Growth of bacterial cells and preparation of cell extracts
Thauera chlorobenzoica was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, No. 18012) and was grown under anaerobic conditions as described (Song et al., 2000b). Benzoate (5 mM), 3-Cl-benzoate or 3-Br-benzoate (each 2 mM) and nitrate (10 mM) were used as carbon and energy sources. For growth analysis on 3-F-benzoate (2 mM), cells previously grown on benzoate were centrifuged in the exponential growth phase, washed twice with medium without substrate prior to carbon source addition.
For the preparation of crude extracts cells were anaerobically harvested in the exponential growth phase by centrifugation (14 000 g) and suspended in 20 mM triethanolamine hydrochloride (TEA)/KOH buffer pH 7.8 (1 g of cells in 2 ml of buffer) and 0.1 mg DNase I. Cell lysates were obtained by French press treatments (137 MPa). After centrifugation at 100 000 g (1 h, 4°C), the supernatant was immediately used or stored at −20°C.
Enzyme assays: halobenzoyl-CoA reductase
The assay (200 µl) contained 5 mM Ti(III)-citrate, 5 mM MgATP, 0.5 mM benzoyl-CoA/halobenzoyl-CoA, respectively, and was carried out under anaerobic conditions in a 100 mM 3-(N-morpholino)propanesulphonic acid (Mops) buffer with 5 mM MgCl2 at pH 7.3 and 30°C. The reaction was started by addition of 15 µl of cell purified BCR from T. aromatica. The latter was obtained as described (Boll and Fuchs, 1995), specific activity was 540 nmol benzoyl-CoA reduced min−1 mg−1. Samples were taken at different time points and stopped by the addition of twofold volumes methanol. After centrifugation the samples were applied to an HPLC column (Waters 2695 separation module, C18 reverse-phase HPLC column, Knauer) using a linear gradient from 5% to 30% acetonitrile in 50 mM potassium phosphate, pH 6.8 (for benzoyl-CoA, 3-Cl-/3-Br-benzoyl-CoA) within 18 min or from 12% to 16% in 20 mM ammonium acetate buffer, pH 6.8 (for 3-F-benzoyl-CoA) within 10 min.
Purification of 3-Cl-benzoate CoA ligase
The 3-Cl-/3-Br-benzoate converting halobenzoate CoA ligase activity was enriched from 7 g of T. chlorobenzoica cells (wet mass) grown on 3-Cl-benzoate/nitrate. All steps were carried out at 4°C. The crude extract obtained after French Press treatment (7 ml) was applied onto a Q-Sepharose column (Fast Flow, volume 25 ml, 16 mm diameter, GE-Healthcare), which had been equilibrated with basic buffer (containing 20 mM TEA-HCl, 4 mM MgCl2, 10% glycerol, 1 mM DTE, pH 7.5). After the column was washed with three bed volume of basic buffer, protein was eluted in a linear gradient from 0 to 150 mM KCl in 125 ml of basic buffer. The fractions collected were tested for benzoate CoA and 3-Cl-benzoate CoA ligase activities. Fractions eluting between 90 and 110 mM KCl contained only benzoate CoA ligase activity, whereas fractions eluting between 110 and 150 mM KCl exhibited both, benzoate and 3-Cl-benzoate CoA ligase activities. The 3-Cl-benzoate CoA ligase activity containing fractions were pooled and loaded onto a hydroxyapatite column (20 ml, 16 mm diameter, ceramic hydroxyapatite, Bio-Rad), equilibrated with basic buffer. The column was run with a linear gradient from 0 to 100 mM potassium phosphate in TEA buffer. The active fractions (40 ml, elution between 10 and 40 mM potassium phosphate) were pooled and subsequently applied onto a Reactive Green cross-linked agarose column (20 ml, 16 mm diameter, Reactive-Green 19-agarose, Sigma-Aldrich), equilibrated with basic buffer. After a washing step of two bed volumes 150 mM KCl in basic buffer, active fractions were eluted by the addition of 1 mM 3-Cl-benzoate plus 150 mM KCl to basic buffer. For the last purification step a MonoQ column (MonoQ, 5/50 GL, 5 mm diameter, bed volume 1 ml, GE-Healthcare) was equilibrated with five bed volumes of basic buffer. The fractions applied eluted in a linear gradient from 0 to 140 mM KCl in 25 ml at 110 mM KCl. The 3-Cl-benzoate CoA ligase activity containing fractions were concentrated in microconcentrators (30 kDa exclusion limit, Vivaspin 6, Sartorius) by centrifugation and stored at −20°C.
Purified 3-Cl-benzoate CoA ligase was separated by one-dimensional SDS gel electrophoresis after it was excised from the gel and subsequently digested by trypsin in-gel. Peptides were analysed by UPLC-LTQ Orbitrap-MS/MS (Bastida et al., 2010). The peptides were eluted with an 8–40% acetonitrile gradient in 0.1% formic acid (30 min). Continuous scanning of eluted peptide ions was carried out between 150 and 2000 m/z, automatically switching to MS/MS CID mode on ions exceeding a minimal signal intensity of 2000.
Raw data were processed for database search using Thermo® Proteome Discoverer software (v1.0 build 43, Thermo Fisher Scientific). Database search was performed by tandem mass spectrometry ion search algorithms from the Mascot house server. The following parameters were selected: bacteria of NCBInr (National Center for Biotechnology Information) as criterion for taxonomy, tryptic cleavage and maximal two missed cleavage sites. A peptide tolerance threshold of ± 10 ppm and an MS/MS tolerance threshold of ± 0.2 Da were chosen. Carbamidomethylation at cysteines was given as static and oxidation of methionines as variable modification.
CoA ester analysis for in vitro assays
Experiments were carried out on an Agilent 1100 series binary HPLC system (Agilent Technologies) coupled with a 4000 QTRAP linear ion trap mass spectrometer (AB Sciex) equipped with a TurboIon spray source. The chromatographic separation of CoA-esters was achieved on a Chromolith SpeedROD RP-18e 50 mm × 4.6 mm from Merck at 40°C with eluent A (95% 20 mM ammonium acetate, 5% acetonitrile) and eluent B (50% 20 mM ammonium acetate, 50% acetonitrile) with a flow rate of 400 µl min−1. A gradient of eluent B was applied as follows: 2 min, 0%; 10 min, 40%; 11 min, 100%; 15 min, 100%; 15.5 min, 0%; 20 min, 0%. The injection volume of each sample was 10 µl. Positive ESI-MS/MS mass spectrometry was performed using the following parameters: ion source temperature 300°C, ion spray voltage 5.0 kV, nebulizer gas 40 AU, auxiliary gas 60 AU, curtain gas 20 AU and collision gas 5 AU. For neutral loss (NL), enhanced product ion (EPI) and selective reaction monitoring (SRM) experiments optimized declustering potential, collision energy, entrance potential and collision cell exit potential were set to 100 V, 55 V, 10 V and 10 V respectively. Neutral loss scans were obtained by scanning from 650 to 1200 m/z in 1000 ms with a neutral loss mass of 507 Da and in case of exceeding an intensity of 5000 cps combined via independent data analysis with an EPI scan from 100 to 500 m/z in 400 ms with an ion trap fill time of 20 ms. Recorded transitions for the SRM-assay were as follows (m/z): benzoyl-CoA, 872.3/365.0; 1,5-dienoyl-CoA, 874.3/367.0; 6-OH-monenoyl-CoA and 3-F-benzoyl-CoA, 892.3/385.0; fluoro-1,5-dienoyl-CoA, 894.3/387.0; fluorocyclohex-1-ene-1-carboxl-CoA, 896.3/389.0.
CoA ester analysis in whole cells
Cells grown on 3-Cl-benzoate, 3-Br-benzoate were harvested in the exponential growth phase at a biomass concentration of approximately 0.3 g l−1 by centrifugation (4°C, 16 000 g). The freshly harvested cell pellet was added to 3 ml of a −20°C quenching solution containing 25 mM formic acid in acetonitrile. Cell extracts were prepared by three consecutive ultrasonication steps for 30 s each. The samples were diluted with 20 ml of ice-cold water (ddH2O) and cooled with liquid nitrogen prior to freeze drying. In case of 3-F-benzoate metabolome analysis, cells were grown on benzoate, harvested in the exponential growth phase and washed twice with medium without a carbon substrate. Afterwards cells were incubated with 2 mM 3-F-benzoate for 30 min at 30°C. Cell extract preparation and extraction of CoA thioesters was as described for cells grown on 3-Cl-/3-Br-benzoate.
Analyses were performed with a Rheos 2200 HPLC system (Flux Instruments, Basel, Switzerland) coupled to an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), equipped with an electrospray ionization probe. Coenzyme A esters were analysed as described previously (Peyraud et al., 2009) with slight modifications. To perform online desalting of samples prior to LC separation two C18 analytical columns (Gemini 50 × 2.0 mm and 100 × 2.0 mm, particle size 3 µm; Phenomenex, Torrance, CA, USA) were used. Flow rate was 220 µl min−1. Solvent A was 50 mM formic acid adjusted to pH 8.1 with NH4OH and solvent B was methanol. Injection volume was 10 µl. For online desalting samples were loaded on 50 × 2.0 mm C18 column and the sample was washed on column for 5 min with 100% solvent A. During desalting the short column was connected to waste via a six-port valve and the 100 × 2.0 mm column was equilibrated with solvent A by an additional pump. After desalting both columns were connected in series and the following gradient of B was applied to separate CoA esters: 5 min, 5%; 15 min, 23%; 25 min, 80%; 27 min, 80%. The LC-MS system was equilibrated for 6 min at initial elution conditions between two successive analyses. The LC was coupled to the mass spectrometer. Sheath gas flow rate was 40, auxiliary gas flow rate was 30, tube lens was 80 V, capillary voltage was 35 V, and ion spray voltage was 4.3 kV. MS analysis was performed in the positive FTMS mode at a resolution of 60 000 (m/z 400). To perform targeted MS/MS experiments of CoA esters, parent masses were selected by inclusion lists. CE voltage for collision induced collision was 25.
CoA thioester identification was performed in two steps. In a first step high-resolution LC-MS analyses were performed, mass lists of potential CoA thioesters were calculated, and [M+H]+ ions were selected as potential CoA thioesters when mass difference between calculated and measured values was below 5 ppm. In a subsequent targeted LC-MS/MS analysis selected mass ions of potential CoA thioesters were fragmented using inclusion lists and fragment spectra were searched for presence of specific CoA ester fragment ions. Potential CoA thioesters mass lists were calculated from a constrained combination of elements C, H, N, O, P and S optionally amended with F, Cl or Br. Constraints consisted of the following assumptions: CoA part: C 21, H 34, N 7, O 16, P 3, S 1 atom; plus residual organic acid C limited to 1–12; H/C 0.2 ≤ H/C ≤ 3. 1 (1 O part of thioester group) ≤ O/C ≤ 1.2 (Kind and Fiehn, 2006); for halogenated CoA esters 1 H was replaced by F, Cl or Br; for identification of chlorinated and brominated CoA thioesters isotopic abundances of 35Cl (75.77%), 37Cl (24.23%) and 79Br (50.69%), 81Br (49.31%) and the resulting mass shifts for mass isotopomers (Δ37Cl,35Cl = 1.9971, Δ81Br,79Br = 1.9980) were taken into account.
To identify CoA thioesters on MS2 level fragment spectra of selected parent ions were searched for presence of two distinct fragment ions (Dalluge et al., 2002): A CoA-specific fragment ion at m/z = 428.0367 remaining unchanged for all CoA esters and a characteristic fragment ion arising from the molecular region containing the CoA thioester that allows determining the elemental composition of the esterified organic acid when using high-resolution mass spectrometry for fragment ion analysis.
To estimate the relative amounts of CoA esters, 95 µl of cell extract were mixed with 5 µl of ethylmalonyl-CoA standard solution prior to analysis. Ethylmalonyl-CoA was chosen as internal standard since it was not present in the cell extracts. Peak intensities of identified CoA esters were normalized to peak intensity of the internal standard and log2 of ratios were classified as follows: +, log2(ICoA/IIS) < −1; ++, −1 ≤ log2(ICoA/IIS) ≤ 1; +++, log2(ICoA/IIS) > 1.