Mass spectrometry-based approaches to study lanthanides and lanthanide-dependent proteins in the phyllosphere

Dynamic character displacement among a pair of bacterial phyllosphere commensals in situ

“…As a reference to the in planta conditions, both Rhizobium Leaf68 and Sphingomonas Leaf257 were each grown on R2A + M-agar (see above) in the plant growth chamber at the settings described above. Bacterial cell pellets were dissolved and lysed using a lysis buffer containing 100 mM ammonium bicarbonate, 8 M urea and 1× cOmplete EDTA-free protease inhibitor cocktail (Sigma-Aldrich, Buchs, Switzerland) and indirect sonication (3 × 1 min, 100% amplitude, 0.8 cycle time) in a VialTweeter (HIFU, Hielscher, Teltow, Germany) 45 , 109 . Insoluble parts were removed by centrifugation at 13,000 g for 15 min at 4 °C.…”

“…After incubation, trypsin was inactivated using heat incubation in a tabletop shaker at 95 °C for 5 min followed by acidification through addition of formic acid to an approximate final concentration of 1%. Insoluble parts were removed by centrifugation at 20,000 g for 10 min and the supernatant was desalted using Sep-Pak Vac C18 reversed phase columns (Waters Corporation, Baden-Dättswil, Switzerland) as previously described 109 and dried under vacuum. The samples were re-solubilized in 3% acetonitrile (ACN) and 0.1% formic acid (FA) to a final concentration of 0.1-1.0 mg mL −1 .…”

Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus

“…For a detailed step-by-step protocol, refer to Ref. ( 48 ). Briefly, M. flagellatus KT was grown as independent precultures and main cultures with or without 20 μM LaCl 3 .…”

“…In total, five independent biological replicates were generated for each condition. Bacterial cell pellets were dissolved and lysed as described previously ( 10 , 48 , 49 ) in lysis buffer containing 100 mM ammonium bicarbonate, 8 M urea, and 1× cOmplete EDTA-free protease inhibitor cocktail (Roche). Lysis was achieved by indirect sonication (3× 1 min, 100% amplitude, 0.8 cycle time) in a VialTweeter (Hielscher).…”

“…After digestion, the trypsin was heat inactivated (95 °C, 5 min), and formic acid was added to an approximate concentration of 1%. Peptide samples were centrifuged (20,000 g , 10 min), and the supernatant was taken and desalted using Sep-Pak Vac C18 reversed phase columns (Waters) as described previously ( 48 , 49 ) and dried under vacuum. Prior to LC–MS/MS analysis, the samples were resolubilized in 3% acetonitrile containing 0.1% formic acid.…”