Apramycin

The bacterial strains and plasmids used in this study are listed in Table 1. All strains were grown in Luria–Bertani (LB) broth (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl) or low-salt LB (reducing NaCl to 5 g/L when hygromycin was needed) with aeration at 37 °C. For plasmid selection in Escherichia coli, apramycin (Duchefa Biochemie), ampicillin and hygromycin (Sigma-Aldrich, Merck Life Science S.L.U., Madrid, Spain) were added at concentrations of 50, 50 and 100 µg/mL, respectively. apramycin and hygromycin were added to the medium at 200 µg/mL and 1250 µg/mL, respectively, to select the recombinants in K. pneumoniae. MGH 78578 ΔmurI was cultivated in LB medium supplemented with 10 mM D-glutamate (Sigma-Aldrich).

All L-form strains were cultured in liquid L phase broth (LPB) and solid L phase medium agar (LPMA). LPB consists of a 1:1 mixture of yeast extract malt extract (YEME) and tryptic soy broth supplemented with 10% sucrose (TSBS) and 25 mM MgCl₂. LPMA consists of LPB supplemented with 1.5% agar, 5% horse serum, and 25 mM MgCl₂ (9 ). P-buffer containing sucrose, K₂SO₄, MgCl₂, trace elements, KH₂PO₄, CaCl₂, and N-tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid (TES) (9 ) was used for transformation and all fusion experiments and was supplemented with 1 mg/mL DNase I (Roche Diagnostics GmbH). The antibiotics apramycin (Duchefa Biochemie) and hygromycin (Duchefa Biochemie) were used for selection and were added at final concentrations of 50 μg/mL and 100 μg/mL, respectively. Growth conditions for all cultures were 30°C in an orbital shaker (New Brunswick Scientific Innova) with 100 rpm for the liquid cultures. Centrifugation (Eppendorf centrifuge 5424) conditions were always 1,000 × g for 10 min (<1 mL) or 30 min (>10 mL), depending on culture volume. The above-mentioned culture conditions and centrifugation settings were applied throughout the study unless mentioned otherwise. All measurements for optical densities (ODs) of samples were done with 200-μL aliquots of culture in a 96-well flat-bottom plate (Sarstedt) using the Tecan Spectramax plate reader.

All L-form strains were cultured in liquid L phase broth (LPB) and solid L phase medium agar (LPMA). LPB consists of a 1:1 mixture of yeast extract malt extract (YEME) and tryptic soy broth supplemented with 10% sucrose (TSBS) and 25 mM MgCl₂. LPMA consists of LPB supplemented with 1.5% agar, 5% horse serum, and 25 mM MgCl₂ (9 ). P-buffer containing sucrose, K₂SO₄, MgCl₂, trace elements, KH₂PO₄, CaCl₂, and N-tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid (TES) (9 ) was used for transformation and all fusion experiments and was supplemented with 1 mg/mL DNase I (Roche Diagnostics GmbH). The antibiotics apramycin (Duchefa Biochemie) and hygromycin (Duchefa Biochemie) were used for selection and were added at final concentrations of 50 μg/mL and 100 μg/mL, respectively. Growth conditions for all cultures were 30°C in an orbital shaker (New Brunswick Scientific Innova) with 100 rpm for the liquid cultures. Centrifugation (Eppendorf centrifuge 5424) conditions were always 1,000 × g for 10 min (<1 mL) or 30 min (>10 mL), depending on culture volume. The above-mentioned culture conditions and centrifugation settings were applied throughout the study unless mentioned otherwise. All measurements for optical densities (ODs) of samples were done with 200-μL aliquots of culture in a 96-well flat-bottom plate (Sarstedt) using the Tecan Spectramax plate reader.