D glucose monohydrate

Food samples
used for the analysis of flazin, including processed tomato and vegetable
products, sauces, processed fruit products, beer, molasses, and honey
(Table 1), were obtained
in local supermarkets. Chemical compounds were obtained as follows: l-tryptophan (l-Trp), 5-(hydroxymethyl)furfural (5-HMF),
and d-(−)-fructose from Sigma; d-(+)-glucose
monohydrate from Merck (Darmstadt, Germany); 3-deoxy-d-glucosone
(3-deoxyglucosone, 3-DG) from Biosynth-Carbosynth; and sucrose from
Scharlau (Barcelona, Spain). Flazin was obtained and isolated from
reactions of l-Trp and d-fructose, as mentioned
below.

The compounds used in the catalyst synthesis as well as in the chemical reactions i.e. chloroacetonitrile (ClCH₂CN, 99%), d ( +)-Glucose monohydrate (C₆H₁₂O₆·H₂O), silver nitrate (AgNO₃, 99%), sodium hydroxide (NaOH, 98%), sodium borohydride (NaBH₄, 98%), were prepared from Merck, Germany, Sigma Aldrich, and Fluka chemical companies. The precursor materials and solvents were all highly purified, without the need for further purification.

Cystobactamids CN-DM-861 and AR351 were synthesized in the lab of the corresponding author according to reference 21 (link). Ciprofloxacin was purchased from AppliChem. Coumermycin A1, levofloxacin, lomefloxacin HCl, novobiocin sodium salt, trimethoprim, nortriptyline HCl, caffeine, (NH₄)₂SO₄, and Fe(II)SO₄ 7×H₂O were obtained from Sigma-Aldrich. Glipizide was purchased from ACROS Organics, and (S)-naproxen was purchased from Cayman Chemical Company. d(+)-Glucose monohydrate and K₂HPO₄ were obtained from Merck Millipore, and MgSO₄ 7×H₂O and casamino acid were obtained from Roth. Acetonitrile ultra LC/MS grade, water ultra LC/MS grade, and methanol ultra LC/MS grade were obtained from Fisher Scientific.

The strains used in this study are shown in Table 1. Bursa aurealis transposon (Tn) insertion mutations encoding functionally non-redundant TCA- and urea cycle enzymes (Figure 1) in USA300-JE2 were obtained from the Nebraska Transposon Mutant Library (NTML, www.beiresources.org) [16 (link)]. Parental strains UAS391, UAS391-Ery^S (erythromycin resistance cured UAS391), and JE2 are all MRSA belonging to the highly virulent and widespread clonal lineage, USA300. These, as well as Tn insertion mutants, were routinely grown on Brain-Heart infusion (BHI; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 0.1% D(+)-glucose monohydrate (Merck Millipore, Billerica, MA, USA) and BHI Bacto™ agar (Becton, Dickinson and Company, USA) for biofilm, transduction and complementation experiments. Lysogeny broth (LB; Becton, Dickinson and Company, USA) was used for Escherichia coli. For the Tn-carrying S. aureus transductants with the erythromycin resistance marker ermB, 5 or 10 µg/mL erythromycin (Sigma-Aldrich^®, Merck KGaA, St. Louis, MO, USA) was supplemented to the growth medium.

Chitinase from S. griseus, HRP, cellulase from A. niger, N-acetyl-D-glucosamine and 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium salt were purchased from Sigma-Aldrich (St Louis, MO, USA). One unit of HRP is defined as the amount of enzyme that will form 1.0 mg of purpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20°C. One unit of chitinase is defined as the amount of enzyme that liberates 1.0 mg of N-acetyl-D-glucosamine from chitin per hour at pH 6.0 at 25°C in a 2-hour assay. One unit of cellulase is defined as the amount of enzyme that liberates 1.0 μmol of glucose from cellulose in 1 hour at pH 5.0 at 37°C. 4-Aminoantipyrine was purchased from Acros Organics (Geel, Belgium), D-glucose monohydrate was purchased from Merck (Darmstadt, Germany) and cellotetraose, chitobiose and chitotetraose were purchased from Dextra (Reading, UK). Cellobiose (purity >98%) was purchased from TCI Europe (Zwijndrecht, Belgium). Whatman filter paper grade 1 was purchased from GE Healthcare Life Sciences (Little Chalfont, UK). E. coli ORIGAMI2 DE3 was purchased from EMD Millipore (Billerica, MA, USA) and pET-SUMO vector was obtained from Invitrogen (Carlsbad, CA, USA).

Methacrylic acid (MAA), N-vinylpyrrolidone (VP), dimethyl sulfoxide (DMSO), potassium chloride (KCl), and agarose powder were purchased from VWR International (Vienna, Austria); N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA), 2,2′-azobis(isobutyronitrile) (AIBN), sodium bromide (NaBr), and Sudan Black B were from Sigma-Aldrich (Steinheim, Germany). Tris(hydroxymethyl)-aminomethane (Tris), ethylenediamine tetraAcetic acid (EDTA), calcium chloride (CaCl₂), magnesium chloride (MgCl₂), urea (CH₄N₂O), and (D+)-glucose monohydrate were purchased from Merck (Darmstadt, Germany). Sodium chloride (NaCl) was obtained from Applichem (Darmstadt, Germany). Acetic acid was purchased from Carl Roth (Karlsruhe, Germany). 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) was obtained from Alfa Aesar (Karlsruhe, Germany). HSA was purchased from Millipore (MA, USA). Brilliant gold paste (gold colloid, 12% gold content) was purchased from Heraeus, Germany. All reagents were of analytical or highest synthetic grade commercially available.