title: CcrR, a TetR family transcriptional regulator, activates the transcription of a gene of the Ethylmalonyl coenzyme A pathway in Methylobacterium extorquens AM1.
abstract: The ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway is one of the central methylotrophy pathways in Methylobacterium extorquens involved in glyoxylate generation and acetyl-CoA assimilation. Previous studies have elucidated the operation of the ethylmalonyl-CoA pathway in C(1) and C(2) assimilation, but the regulatory mechanisms for the ethylmalonyl-CoA pathway have not been reported. In this study, a TetR-type activator, CcrR, was shown to regulate the expression of crotonyl-CoA reductase/carboxylase, an enzyme of the ethylmalonyl-CoA pathway involved in the assimilation of C(1) and C(2) compounds in Methylobacterium extorquens AM1. A ccrR null mutant strain was impaired in its ability to grow on C(1) and C(2) compounds, correlating with the reduced activity of crotonyl-CoA reductase/carboxylase. Promoter fusion assays demonstrated that the activity of the promoter required for ccr expression (the katA-ccr promoter) decreased as much as 50% in the absence of ccrR compared to wild-type M. extorquens AM1. Gel mobility shift assays confirmed that CcrR directly binds to the region upstream of the katA-ccr promoter. A palindromic sequence upstream of katA at positions -334 to -321 with respect to the predicted translational start site was identified, and mutations in this region eliminated the gel retardation of the katA-ccr promoter region by CcrR. CcrR does not appear to regulate the expression of other ethylmalonyl-CoA pathway genes, suggesting the existence of additional regulators.
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, WA, USA. bohu@uw.edu
authors.forename: Bo
authors.initials: B
authors.lastname: Hu
authors.forename: Mary
authors.initials: M
authors.lastname: Lidstrom
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D044944:Acyl-CoA Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D020601:Methylobacterium extorquens / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D015534:Trans-Activators; C102829:ethylmalonyl-coenzyme A; D044944:Acyl-CoA Dehydrogenases; C100154:acyl-CoA dehydrogenase (NADP+)