L methionine

The total superoxide dismutase (SOD) activity was determined by the method outlined by Beauchamp and Fridovich (19 (link)). Briefly, the reaction mixture consisted of 50 mM phosphate buffer (pH=7.8), 0.1 mM EDTA, 13 mM l-methionine (Merck Millipore, Burlington, VT, USA), 75 µM nitro blue tetrazolium chloride (NBT) (SERVA Electrophoresis GmbH, Heidelberg, Germany), 2 µM riboflavin (Supelco, Bellefonte, PA, USA) and an appropriate aliquot of the extract. riboflavin was added last and the reaction was initiated by placing the tubes under the fluorescent light (30 W) for 10 min at room temperature. Absorbance was read at 560 nm using an Ultrospec 3300 pro UV/Vis spectrophotometer (Amersham Biosciences, Amersham, UK). One unit of the SOD activity was defined as the amount of enzyme required for 50% inhibition of NBT reduction and expressed in unit per milligram of protein.

A solution of 1.5 mM l‐methionine (Merck) or water (as control) was sprayed every second day onto the leaves of mock‐ and PVY‐infected plants maintained at 22 or 28 °C. PVY RNA accumulation and content of MTC‐related metabolites in these plants were quantified as described above.

In this study, two monoclonal IgG type-1 antibodies (mAbs) were used: one sourced from the laboratory’s stock (LMU1, Munich, Germany), and the other (LMU2) generously provided by Boehringer Ingelheim Pharma GmbH & Co. KG (Ingelheim am Rhein, Germany). Further, G-CSF (filgrastim) was used as a model protein. L-histidine (cell culture reagent) and L-histidine monohydrochloride monohydrate (99% purity) were purchased from Alfa Aesar (Ward Hill, MA, USA). EMPROVE^® exp sucrose, EMPROVE^® exp di-sodium hydrogen phosphate dihydrate, EMPROVE^® bio sodium chloride, sodium citrate dihydrate (≥99.0%), and L-methionine were purchased from Merck KGaA (Darmstadt, Germany). D(+)-trehalose dihydrate (97.0–102.0% purity) Ph. Eur., NF certified, and D(-)-mannitol (97.0–102.0% purity) Ph. Eur., USP certified were purchased from VWR International (Radnor, PA, USA). Sodium dihydrogen phosphate dihydrate (99%) was purchased from Grüssing GmbH (Filsum, Germany). Trizma^® base and Trizma^® hydrochloride (both in BioXtra grade), anhydrous citric acid BioUltra grade (≥99.5%), and sodium azide (≥99.5%) were purchased from Sigma Aldrich (Burlington, MA, USA). Super Refined™ Polysorbate 20-LQ-(MH) was purchased from Croda (Edison, NJ, USA). All solutions were prepared using ultrapure water from a Sartorius Lab Instruments GmbH Arium^® system (Goettingen, Germany).

In this experimental study at QAU from January,
2013 to March, 2013, ET-1 and NAC were purchased
from Sigma Aldrich (St. Louis, MO, USA).
BNP antibodies, goat anti-rabbit IgG-AP antibody
and 0.45-μm pore-size nitrocellulose membrane were
purchased from Santa Cruz Biotechnology (Dallas,
Texas, USA). Alkaline phosphatase (AP), 5-bromo-
4-chloro-3-indolyl-phosphate (BCIP) and nitro blue
tetrazolium (NBT) were purchased from Tiangen
(Beijing, China). Sodium acetate, N-diethyl-peraphenylenediamine
(DEPPD), ferrous sulphate, NACl,
KH₂PO₄, Na₂HPO₄, KCl, L-methionine, triton X-100,
riboflavin were purchased from Merck Chemicals
(Germany).

S. cerevisiae BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) and the derived deletion mutants pdr18Δ and snq2Δ were obtained from EUROSCARF collection. C. glabrata BPY55 (clinical isolate) and the derived deletion mutant snq2Δ built using the SAT1 flipper system were kindly provided by Professor Dominique Sanglard, Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland.
Cultivation of S. cerevisiae strains was performed in MM4 medium, containing 1.7 g/L yeast nitrogen base without amino acids and ammonium sulfate (Difco, Detroit, MI, United States), 20 g/L glucose (Merck, Darmstadt, Germany), 2.65 g/L (NH₄)₂SO₄ (Panreac AppliChem, CT, United States), 20 mg/L L-methionine, 20 mg/L L-histidine (both from Merck, Darmstadt, Germany), 60 mg/L L-leucine and 20 mg/L L-uracil (both from Sigma, St. Louis, MO, United States). C. glabrata strains were cultivated in MM medium, with the composition of MM4 medium, without supplementation with amino acids and uracil. YPD medium contained 20 g/L glucose, 20 g/L Bacto^TM Peptone and yeast extract (both from BD Biosciences, Franklin Lakes, NJ, United States). Solid media were prepared by the addition of 20 g/L agar (Iberagar, Barreiro, Portugal) to the different liquid media. Media pH were adjusted to 4.5 with HCl. Growth in liquid media was performed at 30°C with orbital agitation (250 rpm).

Saccharomyces cerevisiae parental strain BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) and the derived deletion mutant pdr18Δ were obtained from the EUROSCARF collection (http://web.uni-frankfurt.de/fb15/mikro/euroscarf), as well as plasmid pYCG_PDR18, expressing the PDR18 gene from its natural promoter, and the corresponding cloning vector, pRS416, used for phenotypic complementation assays.
Yeast cells were cultivated at 30 °C with orbital agitation (250 rpm) in liquid minimal growth medium supplemented with the amino acids and the nucleotide to support growth of the auxotrophic strains (MM4). MM4 contained 1.7 g/L yeast nitrogen base without amino acids and ammonium sulphate (Difco, Michigan, USA), 20 g/L glucose (Merck, Darmstadt, Germany), 2.65 g/L (NH₄)₂SO₄ (Panreac AppliChem, Connecticut, USA), 20 mg/L L-methionine, 20 mg/L L-histidine (both from Merck, Darmstadt, Germany), 60 mg/L L-leucine and 20 mg/L L-uracil (both from Sigma, Missouri, USA), adjusted to pH 4.0 with HCl. Solid medium was prepared by the addition of 20 g/L agar (Iberagar, Barreiro, Portugal) and the pH was set to 4.5 with HCl. Cells harboring the cloning vector pRS416 or derived plasmids were grown in the same medium lacking uracil supplementation (MM4-U medium) to maintain selective pressure.

For long-term growth culture, an overnight pre-culture of H. pylori N6 wild type was set up in BHI medium (Oxoid) with 2.5% yeast extract and 5% horse serum. This pre-culture was used to set up a day culture for time course of growth (growth curve), with a starting OD₆₀₀ of ∼0.15. Bacterial pellets were harvested at OD₆₀₀ of 0.5, 1.0, and 1.5. For all other short-term co-incubation conditions, a short-term liquid culture was set up with an initial OD₆₀₀ of ∼0.8 (prepared from a pre-culture on plate), then further cultured with shaking for 4 h, before harvesting pellets for DNA (EM-Seq analyses) and RNA isolation.
In order to grow H. pylori N6 in methionine-depleted versus -replete conditions in liquid culture, bacteria were resuspended in BHI medium, supplemented with 5% dialyzed Fetal Bovine Serum One Shot™ (Thermo Fisher Scientific—Gibco), which is depleted in amino acids, in either the absence or the presence (supplementation) of 50 µg/ml L-methionine (Merck) with an initial OD₆₀₀ of ∼0.8, and then further cultured for 4 h.