Coregulation by phenylacetyl-coenzyme A-responsive PaaX integrates control of the upper and lower pathways for catabolism of styrene by Pseudomonas sp. strain Y2

Abstract

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.

FIG. 1.

Styrene catabolic genes and enzymatic pathway of Pseudomonas sp. strain Y2. (A) Genetic map of the sty and paa1 gene clusters of Pseudomonas sp. strain Y2. The P_styA promoter controlled by the StySR proteins regulates expression of the sty catabolic genes (styABCD) that encode the enzymes responsible for the transformation of styrene to phenylacetate; styE encodes a putative porin (38). The paaF2N2ABCDEFGHIJKPLN genes encode the enzymes that transform phenylacetate into metabolites of the Krebs cycle; paaXY genes encode regulatory proteins. The consensus nomenclature for the paa genes proposed by Luengo et al. (23) has been used. Arrows indicate the direction of transcription (1). (B) Styrene upper and lower catabolic pathways. Compounds are as follows: 1, styrene; 2, styrene oxide; 3, phenylacetaldehyde; 4, phenylacetate; 5, phenylacetyl-CoA; 6, hydroxy derivative of phenylacetyl-CoA; 7, aliphatic derivative of phenylacetyl-CoA; 8, intermediate metabolites of tricarboxylic acids cycle. Enzymes and proteins are as follows: StyAB, styrene monooxygenase; StyC, epoxystyrene isomerase; StyD, phenylacetaldehyde dehydrogenase; PaaL, permease; PaaP, membrane protein; PaaF and PaaF2, phenylacetyl-CoA ligases; PaaGHIJK, putative multicomponent phenylacetyl-CoA oxygenase; PaaN, PaaN2 and PaaABCDE, putative enzymes involved in ring-cleavage- and β-oxidation-like reactions of the aliphatic-CoA intermediate. “Out” and “in” refer to the periplasmic and cytoplasmic spaces, respectively (1, 4).

FIG. 2.

Sequence of the P_styA region of Pseudomonas sp. strain Y2. The 3′ end of styR and the 5′-end region of styA are in boldface letters. The StyR binding site proposed for Pseudomonas sp. strain Y2 (38) is underlined. The high- (STY2), medium- (STY1), and very-low-affinity (STY3) StyR binding sites of P. fluorescens ST proposed by Leoni et al. (22) are overlined. The IHF binding site, the extended −10 box of P_styA promoter, the transcription start site (+1) for styA, and the PaaX binding site (PaaX box) are underlined. The locations and sequences of styAXR and styAXL oligonucleotides used to amplify the STY probe and the E1 and B1 oligonucleotides used to amplify the styR-styA intergenic region are indicated in lowercase letters.

FIG. 3.

PaaX binding sites. Comparison of PaaX binding sites of several promoters of E. coli W (GenBank accession number X97452) and Kluyvera citrophila (M15418) with putative PaaX binding sites found in the paa promoters of P. putida KT2440 (AE015451), P. putida U (AF029714), P. fluorescens Pf-5 (NC004129), and Pseudomonas sp. strain Y2 (AJ000330 and AJ579894) and with the PaaX binding site identified in P_styA. P_G (formerly P_a), P_N (formerly P_z), and P_X refer to the promoters that control transcription of the three paa operons of E. coli. P_pac is the promoter that drives the transcription of the penicillin G acylase gene. P_paaA, P_paaG, P_paaA2, P_paaG2, and P_paaB are promoters of the corresponding paa genes of Pseudomonas species. P_styA is the promoter that controls the transcription of the styABCD styrene catabolic operon of Pseudomonas sp. strain Y2. The location of the region encompassing both 6-bp inverted repeats relative to the putative transcription start point is given in parentheses at right. Consensus sequences of putative PaaX binding sites are also displayed. Nucleotides identical to the consensus are shown in uppercase bold letters. Conserved nucleotides between the E. coli and Pseudomonas consensus sequences are underlined. The region of P_styA protected by PaaX1 of Pseudomonas sp. strain Y2 against DNase I is displayed as a box (see the text).

FIG. 4.

Electrophoretic mobility shift assays of the STY probe with cell extracts from different Pseudomonas species. Lane 1, free STY probe; lane 2, cell extracts of Pseudomonas sp. strain Y2K1 (Δpaa1) (4.4 μg/μl); lane 3, cell extracts of Pseudomonas sp. strain Y2T2 (Δpaa2) (4.2 μg/μl); lane 4, cell extracts of Pseudomonas sp. strain Y2K1T2 (Δpaa1 Δpaa2) (3.5 μg/μl); lane 5, cell extracts of P. putida KT2442 (2.3 μg/μl); lane 6, cell extracts of P. putida U (5.5 μg/μl); lane 7, cell extracts of Pseudomonas sp. strain Y2 (pVLTX1) (3.3 μg/μl).

FIG. 5.

Electrophoretic mobility shift assays of the STY probe with purified MBP-PaaX1 protein. (A). Lanes 1 to 7, retardation assays of the STY probe with 0, 5, 10, 25, 50, 100, and 150 nM MBP-PaaX1, respectively. (B). Lane 1, free STY probe. Lanes 2 to 7, retardation assays produced by 150 nM purified MBP-PaaX1 in the presence of 0, 25, 50, 100, 500, and 1,000 μM PA-CoA, respectively. Lane 8, retardation assay produced in the presence of 150 nM MBP-PaaX1 and 1 mM PA. Lane 9, retardation produced by 150 nM MBP-PaaX1 in the presence of 10 nM unlabeled STY probe.

FIG. 6.

DNase I footprinting analysis of the interaction of purified MBP-PaaX1 with the STY probe. Lanes 1 to 5 show footprints with 0, 25, 50, 150, and 300 nM purified MBP-PaaX1, respectively. Lane 6, A+G Maxam and Gilbert sequencing reaction. The nucleotide sequence of the protected region, complementary to that shown in Fig. 2 and 3, is shown within brackets.

FIG. 7.

Repression of in vitro transcription from P_styA promoter by MBP-PaaX1 and effect of the presence of PA-CoA. (A). Transcripts from pTE-E1B1 in the presence of phosphorylated StyR and different amounts of MBP-PaaX1. Lane 1 to lane 6, 0, 25, 75, 150, 300, and 600 nM MBP-PaaX1, respectively. Arrows point to the styA transcript (styA) and to the internal control transcript (RNA-1). (B). styA transcript levels (in arbitrary units) produced from pTE-E1B1 template in the presence of 0 to 600 nM purified MBP-PaaX1. The results are the normalized averages of six independent experiments. Error bars, standard errors. The inset shows the autoradiography of styA transcripts produced in the absence of MBP-PaaX1 (lane 1), in the presence of 600 nM MBP-PaaX1 (lane 2), and in the presence of 600 nM MBP-PaaX1 challenged with 1 mM PA-CoA (lane 3).

FIG. 8.

RT-PCR quantification of styA mRNA levels in different Pseudomonas and E. coli strains. The styA transcript level was determined in cultures of the indicated strains grown under either noninduced (gray bars) or styrene-induced (black bars) conditions as described in Materials and Methods. Bars represent the means of styA transcript levels from three independent experiments, each performed in duplicate. Error bars, standard errors.