All bacterial strains and plasmids used in this study are listed in Table S1 in the supplemental material. Escherichia coli strains were used as a general cloning host and cultured at 37°C in lysogeny broth (LB; Teknova). LAB were grown as static cultures in deMan, Rogosa, and Sharpe (MRS) medium (Difco; BD BioSciences) under hypoxic conditions (5% CO2, 2% O2) at 37°C (Lactobacillus casei , Lactobacillus fermentum , Lactobacillus plantarum , Lactobacillus reuteri , Lactobacillus rhamnosus , and Lactobacillus salivarius ) or under static conditions at 30°C in a conventional aerated incubator (Lactobacillus brevis and L. sakei , Fructobacillus fructosus , Pediococcus acidilactici , Weissella paramesenteroides , and Leuconostoc mesenteroides ). Lactobacillus electrocompetent cells were prepared as described previously (7 (link)). The protocol for E. coli EC1000 competent cells preparation was adapted from reference 45 (link). When applicable, erythromycin was supplemented at 5 μg/ml for Lactobacillus strains and at 300 μg/ml for E. coli EC1000. Chloramphenicol was supplemented at 5 μg/ml for Lactobacillus .
Lysogeny broth lb
Manufactured by Teknova
Lysogeny broth (LB) is a commonly used microbial growth medium. It provides essential nutrients to support the growth and proliferation of a variety of bacterial species.
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3 protocols using lysogeny broth lb
1
Cultivation and Transformation of Lactobacillus Strains
2
Genetic Manipulation of Lactobacillus reuteri
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All bacterial strains, plasmids, and oligonucleotides used in this study are listed in Table 1 , Table 5 , and Table 6 , respectively. Lactobacillus reuteri strains were cultured in De Man-Rogosa-Sharpe (MRS) medium (Difco, BD Biosciences). Unless stated otherwise, we prepared bacterial cultures as follows. Lactobacilli were incubated at 37°C under hypoxic conditions (5% CO2, 2% O2). Escherichia coli EC1000 was used as a general cloning host and cultured at 37°C in lysogeny broth (LB; Teknova). Electrocompetent E. coli EC1000 cells were prepared as described previously (41 (link)). Electrocompetent L. reuteri cells were prepared as described previously (42 (link)), with slight modifications. Briefly, L. reuteri cells were grown to an optical density at 600 nm (OD600) of 0.6 and harvested by centrifugation (4°C at 3,200 × g for 5 min). Cell pellets were washed twice with wash butter (0.5 M sucrose, 10% [vol/vol] glycerol). If applicable, erythromycin was supplemented at 5 μg/ml for Lactobacillus strains and at 300 μg/ml for E. coli EC1000 strains.
3
Coculture Protocol for Evolution Experiments
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Cocultures for evolution experiments and phenotyping were conducted on TSA. After each coculture period each species was isolated on their respective isolation agar, Pseudomonas Isolation agar (PIA; BD Difco) and Staphylococcus isolation agar (SIA; BD Difco TSA with 7.5% NaCl). For liquid cultures, bacteria were cultured in lysogeny broth (LB; Teknova) which was supplemented with erythromycin (25 µg/mL) to select for transposon mutants and/or chloramphenicol (10 µg/mL) to maintain fluorescent plasmids. Chemically defined media with glucose (CDMG) was made according to Hussain et al. (1991), with varying levels of aspartate (1.1 mM or 2.2 mM) and glutamate (1.0 mM or 2.0 mM), as needed. CDMG batches were always used within 5 days and stored at room temperature, in the dark. Depleted Trypticase Soy Broth (TSB) medium, used for single-cell microscopy, was prepared by diluting an overnight culture of PAO1 1:100 into 10 mL TSB and growing the culture for either 3 or 16 hours before filter sterilizing (0.2 µm filter, Sarstedt) to remove cells from the supernatant.
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