D glucose

A glucose tolerance test (GTT) was performed at day 0, 1, 7, 14, 21, 28, 35 as described in Pierard et al. (2016) (link). Briefly, an intraperitoneally administration of a dose of 2 g/kg body weight of D-glucose (Roth, Karlsruhe, Germany) to fasting animals was realized. Blood glucose level was measured 0, 30, 60, and 120 min after glucose injection using a One Touch^®Vita^®glucometer (Zug, Switzerland).

After an overnight fast and 18 h after the last exercise session, a glucose tolerance test (GTT) was performed before the protocol, after week 2 of the protocol and at the end of the protocol. A dose of 2 g/kg body weight of D-glucose (Roth, Karlsruhe, Germany) was administered intraperitoneally. Blood samples were then obtained from the caudal vein, and the blood glucose level was measured 0, 30, 60, and 120 min after glucose injection using a One Touch® Vita® glucometer (Zug, Switzerland).

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).

Chemicals for cultivation of Escherichia coli and Thermococcus sp. strain 2319x1E, such as yeast extract, lysogeny broth (LB), beechwood xylan, D-glucose and D-xylose were obtained from Carl Roth (Germany). Avicel^® cellulose, carboxymethyl cellulose (CMC), maltodextrin and soluble starch were purchased from Sigma-Aldrich (United States), xylobiose, ortho-nitrophenyl-β-D-galactopyranoside (ONPG), para-nitrophenyl acetate (PNPA), para-nitrophenyl-β-D-xylopyranoside (PNPX) and para-nitrophenyl- β-D-glucopyranoside (PNPG) from Megazyme (Ireland). Other chemicals which were used in this work are 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, United States), urea (GE Healthcare Life Sciences, Germany), ammonium bicarbonate (ABC; Sigma-Aldrich, United States), dithiothreitol (DTT; Sigma-Aldrich, United States), iodoacetamide (IAM; Sigma-Aldrich, United States), formic acid (FA; Fischer Chemical, Germany) and bovine serum albumin (BSA; VWR Chemicals, United States).

Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed using intraperitoneal (IP) injections of D-glucose (Roth, Karlsruhe, Germany) (2 g of glucose/kg total body mass) and insulin (0.75 U insulin/kg total body mass) after a 4-h fast. Blood samples were then obtained from the caudal vein, and the blood glucose level was measured 0, 30, 60, and 120 min after glucose injection using a One Touch® Vita® glucometer (Zug, Switzerland). Four to six mice were used from each group.

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.

Cells were washed once with loading buffer containing in mM: 2 CaCl₂, 135 NaCl, 5 KCl, 1 MgCl₂, 1 HEPES, 2.6 NaHCO₃, 0.44 KH₂PO₄, 0.34 Na₂HPO₄, 10 d-glucose (Carl Roth, Karlsruhe, Germany), 0.1% vitamins, 0.2% essential amino acids and 1% penicillin/streptomycin at pH 7.4. Cells were incubated in loading buffer containing 1000, 500, 200, 81, 40.5, 13.5, 5.4, 2.7, or 1.35 nM TMRM (tetramethylrhodamine methyl ester, Invitrogen™) for 30 min. As TMRM might degrade over time in storage, TMRM concentrations were measured regularly. Therefore, after TMRM was dissolved in methanol the absorption at 550 nm was measured and the concentration was calculated using Eq. 1. $c=A/\left(\epsilon \cdot l\right)$ c is the molar concentration of TMRM, A the absorption of TMRM at 550 nm, ε is the specific extinction coefficient of TMRM at 550 nm in methanol, and l is the pathlength the light has to path through the TMRM solution.