Genetic Manipulation of Lactobacillus reuteri

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.