characterization and spreadability of protocatechuate 4,5-cleavage

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Appl Microbiol Biotechnol Table 1 Bacterial strains and plasmids used in this study Strain/plasmid/ oligonucleotide Description Sources/ references Strains Comamonas testosteroni CNB-1 Wu et al. (2005) CNB-1ΔpmdF A fragment of DNA coding for amino acids 105 to 215 of pmdF was deleted This study CNB-1ΔpmdF/ This study pBBR1MCS3-pmdF CNB-1ΔpmdE A fragment of DNA coding for amino acids 32 to 296 of pmdE was deleted This study CNB-1ΔpmdE/ This study pBBR1MCS2-pmdE CNB-1ΔpmdE/ This study pBBR1MCS2-galB CNB-1ΔpmdU A fragment of DNA coding for amino acids 6 to 226 of pmdU was deleted This study CNB-1ΔpmdU/ This study pBBR1MCS2-pmdU CNB-1ΔpmdU/ This study pBBR1MCS2-galD E. coli strains This study BL21(DE3) F − ompT hsdSB(rB − mB − ) gal dcm λDE3 (harboring gene 1 of the RNA polymerase from the phage T7 under the PlacUV5 promoter) Maniatis et al. (1982) DH5α (f80lacZΔM15)endA1 recA1 hsdR17 (rK − mK − ) supE44ΔlacU169 Maniatis et al. (1982) Pseudomonas putida KT2440 Plasmid Franklin et al. (1981) pK18mobSacB Mobilizable vector, allows for selection of double crossover in CNB-1 Schäfer et al. (1994) pK18mobSacB-ΔpmdE pK18mobSacB-ΔpmdU pK18mobSacB-ΔpmdF pBBR1MCS3 Tc r , lacPOZ′ broad host vector with R type conjugative origin Kovach et al. (1995) pBBR1MCS2 Km r , lacPOZ′ broad host vector with R type conjugative origin Kovach et al. (1995) pBBR1MCS3-pmdF Carrying pmdF (to generate complementation for pmdF) This study pBBR1MCS2-pmdE Carrying pmdE (to generate complementation for pmdE) This study pBBR1MCS2-pmdU Carrying pmdU (to generate complementation for pmdU) This study pBBR1MCS2-galD Carrying galD (to generate expression for galD) This study pBBR1MCS2-galB Carrying galB (to generate expression for galB) This study pET-28a(+) Expression vector Novagen pET-28a-pmdAB pET28a derivative for expression of pmdAB This study pET-28a-pmdU pET28a derivative for expression of pmdU This study pET-28a-pmdE pET28a derivative for expression of pmdE This study pET-28a-pmdF pET28a derivative for expression of pmdF This study pET-28a-galB pET28a derivative for expression of galB This study pET-28a-galD pET28a derivative for expression of galD This study of the target genes in pK18mobsacB derivatives and in CNB-1 mutants were verified by PCR amplification. The complementation of these genes was conducted by introducing pBBR1MCS2 or pBBR1MCS3 derivatives into the mutants. Cloning, expression, and purification of pmd and gal genes in E. coli Each gene of the pmd gene cluster of strain CNB-1 and gal cluster of strain KT2440 was amplified by PCR from the
Appl Microbiol Biotechnol genomes of strains CNB-1 and KT2440. Purified PCR products were treated with restriction enzymes and then ligated into the similarly treated pET28a (+). The resulting plasmids were transformed in E. coli BL21 (DE3) for expression of the genes. Expression of the genes in E. coli was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C for 10– 12 h, when the optical density at 600 nm reached 0.6. Cellular lysates of recombinant E. coli were prepared by sonication of cell suspensions in 50 mM phosphate buffer (pH 8.0) or in 50 μM Fe(NH 4 ) 2 (SO 4 ) 2 solution when purifying PmdAB or 20 μM ZnSO 4 solution when purifying PmdU and GalD. Sonication was conducted (at 300 W, 3 s, interval of 5 s, for 90 cycles). Cell debris was removed by centrifugation at 12,000 rpm for 20 min, and the supernatant was used for further purification. Proteins were purified with His-affinity resin chromatography by following the instruction from the manufacturers (Novagen). Enzymatic synthesis of OMA keto , OMA enol , and CHA OMA keto ,OMA enol , and CHA were prepared as previously described (Nogales et al. 2011) with the following modification. OMA keto was freshly prepared from gallic acid in a system containing (totally 2 mL): 0.2 mM gallic acid, 50 μM Fe(NH 4 ) 2 (SO 4 ) 2, 24 μg recombinant PmdAB from E. coli, 50 mM potassium phosphate buffer, pH 7.0. After incubation at room temperature for 5 min, this mixture solution was used as the crude OMA keto preparation without any further purification. OMA enol was prepared by incubating the freshly prepared OMA keto with GalD and 20 μM ZnSO 4 for 5 min. CHA was prepared by adding PmdE or GalB (Nogales et al. 2011) into the freshly prepared OMA enol solution and incubated at room temperature for another 5 min. Enzyme activity assays The OMA hydratase activity of PmdE was assayed spectrophotometrically at room temperature by measuring the decrease of A 265 due to the disappearance of OMA enol (Nogales et al. 2011). The OMA tautomerase activity of PmdU was qualitatively determined at room temperature by adding 0.8 μg purified PmdU in the 2.0 mL freshly prepared OMA keto .WhenPmdU was added in the system, an increase in the absorbance at 265 nm can be observed. The CHA aldolase activity of PmdF was assayed at room temperature by monitoring the decrease in A 340 derived from NADH oxidation in a coupled assay by adding 1 mM MgSO 4 ,140μM NADH, 30 U lactate dehydrogenase (Sigma), 30 U malate dehydrogenase (AMRESCO), and 2.6 μg recombinant PmdF from E. coli (Hara et al. 2003; Nogales et al. 2011). Catechol 2,3-dioxygenase (C23D) activity was assayed by monitoring the absorbance changes at 375 nm to monitor the formation of catechol ring cleavage product (Cerdan et al. 1994), and PCA P45D activity was determined spectrophotometrically by measuring the increase in the absorbance at 410 nm due to the formation of 4-carboxy-2-hydroxymuconate-6-semialdehyde (Kamimura et al. 2010a;Mampelet al. 2005) and gentisate 1,2-dioxygenase (G12D) was measured spectrophotometrically by measuring the increase of absorption at 330 nm derived from maleylpyruvate (Shen et al. 2005). Results C. testosteroni strain CNB-1 grew on various aromatic compounds Strain CNB-1 had been isolated for 4CNB degradation, and it takes 4CNB as sole carbon and nitrogen sources (Wu et al. 2005). Previous results showed that CNB-2 grew on benzoate, phenol, 3- and 4HB, PCA, and vanillate (Ma et al. 2009). In this study, the ability of strain CNB-1 (the parent of strain CNB-2) to grow on a range of aromatic compounds was tested. Results showed that CNB-1 also grew on the following aromatic compounds, including phenol, benzoate, 3HB, 4HB, vanillate, and gentisate, but not on 4-cresol, 2,4-dihydroxybenzoate, resorcinol, and phenylacetate. Further, aromatic ring cleavage dioxygenase activities were determined. It was found that C23D activity was associated with cells grown on phenol. P45D activity was associated with cells grown on vanillate, 3HB, and 4HB. G12D activity was associated with cells grown on gentisate, benzoate, and 3HB (Table 2). These results suggested that strain CNB-1 possessed multiple degradative pathways for various aromatic compounds. Identification of proteins involving in vanillate, 3HB, and 4HB degradation In order to identify enzymes/proteins involved in the degradation of these compounds, comparative proteomic analyses were carried out on CNB-1 cells grown with vanillate, 3HB, or 4HB versus cells grown with succinate. With cells grown on vanillate, 3HB, and 4HB, 704±5, 705±12, and 692±17 spots were recognized, respectively (Supplementary Fig. S1). Compared with cells grown on succinate, 40 proteins in total were differently synthesized, of which 30 were induced and ten were upregulated. Thirty-seven out of the 40 protein spots were successfully identified with MALDI- TOF mass spectrometry (Table 3). Most of the identified proteins were functionally related to degradation of vanillate, 3HB, and 4HB. They are either involved in the peripheral routes leading to PCA or involved in the PCA 4,5-cleavage pathway (Fig. 1a). The following enzymes involved in the peripheral routes were detected: vanillate monooxygenase (V2 and V3) and vanillate O- demethylase subunit (V5), 4HB-3-hydroxylase (O3 and O4), and 3HB hydroxylase (M1). Four enzymes of the
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