Genomic DNA isolation was performed using DNeasy blood and tissues Kit (Qiagen, France) following manufacturer’s instructions. Cell pellets were resuspended in 180 μL of ATL lysis buffer and incubated for 2 h at 37 °C with 2 mg/mL lysozyme (Euromedex, France). Residual co-extracted RNA was eliminated by adding 100 mg/mL RNase A, for 2 min at room temperature, then isolated DNA was eluted in 30 μL of DNase-free water. To isolate total RNA, cell pellets were crushed in 350 μL RLT lysis buffer of RNeasy Mini Kit (Qiagen, France) using RNase-free piston pellet (Kontes, USA) and following manufacturer’s recommendations. Then RNA was eluted in 37 μL of RNase-free water and treated with DNase using the TURBO-DNA free kit (Ambion, USA) in 50 μL final volume following the manufacturer’s instructions. DNA and RNA were quantified using a UV-mc2 spectrophotometer and diluted to 5 ng/μL, then frozen at −20 °C (DNA) or −80 °C (RNA) until use.
Lysozyme
Manufactured by Euromedex
Sourced in France
Lysozyme is an enzyme found in a variety of biological sources, including egg whites, human tears, and saliva. It is a naturally occurring antimicrobial agent that can disrupt the cell walls of certain bacteria, making it a useful tool in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy blood and tissues kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Resource s, by GE Healthcare (1 mentions)
Superose 12, by GE Healthcare (1 mentions)
Dnase 1 grade 2, by Roche (1 mentions)
Hitrap chelating, by GE Healthcare (1 mentions)
Rnase, by Roche (1 mentions)
Benzonase, by Merck Group (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Chapso, by Anatrace (1 mentions)
Chaps, by Anatrace (1 mentions)
Dextran, by Merck Group (1 mentions)
Ambion turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Atl lysis buffer, by Qiagen (1 mentions)
Uvmc2 spectrophotometer, by Safas (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
8 protocols using lysozyme
1
Genomic DNA and Total RNA Isolation
2
Recombinant Expression and Purification of P. abyssi Q9UZY3
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Four recombinant versions of P. abyssi Q9UZY3 were synthesized and cloned (Genecust) as described in Figure S2 . Following the same protocol as for PAN expression, proteins were overexpressed in E. coli BL21(DE3) and cell pellets were resuspended into Buffer 2 (20 mM Tris–HCl, pH 8.0, 150 mM NaCl), complemented with 0.1% Triton X‐100, 25 mM MgSO4, 0.25 mg ml−1 lysozyme (Euromedex), 0.05 mg ml−1 DNase I grade II (Roche), 0.2 mg ml−1 RNase (Roche) and EDTA‐free protease inhibitor (cOmplete™, Roche). The cells were disrupted by sonication and incubated at 25°C for 30 min. Then the cell extract was treated by heating at 70°C for 15 min and the lysate was clarified by centrifugation at 10,000×g for 1 h. His‐tagged Q9UZY3 was purified by using (i) an affinity column (5‐ml HiTrap Chelating, GE Healthcare) with a linear gradient of 20–250 mM imidazole; (ii) a cation exchange column (Resource S, GE Healthcare) with a linear gradient of 50–500 mM NaCl and (iii) a size exclusion column (Superose 12, GE Healthcare) with elution in Buffer 2. For the untagged Q9UZY3, only the purification steps (ii) and (iii) were performed. The final Q9UZY3 concentration was calculated by measuring the absorbance at 280 nm using the predicted extinction coefficient (ProtParam, ExPASy) listed in Table S1 . The primers and cloning strategies for expression in bacteria cells are listed in Table S2 .
3
Isolation of Bacterial Inner Membrane Proteins
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Bacteria were grown in the CAA medium in the presence of 10 µM enterobactin as described above for the 55Fe uptake assays. Bacterial cultures were centrifuged, cell pellets were washed twice with 50 mM Tris-HCl pH 8.0 and then resuspended in 1.5 ml of buffer A (200 mM Tris-HCl pH 8.0, 20% (w/v) sucrose). Lysozyme (Euromedex) was added to the suspension to a final concentration of 200 µg.ml−1, and the mixture incubated at room temperature for 30 min. Spheroplasts were pelleted by centrifugation (10 min at 8,500 × g), cold water (1 ml) was added and the resulting suspension was incubated for 1 h at 37 °C with benzonase (1 μl of benzonase ≥ 250 units/μl from Sigma). Membranes were isolated by ultracentrifugation (40 min at 100,000 × g). Membrane pellets were washed twice with ultra-pure water and resuspended in 200 mM Tris-HCl pH 8.0, 1% (w/v) N-lauroylsarkosine buffer and incubated during 1 h at room temperature to solubilise specifically all inner-membrane proteins. Afterwards, outer membranes were pelleted by ultracentrifugation (40 min at 100,000×g).
4
Lipid Reconstitution Protocol Optimization
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5
Bacterial DNA Extraction and Normalization
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For each strain, a bacterial pellet of 48 -h bacterial cultures (10 μL) was resuspended in 100 μL of RES buffer (Macherey NagelTM, Düren, Germany) and supplemented with lysozyme (Euromedex, Souffelweyersheim, France, 20 mg·mL−1). After it was shaken and incubated for 10 min at room temperature, 200 μL of sodium dodecyl sulfate (0.1%) and 300 μL of phenol-chloroform-isoamyl alcohol (25:24:1) were added. The suspension was homogenized by successive pipetting and centrifuged at 18,000× g and 5 °C for 5 min. The aqueous phase was recovered and then supplemented with 300 μL of phenol-chloroform-isoamyl alcohol (25:24:1). After it was shaken and centrifuged at 18,000× g and 5 °C for 10 min, the aqueous phase was then supplemented with 1/10 of its volume of sodium acetate and 1 volume of isopropanol. The suspension was mixed by turning it several times and stored at −20 °C for 15 h. This suspension was centrifuged for 30 min at 18,000× g and 5 °C, and then the supernatant was removed. The DNA pellet was dried for a few minutes in the open air and resolubilized in 100 μL elution buffer (TRIS-HCl pH 8.5). The concentration and quality of the extracted DNA were estimated by a Nanodrop® spectrophotometer (Thermo Scientific NanoDrop™ 1000 Spectrophotometer, Waltham, MA, USA). The bacterial DNA of the 58 strains was normalized to 20 ng·μL−1.
6
Genomic DNA and Total RNA Isolation
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Genomic DNA isolation was performed using DNeasy blood and tissues kit (Qiagen, France) following manufacturer's recommendations. After lysis in 180 μL of ATL buffer, samples were incubated for 2 h a 37°C with lysozyme (Euromedex, France) at a final concentration of 2 mg/mL. Residual co-extracted RNA was eliminated by adding 100 mg/mL of RNase A, for 2 min at room temperature. The isolated DNA was eluted in 30 μL of DNase-free water. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, France) as recommended by supplier. Cell pellets were crushed in 350 μL RLT lysis buffer using RNase-free piston pellet (Kontes, USA), and RNA was eluted in 37 μL of RNase-free water. RNA solution was treated with DNase using the Ambion TURBO-DNA free kit (Ambion, USA) in 50 μL final volume following the manufacturer’s instructions. DNA and RNA were quantified using a UV-mc2 spectrophotometer and diluted to 5 ng/μL, then frozen at -20°C (DNA) or -80°C (RNA) until use.
7
Gut Microbiome DNA Extraction
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Samples, containing 50 guts, were centrifuged at 13,200 rpm for 30 min. Pellets were resuspended in 180 μL of ATL lysis buffer (QIAGEN, Hilden, Germany) containing 0.44 μg.mL−1 lysozyme (Euromedex, Strasbourg, France). Genomic DNA was extracted as previously described [22 (link)] using the DNeasy Blood and Tissue kit (QIAGEN) and stored at −20 °C until further procedure. The DNA concentration was measured using the UVmc2 spectrophotometer (SAFAS, Monaco).
8
Bacterial Iron Uptake and Fractionation
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Iron uptake and cell fraction were carried out as previously described (Roche et al., 2021) (link). Cells were cultured overnight in LB medium at 30 C, and afterwards at 30 C overnight in CAA medium. Then, they were diluted in fresh CAA medium and grown at 30 C overnight. Bacteria were washed with 50 mM Tris-HCl at pH 8 and diluted to an OD 600 nm of 1. Cells were incubated 30 min with 200 nM PCH-55 Fe at 30 C. Cells were centrifuged for 8 min at 6700g to obtain the supernatant (extracellular medium), and the cell pellet was resuspended in Tris-sucrose EDTA (Tris-HCl 0.2 M pH 8, EDTA 1 mM, sucrose 20%). Spheroplasts were obtained by adding 200 μg ml À1 lysozyme (Euromedex, Souffelweryersheim, France) and incubated at 4 C for 1 h. The suspension was centrifuged for 8 min at 6700g at 4 C to obtain the spheroplast pellet and the periplasmic fraction (supernatant). The supernatant was ultracentrifuged (120,000g, 40 min, 4 C) to clarify the periplasmic fraction. Spheroplast pellet was washed with Tris-HCl 0.2 M pH 8, sucrose 20%, and resuspended in cold water by vortexing. The suspension was then incubated with benzonase for 1 h at 37 C. The cytoplasmic fraction was isolated by ultracentrifugation (120,000g, 40 min, 4 C). Pellet was washed twice with cold water and resuspended in Tris-HCl 0.2 M pH 8, sucrose 20% to obtain the bacterial membranes.
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