Antibiotics, most carbon sources, CCCP, NaN3, malonate, furfural, DMSO, K2CO3, and N,N-dimethylformamide were purchased from Sigma-Aldrich. Arabinose and gluconic acid potassium were purchased from Acros Organics. D-Glucose, disodium citrate, mannitol, LB (Miller formulation), and PBS were purchased from Fisher Scientific. M9 minimal medium (parts A and B, containing 7 g/L K2HPO4, 3 g/L KH2HPO4, 1 g/L (NH4)2SO4, 0.1 g/L MgSO4, 0.588 g/L sodium citrate) was purchased from MP Biomedicals. DiBAC4(3) and Texas red sulfonyl chloride were purchased from Life Technologies.
M9 minimal medium
Manufactured by MP Biomedicals
Sourced in United Kingdom
M9 minimal medium is a basic growth medium used for culturing bacteria, particularly Escherichia coli (E. coli). It provides the essential nutrients and salts required for bacterial growth and maintenance. The medium consists of a defined set of inorganic compounds, including sodium and potassium salts, ammonium sulfate, and phosphates, as well as a carbon source, typically glucose.
Automatically generated - may contain errors
Lab products found in correlation
Ampicillin, by Merck Group (3 mentions)
Neomycin, by Merck Group (3 mentions)
Bacteriological agar, by Thermo Fisher Scientific (2 mentions)
Tetracycline, by Merck Group (2 mentions)
Quarter strength 1 4 ringer s buffer, by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (2 mentions)
Malonate, by Merck Group (1 mentions)
Furfural, by Merck Group (1 mentions)
Arabinose, by Thermo Fisher Scientific (1 mentions)
Mannitol, by Thermo Fisher Scientific (1 mentions)
K2co3, by Merck Group (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Dibac4 3, by Thermo Fisher Scientific (1 mentions)
Texas red sulfonyl chloride, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Magnesium sulfate, by Merck Group (1 mentions)
Calcium chloride, by Merck Group (1 mentions)
6 protocols using m9 minimal medium
1
Microbial Metabolism Evaluation Protocol
2
Cultivating P. atrosepticum and E. coli Strains
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P. atrosepticum SCRI1043 was grown at 28 °C with aeration (140 rpm) in tryptic soy broth (TSB; Oxoid, Basingstoke, UK) or in M9 minimal medium (MP Biomedicals, Thüringen, Germany) prepared as described by Czajkowski et al. [61 (link)] containing 2 mM magnesium sulfate (Sigma-Aldrich, Darmstadt, Germany), 0.1 mM calcium chloride (Sigma-Aldrich, Darmstadt, Germany), and 0.4% glucose (Sigma-Aldrich, Darmstadt, Germany) as a carbon source, pH 7. To solidify the media, 15 g L−1 agar (Oxoid, Basingstoke, UK) was added. Escherichia coli S17-1 λ-pir strain carrying plasmid pCAM140 with mini-Tn5 transposon [64 (link)] was cultured with aeration (120 rpm) in TSB or on tryptic soy agar (TSA; Oxoid, Basingstoke, UK) supplemented with ampicillin (Sigma-Aldrich, Darmstadt, Germany) to a final concentration of 100 μg mL−1 at 37 °C. When required, the bacterial media were supplemented with streptomycin (Sigma-Aldrich, Darmstadt, Germany) to a final concentration of 50 μg mL−1 and with X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid; GeneON, Ludwigshafen am Rhein, Germany) to a final concentration of 20 μg mL−1. For long-term usage, bacterial strains were stored in 40% (v/v) glycerol at −80 °C.
3
E. coli Growth Conditions and Imaging
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All strains in this study were derivatives of E. coli MG1655 (Supplementary Table S1). In slow growth conditions, cells were cultivated in M9 minimal medium (MP Biomedicals) supplemented with magnesium sulfate and 0.3% glycerol. LB medium (Fischer Scientific) was used for fast growth. 20 μg/ml kanamycin and 34 μg/ml chloramphenicol were added to the growth medium for strains with respective resistance markers. All bacteria were grown and imaged at 28°C.
For still imaging, we used M9 and LB agarose pads, and for time lapse imaging, home-made glass bottom dishes. In time lapse imaging, cells were pipetted to #1.5 glass coverslips on the bottom of the dish and covered with a (about 0.5 cm thick) slab of M9 agarose with magnesium sulfate and 0.3% glycerol. No antibiotics were used in agarose during imaging.
For still imaging, we used M9 and LB agarose pads, and for time lapse imaging, home-made glass bottom dishes. In time lapse imaging, cells were pipetted to #1.5 glass coverslips on the bottom of the dish and covered with a (about 0.5 cm thick) slab of M9 agarose with magnesium sulfate and 0.3% glycerol. No antibiotics were used in agarose during imaging.
4
Propagation and Characterization of Lytic Phage ΦD5
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The lytic bacteriophage vB_Dsol_D5 (ΦD5) was previously described46 ,47 ,91 . For this work, ΦD5 was propagated on its wild-type host, D. solani strain IPO 222238 , and quantified as described earlier46 . A stock of ΦD5 phage particles (ca. 108—109 plaque-forming units (PFU) mL−1 in tryptone soya broth (TSB, Oxoid) or quarter-strength (1/4) Ringer’s buffer (Merck) was used in all experiments unless otherwise stated. Bacterial strains used in this study are listed in Supplementary Table 1. The pool of 1000 mutants from a collection of 10,000 D. solani Tn5 mutants previously generated84 (link),92 was interrogated as a source of ΦD5-resistant mutants. The D. solani wild type (WT) strain was cultivated for 24–48 h at 28 °C on tryptic soy agar (TSA, Oxoid), in tryptone soy broth (TSB, Oxoid) or M9 minimal medium (MP Biomedicals) supplemented with glucose (0.4%) (Sigma-Aldrich). Bacteriological agar (Oxoid) (15 g L−1) was added to solidify the media. As appropriate, media were supplemented with neomycin (Sigma-Aldrich) (50 µg mL−1), ampicillin (Sigma-Aldrich) (150 µg mL−1) or tetracycline (Sigma-Aldrich) (40 µg mL−1). Bacterial cultures were agitated during incubation (120 rpm). To prevent fungal growth, cycloheximide (Sigma-Aldrich) was added to the growth medium at a final concentration of 200 µg mL−1.
5
Characterization of Bacteriophage vB_Ppp_A38
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The lytic bacteriophage vB_Ppp_A38 (ϕA38) was isolated and characterized in detail previously [24 (link),25 ]. For this work, ɸA38 was propagated on its wild-type bacterial host, P. parmentieri SCC 3193 [57 (link)], and titered as described earlier [15 (link)]. The adjusted stock concentration of ϕA38 phage particles was 108–109 plaque-forming units (PFU) mL−1 in tryptone soya broth (TSB, Oxoid, Basingstoke, UK) or quarter-strength (1/4) Ringer’s buffer (Merck, Warsaw, Poland) unless stated otherwise. P. parmentieri strain SCC 3193 (wild-type: WT) was grown for 24–48 h at 28 °C on tryptic soya agar (TSA, Oxoid, Basingstoke, UK), in tryptone soya broth (TSB, Oxoid, Basingstoke, UK) or in M9 minimal medium (MP Biomedicals, Santa Ana, CA, USA) supplemented with glucose (Sigma-Aldrich, Darmstadt, Germany) to a final concentration of 0.4%.When required, 15 g L−1 bacteriological agar (Oxoid, Basingstoke, UK ) was added to solidify the media and if needed the growth media were supplemented with neomycin (Sigma-Aldrich, Darmstadt, Germany) to a final concentration of 50 µg mL−1. The liquid bacterial cultures were agitated at 120 rpm during incubation. The phage-resistant P. parmentieri Tn5 mutants characterized in this study are listed in Table 1 .
6
Propagation and Characterization of Lytic Bacteriophage ΦD5
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The lytic bacteriophage vB_Dsol_D5 (ΦD5) was previously described 46, 47, (link)91 (link) . For this work, ΦD5 was propagated on its wildtype host, D. solani strain IPO 2222 38 , and quanti ed as described earlier 46 . A stock of ΦD5 phage particles (ca. 10 8 - 10 9 plaque-forming units (PFU) mL - 1 in tryptone soya broth (TSB, Oxoid) or quarter-strength (1/4) Ringer's buffer (Merck) was used in all experiments unless otherwise stated. Bacterial strains used in this study are listed in Supplementary Table 1. The pool of 1000 mutants from a collection of 10,000 D. solani Tn5 mutants previously generated 84, 92 (link) was interrogated as a source of ΦD5-resistant mutants. The D. solani wild type (WT) strain was cultivated for 24-48 h at 28 ºC on tryptic soy agar (TSA, Oxoid), in tryptone soy broth (TSB, Oxoid) or M9 minimal medium (MP Biomedicals) supplemented with glucose (0.4%) (Sigma-Aldrich). Bacteriological agar (Oxoid) (15 g L - 1 ) was added to solidify the media. As appropriate, media were supplemented with neomycin (Sigma-Aldrich) (50 µg mL - 1 ), ampicillin (Sigma-Aldrich) (150 µg mL - 1 ) or tetracycline (Sigma-Aldrich) (40 µg mL - 1 ). Bacterial cultures were agitated during incubation (120 rpm). To prevent fungal growth, cycloheximide (Sigma-Aldrich) was added to the growth medium at a nal concentration of 200 µg mL - 1 .
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