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 C1 and C2 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 C1 and C2 compounds in Methylobacterium extorquens AM1. A ccrR null mutant strain was impaired in its ability to grow on C1 and C2 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 wildtype 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. © 2012, American Society for Microbiology.