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J Bacteriol. 2012 Jun;194(11):2802-8. doi: 10.1128/JB.00061-12. Epub 2012 Mar 23.

CcrR, a TetR family transcriptional regulator, activates the transcription of a gene of the Ethylmalonyl coenzyme A pathway in Methylobacterium extorquens AM1.

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1
Department of Chemical Engineering, University of Washington, Seattle, WA, USA. bohu@uw.edu

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.

PMID:
22447902
PMCID:
PMC3370644
DOI:
10.1128/JB.00061-12
[Indexed for MEDLINE]
Free PMC Article
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