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

ThePstyA promoter ofPseudomonas sp. strain Y2 controls expression of thestyABCD genes, which are required for the conversion of styrene to phenylacetate, which is further catabolized by the products of twopaa gene clusters. Two PaaX repressor proteins (PaaX1 and PaaX2) regulate transcription of thepaa gene clusters of this strain. In silico analysis of thePstyA promoter region revealed a sequence located just withinstyA that is similar to the reported PaaX binding sites ofEscherichia coli and the proposed PaaX binding sites of thepaa genes ofPseudomonas species. Here we show that protein extracts from somePseudomonas strains that havepaaX genes, but not from apaaX mutant strain, can bind and retard the migration of aPstyA specific probe. Purified maltose-binding protein (MBP)-PaaX1 fusion protein specifically binds thePstyA 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 thePstyA promoter in a phenylacetyl-coenzyme A-dependent manner in vitro. Finally, the inactivation of bothpaaX gene copies ofPseudomonas sp. strain Y2 leads to a higher level of transcription from thePstyA promoter, while heterologous expression of the PaaX1 inE. coli greatly decreases transcription from thePstyA 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 thepaa -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.