Tracking the Reversed Oxidative Tricarboxylic Acid Cycle in Bacteria

Materials and Reagents

1. Cultivation experiments of anaerobic organisms

   1. Serum bottle N20, 120 mL (MediPac, catalog number: 10113100) or equivalent

   2. Infusion bottles, 1 L (VWR, catalog number: 215-9243) or equivalent

   3. Butyl rubber stopper N20 (Glasgerätebau Ochs, catalog number: 102049) or equivalent

   4. Rubber stoppers for 1 L infusion bottles (ERIKS)

   5. Crimp aluminum caps (WICOM, catalog number: WIC 44580) or equivalent

   6. Crimper (VWR, catalog number: 548-0073)

   7. Decapper (VWR, catalog number: 548-0074)

   8. Screw top, with hole Ø 19 mm (VWR, catalog number: MUEL14.076.04)

   9. Duran^® flasks, 1 L (VWR, catalog number: SCOT818015403)

   10. Rubber stoppers for Duran^® flasks (Glasgerätebau Ochs, catalog number: 444704)

   11. Lecture-bottle control valve (Sigma, catalog number: Z146951)

   12. Sterile syringes, male Luer-Lock (Fisher Scientific, catalog number: 14955461) or equivalent

   13. Luer-to-tubing connector, female Luer-Lock (Sigma, catalog number: Z261580)

   14. Sterile ERSTA syringes for single use, 1 mL (Th. Geyer, catalog number: 6075936) or equivalent

   15. Disposable needles (Carl Roth, catalog number: X132.1) or equivalent

   16. Sterile syringe filters, 0.2 µm pore size (VWR, catalog number: 514-0061)

   17. Na₂S·9H₂O (Sigma, catalog number: 1313-84-4); store at 4°C

   18. Sulfur powder (Carl Roth, catalog number: 9304.1); store at room temperature (RT)

   19. Resazurin sodium salt (Sigma, catalog number: R7017); store at RT

   20. Hydrogen gas (Air Liquide Deutschland, catalog number: P0231L50R2A001)

   21. Carbon dioxide gas (Air Liquide Deutschland, catalog number: P0760L50R0A001)

   22. Carbon-13 dioxide gas, 99% ¹³C (Sigma, catalog number: 364592)

   23. L-Glutamic acid-1-¹³C, 99% ¹³C (Sigma, catalog number: 604968)

   24. H. maritima medium (modified from DSMZ 854) (see Recipe 1)

   25. D. acetivorans, D. multipotens, and D. propionica medium (modified from DSMZ 480) (see Recipe 1)

   26. SL-10 trace element solution (see Recipe 2)

   27. Wolfe vitamin solution (see Recipe 3)

   28. Trace element solution (DSMZ 141) (see Recipe 4)

       
       

2. Harvesting of biomass and preparation of cell extracts

   1. Folded filters, Ø 240 mm (Whatman Schleicher & Schuell, catalog number: 10311651) or equivalent

   2. Funnels with short stem (VWR, catalog number: 221-0180) or equivalent

   3. Centrifuge bottles with sealing closure, 1 L (Thermo Fisher Scientific, catalog number: 05-564-26) or equivalent

   4. Centrifuge tubes, 50 mL (VWR, catalog number: 21008-940) or equivalent

   5. Microcentrifuge tubes, 1.5 and 2 mL (VWR, catalog number: 72.690.001, 72.689) or equivalent

   6. Serological pipettes (Thermo Fischer Scientific, catalog number: 170350) or equivalent

   7. Liquid nitrogen

   8. 0.9% [w/v] NaCl solution (saline)

   9. Aluminum cooling block (Sigma, catalog number: Z743486)

   10. Cell lysis buffer (see Recipe 5)

       
       

3. Enzyme assays

   Reaction mixtures are listed. While working, we recommend keeping all chemicals and helping enzymes on ice. All listed components were solved or diluted with dH₂O, unless mentioned otherwise. When special attention is required due to their stability, recommended storage conditions or special notes are reported (e.g., freshly solved before the assays). The pH of organic acids and ATP solutions should be adjusted to neutral; the pH of Tris-HCl buffers can be adjusted at RT, but please note that when the final test temperature differs from RT, the pH also changes according to the following formula: ΔpKa/°C≈-0.028 (e.g., the pH of Tris solutions decreases 0.028 pH units for each 1°C increase in temperature). For long-term storage, organic compounds and CoA esters solutions should be kept at -20°C or -80°C, if not mentioned otherwise; all buffers and salt solutions could be kept at RT. Acetyl-CoA was chemically synthesized from the corresponding anhydrides and CoA, according to a previously described method (Simon et al., 1953).

   In our case, the concentrations of stock solutions were as follows:

   1. Citrate synthase (forward reaction, spectrophotometric continuous assay)

      1. 1 M Tris-HCl (pH 7.5) (Carl Roth, catalog number: 9090.4)

      2. 50 mM oxaloacetate, store at -20°C or -80°C for ≤2 months (Sigma, catalog number: 04126)

      3. 10 mM acetyl-CoA

      4. 10 mM 5,5-dithiobis-2-nitrobenzoic acid (DTNB), freshly solved in 100 mM Tris-HCl (pH 7.5) (Sigma, catalog number: D8130)

   2. Citrate synthase (backward reaction, spectrophotometric continuous assay)

      1. 1 M Tris-HCl (pH 7.8) (Carl Roth, catalog number: 9090.4)

      2. 50 mM MgCl₂

      3. 50 mM dithioerythritol (DTE), freshly solved (Sigma, catalog number: D8255)

      4. 5 mM CoA (Neofroxx, catalog number: 2100GR005)

      5. 200 mM citrate (Sigma, catalog number: C8532)

      6. 5 mM NADH, freshly solved (Acros organics, catalog number: 10711911)

      7. 200 U/mL porcine malate dehydrogenase, store at 4°C, freshly diluted (Sigma, catalog number: M1567)

   3. Citrate synthase (backward reaction, UHPLC discontinuous assay)

      1. 1 M Tris-HCl (pH 7.8) (Carl Roth, catalog number: 9090.4)

      2. 50 mM MgCl₂

      3. 50 mM DTE, freshly solved (Sigma, catalog number: D8255)

      4. 5 mM CoA (Neofroxx, catalog number: 2100GR005)

      5. 200 mM citrate (Sigma, catalog number: C8532)

      6. 5 mM NADH, freshly solved (Acros organics, catalog number: 10711911)

      7. 200 U/mL porcine malate dehydrogenase, store at 4°C, freshly diluted (Sigma, catalog number: M1567)

      8. Vial insert, 250 µL, pulled point glass (Agilent, catalog number: 5183-2085)

      9. Screw top vials, 2 mL, and screw caps (Agilent) or equivalent

      10. Microplate, sterile, 96 well (Greiner Bio-One, catalog number: 655161) or equivalent

      11. Membrane filter, 0.22 µm pore size (Sigma, catalog number: GSWP04700)

      12. Acetonitrile anhydrous, 99.8% (Sigma, catalog number: 271004)

      13. Stop solution (see Recipe 6)

      14. UHPLC-grade double distilled water (see Recipe 7)

      15. Potassium phosphate buffer (see Recipe 7)

   4. ATP-citrate lyase

      1. 1 M Tris-HCl (pH 7.5) (Carl Roth, catalog number: 9090.4)

      2. 100 mM MgCl₂

      3. 10 mM ATP, freshly solved (Sigma, catalog number: A26209)

      4. 50 mM DTE, freshly solved (Sigma, catalog number: D8255)

      5. 50 mM CoA (Neofroxx, catalog number: 2100GR005)

      6. 100 mM citrate (Sigma, catalog number: C8532)

      7. 5 mM NADH, freshly solved (Acros Organics, catalog number: 10711911)

      8. 200 UF/mL porcine malate dehydrogenase, store at 4°C, freshly diluted (Sigma, catalog number: M1567)

   5. Citrate lyase

      1. 1 M Tris-HCl (pH 7.5) (Carl Roth, catalog number: 9090.4)

      2. 100 mM MgCl₂

      3. 50 mM DTE, freshly solved (Sigma, catalog number: D8255)

      4. 100 mM citrate (Sigma, catalog number: C8532)

      5. 5 mM NADH, freshly solved (Acros Organics, catalog number: 10711911)

      6. 200 U/mL porcine malate dehydrogenase, store at 4°C, freshly diluted (Sigma, catalog number: M1567)

   6. Malate dehydrogenase

      1. 1 M Tris-HCl (pH 8) (Carl Roth, catalog number: 9090.4)

      2. 50 mM MgCl₂

      3. 50 mM DTE, freshly solved (Sigma, catalog number: D8255)

      4. 25 mM oxaloacetate, store at -20°C or -80°C for ≤2 months (Sigma, catalog number: 04126)

      5. 5 mM NADH, freshly solved (Acros organics, catalog number: 10711911)

   7. Pyruvate synthase

      1. 1 M Tris-HCl (pH 7.5) (Carl Roth, catalog number: 9090.4)

      2. 100 mM MgCl₂

      3. 50 mM DTE, freshly solved (Sigma, catalog number: D8255)

      4. 50 mM CoA (Neofroxx, catalog number: 2100GR005)

      5. 100 mM pyruvate (Sigma, catalog number: P2256)

      6. 10 mM methyl viologen (Sigma, catalog number: 856177)

   8. 2-Oxoglutarate synthase

      1. 1 M Tris-HCl (pH 7.5) (Carl Roth, catalog number: 9090.4)

      2. 100 mM MgCl₂

      3. 50 mM DTE, freshly solved (Sigma, catalog number: D8255)

      4. 50 mM CoA (Neofroxx, catalog number: 2100GR005)

      5. 100 mM 2-oxoglutarate (Sigma, catalog number: 75890)

      6. 20 mM benzyl viologen (Sigma, catalog number: D8130)

         
         

4. Isotopologue profiling (based on GC-MS analysis)

   1. Glass vials, 1.5 mL (VWR, catalog number: 548-0018)

   2. Screw caps for GC/MS vials (VWR, catalog number: 548-0814)

   3. Glass inlets, 200 µL (VWR, catalog number: 548-0780)

   4. Microcentrifuge tubes, 2 mL (VWR, catalog number: 525-0228)

   5. Glass Pasteur pipettes, short form (Glasswarenfabrik Karl Hecht GmbH & Co. KG, catalog number: 40567001)

   6. Glass wool, superfine (Glasswarenfabrik Karl Hecht GmbH & Co. KG, catalog number: 41408003)

   7. Hydrochloric acid, 37% (Sigma, catalog number: 320331)

   8. Acetic acid, ≥99% (Sigma, catalog number: A6283)

   9. Dowex 50WX8 200-400 (H^±) (Alfa Aesar, catalog number: L13922.18)

   10. Methanol, 99.8% (Sigma, catalog number: 322415)

   11. Ammonia solution in water, 25% (Merck, catalog number: 1054321000)

   12. Acetonitrile anhydrous, 99.8% (Sigma, catalog number: 271004)

   13. N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide ≥95% containing 1% tert-butyldimethylchlorsilane (MTBSTFA) (Sigma, catalog number: 375934), store at -20°C

Equipment

1. Cultivation experiments of anaerobic organisms

   1. Scales

   2. Gas piping system

   3. Membrane vacuum pump (Vacuubbrand, catalog number: 19137003)

   4. Vinyl anaerobic chamber (Coy laboratory products) or equivalent

   5. Bottle-top dispensers (Brand, catalog number: 4600360) or equivalent

   6. pH Meter SevenCompact S210 (Mettler Toledo, catalog number: 30130862) or equivalent

   7. Incubator shaker New Brunswick Innova^® 44R (Eppendorf, catalog number: M1282-006) or equivalent temperature-controlled shaker

   8. Optical microscope (Zeiss, model: Axiostar) or equivalent

   9. Thoma cell counting chamber (VWR, catalog number: 631-0697)

      
      

2. Harvesting of biomass and preparation of cell extracts

   1. Sorvall centrifuge RC6+ (Thermo Fisher Scientific, catalog number: 46915) or equivalent

   2. Fixed-angle rotor F9-4x1000y (Thermo Fisher Scientific, catalog number: 096-041032)

   3. Table centrifuge 5810/5810 R (Eppendorf, catalog number: 5811000015) or equivalent

   4. Rotor A-4-81 (Eppendorf, catalog number: 5810718007)

   5. Table microcentrifuge 5417R (Eppendorf) or equivalent

   6. Rotor F45-30-11 (Eppendorf)

   7. Electronic pipette (Eppendorf, model: Easypet) or equivalent

   8. Vinyl anaerobic chamber (Coy laboratory products) or equivalent

   9. Optical microscope (Zeiss, model: Axiostar or equivalent)

   10. Ultrasonic homogenizer Sonopuls (BANDELIN electronic, catalog number: 2451) or equivalent

   11. Aluminum cooling block

   12. -80°C Freezer

       
       

3. Enzyme assays

   1. Cary 3500 UV-Vis spectrophotometer (Agilent) or equivalent; please note that working with thermophiles requires a temperature-controlled cuvette holder (air-cooled Peltier temperature control in our case)

   2. Cuvettes, 400 µL, light path 10 mm (Hellma, catalog number: 115-10-40) or equivalent

   3. Syringe Hamilton, 50 µL (Carl Roth, catalog number: X048.1) or equivalent

   4. Rubber stoppers for cuvettes

   5. Vinyl anaerobic chamber (Coy laboratory products) or equivalent

   6. Thermomixer (Eppendorf, model: comfort) or equivalent

   7. Vacuum pump

   8. Ultrasonic bath (BANDELIN electronic, model: DT 100) or equivalent

   9. Ultra-high-performance liquid chromatography instrument (UHPLC) (Agilent, model: 1290 Infinity II LC System) or equivalent

   10. Column for reversed-phase LC separations (phase EC-C18) (Agilent, catalog number: 699675-902) or equivalent

   11. Pipette set (RAININ) or equivalent

       
       

4. Isotopologue profiling (based on GC-MS analysis)

   1. Freeze-dryer Alpha 2-4 LDplus (Martin Christ, Osterode, Germany) or equivalent

   2. Oven (Binder, model E28, Tuttlingen, Germany,) or equivalent

   3. Heating block Techne^® DRI Block DB 2A (Merck, Darmstadt, Germany) or equivalent

   4. GCMS-QP 2010 plus with autosampler AOC20i (Shimadzu, Duisburg, Germany) or equivalent

   5. Silica capillary column (equity TM-5; 30 m by 0.25 mm; 0.25 µm film thickness; Sigma-Aldrich, Supelco, catalog-number 28098-U)

Software

1. Interactive Codon Analysis INCA software (Supek and Vlahovicek, 2005; http://bioinfo.hr/software-tools/)

2. National Center for Biotechnology Information (NCBI) BLAST (http://www.ncbi.nlm.nih.gov/BLAST/)

3. Labsolutions (Shimadzu, https://www.shimadzu.de/labsolutions%E2%84%A2-lcms-software-simple-analytical-platform)

4. Isotopo (Ahmed et al., 2014, https://www.uni-wuerzburg.de/tr34/software-developments/isotopo/)

5. Microsoft Excel (Microsoft, 2016, https://www.microsoft.com/en-us/microsoft-365/excel)

Procedure

1. Cultivation experiments with anaerobic organisms

   Please note that the use of gases is described according to the specifications of our piping system. In particular, the manual addition of CO₂ required a sterile syringe (male Luer-lock), a Luer-to-tubing connector (female Luer Lock), and, specifically for the ¹³CO₂ gas, a lecture-bottle control valve to connect to the carbon steel lecture bottle (see Materials and Reagents A).

   1. Growth with different CO₂ concentrations

      1. Here we describe the cultivation of anaerobic microorganisms in serum bottles (117 mL total volume; 20 mL, 5 mL, and 2 mL medium).

      2. Solve the ingredients (see Recipe 1 for cultivation of Desulfurellaceae spp). Please remember that the reported list of ingredients may differ from the one suited for the cultivation of other autotrophs.

      3. Make the solution anoxic by performing vacuum-gas (usually 1 min each) cycles with 100% N₂ (50–100 kPa overpressure) for approximately 40 min (see Note 5).

      4. Reduce the solution by the addition of Na₂S9H₂O at a final concentration of 0.05% (w/v).

      5. Adjust the pH of the medium to the growth optimum of the organism in question. Indeed, since the addition of sodium sulfide makes the medium pH alkaline (pH of ~8 in our case), it is necessary to adjust it by addition of an anoxic HCl solution (see Note 5). Other acids could be used, if compatible with the growth medium. Please consider that different medium compositions might influence the resulting pH and differ from our experience.

      6. Get rid of the overpressure in the medium bottle, then transfer it to the anaerobic chamber (see Note 3 for an alternative way of aliquoting without using the anaerobic chamber).

      7. Aliquot the medium (20 mL, 5 mL, and 2 mL) with the help of a bottle-dispenser. When cultivating sulfur reducers, distribute the elemental sulfur powder (10 g L^-1) in advance into the serum bottles.

      8. Close the serum bottles with butyl-rubber stoppers, then transfer them out of the anaerobic chamber, and seal them with crimp aluminum caps.

      9. Autoclave sulfur-containing media at 110°C for 40 min. The autoclaved media can be stored at RT long-term. Please remember that the medium for the cultivation of other autotrophic microbes may require different sterilization temperatures.

      10. Prior to inoculation, add the sterile filtered vitamins (see Recipe 3) to the cultures and replace the gas phase. Due to the heat sensitivity, vitamins are usually not added to the medium before sterilization. Transfer 40 mL of vitamin solution to a serum bottle, make it anoxic (see Note 5), then add the vitamins to the culture medium anaerobically using sterile disposable syringes and needles, to a final concentration of 10 mL L^-1 .

      11. Replace the gas phase of fresh cultures by performing vacuum-gas cycles with 100% H₂. Before adding the CO₂, release any overpressure present in the cultures.

      12. Add CO₂ manually with a syringe at the respective final partial pressures: 80 kPa or 40 kPa for 20 mL cultures, 10 kPa for 5 ml cultures, 4 kPa for 5 mL cultures, and 2 kPa for 2 mL cultures.

      13. Fill up with H₂ to 100 kPa overpressure.

      14. If necessary, adjust the pH to the organism optimum with anoxic 1 M NaOH (see Note 5). This is important because the addition of CO₂ acidifies the medium (CO₂ + H₂O ⇋ H₂CO₃ ⇋ + H⁺).

      15. For the inoculation, use actively growing cultures during late exponential phase, to minimize the lag phase. In our case, the starting cell density was 10⁶ [cells/mL]. Incubate the main cultures (Figure 1A) at the optimum growth temperature with agitation.

          
          

   2. Carbon-13 labelling experiments

      1. Here, we describe the cultivation of anaerobic microorganisms in the presence of carbon-13 compounds in 1 L infusion bottles (200 mL medium).

      2. Solve the ingredients (see Recipe 1 for cultivation of Desulfurellaceae spp). Please remember that the reported list of ingredients may differ from the one suited for the cultivation of other autotrophic microbes.

      3. Aliquot the medium (200 mL) with the help of a measuring cylinder. When cultivating sulfur reducers, distribute the elemental sulfur powder (10 g L^-1) into the 1 L infusion bottles in advance. Close the bottles with rubber stoppers and aluminum caps.

      4. Make the cultures anoxic by performing vacuum-gas cycles (usually 1 min each) with 100% N₂ (50–100 kPa overpressure) for approximately 15–20 min (see Note 5).

      5. Reduce the cultures by the addition of Na₂S9H₂O at a final concentration of 0.05% (w/v).

      6. Adjust the pH of the medium to the growth optimum of the organism in question. Indeed, since the addition of sodium sulfide makes the medium pH alkaline (pH of ~8 in our case), it is necessary to adjust it by addition of an anoxic HCl solution (see Note 5). Other acids could be used, if compatible with the growth medium. Please consider that different medium compositions might influence the resulting pH and differ from our experience.

      7. Autoclave sulfur-containing media at 110°C for 40 min. The autoclaved media can be stored at RT long-term. Please remember that the medium for the cultivation of other autotrophic microbes may require different sterilization temperatures.

      8. Prior to inoculation, add the sterile filtered vitamins (see Procedure A1j) to the cultures.

      9. Add the tracer (i: ¹³CO_2, or ii: [1-¹³C]glutamate), according to the following instructions.

         1. ¹³CO₂:

            Replace the gas phase of fresh cultures by performing vacuum-gas cycles with 100% H₂. Before adding the ¹³CO₂, release any overpressure present in the cultures. Manually add ¹³CO₂ (99% carbon-13) with a syringe to a final partial pressure of 40 kPa, and fill up with H₂ to 100 kPa overpressure.

         2. [1-¹³C]glutamate:

            Replace the gas phase with H₂:CO₂ (80:20; carbon-12) and add [1-¹³C]glutamate to the medium to a final concentration of 14 mg L^-1.

         1. If necessary, adjust the pH to the organism optimum with anoxic 1 M NaOH (see Note 5). It is important because the addition of CO₂ acidifies the medium (CO₂ + H₂O ⇋ H₂CO₃ ⇋ + H⁺).

      10. For the inoculation, use actively growing cultures during late exponential phase to minimize the lag phase. Please note that in our case the starting cell density was 10⁶ [cells/mL]. Incubate the main cultures (Figure 1B) at the optimum growth temperature with agitation.

          
          

   
   Figure 1. Anaerobic cultures in the incubator shaker. A. Serum bottle with 20 mL of medium. B. Infusion bottle with 200 mL of medium. Both cultures contain a suspension of elemental sulfur.

   
   

2. Harvest of biomass and preparation of cell extracts

   1. Harvest the cells during mid-exponential growth for enzyme activity measurements, and during late-exponential growth for the GC-MS analysis. To identify the late-exponential growth phase, we counted the cells in the medium using a Thoma cell counting chamber (see Note 7 and Data analysis A2) and compared the results with the growth curves measured for the corresponding organism. In our case, the cultures typically reached the late-exponential growth phase after 48–96 h of growth.

   2. To separate elemental sulfur and cells before centrifugation, release the overpressure, open the bottle, and filter the medium with a folded filter and a funnel under aerobic conditions. Please note that performing this procedure under the fume hood is preferable, especially in the case of sulfur reducers producing H₂S as a metabolic by-product.

   3. Centrifuge the filtered culture (15,000 × g, 4°C, 20 min) and carefully discard the supernatant. Please note that cell pellets of some organisms might get resuspended very easily. For better results, it is possible to use electronic pipettes to discard the supernatant in this procedure.

   4. Resuspend the cell pellet with approximately 20 mL of leftover medium and transfer the cell suspension to a 50 mL centrifuge tube.

   5. Centrifuge the cell suspension (3,000 × g, 4°C, 40 min) and carefully discard the supernatant.

   6. Resuspend the cell pellet with 1 mL of 0.9% [w/v] NaCl solution (saline) and transfer the cell suspension to a 2 mL microcentrifuge tube.

   7. Centrifuge the cell suspension (21,000 × g, 4°C, 20 min) and carefully discard the supernatant.

   8. Freeze the cell pellet with liquid nitrogen and store it at -80°C, or disrupt the cells for further tests.

   9. In the latter case, transfer the microcentrifuge tube with the cell pellet to the anaerobic chamber.

   10. Resuspend the cell pellet with 500 µL of anoxic cell lysis buffer (see Recipe 5). Keep the solution cool with a cooled aluminum block.

   11. Lyse the cells with an ultrasonic homogenizer (60% amplitude, 4 min run, 1-s pulse, 2-s break; total energy input of 2,000 kJ). Especially for oxygen sensitive enzymes (i.e., pyruvate synthase, and 2-oxoglutarate synthase), it is crucial to perform the cell opening anoxically, either in the anaerobic chamber or in an anaerobic environment.

   12. Remove the insoluble cell debris by centrifugation (21,000 × g, 4°C, 20 min).

   13. Determine the protein concentration (Bradford, 1976), and handle on ice. Cell extracts are suitable for approximately 3–4 h of work. Long-term storage at -20°C compromises enzyme stability.

       
       

3. Enzyme assays

   In the following procedures, the final concentrations of the reaction components are given. The suggested stock concentrations are listed in the Materials and Reagents section C. Before performing the assays, please take into account the following practical recommendations: start the enzymatic reactions with the substrates; choose the test temperature in accordance with the optimum growth temperature of the organism in question, and assume that a 10°C rise in temperature doubles the reaction rate; high temperatures might compromise the stability of the chemicals present in the reaction mixture; in case of spectrophotometric measurements, notice the maximum absorbance limit of the device in use.

   Test at least two different concentrations of cell extract to obtain a linear dependence of activity on the amount of the added enzyme. For the measurement of highly active enzymes of the roTCA cycle (i.e., citrate synthase and malate dehydrogenase), only small amounts of cell extract should be added to the reaction mixture, if studied organisms possess this cycle. In our case, 0.5–1 (µg proteinmL^-1) of cell extract was sufficient to obtain a linear activity. This corresponded to a difference of 0.3 to 0.7 absorption units per minute (Δabs min^-1) in the assays of citrate synthase in the direction of citrate synthesis (oxaloacetate + acetyl-CoA + H₂O → citrate + CoA), or malate dehydrogenase in the direction of oxaloacetate reduction (oxaloacetate + NADH + H⁺ → malate + NAD⁺). However, citrate synthase is much less active in the direction of citrate cleavage (citrate + CoA → acetyl-CoA + oxaloacetate + H₂O), thus requiring high protein concentration in the mixture (in our case, at least 20-fold higher). If the results of the assays are ambiguous, it may be useful to perform additional negative controls, by replacing the cell extract or the substrates with water.

   In our case, the final volume of the reaction mixture was 300 µL.

   1. Citrate synthase (forward reaction, spectrophotometric continuous assay, λ=412 nm)

      1. Activity of citrate synthase was measured in a reaction mixture consisting of:

         1. 100 mM Tris-HCl (pH 7.5)

         2. 1 mM DTNB

         3. 0.5 mM acetyl-CoA

         4. 5 mM oxaloacetate

         5. Cell extract

         6. dH₂O

      2. Preheat the cuvette holder of the spectrophotometer to the test temperature.

      3. Add all the reaction mixture components to the cuvette, except oxaloacetate, to reach a final volume of 300 μL.

      4. Mix the reaction mixture thoroughly with a pipette.

      5. Start the measurement and get a stable baseline.

      6. As the baseline is stabilized, wait 1 min to record the background activity, and then start the reaction by adding 5 mM oxaloacetate. Monitor the oxaloacetate-dependent formation of free CoA from acetyl-CoA with DTNB (ϵ₄₁₂=14.2 mM^-1 cm^-1 [Riddles et al., 1979]) as reagent.

   2. Citrate synthase (backward reaction, spectrophotometric continuous assay, λ=365 nm)

      1. Activity of citrate synthase in the backward reaction was measured in a reaction mixture consisting of:

         1) 100 mM Tris-HCl (pH 7.5)

         2) 5 mM DTE

         3) 5 mM MgCl₂

         4) 0.5 mM CoA

         5) 0.5 mM NADH

         6) 20 mM citrate

         7) 20 U/mL porcine malate dehydrogenase

         8) Cell extract

         9) dH₂O

      2. Preheat the cuvette holder of the spectrophotometer to the test temperature.

      3. Add all the reaction mixture components to the cuvette, except citrate, to reach a final volume of 300 μL.

      4. Mix the reaction mixture thoroughly with a pipette.

      5. Start the measurement and get a stable baseline.

      6. As the baseline is stabilized, wait 1 min to record the background activity, and then start the reaction by adding 20 mM citrate. Monitor the citrate-dependent oxidation of NADH (ϵ₃₆₅=3.4 mM^-1 cm^-1 [Bergmeyer, 1975]) due to the activity of the helping enzyme malate dehydrogenase, with oxaloacetate forming in the citrate synthase backward reaction.

   3. Citrate synthase (backward reaction, UHPLC discontinuous assay, λ=260 nm)

      1. Performing the assay

         1. Activity of citrate synthase in the backward reaction was measured in a reaction mixture consisting of:

            1) 100 mM Tris-HCl (pH 7.5)

            2) 5 mM DTE

            3) 5 mM MgCl₂

            4) 0.5 mM CoA

            5) 0.5 mM NADH

            6) 20 mM citrate

            7) 20 U/mL porcine malate dehydrogenase

            8) Cell extract

            9) dH₂O

         2. Preheat the thermomixer for 1.5 mL microcentrifuge tubes to the test temperature.

         3. In this case, mix all the reaction mixture components except citrate. The final volume should ensure the correct sampling of four time points (e.g., final volume of 100 μL for four 20 μL samples).

         4. Mix the reaction mixture thoroughly and spin it down.

         5. Preincubate the reaction mixture for 1 min at the test temperature, then start the reaction with 20 mM citrate.

         6. Take a sample (t0) and stop the reaction immediately (sample:stop solution, 1:1 [v/v]).

         7. Similarly, take samples after 1 min (t1), 2 min (t2), and 10 min (t10), and each time stop the reaction immediately (sample:stop solution, 1:1 [v/v]).

         8. During the assay, keep the samples cooled, then freeze them with liquid nitrogen and store at -20°C.

      2. UHPLC run

         1. Centrifuge the samples twice (21,000 × g, 4°C, 5 min) to get rid of insoluble proteins and particles.

         2. Prepare the samples for the UHPLC in 2 mL screw top vials or in 96-well microplates. If necessary, dilute the samples only with UHPLC-grade ddH₂O.

         3. In our case, the CoA-esters were analysed with a 1290 Infinity II UHPLC system equipped with a EC-C18 column for reversed-phase LC separations (Agilent InfinityLab Poroshell 120 EC-C18 1.9 µm 2.1 × 50 mm). The diode array detector (Agilent, lamp catalog number: G7117B) allowed the UV detection of CoA and of different CoA-esters at 260 nm.

         4. Before the run, wash the pumps with UHPLC-grade ddH₂O and potassium phosphate buffer, and equilibrate the column to the starting conditions (2% acetonitrile – 98% potassium phosphate buffer).

         5. For the actual sample analysis, use the following chromatographic solvents: solvent A, acetonitrile, and solvent B, potassium phosphate buffer in water (pH 7) (see Recipe 7). Perform chromatography using a gradient elution: after injection, increase the level of acetonitrile from 2 to 8% over 2.66 min, up to 30% in 3.33 min, then reduce it to 2% in 3.68 min, and maintain it isocratic up to 5.00 min. Keep the flow rate constant at 0.55 mL min^-1.

   4. ATP-citrate lyase (spectrophotometric continuous assay, λ=365 nm)

      1. Activity of ATP-citrate lyase was measured in a reaction mixture consisting of:

         1) 100 mM Tris-HCl (pH 7.5)

         2) 5 mM MgCl₂

         3) 5 mM DTE

         4) 0.5 mM CoA

         5) 0.4 mM NADH

         6) 20 mM citrate

         7) 0.5 mM ATP

         8) 20 U/mL porcine malate dehydrogenase

         9) Cell extract

         10) dH₂O

      2. Preheat the cuvette holder of the spectrophotometer to the test temperature.

      3. Add all the reaction mixture components except ATP to the cuvette, to reach a final volume of 300 μL.

      4. Mix the reaction mixture thoroughly with a pipette.

      5. Start the measurement and get a stable baseline.

      6. As the baseline is stabilized, wait 1 min to record the background activity, and then start the reaction by adding 0.5 mM ATP. Measure oxaloacetate formation by monitoring NADH oxidation (ϵ₃₆₅=3.4 mM^-1 cm^-1 [Bergmeyer, 1975]) due to the activity of the helping enzyme malate dehydrogenase that is present in the mixture.

   5. Citrate lyase (spectrophotometric continuous assay, λ=365 nm)

      1. Activity of citrate lyase was measure in a reaction mixture consisting of:

         1) 100 mM Tris-HCl (pH 7.5)

         2) 5 mM DTE

         3) 5 mM MgCl₂

         4) 0.5 mM NADH

         5) 20 mM citrate

         6) 20 U/mL porcine malate dehydrogenase

         7) Cell extract

         8) dH₂O

      2. Preheat the cuvette holder of the spectrophotometer to the test temperature.

      3. Add all the reaction mixture components except citrate to the cuvette, to reach a final volume of 300 μL.

      4. Start the measurement and get a stable baseline.

      5. As the baseline is stabilized, wait 1 min to record the background activity, and then start the reaction by adding 20 mM citrate. Measure oxaloacetate formation monitoring NADH oxidation (ϵ₃₆₅=3.4 mM^-1 cm^-1 [Bergmeyer, 1975]) due to the activity of the helping enzyme malate dehydrogenase that is present in the mixture.

   6. Malate dehydrogenase (spectrophotometric continuous assay, λ=365 nm)

      1. Activity of malate dehydrogenase was measured in a reaction mixture consisting of:

         1) 100 mM Tris-HCl (pH 8)

         2) 5 mM DTE

         3) 5 mM MgCl₂

         4) 0.5 NADH

         5) 2.5 mM oxaloacetate

         6) Cell extract

         7) dH₂O

      2. Preheat the cuvette holder of the spectrophotometer to the test temperature.

      3. Add all the reaction mixture components except oxaloacetate to the cuvette, to reach a final volume of 300 μL.

      4. Mix the reaction mixture thoroughly with a pipette.

      5. Start the measurement and get a stable baseline.

      6. As the baseline is stabilized, wait 1 min to record the background activity, and then start the reaction by adding 2.5 mM oxaloacetate. Measure the oxidation of NADH (ϵ₃₆₅=3.4 mM^-1 cm^-1 [Bergmeyer, 1975]).

   7. Pyruvate synthase (spectrophotometric anaerobic continuous assay, λ=578 nm)

      The pyruvate synthase reaction requires reduced conditions. Add dithionite to reduce the viologens until the solution is slightly blue (absorbance at 578 nm of ~0.8), then proceed. There is an alternative way to perform this test without the use of the anaerobic tent (see Note 6).

      1. Activity of pyruvate synthase was measured in a reaction mixture consisting of:

         1) 50 mM Tris-HCl (pH 7.5)

         2) 5 mM MgCl₂

         3) 2.5 mM DTE

         4) 0.5 mM CoA

         5) 1 mM methyl viologen

         6) 10 mM pyruvate

         7) Cell extract

         8) dH₂O

      2. Preheat the cuvette holder of the spectrophotometer to the test temperature.

      3. While working in an anaerobic tent, add all the anoxic reaction mixture components except pyruvate to the cuvette, to reach a final volume of 300 μL.

      4. Mix the reaction mixture thoroughly with a pipette.

      5. Close the cuvette with a rubber plug and transfer it out of the tent.

      6. Replace the gas phase of the cuvette with N₂.

      7. Start the measurement and get a stable baseline.

      8. As the baseline is stabilized, wait 1 min to record the background activity, and then start the reaction by adding 10 mM pyruvate using a Hamilton syringe. Monitor the pyruvate-dependent reduction of methyl viologen (ϵ₅₇₈ nm=9.7 mM^-1 cm^-1 [Dawson et al., 1986]).

   8. 2-oxoglutarate synthase (spectrophotometric anaerobic continuous assay, λ=578 nm)

      The 2-oxoglutarate synthase reaction requires reduced conditions. Add dithionite to reduce the viologens until the solution is slightly blue (absorbance at 578 nm of ~0.8), then proceed. Please note that there is an alternative way to perform this test without the use of an anaerobic tent (see Note 6).

      1. Activity of 2-oxoglutarate synthase was measured in a reaction mixture consisting of:

         1) 100 mM Tris-HCl (pH 7.5)

         2) 5 mM MgCl₂

         3) 2.5 mM DTE

         4) 0.5 mM CoA

         5) 1 mM benzyl viologen

         6)10 mM 2-oxoglutarate

         7) Cell extract

         8) dH₂O

      2. Preheat the cuvette holder of the spectrophotometer to the test temperature.

      3. While working in the anaerobic tent, add all the anoxic reaction mixture components except 2-oxoglutarate to the cuvette, to reach a final volume of 300 μL.

      4. Mix the reaction mixture thoroughly with a pipette.

      5. Close the glass cuvette with a rubber plug and transfer it out of the tent.

      6. Replace the gas phase of the cuvette with N₂.

      7. Start the measurement and get a stable baseline.

      8. As the baseline is stabilized, wait 1 min to record the background activity, and then start the reaction by adding 10 mM 2-oxoglutarate. Monitor 2-oxoglutarate-dependent reduction of benzyl viologen (ϵ₅₇₈ nm=9.7 mM^-1 cm^-1 [Dawson et al., 1986]).