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Coregulation by Phenylacetyl-Coenzyme A-Responsive PaaX Integrates Control of the Upper and Lower Pathways for Catabolism of Styrene by Pseudomonas sp. Strain Y2

Department of Biochemistry and Molecular Biology, I, Facultad de Ciencias Biológicas, Universidad Complutense, 28040 Madrid, Spain,¹ Department of Molecular Microbiology, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid, Spain,² The Burnham Institute for Medical Research, La Jolla, California 92037,³ Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden⁴

Received 2 February 2006/ Accepted 6 April 2006

The P_styA promoter of Pseudomonas sp. strain Y2 controls expression of the styABCD genes, which are required for the conversion of styrene to phenylacetate, which is further catabolized by the products of two paa gene clusters. Two PaaX repressor proteins (PaaX1 and PaaX2) regulate transcription of the paa gene clusters of this strain. In silico analysis of the P_styA promoter region revealed a sequence located just within styA that is similar to the reported PaaX binding sites of Escherichia coli and the proposed PaaX binding sites of the paa genes of Pseudomonas species. Here we show that protein extracts from some Pseudomonas strains that have paaX genes, but not from a paaX mutant strain, can bind and retard the migration of a P_styA specific probe. Purified maltose-binding protein (MBP)-PaaX1 fusion protein specifically binds the P_styA promoter proximal PaaX site, and this binding is eliminated by the addition of phenylacetyl-coenzyme A. The sequence protected by MBP-PaaX1 binding was defined by DNase I footprinting. Moreover, MBP-PaaX1 represses transcription from the P_styA promoter in a phenylacetyl-coenzyme A-dependent manner in vitro. Finally, the inactivation of both paaX gene copies of Pseudomonas sp. strain Y2 leads to a higher level of transcription from the P_styA promoter, while heterologous expression of the PaaX1 in E. coli greatly decreases transcription from the P_styA promoter. These findings reveal a control mechanism that integrates regulation of styrene catabolism by coordinating the expression of the styrene upper catabolic operon to that of the paa-encoded central pathway and support a role for PaaX as a major regulatory protein in the phenylacetyl-coenzyme A catabolon through its response to the levels of this central metabolite.

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