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Fig. S1. Aerobic phenylacetate degradation pathway. Reactions and intermediates are according to Teufel and colleagues (2012). The Paa enzymes involved in the different enzymatic steps (solid arrows) are shown in bold. The spontaneous formation of 2-hydroxycyclohepta-1,4,6-triene-1-carboxyl-CoA, a side product of the pathway that is likely a precursor for primary and secondary metabolites, e.g, antibiotics and ω-cycloheptyl fatty acids (grey shadow), is indicated by a dashed arrow. The thioesterase activity of PaaY is also shown.

Fig. S2. The paaX and paaY genes constitute an operon.

A. RT-PCR analysis of the expression of paaX and paaY genes in PA-grown E. coli W cells was performed as indicated in Experimental procedures. The schematic representation of the paaX-paaY region, the Px promoter, and the localization of the primers used for PCR amplification are shown at the bottom. Lane M, HaeIII-digested ΦX174 DNA ladder (in bp). Lane 1, amplification of a paaX internal fragment (primers IX5 and IX3). Lane 3, amplification of a paaY internal fragment (primers IY5 and IY3). Lane 5, amplification of a paaXY intergenic fragment (primers IX25 and IY25). Lanes 2, 4 and 6, control reactions of paaX, paaY and paaXY expression, respectively, in which reverse transcriptase was omitted from the reaction mixture.

B. Western blot analysis of PaaY content in different PA-grown E. coli cells. Lane 1, cell extract of E. coli W14 (pAAD; contains a wild-type paa cluster); lane 2, cell extract of E. coli W14 (pAAD::Tn1000-6; contains a disrupted paaX gene); lane 3, cell extract of E. coli W14 (pAAD::Tn1000-84; contains a disrupted paaY gene); lane 4, cell extract of E. coli W14 (pCK01; control plasmid); lane M, molecular mass markers. Approximately 10 μg of total protein and an anti-PaaY antiserum was used to detect the presence of PaaY (indicated by an arrow) in each cell extract.

Fig. S3. A. Nucleotide sequence of the Px promoter region of E. coli W. The sequence is numbered relative to the Px transcription start site (+1). The −10 extended promoter box is indicated in grey. The ATG start codon of paaX and TGA stop codon of paaK are shown in lowercase letters. Direction of transcription is indicated by arrows. The PX5 and PX3 primers used to amplify the PX DNA fragment (200 bp) are underlined. The PaaX-mediated protection from DNase I digestion is boxed in the non-coding strand, and the nucleotides matching the consensus PaaX-binding sequence are shown in bold letters.

B. Comparison of PaaX operator regions in different promoters. Pa, Pz and Px refer to the promoters that control the transcription of the three paa operons of E. coli W. Ppac is the promoter of penicillin G acylase gene from E. coli W or Kluyvera citrophila. Nucleotides matching the consensus sequence are shown in uppercase bold letters. N, indicates the distance (in nt) between the inverted repeats of the operator. The − 35 and − 10 boxes for RNAP binding to the promoters are underlined, except for the boxes of Ppac promoters which are located further downstream the operator region.

Fig. S4. Purification of the PaaX protein. Analysis on a 12.5% SDS-PAGE of the purification process of PaaX from E. coli JM109 (pUCX2) cells as detailed in Experimental procedures.

A. Lane M, ‘Broad Range’ (Bio-Rad) molecular mass markers; lane 1, soluble fraction of the crude extract of E. coli JM109 containing pUC18 as a negative control; lane 2, soluble fraction of the crude extract from E. coli JM109 (pUCX2) cells; lane 3, soluble fraction after polyethylenimine precipitation; lane 4, soluble fraction after dialysis; lane 5, protein fraction resuspended after ammonium sulfate precipitation; lane 6, protein fraction resuspended after dialysis.

B. Lane M, molecular mass markers; lane 1, purified PaaX protein after Sephadex G-100 chromatography. The PaaX protein is indicated with an arrow.

Fig. S5. PaaX competes with the RNAP for the interaction at the Px promoter. The interaction of RNAP and PaaX to the Px promoter was monitored by gel retardation assays as detailed in Experimental procedures. Lane 1, PX probe. Lane 2, PX probe and RNAP (150 nM). Lanes 3–5, increasing concentrations of PaaX (10, 50 and 100 nM, respectively), the PX probe and RNAP (150 nM) were added simultaneously. Lane 6, PaaX (100 nM), PX probe and RNAP (150 nM) were incubated in the presence of 500 μM PA-CoA. Lane 7, PX probe and PaaX (100 nM). − and +, indicate the absence or presence of purified proteins, respectively. The unbound PX probe and the PX/RNAP and PX/PaaX complexes are indicated by arrows.

Fig. S6.In vitro binding of purified PaaX, RNAP and CRP to the Pz promoter. Gel retardation analyses were performed as indicated under Experimental procedures. − and + , indicate the absence or presence of purified proteins and PA-CoA. The unbound PZ probe and the PZ/RNAP/CRP and PZ/PaaX complexes are indicated by arrows. Lane 1, PZ probe. Lanes 2 to 7 contain 150 nM of RNAP. Lanes 3 to 7, contain 100 nM CRP. Lanes 4 to 6, contain 10, 50 and 200 nM of PaaX, respectively; in the rest of cases PaaX was added at 200 nM. Lane 7, PA-CoA was added at 500 μM. Proteins were incubated simultaneously. Increasing concentrations of PaaX cause a decrease in the yield of the RNAP-PZ complex and the appearance of a gel retardation band corresponding to the PaaX-PZ complex. We performed the retardation assays in the presence of CRP and cAMP, although we have observed that RNAP can bind to the Pz promoter even in the absence of CRP. The addition of PA-CoA to the retardation assays abolished the binding of PaaX and restored the formation of the RNAP-PZ complex.

Fig. S7.In vitro binding of purified PaaX, RNAP and CRP to the Pa promoter. Gel retardation analyses were performed as indicated under Experimental procedures. − and +, indicate the absence or presence of purified proteins and PA-CoA. The unbound PA probe and the PA/RNAP/CRP/PaaX, PA/RNAP/CRP, PA/CRP and PA/PaaX complexes are indicated by arrows. Lanes 2 to 7, contain 150 nM of RNAP. Lanes 3 to 7, contain 100 nM CRP. Lanes 4 to 6, contain 10, 50 and 200 nM of PaaX, respectively; in the rest of cases PaaX was added at 200 nM. Lane 7, PA-CoA was added at 500 μM. Lane 9, PA probe and 200 nM CRP. Binding of RNAP to the Pa promoter depends on the presence of cAMP-CRP. RNAP is able to bind to the Pa promoter even in the presence of increasing concentrations of PaaX, and a low mobility complex due to the interaction of the PA probe with RNAP, PaaX and cAMP-CRP is observed.

Fig. S8. The paaY mutation causes a change in cell morphology. Cultures of E. coli W14 harboring plasmid pAAD (paa wild type) or pAAD::Tn1000-84 (paaY mutant) grown in PA-containing minimal medium at an A600 of 0.7 were observed by phase contrast microcopy.

Fig. S9. Gel retardation analysis of PaaX binding to the Pa-Pz promoter region in the absence (A) or presence (B) of the PaaY protein. Protein extracts of E. coli W14 (pAFX), that overexpresses paaX, and E. coli W14 (pQEYES), that overexpresses paaY (Table S1), were used. The PA-PZ probe used contains the divergent Pa and Pz promoters. The positions of the free probe and the PA-PZ/PaaX complexes I and II are shown with arrows. (A) 0.08 μg of crude extract from E. coli W14 (pAFX) was used. Increasing concentrations of PA-CoA, 10, 25, 50, 250, 500 and 1000 μM, were added in lanes 3 to 8, respectively. (B) 0.08 μg of crude extract from E. coli W14 (pAFX) and 0.03 μg of crude extract from E. coli W14 (pQEYES) were used. Increasing concentrations of PA-CoA, 10, 25, 50, 250, 500 and 1000 μM, were added in lanes 3 to 8, respectively.

Fig. S10. Structure of PaaY.

A. 3D structural model of the PaaY trimer, viewed parallel to the molecular threefold axis, emphasizing the triangular LβH domain. The positions of the putative metal atoms are indicated by small pink spheres.

B. Ribbon diagram of the PaaY monomer viewed perpendicular to the molecular threefold axis.

C. Structure-based sequence alignment of the LβH domain of PaaY. Residues corresponding to seven complete or partial coils (C1 to C7) are aligned. Each complete coil is composed of three flat β-strands (PB1, PB2 and PB3) separated by three turns (T1, T2 and T3). The most highly conserved residues are shaded in blue [LIV], yellow [GAED] and purple [STAV]. The three conserved histidines (H65, H82 and H87) are marked in red.

Fig. S11. Purification process of the PaaY protein. SDS-PAGE (12.5%) analysis of the fractions obtained in the purification process as described in Experimental procedures. Lane M, ‘Broad Range’ (Bio-Rad) molecular mass markers; lane 1, 10 μg of protein extract of E. coli M15 (pREP4, pQE32); lane 2, 15 μg of protein extract of E. coli M15 (pREP4, pQEYES) after centrifugation at 14000 rpm for 15 min; lane 3, 12 μg of extract after precipitation with 60% ammonium sulfate; lane 4, 6 μg of the protein fraction recovered from the Phenyl-Sepharose column; lane 5, 1.5 μg of protein recovered from the Sephadex G-100 column; lane 6, 1 μg of protein recovered from the DEAE cellulose column. The PaaY protein is indicated with an arrow.

Fig. S12. Study of the oligomerization state of the PaaY protein in solution by analytical ultracentrifugation experiments. The symbols (●) represent the experimental data obtained at 13000 rpm, 20°C, of a PaaY protein concentration of 47 μM in 25 mM Tris-HCl buffer pH 8, 100 mM NaCl. Data did not change significantly with protein concentration over the range examined (6–47 μM). The solid line represents the best fit of the experimental data to the sedimentation equilibrium gradient of a single protein trimer species (Mw = 63 740). The upper plot shows the residuals expressed as the difference between the experimental and the fitted data.

Table S1. Bacterial strains and plasmids used in this study.

Table S2. PaaY thioesterase activity. % activity represents the percentage of activity considering 100% that obtained with acetoacetyl-CoA. *ND, not detectable.

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