Table 1 lists all strains and plasmids utilized. E. coli and L. bulgaricus were cultured in LB and Man-Rogosa-Sharpe (MRS) (Beijing Land Bridge Technology Co., Ltd. CM187), respectively, at 37°C with no shaking. Saccharomyces cerevisiae Y2HGOLD cells, carrying four reporter genes (HIS3, ADE2, AUR1-C, and MEL1) controlled by the GAL4 promoter (Xu et al., 2015 (link)), were cultured in Yeast Peptone Dextrose (10 g yeast extract, 20 g peptone, 20 g dextrose per liter) or synthetic defined (SD) medium (BD Difco Ltd., USA) at 28°C. Ampicillin (Amp, Sigma Chemical Co, USA), was used for E. coli at 100 μg/mL. Erythromycin (Em, Sigma) and Amp were used for L. bulgaricus at 50 μg/mL each. Chloramphenicol was used for L. bulgaricus at 10 μg/mL.
Erythromycin em
Manufactured by Merck Group
Sourced in United States
Erythromycin (Em) is a macrolide antibiotic produced by the bacterium Saccharopolyspora erythraea. It functions as a protein synthesis inhibitor, preventing the growth and replication of certain bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Chloramphenicol cm, by Merck Group (3 mentions)
Ampicillin amp, by Merck Group (1 mentions)
Mrs broth, by BD (1 mentions)
5 fluorouracil 5 fu, by Merck Group (1 mentions)
Spectinomycin, by MP Biomedicals (1 mentions)
Tetracycline tc, by Merck Group (1 mentions)
Kanamycin kn, by Merck Group (1 mentions)
Ampicillin ap, by Merck Group (1 mentions)
Spectinomycin sp, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Kanamycin km, by Merck Group (1 mentions)
E coli dh5α, by Thermo Fisher Scientific (1 mentions)
E coli pir2, by Thermo Fisher Scientific (1 mentions)
Carbenicillin carb, by IBI Scientific (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
8 protocols using erythromycin em
1
Culturing Bacterial and Yeast Strains
2
Bacterial Propagation and Strain Selection
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The bacterial strains, plasmids, and primers used in this study are listed in Table 1 . Strains of L. acidophilus were propagated statically in de Man-Rogosa-Sharpe (MRS) broth (Difco Laboratories, Inc., Detroit, MI) or on MRS agar (1.5% [wt/vol]; Difco) under aerobic conditions at 37°C or 42°C. Transformants were selected in the presence of 2 μg/ml erythromycin (Em) (Sigma-Aldrich, St. Louis, MO) and/or 2 to 5 μg/ml chloramphenicol (Cm) (Sigma). Escherichia coli was grown in brain heart infusion (BHI) medium (Difco) at 37°C with shaking aeration. E. coli EC101 was propagated in the presence of 40 μg/ml kanamycin (Km) (39 (link)). For upp-based counterselective gene replacement procedures, plasmid-free double recombinants were selected on a glucose semidefined agar medium containing 100 μg/ml 5-fluorouracil (5-FU) (Sigma), as previously described (38 (link)).
3
Engineered Lactococcus and Lactobacillus Strains
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The LAB strains used in this work as recipients for the lux genes were LactococcuslactisMG1614, a laboratorial strain widely used in studies dealing with molecular biology of lactococci [13 (link)], and Lactobacillus salivarius PS2, a probiotic strain originally isolated from human milk [14 (link),15 (link),16 (link)]. L. lactisMG1614 was routinely grown in M17 (Oxoid, Basingstoke, UK) supplemented with 0.5% (w/v) glucose (GM17 medium) and incubated at 30 °C, while L. salivariusPS2 was grown in Man, Rogosa, and Sharpe (MRS) (Oxoid) medium and incubated at 37 °C. Competent Escherichia coli cells were purchased from Bioline (BIO-85027; Bioline Reagents Ltd., London, UK). E. coli was grown in Luria Bertani (LB) medium and incubated at 37 °C. When required, erythromycin (Em) (Sigma-Aldrich, St. Louis, MI, USA) was added to the cultures at the following concentrations: 150 μgmL−1 for E. coli, 2.5 μg mL−1 for L. lactisMG1614, and 5 μg mL−1 for L. salivarius PS2. Previously, the Em resistance of L. salivarius PS2 had been tested in MRS broth containing Em concentrations ranging from 0.25 to 5 μg mL−¹ at 37 °C for 24 h and bacterial growth was detected by measuring the OD600 of the cultures.
4
Cultivation and Preservation of L. acidophilus
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All bacterial strains and plasmids are listed in Table 1 . Bacterial cultures were cryopreserved in their respective media with a 15% glycerol concentration (vol/vol) and stored at -80°C. L. acidophilus was propagated in de Mann, Rogosa and Sharpe (MRS) (Difco Laboratories, Inc., Detroit, MI, USA) broth under static aerobic conditions at 37°C, or on MRS agar (1.5% wt/vol agar, Difco) incubated anaerobically at 37°C for 48 h. Concentrations of 2 μg/mL of erythromycin (Em) (Sigma-Aldrich, St. Louis, MO, USA) and 2–5 μg/mL of chloramphenicol (Cm) (Sigma) were used for plasmid selection in L. acidophilus NCFM, when appropriate. Selection for 5-fluorouracil resistant L. acidophilus was performed by supplementing glucose semi-defined (GSDM) (Kimmel and Roberts, 1998 (link)) agar with a final concentration of 100 μg/mL of 5- fluorouracil (Sigma) (Goh et al., 2009 (link)). Escherichia coli EC1000 was propagated aerobically in Luria-Bertani (Difco) broth at 37°C, or on brain-heart infusion (Difco) solid medium supplemented with 1.5% agar. Antibiotic selection of E. coli was maintained with 40 μg/mL kanamycin (Kn) and 150 μg/mL of Em for recombinant E. coli, when appropriate.
5
Genetic Manipulation of S. aureus Strains
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The S. aureus strains tested in the murine gastrointestinal colonization model are listed in Table 3 ; each was resistant to Sm. Mutations in the S. aureus ica locus, spa, and atl were moved into Sm-resistant Newman by transduction with phage 80α or phage 85 from the original antibiotic marked mutant strains [42 (link)–44 (link)]. The tagO::tet mutation was moved from S. aureus RN4220 into Newman Δatl as described previously [22 (link)]. Mutants were confirmed by PCR or Southern blot analysis and were phenotypically identical to the parental strains in terms of the growth rate, hemolysis on sheep blood agar plates, and the metabolic profile on API Staph test strips (Biomerieux, Inc., Durham, NC). S. aureus strains were cultivated in TSB to the logarithmic growth phase, unless otherwise noted. Sm (0.5 mg/ml; Sigma Chemical Co., St. Louis, Mo.), erythromycin (Em; 5 μg/ml; Sigma), tetracycline (Tc; 2.5 μg/ml; Sigma), or spectinomycin (Spc; 100 μg/ml; MP Biomedicals, Solon, OH) was added to culture medium for selection where appropriate.
6
Bacterial Cultivation and Genetic Manipulation
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Bacterial strains and plasmids used in this study are listed in Table 1 . L. acidophilus strains were propagated in de Man Rogosa and Sharpe (MRS) broth (Difco) under ambient atmospheric conditions, statically or on MRS solid medium containing 1.5% (w/v) agar (Difco) under anaerobic conditions at 37°C, or at 42°C where indicated. Recombinant strains were selected in the presence of 2 μg/ml of erythromycin (Em; Sigma–Aldrich) and/or 2–5 μg/ml of chloramphenicol (Cm; Sigma). Escherichia coli strains were grown in brain heart infusion (Difco) medium at 37°C with aeration. E. coli EC101 was grown in the presence of 40 μg/ml kanamycin (Kn; Sigma–Aldrich) while NCK1911 and transformants were grown with 40 μg/ml Kn and 150 μg/ml Em. Counterselection of plasmid-free excision recombinants was performed using 5-fluorouracil-supplemented glucose semi-defined medium, as previously described (Goh et al., 2009 (link)).
7
Streptococcus agalactiae Isolation and Characterization
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Streptococcusagalactiae GD201008-001 was isolated in 2010 from tilapia with meningoencephalitis in Guangdong Province, China [21 (link)]. GD201008-001 wild-type strain (WT) and its derived luxS mutant strain (ΔluxS) and the luxS complemented strain (CΔluxS) [13 (link)] were maintained in Todd-Hewitt broth (THB) or in chemically defined medium (CDM) [22 (link)]. Escherichia coli was cultured in Luria–Bertani (LB) medium. For plasmids screening required, media were supplemented with antibiotics using the concentration below: 100 μg/mL spectinomycin (Sp, Sigma, St. Louis, MO, USA), 10 μg/mL erythromycin (Em, Sigma), 100 μg/mL kanamycin (Km, Sigma) or 100 μg/mL ampicillin (Ap, Sigma). The details of bacterial strains and plasmids are listed in Additional file 1 . Macrophage cell line RAW 264.7 were maintained in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco).
8
Anaerobic Culturing and Genetic Manipulation
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Detailed information of the strains used in this study is provided in Table S1. All anaerobes were cultured in an anaerobic chamber with an atmosphere of 83% N2, 2% H2 and 15% CO2 at 37 ºC.
For most experiments, Bacteroides strains, AC, CC were grown in Anaerobe Basal Broth (ABB, Oxoid), ER was grown in ABB media with the addition of 3.3 mM sodium acetate (Sigma) and RI was grown in Brain Heart Infusion Broth (BHI, Sigma). We used E. coli pir2 (Invitrogen) for cloning and maintenance of plasmids with R6K origin (pNBU2-ermGb derivatives and pFW1000 derivatives, Table S2). E. coli DH5α (Thermo Fisher Scientific) was used for cloning and maintenance of plasmids with p15A, pSC101ts and ColE1 origins (pFW2000 derivatives, pFW3000 and pFW4000). We used E. coli BW29427 (E. coli Genetic Stock Center, CGSC) for E. coli-Bacteroides conjugations. All E. coli strains were grown aerobically in Luria Bertoni (LB) media. To support the growth of E. coli BW29427, we supplemented LB media with 25 nM of 2,6-Diaminopimelic acid (DAP, Sigma). We used the following antibiotics when required including 100 µg mL -1 carbenicillin (Carb, IBI Scientific), 25 µg mL -1 erythromycin (Em, Sigma) and 200 µg mL -1 gentamicin (Gm, Sigma). We used 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG, Gold Biotechnology) for the induction of FnCpf1.
For most experiments, Bacteroides strains, AC, CC were grown in Anaerobe Basal Broth (ABB, Oxoid), ER was grown in ABB media with the addition of 3.3 mM sodium acetate (Sigma) and RI was grown in Brain Heart Infusion Broth (BHI, Sigma). We used E. coli pir2 (Invitrogen) for cloning and maintenance of plasmids with R6K origin (pNBU2-ermGb derivatives and pFW1000 derivatives, Table S2). E. coli DH5α (Thermo Fisher Scientific) was used for cloning and maintenance of plasmids with p15A, pSC101ts and ColE1 origins (pFW2000 derivatives, pFW3000 and pFW4000). We used E. coli BW29427 (E. coli Genetic Stock Center, CGSC) for E. coli-Bacteroides conjugations. All E. coli strains were grown aerobically in Luria Bertoni (LB) media. To support the growth of E. coli BW29427, we supplemented LB media with 25 nM of 2,6-Diaminopimelic acid (DAP, Sigma). We used the following antibiotics when required including 100 µg mL -1 carbenicillin (Carb, IBI Scientific), 25 µg mL -1 erythromycin (Em, Sigma) and 200 µg mL -1 gentamicin (Gm, Sigma). We used 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG, Gold Biotechnology) for the induction of FnCpf1.
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