Thiamine pyrophosphate

Neisseria gonorrhoeae was grown overnight at 37°C and 5% CO₂ on agar plates containing gonococcal base (GC) agar [10 g/l Bacto agar (BD Biosciences, Bedford, MA, United States), 5 g/l NaCl (Roth, Darmstadt, Germany), 4 g/l K₂HPO₄ (Roth), 1 g/l KH₂PO₄ (Roth), 15 g/l Proteose Peptone No. 3 (BD), 0.5 g/l soluble starch (Sigma-Aldrich, St. Louis, MO, United States)] supplemented with IsoVitaleX (IVX): 1 g/l D-Glucose (Roth), 0.1 g/l L-glutamine (Roth), 0.289 g/l L-cysteine-HCL × H₂0 (Roth), 1 mg/l thiamine pyrophosphate (Sigma-Aldrich), 0.2 mg/l Fe(NO₃)₃ (Sigma-Aldrich), 0.03 mg/l thiamine HCl (Roth), 0.13 mg/l 4-aminobenzoic acid (Sigma-Aldrich), 2.5 mg/l β-nicotinamide adenine dinucleotide (Roth) and 0.1 mg/l vitamin B₁₂ (Sigma-Aldrich). GC medium is identical to the base agar composition but lacks agar and starch.
E. coli was grown in LB (Lysogeny Broth, Roth) medium or on LB agar plates (15 g/l Bacto agar (BD Biosciences, Bedford, MA, United States) at 37°C.
For N. gonorrhoeae antibiotics were used at the following concentrations: 2.5–5 μg/ml erythromycin (Thermo-Fisher), 100 μg/ml streptomycin (Sigma-Aldrich), 10 μg/ml chloramphenicol (Sigma-Aldrich). For E. coli antibiotics were used at the following concentrations: 50 μg/mL kanamycin (Roth).

NADH, NAD⁺, FMN, FAD, pyruvate, thiamine pyrophosphate, methyl viologen, and coenzyme A were purchased from Sigma-Aldrich (St. Louis, MO, United States).

Gonococcal base agar was made from 10 g/l dehydrated agar (BD Biosciences, Bedford, MA), 5 g/l NaCl (Roth, Darmstadt, Germany), 4 g/l K₂HPO₄ (Roth), 1 g/l KH₂PO₄ (Roth), 15 g/l Proteose Peptone No. 3 (BD Biosciences), 0.5 g/l soluble starch (Sigma-Aldrich, St. Louis, MO), and supplemented with 1% IsoVitaleX (IVX): 1 g/l D-glucose (Roth), 0.1 g/l L-glutamine (Roth), 0.289 g/l L-cysteine-HCL x H₂O (Roth), 1 mg/l thiamine pyrophosphate (Sigma-Aldrich), 0.2 mg/l Fe(NO₃)₃ (Sigma-Aldrich), 0.03 mg/l thiamine HCl (Roth), 0.13 mg/l 4-aminobenzoic acid (Sigma-Aldrich), 2.5 mg/l β-nicotinamide adenine dinucleotide (Roth), and 0.1 mg/l vitamin B12 (Sigma-Aldrich). GC medium is identical to the base agar composition but lacks agar and starch.

Thiamine pyrophosphate (TPP), pyruvate, D,L-glyceraldehyde-3-phosphate (GAP), 1-deoxy-D-xylulose-5-phosphate sodium salt, bovine serum albumin, and LB-broth were purchased from Sigma Aldrich. NADPH was purchased from Alexis Biochemical, Ni-NTA resin was purchased from Invitrogen, and β-mercaptoethanol (β-Me) was purchased from Fisher. E. coli XL-10 cells, deoxynucleotide mix PCR grade, pfuUltra Hotstart DNA polymerase, QuikChange II site directed mutagenesis kit and acetonitrile (HPLC grade) were purchased from Agilent. The DNA vectors, pET28a (+) and pET15b (+), and E. coli BL-21 B (DE3) cells were purchased from EMD Biosciences. DNA sequencing services and primers were purchased from MWG operon. All the other reagents were of the highest quality commercially available.

Highly purified CPI-613 and CPI-157 were synthesized from D,L lipoate as described previously
[18 (link)]. N-acetylcysteine (NAC), auranofin, resazurin, diaphorase, glutaredoxin-1, reduced glutathione, Triton X-100, digitonin, lauryl maltoside, dithiothreitol (DTT), NAD⁺, ADP, thiamine pyrophosphate, coenzyme-A (CoA), and N-ethylmaleimide (NEM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Biotin-HDPD and gel filtration columns (PD10) were from Thermo Scientific (Waltham, MA, USA). 2',7'-dichlorodihydrofluorescein diacetate (DCF), dihydroethidium (DHE), and Amplex Red were from Life Technologies. Antibodies to Prx1, Prx3 and reduced lipoate were purchased from AbCam (Cambridge, MA, USA). Antibodies against dihydrolipoamide dehydrogenase (E3) were from Rockland Immunochemicals (Gilbertsville, PA, USA) and KGDH dihydrolipoamide succinyltransferase (E2) antibodies were from Cell Signaling (Danvers, MA, USA).