Mnso4

Survival of strains in pooled human serum and pooled human urine (Lee Biosolutions) was monitored as previously described [6 (link)]. Briefly, overnight cultures grown in BHI+Mn were washed in sterile PBS as indicated above to remove excess Mn and inoculated 1:200 in human serum or urine. Cultures were incubated aerobically at 37°C and, at selected time points, aliquots were serially diluted and plated on Mn-supplemented BHI plates for colony-forming unit (CFU) determination. For determination of growth in human urine, overnight cultures were normalized to an OD₆₀₀ of 1.0 in BHI+Mn. Cells were washed 3 times with 1X PBS followed by 1:1000 dilution into undiluted pooled female urine supplemented with 20 mg ml^-1 of BSA and incubated at 37°C. Bacterial growth was monitored by quantifying CFU as described above. When indicated, serum and urine were supplemented with a final concentration of 1 mM FeSO₄ (99%, Sigma), 1 mM MnSO₄ (99%, Sigma) or with increasing concentrations (0.5 μM, 0.5 mM or 1 mM) of CuSO₄ (99%, Acros Organics). Urine was pooled from 3 healthy female donors, clarified by centrifugation, filter-sterilized, and adjusted to pH 6.5 prior to use. All urine samples were collected after obtaining written consent as per the study approval from the Washington University School of Medicine Internal Review Board (approval ID #201207143).

The chemicals CuSO₄, MnSO₄, benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), dicyclohexyl phthalate (DCP), diethyl phthalate (DEP), di-(2-ethylhexyl) phthalate (DEHP), acetosyringone (AS), syringaldehyde (SA), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), vanillin (VA), N-hydroxyphthalimide (HPI), (2,2,6,6-tetramethylpiperidin-1-yl) oxyl (TEMPO), and hexane, along with other solvents (analytical grade, 99.0% purity), were purchased from Sigma-Aldrich (Merck/Millipore Sigma, St. Louis, Missouri, USA). The chemical structures of the compounds used in this study are shown in Figure 1A and Supplementary Table S1. Laccases from three fungi (Aspergillus sp., Rhus vernicifera, and Trametes versicolor) were purchased from Sigma-Aldrich (Merck/Millipore Sigma, St. Louis, Missouri, USA).

Modified MRS broth (MMRS) was made by the omission of beef extract and any other additional sugar source and was subsequently used as a medium to examine the growth of three strains in the presence of different carbohydrate substrates. MMRS contained the following: bacteriological peptone (Oxoid, Basingstoke, UK) 10 g, yeast extract (Merck, Darmstadt, Germany) 10 g, Tween® 80 (SigmaAldrich, St. Louis, MO, USA) 1 g, ammonium citrate 2 g, CH₃COONa 5 g, MgSO₄ 0.1 g, MnSO₄ 0.05 g, Na₂HPO₄ 2 g (all products of SigmaAldrich) per 1 L of the medium. The pH of the media was adjusted to 6.4 and sterilised by autoclaving at 121°C for 15 min.

Four OD₆₀₀ unit of cells at an OD₆₀₀ of ~1.0 were serially diluted up to 10⁻⁵ dilution, and 3 µL were spotted on YMM-agar medium containing a variety of stress factors at a final concentration of 0.3 mM CuSO₄ (Sigma-Aldrich, Hamburg, Germany), 20 mM MnSO₄ (Sigma-Aldrich, Hamburg, Germany), 25 mM MnCl₂ (Merck, Darmstadt, Germany), 10 mM ZnCl₂ (Sigma-Aldrich, Hamburg, Germany), 2mM CrCl₃ (Acros organics, Geel, Belgium) , 25 mM FeCl₂ (Sigma-Aldrich, Hamburg, Germany), 0.3 mM NiCl₂ (Merck, Darmstadt, Germany), 2 mM CoCl₂ (Sigma-Aldrich, Hamburg, Germany), 50 mM (NH₄)₂Fe(SO₄)₂ (Merck, Darmstadt, Germany), 250 µM bathophenanthroline sulphonate (BPS) (Alfa Aesar Karlsruhe, Germany), 0.5 mM H₂O₂ (Merck, Darmstadt, Germany) and 2 mL/L phenylethanol (Sigma-Aldrich, Hamburg, Germany). The plates were observed and photographed after incubation at 30 °C for 48 h.

Batch fermentations were performed in synthetic grape juice medium containing (per litre) 100 g glucose, 100 g fructose (Merck), 1 g yeast extract (Oxoid), 0.3 g citric acid, 5 g l-malic acid, 5 g l-tartaric acid, 0.4 g MgSO₄, 5 g KH₂PO₄, 0.2 g NaCl, 0.05 g MnSO₄ (Sigma-Aldrich) and anaerobic factors (ergosterol 10 mg (Sigma-Aldrich), tween 80 0.5 mL (Merck)) [34 , 35 ]. Fermentations were conducted in 1.3 L BioFlo 110 bench top bioreactors (New Brunswick, NJ, USA) using 900 mL of final working volume, a temperature of 25 °C and an agitation speed of 200 rpm. Fermentations were performed under two conditions: anaerobic and aerobic at 5% (0.41 mg L^− 1) dissolved oxygen (DO). The anaerobic conditions were created by initially sparging N₂ to bring down the DO level to 0%, and then to minimize diffusion of atmospheric oxygen into the cultures, the entire fermentation set-up was equipped with Norprene tubing. The 5% DO level was maintained through the supplementary addition of 4 gasses (CO₂, N₂, O₂ and compressed air) whenever required, using an automated gas flow controller. The DO levels in the cultures were monitored with an oxygen electrode.

All bacterial strains and plasmids used in this study are listed in Table 1. Bacterial strains were grown on nutrient agar or in liquid Luria-Bertani (LB) broth and in M9 minimum medium at 37^°C [29 ]. Where necessary, growth media were supplemented with either100 μg/ml Ampicillin (Sigma-Aldrich), 50 μg/ml Kanamycin (Sigma-Aldrich) or 10 mM MnSO₄(Sigma-Aldrich).

Glycerol, NAD⁺, poly (2-ethyl-2-oxazoline) (500kDa), CuSO₄, NiSO₄, ZnSO₄, MnSO₄, MgSO₄ and CaCl₂ were purchased from Sigma-Aldrich (Tianjin, China). Ampicillin and isopropyl-β-d-thiogalactoside (IPTG) were purchased from TransGen Biotech (Beijing, China). All other chemicals were of analytical grade and were purchased from Sangon Biotech (Shanghai, China). E. coli BL21(DE3) pET-32a-GDH was constructed in our laboratory (not published, detailed information was listed in Supplementary information).