex formation in vitro. A binding stoichiometry of one effector molecule per MeqR2 monomer and a K(D) of 22 nM were determined for the effector-protein complex by isothermal titration calorimetry (ITC). Quantitative reverse transcriptase PCR analyses suggested that MeqR2 is a potent regulator of the meqDEF operon; however, additional regulatory systems have a major impact on transcriptional control of the catabolic operons and of meqR2.
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms-University Münster, Münster, Germany.
authors.forename: Heiko
authors.initials: H
authors.lastname: Niewerth
authors.forename: Katja
authors.initials: K
authors.lastname: Parschat
authors.forename: Melanie
authors.initials: M
authors.lastname: Rauschenberg
authors.forename: Bart Jan
authors.initials: BJ
authors.lastname: Ravoo
authors.forename: Susanne
authors.initials: S
authors.lastname: Fetzner
mesh_terms: D001173:Arthrobacter / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003065:Coenzyme A / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011798:Quinaldines / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D062367:ortho-Aminobenzoates / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011798:Quinaldines; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; C037073:2-methylquinoline; D003065:Coenzyme A