MATERIALS AND METHODS Chemicals

Standard chemicals were obtained from Sigma-Aldrich, Fluka or Merck. 4-Sulfoacetophenone (4-acetylbenzenesulfonate) was purchased from ABCR (Karlsruhe, Germany), 4-sulfophenyl acetate (1-phenol-4-sulfonate-acetate) from SYNCHEM (Felsberg-Altenburg, Germany), and biochemicals (NADH, NADPH, NAD⁺ and NADP⁺) from Biomol (Hamburg, Germany).

Racemic 3-(4-sulfophenyl)butyrate was synthesized as described previously (Schleheck et al.

2004a). 4-Sulfocatechol was a gift of B. J. Feigel (Stuttgart).

Growth conditions

C. testosteroni KF-1 (DSM 14576) was grown in a phosphate-buffered, mineral-salts medium (Thurnheer et al. 1986) supplemented with 6 mM 3-(4-sulfophenyl)butyrate, 10 mM 4-sulfophenol or 15 mM succinate as sole carbon source. Cultures were incubated in glass tubes (Corning, 3-ml scale) in a roller, or in Erlenmeyer flasks (0.1 – 2-l scale) on a shaker at 30°C in the dark. Cultures were inoculated (1%) with cultures pre-grown with the same substrate.

Preparation of cell extracts and enrichment of hydroxyquinol dioxygenase and superoxide dismutase

C. testosteroni KF-1 cells were harvested in the exponential growth phase by centrifugation (15,000 x g, 20 min, 4°C) and stored frozen (-20°C). Cells were resuspended in 20 mM Tris-HCl buffer (pH 8.0) containing 0.03 mg ml^-1 DNase I (Sigma) and disrupted by four passages through a chilled French pressure cell (140 MPa, 4°C) (Aminco, Silver Spring, USA). Whole cells and debris were removed by centrifugation (15,000 x g, 20 min, 4°C) to obtain crude extract, and membranes were removed by ultracentrifugation (60,000 x g, 60 min, 4°C) to obtain the soluble fraction.

The soluble protein solution was subjected to anion exchange chromatography on, either a MonoQ HR (high-resolution) 10/10 column (Pharmacia), or on a DEAE Sepharose fast flow column (Pharmacia). The MonoQ column was pre-equilibrated with 20 mM Tris-HCl containing 10% glycerol at a flow rate of 1 ml min^-1. Bound proteins were eluted with a NaCl solution gradient (10 min without NaCl, in 50 min to 0.3 M NaCl, and in further 30 min to 1 M NaCl). The fractions were collected in 3 ml volumes; the hydroxyquinol dioxygenase activity eluted at about 350 mM NaCl as assayed photospectrometrically by an increase of its proposed product maleylacetate or by oxygen consumption in a Clark-type electrode (see below).

The DEAE column was pre-equilibrated with 20 mM Tris-HCl containing 10% glycerol at a flow rate of 1 ml min^-1. Bound proteins were eluted using a linear NaCl gradient (20 min without NaCl, in 30 min to 1 M NaCl), and fractions (3 ml) were collected; the ʻhydroxyquinol protecting activityʼ (see below) eluted at about 300 mM NaCl.

Analytical chemistry

Total cellular protein was determined following a Lowry-based protocol (Kennedy and Fewson 1968) and soluble protein by protein dye binding (Bradford 1976); bovine serum albumin was used as a reference standard. The release of sulfate during growth was quantified turbidimetrically (Sörbo 1987) as a suspension of BaSO4.

124 CHAPTER 7 Protein gel electrophoresis and proteomics

Proteins of whole cells, soluble protein fractions, or proteins obtained from heterologous gene expressions, were analyzed on 12% SDS-PAGE with Coomassie brilliant blue R-250 staining (Laemmli 1970). Proteins in the soluble fraction of C. testosteroni KF-1 cells were analyzed by two dimensional gel elctrophoresis (2D-PAGE; IEF/SDS-PAGE) as described previously (Schmidt et al. 2013). Protein spots of interest were excised and analyzed by PF-MS at the Proteomics Facility of the University of Konstanz. The MASCOT engine (Matrix Science, London, UK) was used to search against an amino-acid sequence database of C. testosteroni KF-1 genes (IMG version 2011-08-16). The parameters for searching and scoring were set as described previously (Schmidt et al. 2013).

Heterologous expression of the two dioxygenase candidate genes in E. coli and purification of the recombinant proteins

Chromosomal DNA of C. testosteroni KF-1 was isolated using the Illustra bacteria genomicPrep Mini Spin Kit (GE Healthcare). The genes were amplified by PCR using Phusion HF DNA Polymerase (Finnzymes). The PCR primers were purchased from Microsynth (Balgach, Switzerland). The sequences of the primer pairs were: PD5469 forward and PD5471 forward, 5´-CACCATGCGCAACATCAACGAAGACACC-3´, PD5469 reverse, 5´-TCATGATTGCTGCGCGCTCGGCTTGGTGGG-3´, and PD5471 reverse, 5´-TCAAGTGGAAACCTTGAGTGGG-3´.

For CtesDRAFT_PD5469 and CtesDRAFT_PD5471, the PCR conditions were: 90 s initial denaturation at 98°C; 30 cycles of 15 s denaturation at 98°C, 20 s annealing at 61.6°C, and 60 s elongation at 72°C.

The PCR products were separated by agarose gel electrophoresis, excised, and purified using a QIAquick gel extraction kit (Qiagen) and ligated into pET100, an N-terminal His6-tag expression vector, by using the Champion pET100 Directional TOPO Expression Kit and OneShot TOP10 E. coli (Invitrogen); correct constructs were confirmed by sequencing (GATC-Biotech, Konstanz). For the gene expression, BL21 Star (DE3) OneShot E. coli (Invitrogen) cells were transformed with the construct and grown in LB medium (100 mg l^-1 ampicillin) at 37°C. At OD580nm ≈ 0.5, the cultures were induced (0.5 mM Isopropyl β-D-1-thiogalactopyranoside; IPTG) and grown for additional 5 h at 20°C; cells were harvested by centrifugation (15,000 x g, 20 min, 4°C) and stored at -20°C.

Cells were resuspended in buffer A (20 mM Tris-HCl pH 8.0, 100 mM KCl, 10% (v/v) glycerol) containing 0.03 mg ml^-1 DNase I (Sigma), and disrupted by four passages through a pre-cooled

French pressure cell (140 MPa; Aminco, Silver Spring, USA). Cell debris was removed by centrifugation (15,000 x g, 10 min, 4°C), and membranes by ultracentrifugation (60,000 x g, 60 min, 4°C). The soluble fraction was loaded on a Ni²⁺-chelating Agarose affinity column (1-ml column volume, Macherey-Nagel, Germany), pre-equilibrated with buffer A, followed by a washing step using 30 mM imidazole in buffer A. The His-tagged protein was eluted using 250 mM imidazole in buffer A, concentrated using Vivaspin (10 kDa cut-off; Sartorius, Germany), and stored at -20°C after addition of glycerol to 30% (v/v) final concentration.

Enzyme assays

The activities of native and recombinant hydroxyquinol dioxygenase were measured as substrate dependent oxygen consumption with a Clark-type oxygen electrode (Schleheck et al.

2010) in either 20 mM Tris-HCl buffer (pH 8.0) or in 20 mM MES-NaOH buffer (pH 6.4).

Specific activities of native and recombinant hydroxyquinol dioxygenase were determined spectrophotometrically as decrease of hydroxyquinol at 289 nm (ε = 3.3 x 10³ M^-1 cm^-1; Perry and Zylstra 2007) and as increase of absorption of the putative reaction product maleylacetate at 242 nm (ε = 4.74 x 10³ M^-1 cm^-1; Halak et al. 2007). The activities were plotted using hyperbolic fit in Origin (Microcal Software Inc.). The ʻhydroxyquinol protecting activityʼ was determined as qualitative method; therefore protein samples were added to 50 mM Tris-HCl (pH 8.0) and 1 mM freshly prepared hydroxyquinol. The oxidation of hydroxyquinol, or its protection, was judged visually, by the appearance or absence of a red color.