N. gonorrhoeae strains used in this study are derived from the laboratory strains FA19 and F62 and are described in S4 Table . Gonococcal strains were grown overnight at 37°C under 5% (v/v) CO2 on GC agar plates containing Kellogg’s supplements I and II [57 (link)]. When indicated glucose in supplement I was replaced by L-lactate or pyruvate at indicated concentrations. Growth in liquid medium was at 37°C with agitation (225 r.p.m.) in GC broth containing Kellogg’s supplements I and II and 0.042% (w/v) sodium bicarbonate. When necessary, culture media were supplemented with ampicillin (Amp; 100 μg/mL), chloramphenicol (Cm; 0.5–1.0 μg/mL), kanamycin (Km; 50 μg/mL), erythromycin (Erm; 1 μg/mL), isopropyl-β-D-thiogalactopyranoside (IPTG; 0.5 to 1.0 mM as indicated) or 5-bromo-4-chloro-3- indolyl-β-D-galactopyranoside (X-gal; 20 μg/mL). E. coli TOP10 (Life Technologies, Carlsbad, CA) and ER2566 (New England BioLabs, NEB) were used for cloning and protein expression purposes respectively and grown on LB medium.
Er2566
Manufactured by New England Biolabs
Sourced in United States
ER2566 is a laboratory strain of Escherichia coli (E. coli) that is commonly used for the expression and purification of recombinant proteins. This strain is designed for high-level protein production and is suitable for a variety of molecular biology applications.
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Lab products found in correlation
Bl21 de3, by Merck Group (2 mentions)
Escherichia coli dh5α, by New England Biolabs (2 mentions)
E coli top10, by Thermo Fisher Scientific (1 mentions)
Top10f, by Thermo Fisher Scientific (1 mentions)
Puc19, by Thermo Fisher Scientific (1 mentions)
Pet30a, by Thermo Fisher Scientific (1 mentions)
Bl21 de3, by Thermo Fisher Scientific (1 mentions)
Escherichia coli top10, by Thermo Fisher Scientific (1 mentions)
Bth101, by Euromedex (1 mentions)
D glucose, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Ampicillin, by New England Biolabs (1 mentions)
Arcticexpress de3, by Agilent Technologies (1 mentions)
Ptargetf, by Addgene (1 mentions)
Ni nta spin column, by Qiagen (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
17 protocols using er2566
1
Cultivation of Neisseria gonorrhoeae
2
Neisseria and Escherichia Strain Growth
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Strains, plasmids and constructs employed in the study are listed in Table 1 . N. meningitidis strains MC58 and M1080 were grown on blood agar plates in a 5% CO2 atmosphere at 37°C. E. coli strain ER2566 (New England Biolabs), used for plasmid propagation and recombinant protein expression, was grown in LB medium or on LB agar plates containing kanamycin (50 μg ml-1) at 37°C.
3
Isolation and Cultivation of Serratia Phage Hosts
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The bacterial host of investigated phages Serratia sp. OS31 was isolated from rock biofilms of the Zloty Stok gold and arsenic mine (SW Poland) using the methods described previously [93 (link)] and was kindly provided by Lukasz Drewniak. The strain was grown under aerobic conditions in lysogeny broth (LB) or R2A media [94 ] at 20 °C. Using the same methodology [93 (link)], we isolated two other Serratia spp. strains (BZSmr3 and BZSmr6) from biofilms of the walls of the Gertruda Adit in the Zloty Stok in April 2018. They were used in host range testing assay. Bacteria used in host range testing (Table S8 ) were grown under aerobic conditions in LB at 30 °C, except Yersinia eneterolitica 2/O:9 (at room temperature, RT) and environmental Serratia spp. strains from Zloty Stok (at 20 °C).
The following Escherichia coli strains were used in this study: TOP10F’ (Invitrogen, Waltham, MA, USA), ER2566 (New England BioLabs, Ipswich, MA, USA), and ER2929 Dam− strain lysogenized with λDE3 element, which carried the gene for T7 RNA polymerase under control of the lacUV5 promoter [95 (link)]. These were cultured under standard conditions in LB medium at 37 °C. When required, the media were supplemented with kanamycin at 50 μg·mL−1, and ampicillin at 100 μg·mL−1. Plasmids pUC19 (Thermo Fisher Scientific, Waltham, MA, USA) and pET30a (Invitrogen) were used as cloning or expression vectors.
The following Escherichia coli strains were used in this study: TOP10F’ (Invitrogen, Waltham, MA, USA), ER2566 (New England BioLabs, Ipswich, MA, USA), and ER2929 Dam− strain lysogenized with λDE3 element, which carried the gene for T7 RNA polymerase under control of the lacUV5 promoter [95 (link)]. These were cultured under standard conditions in LB medium at 37 °C. When required, the media were supplemented with kanamycin at 50 μg·mL−1, and ampicillin at 100 μg·mL−1. Plasmids pUC19 (Thermo Fisher Scientific, Waltham, MA, USA) and pET30a (Invitrogen) were used as cloning or expression vectors.
4
Routine Cultivation of E. coli Strains
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E. coli DH5α (Invitrogen), BL21 (DE3) (Invitrogen) and ER2566 (New England Biolabs) were routinely grown at 37°C in Luria-Broth (LB), M9 minimal (M9M), or 2× YT media supplemented when necessary with ampicillin (Ap, 50 µg/ml), spectinomycin (Sp, 100 µg/ml), tetracycline (Tc, 10 µg/ml), isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM), L-arabinose (L-ara, 0.1% w/v) or D-glucose (glu, 0.2% w/v). Bacterial cell density was measured using a WPA Biowave C08000 at 600 nm (OD600).
5
Recombinant Protein Expression in E. coli
6
Isolation and Characterization of Antarctic Pseudomonas
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Pseudomonas sp. ANT_H14 was isolated from a soil sample collected near the Arctowski Polish Antarctic Station located on King George Island, the largest of the South Shetland Islands, situated 120 km off the coast of Antarctica. The strain was identified based on the 16S rRNA gene sequencing using universal primers 27f and 1492r [18 ]. No permission was required for soil samples collection, as the Arctowski Polish Antarctic Station is located outside the Antarctic Specially Protected Area. Moreover, the field study did not involve endangered or protected species.
Other strains used in this study were: Escherichia coli TOP10 (Thermo Fisher Scientific, Waltham, MA, USA) and ER2566 (New England BioLabs, Ipswich, MA, USA), Pseudomonas aeruginosa PAO1 [19 (link)] and seven Pseudomonas spp. strains isolated from the same environment as Pseudomonas sp. ANT_H14. The stains were cultured under standard conditions in LB medium at 37°C (E. coli and P. aeruginosa PAO1) or 22°C (Pseudomonas environmental isolates).
When required, growth media were supplemented with kanamycin (Km, 50 μg ml−1) and glucose (1%). The vector pET30a (Kmr, Novagen, Inc., Madison, WI, USA) was used in recombinant protein expression experiments.
Other strains used in this study were: Escherichia coli TOP10 (Thermo Fisher Scientific, Waltham, MA, USA) and ER2566 (New England BioLabs, Ipswich, MA, USA), Pseudomonas aeruginosa PAO1 [19 (link)] and seven Pseudomonas spp. strains isolated from the same environment as Pseudomonas sp. ANT_H14. The stains were cultured under standard conditions in LB medium at 37°C (E. coli and P. aeruginosa PAO1) or 22°C (Pseudomonas environmental isolates).
When required, growth media were supplemented with kanamycin (Km, 50 μg ml−1) and glucose (1%). The vector pET30a (Kmr, Novagen, Inc., Madison, WI, USA) was used in recombinant protein expression experiments.
7
Bacterial Strain Handling and Induction
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Escherichia coli strain ER2566 (NEB, Ipswich, United States) was used for all fractionation studies, and E. coli XL1-Blue Mrf’ Kan or Tet (Stratagene, La Jolla, CA, United States), or DH5α were used for cloning. Cells were grown aerobically in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) at 37°C with the appropriate antibiotics (100 μg/ml ampicillin, 25 μg/ml chloramphenicol, 50 μg/ml kanamycin). 0.5 mM IPTG or 0.1% rhamnose were used to induce PlacZ- or PrhaB-dependent protein production at indicated time points. E. coli strain BTH101 (Euromedex, Souffelweyersheim, France) was used for bacterial-2-hybrid (B2H) studies. For growth curves, 25-ml cultures were inoculated with OD600 of 0.1 and grown aerobically. The OD600 was determined in 30-min intervals. For induction of gene expression, 0.01% rhamnose was added at indicated time points. 1% glucose was added for repression of rhamnose-dependent gene expression.
8
Growth and Cultivation of Haemophilus parasuis
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H. parasuis strain 174, the reference strain for serovar 7 (SV7), which was isolated from the nose of a healthy pig36 (link) was selected for the challenge experiment. H. parasuis was grown on chocolate plate or in PPLO [pleuropneumonia-like organisms (Oxoid, USA)] supplemented with 60 µg/mL nicotinamide adenine dinucleotide (β-NAD, Sigma-Adrich, USA) and 2.5 mg/mL D-glucose (Sigma-Aldrich, USA) and incubated at 37 °C in presence of 5% CO2. Escherichia coli strains TOP10 (Invitrogen, USA) and ER 2566 (NEB, USA) were grown in LB broth or agar with ampicillin (100 µg/ml) to select for recombinant plasmids. E. coli strain ER2566 was cultured in auto-induction media40 (link) containing ampicillin (100 µg/ml) (Sigma-Aldrich, USA) for protein production.
9
Genetic Regulation Analysis in E. coli and B. subtilis
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The E. coli strains DH5α (Invitrogen) and ER2566 (New England Biolabs) and the B. subtilis strains BG214, BG508 (4 (link)) and NIG2001 (30 (link)) were used. The BG508 strain carries Pδ fused to a promoter-less lacZ gene. This construct was integrated as a unique copy into the amyE locus of the B. subtilis chromosome (4 (link)). In the NIG2001 strain, the wild-type (wt) rpoC gene was substituted in the B. subtilis genome with a version that had a His-tag coding sequence fused to the 3′-end (30 (link)). The Pω bearing pCB30 plasmid was used for promoter analysis, and pHP14 was used for cloning purposes (4 (link)). The plasmids used for gene over-expression were pT712ω bearing ω, pCB746 bearing δ (4 (link),11 (link),16 (link)), and pT712ωD56A bearing the ωD56A gene (this work). The single mutations in the ω gene were obtained by gene synthesis (Genscript). BG508 cells bearing pHP14 carrying either the ω, δ, ωδ, ωΔN19, ω2, ωK52A, ωE53A, ωD56A, ωR64A or ωK70A genes were used for the β-galactosidase assays. The native promoters of these genes were also incorporated into the constructs.
10
Bacteriophage RaK2 Isolation and Protein Production
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Bacteriophage RaK2 and its host K. pneumoniae KV-3 were isolated as described previously. For all phage experiments, CsCl-purified phage stock with a titer of 109–1011 pfu/mL was used.
Escherichia coli strain DH10B (Thermo Fisher Scientific, Vilnius, Lithuania) was used for plasmid propagation, whereas strains BL21 DE3 (Novagene, Madison, WI, USA), Rosetta DE3 (Novagene, Madison, WI, USA), HMS147 DE3 (Novagene, Madison, WI, USA), Arctic Express DE3 (Agilent Technologies, Santa Clara, CA, USA), and ER2566 (New England Biolabs, Ipswich, MA, USA) were used for the production of recombinant proteins. All bacterial cultures were propagated in Luria–Bertani (LB) medium at 37 °C, unless stated otherwise.
Escherichia coli strain DH10B (Thermo Fisher Scientific, Vilnius, Lithuania) was used for plasmid propagation, whereas strains BL21 DE3 (Novagene, Madison, WI, USA), Rosetta DE3 (Novagene, Madison, WI, USA), HMS147 DE3 (Novagene, Madison, WI, USA), Arctic Express DE3 (Agilent Technologies, Santa Clara, CA, USA), and ER2566 (New England Biolabs, Ipswich, MA, USA) were used for the production of recombinant proteins. All bacterial cultures were propagated in Luria–Bertani (LB) medium at 37 °C, unless stated otherwise.
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