1290 chromatography system

Disaccharide-depsipentapeptide (GlcNAc-MurNAc-L-Ala-D-Gln-mDAP-L-Ala-L-Lac) was purified from PG of dacA1dacA2 double mutant of L. plantarum^NC8. PG was digested with mutanolysin (Sigma-Aldrich), and the resulting soluble muropeptides were reduced by NaBH₄ as described previously (Bernard et al., 2011 (link)). The reduced muropeptides were separated by reverse-phase ultra-high-pressure liquid chromatography (RP-UHPLC) with a 1290 chromatography system (Agilent Technologies) and a Zorbax RRHD Eclipse Plus C18 column (100 by 2.1 mm; particle size, 1.8 µm; Agilent Technologies) at 50°C using ammonium phosphate buffer and methanol linear gradient. The eluted muropeptides were detected by UV absorbance at 202 nm. The peak corresponding to the disaccharide-depsipentapeptide was collected. Purified muropeptide was then incubated with 10 µg of purified DltE_extra or DacA1 in 50 mM Tris-HCl, 100 mM NaCl buffer overnight at 37°C. The reaction mixtures were analyzed by RP-UHPLC as described above and by LC-MS with an UHPLC instrument (Vanquish Flex, Thermo Scientific) connected to a Q Exactive Focus mass spectrometer (Thermo Scientific). Mass spectra were collected over the range m/z = 380–1400. Data were processed using Xcalibur QualBrowser v2.0 (Thermo Scientific).

Peptidoglycan was extracted from exponential-phase cells (OD₆₀₀ of ∼0.8) and then digested with mutanolysin as described previously (50 (link)). The resulting soluble muropeptides were reduced with sodium borohydride and separated by reverse-phase UHPLC (RP-UHPLC) with a 1290 chromatography system (Agilent Technologies) and a Zorbax Eclipse Plus C₁₈ Rapid Resolution High Definition column (100 by 2.1 mm with a particle size of 1.8 μm; Agilent Technologies) at 50°C using ammonium phosphate buffer and a methanol linear gradient as described previously (51 (link)). Muropeptides were identified according to their retention times by comparison with an L. lactis muropeptide reference chromatogram (51 (link)). The different muropeptides were quantified by integration of the peak areas, and the percentage of each peak was calculated as the ratio of its area over the sum of all peak areas. The peptidoglycan cross-linking index was calculated according to methods described previously (52 (link)), as follows: (1/2 Σ dimers + 2/3 Σ trimers + 3/4 Σ tetramers)/Σ all muropeptides.