Cyanocobalamin

NaNO₃ (PanReac, Barcelona, Spain) and NaH₂PO₄·2H₂O (Honeywell, Tokyo, Japan) were used as nitrogen and phosphorus sources, respectively, while the trace elements Na₂EDTA (Sigma, St. Luis, MO, USA), FeCl₃·6H₂O (Acros Organics, Geel, Belgium), CuSO₄·5H₂O (Sigma), ZnSO₄·7H₂O (Sigma), CoCl₂·6H₂O (Fisher Scientific, Waltham, MA, USA), MnCl₂·4H₂O (Acros Organics), and Na₂MoO₄·2H₂O (Chem-Lab NV, Zedelgem, Belgium) were used for media preparation. Cyanocobalamin, Thiamine HCl, and Biotin were procured from Sigma-Aldrich. Chemicals used for analysis included ammonium bicarbonate (Sigma), HPLC-grade chloroform (Honeywell) and HPLC-grade methanol (Fisher Scientific).

The strain S. haloaromaticamans used in the current study was isolated from soil of a wastewater disposal site and it was able to degrade the fungicide OPP only when supplemented with CA (Perruchon et al., 2016 (link)). The bacterium was routinely cultivated in minimal salts media supplemented with nitrogen (MSMN), OPP (30–50 mg L^–1) and CA in a shaking incubator at 27°C in the dark. MSMN preparation and OPP chromatographic analysis was as described by Perruchon et al. (2016) (link), while bacterial growth was determined by measurement of the optical density at 600 nm (OD₆₀₀). Inoculation of flasks was performed by fresh bacterial cells grown at the mid-log phase, pelleted by centrifugation, washed three times with sterile ddH₂O and resuspended with MSMN to an OD₆₀₀ of 0.1.
L-methionine, L-isoleucine, L-tyrosine, L-phenylalanine, L-homoserine, O-succinyl-L-homoserine, L-cystathionine, L-homocysteine and cyanocobalamin (synonym to vitamin B12 in the manuscript), were purchased by Sigma-Aldrich (Taufkirchen, Germany) and they were used for the preparation of aqueous solutions (0.05 mM). These were filter sterilized and used for the preparation of MSMN containing amino acids, B12 and intermediates of methionine biosynthesis at the desired concentrations.

The pattern of protein hydrolysis and molecular weight of enzyme-treated chicken foot collagen were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC), respectively. To analyze the pattern of protein hydrolysis, we used the SDS-PAGE protocol described by Laemmli [21 (link)] with 12% separating and 5% stacking gels. Samples were prepared for electrophoresis by boiling in a loading buffer containing 0.1% Coomassie Brilliant Blue R 250 dye (Sigma-Aldrich, USA). Molecular weight distribution and average molar mass were evaluated using an LC-2000 Plus HPLC system (Jasco, Japan). For evaluation, the collagen samples were prepared by filtration through a 0.45-μm membrane using a Shodex Protein KW-802.5 column (I.D. 8 mm × 300 mm; Shodex, Japan). The mobile phase used was 50 mM phosphate buffer containing 0.3 M NaCl at a flow rate of 0.9 ml/min, and the detector recorded absorbance at 220 nm. We used thyroglobulin (669 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa), aprotinin (6.5 kDa), and cyanocobalamin (1.3 kDa) as protein standards (all obtained from Sigma-Aldrich) for standard curve construction and evaluation of the average molecular weight of enzyme-hydrolyzed collagen samples.

Lobomonas rostrata (SAG 45‐1) was obtained from the Experimental Phycology and Culture Collection of Algae at the University of Goettingen (EPSAG), Germany. It was grown autotrophically on TP⁺ medium (Kazamia et al., 2012). Vitamin B₁₂ was provided as cyanocobalamin (Sigma‐Aldrich) at 100 ng l⁻¹, as this supports the maximum carrying capacity of L. rostrata (Kazamia et al., 2012). The L. rostrata–M. loti coculture was an established coculture that had been maintained over many generations without a source of organic carbon or vitamin B₁₂. Cultures were maintained on a 16 : 8 h, light : dark cycle, with shaking (140 rpm) at 25°C. M. loti (MAFF 303099) was a gift from Prof. Allan Downie at the John Innes Centre, Norwich, UK. It was maintained axenically in TP+ with 0.1% v/v glycerol at 28°C. Cells were harvested by centrifugation, and, if not analysed immediately, cell pellets were frozen in liquid N₂ and stored at −80°C. Algal cell counts were determined using a Dual Threshold Beckman Coulter (Z2) Particle Counter and Size Analyser (Indianapolis, IN, USA). Bacterial cell numbers were determined by plating on solid media.

Chemicals were obtained from the sources indicated: 5′-chloro-5′-deoxyadenosine, Santa Cruz Biotechnology; 7-methylbenzimidazole, Accela; 5-methyl-1H-benzimidazole, Acros Organics; phenol, J. T. Baker; zinc metal, Fisher Scientific; 5-methoxybenzimidazole, purine, and para-cresol, Alfa Aesar; methylmalonyl-CoA, methylmalonic acid, coenzyme A, adenosylcobalamin (coenzyme B₁₂), cyanocobalamin, dicyanocobinamide, 6-methylpurine, 1H-imidazo[4,5-c]pyridine-4-amine (3-deazaadenine), benzimidazole, adenine hemisulfate, 5-azabenzimidazole, 1H-benzo[d]imidazol-7-amine (7-aminobenzimidazole), 2-methyl-1H-purine-6-amine (2-methyladenine), and bovine serum albumin (BSA), Sigma.

The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO): methanol and acetonitrile (MS grade), ammonium acetate, ascorbic acid, β-mercaptoethanol, formic acid and standards, 5-adenosyl-methionine, 5-adenosyl-homocysteine, ATP, betaine, choline, cyanocobalamin, cystathionine, cysteine, dihydrofolate, dimethylglycine, dUMP, folic acid, folinic acid, glycine, homocysteine, methionine, methylcobalamine, methyl-tetrahydrofolate, NADPH, pyridoxal 5-phosphate, riboflavin, serine, taurine, tetrahydrofolate, thymidine 5-phosphate and Dulbecco's modified Eagle's medium mixed 1:1 with Ham's F-12 (DMEM/F12). Ultrapure type 1 water was obtained from a Milli Q water system (Merck Millipore, Darmstadt, Germany). Matrigel (growth factor-reduced) was purchased from BD Biosciences (San Jose, CA). Recombinant basic fibroblast growth factor (bFGF) was purchased from Mylteni (San Diego, USA).