Ampicillin

The materials used in the study were sodium hydroxide (NaOH), ethanol (99%), hydrochloric acid (37%), hydrogen peroxide (6%), silver nitrate 99.9+% (metal basis), and distilled water. All the chemicals used in this research were of analytical grade and applied without further purification. Chemical materials were purchased from the Saudi Chemical Company (PanReac AppliChem, Ar Riyad, Saudi Arabia). Hama fluoride topical gel (1.23% fluoride ion) was obtained from Kal-AlHamaya, Saudi Arabia, antibiotic susceptibility disks (penicillin G (PG), Augmentin (AUG), metronidazole (MZ), ampicillin (AP), ciprofloxacin (CIP), and cotrimoxazole (TS)) from PanReac AppliChem, Riyadh, Saudi Arabia, and Gram-positive cocci ID cards (ID-GPC cards; BioMérieux, Craponne, France.

T. virens Gv 29.8 (Kindly provided by Charles Kenerley, Texas A&M University, United States), T. sp. “atroviride B” LU132 (Braithwaite et al., 2017 (link)), and mutants were grown on potato dextrose agar (PDA) (Difco, Fisher Scientific, United States) at 25°C under a cycle of 12 h light and 12 h dark for 7 days to induce conidiation. Conidia were collected using sterile nanopure water and filtered through a double layer of sterile Miracloth (Millipore Merck, United States). The conidial concentration was determined using a Neubauer chamber. Escherichia coli cells transformants were grown on Luria Bertani (LB) medium supplemented with either 100 μg/mL ampicillin (PanReac AppliChem, Germany) or 50 μg/mL kanamycin (Sigma Aldrich, United States) at 37°C depending on the vector. Agrobacterium tumefaciens EHA105 cells transformants were grown on LB medium containing 25 μg/mL kanamycin and 25 μg/mL rifampicin (Sigma Aldrich, United States) at 28°C.

Antibiotic treatment was given in the drinking water and contained ampicillin (PanReac AppliChem A0839, 0025) 1 g/L + neomycin (Biological Industries 1405-10-3) 0.5 g/L. ampicillin and neomycin were chosen because ampicillin is active against Gram-positive and some Gram-negative bacteria, while neomycin has excellent activity against Gram-negative bacteria and has partial activity against Gram-positive bacteria. In addition, both antibiotics are poorly absorbed (or unabsorbed as in the case of neomycin) and thus have no systemic effects [22 (link)].

SpOpB-expressing plasmid (pET-6HisOpB) was obtained as described in [43 (link)]. Expression of the recombinant protein was carried out in E. coli BL21(DE3)RIPL (Novagen, Madison, WI, USA). Freshly transformed cells were grown in high salt LB medium with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol (Panreac-AppliChem, Darmstadt, Germany) at 37 °C, until the OD₆₀₀ value reached 0.8. Then, expression was induced with 0.2 mM IPTG. After 20 h incubation at 25 °C, the cells were harvested by centrifugation, resuspended in (50 mM TrisHCl and 500 mM NaCl, pH 8.0 buffer supplemented with 0.1% (v/v) Triton X-100, 10% (v/v) glycerol, 20 mM imidazole, and 1 mM PMSF), and disrupted by sonication. After centrifugation (20,000× g, 30 min, 4 °C), the supernatant was filtered and applied to a 5 mL HisTrap HP column (GE healthcare, Chicago, IL, USA) equilibrated with the same buffer. The column was washed with Tris/NaCl buffer pH8.0 supplemented with 50 mM imidazole, and SpOpB was eluted by Tris/NaCl buffer pH8.0 supplemented with 300 mM imidazole. The 30 kDa cutoff centrifugal filter devices (Millipore, MA, USA) were used for buffer exchange and protein concentration. Protein size, purity, and oligomeric state were controlled by electrophoresis in SDS-PAAG (Supplementary Figure S5). Protein concentration was determined by the Bradford method.

1 mL of exponentially growing culture was inoculated in F/2 medium containing final concentrations of 0.1 mg/mL Streptomycin (PanReac Applichem, A1852), 0.06 mg/mL Penicillin (PanReac Applichem, A1837), 1 mg/mL Ampicillin (PanReac Applichem, A0839), 0.1 mg/mL kanamicine (PanReac Applichem, A1493) and 0.02 mg/mL cefotaxime (Sigma-Aldrich, C7039) and allowed to grow for 5–6 days under standard growth conditions. Bacterial contamination was checked in two ways (SI1: Supplementary Fig. 6): (i) by staining DNA with DAPI and examining cultures under the microscope to check for the presence/absence of bacterial nucleoids; (ii) by performing peptone tests. For DAPI staining, 1 µL of DAPI stock solution (4′,6-diamidino-2-phenylindole, 1 mg/mL, Roche, Basel, Switzerland) was added to 1 mL of formalin preserved culture, incubated for 10 minutes and observed under the epifluorescence microscope. For peptone tests, 1 mL of diatom culture was added to a tube containing a peptone solution (1 mg/mL), incubated in the dark and checked after 2–3 days and 1–2 weeks; growth of bacteria in the tubes indicated contamination. If bacterial contamination persisted, the treatment was repeated. Large volume cultures used for DNA extraction were grown with antibiotics and the contamination tests were always performed on an aliquot of the culture.

Escherichia coli DH5α was used for transformation and propagation of the recombinant plasmid. LB medium [0.5% (w/v) yeast extract (BD), 1% (w/v) Bacto-tryptone (BD), 1% (w/v) sodium chloride (BDH Prolabo)] supplemented with 100 μg/ml ampicillin (PanReac Applichem) was used for selection. Bacterial cultures were maintained at 37 °C.
Yeast BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), used as a background strain for yeast chromosomal gene modifications, yeast ASN1::GFP, used as a base strain for colocalization assay, and yeast ASN2::GFP, used as a base strain for assembly dependence assay, were purchased from Thermo Fisher Scientific. YPD [1% (w/v) yeast extract (BD), 2% (w/v) Bacto-peptone (BD), and 2% (w/v) dextrose (Sigma-Aldrich)] medium was used for general growth. G418 (PanReac Applichem) (400 μg/ml final concentration) and hygromycin B (Merck) (200 μg/ml final concentration) were used for selecting yeast transformants. All yeast strains were maintained at 30 °C.

Reduced GSH and albumin (BSA (fraction V)) were obtained from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO, USA). Chloramphenicol was purchased from Fluka (Steinheim, Germany). Fosfomycin (disodium salt) was obtained from Apollo Scientific (Whitefield Rd, Bredbury, Stockport, UK). 5,5′-Dithio-bis(-2-Nitrobenzoate) (DTNB) was obtained from Boehringer Mannheim. Isopropyl 1-thio-β-galactopyranoside (IPTG) and ampicillin (sodium salt) were obtained from PanReac AppliChem, (Darmstadt, Germany). The KAPA HiFi PCR kit and KAPA Taq PCR kit were obtained from Kapa Biosystems (KAPA Biostystems Pty, Cape Town, South Africa). The plasmid isolation kit was purchased from Macherey-Nagel (Macherey-Nagel GmbH & Co., Düren, Germany). The In-Fusion HD Cloning Plus package (including the cloning enhancer, Infusion HD cloning kit, Clone Amp Hifi PCR Premix, and T4 DNA ligase) was obtained from Takara Bio (Takara Bio USA Inc., Mountain View, CA, USA). Yeast extract and peptone were purchased from Scharlau (Sentmenat, Spain). The protein thermal shift dye kit was obtained from Thermo Fischer Scientific (Applied Biosystems, Foster City, CA, USA).