Rrlc 1200 system

Peptidoglycan analysis was done as described earlier (Glauner et al., 1988 (link)). Essentially, the sacculi were digested with 10 U mutanolysin (Sigma-Aldrich, St. Louis, MO, United States) at 37°C in 25 mM Tris–HCl (pH 8.0) for 16 h. mutanolysin hydrolyzes β-1→4 glycosidic bond between MurNAc and GlcNAc residues in PG to form soluble muropeptide fragments. After centrifugation at 30,000 × g for 15 min, soluble muropeptide fragments in supernatant fraction were collected and reduced with 1 mg of sodium borohydride in 50 mM sodium borate buffer (pH 9.0) for 30 min and excess borohydride was destroyed by adding 20% phosphoric acid. pH was adjusted to 3–4 and the samples were loaded onto a reverse-phase C18 column (Zorbax 300 SB; 250 mm × 4.6 mm, 5 mm) connected to Agilent technologies RRLC 1200 system. Column temperature was 55°C and binding was done at a flow rate of 0.5 ml/min with 1% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA) for 10 min. Muropeptides were eluted in a gradient of 1–10% acetonitrile containing 0.1% TFA at a flow rate of 0.5 ml/min for the next 60 min (using RRLC online software called Chemstation). The absorbance of muropeptides was detected at 205 nm.

An Agilent RRLC 1200 system was utilized as the liquid chromatography (LC) delivery system with the use of Agilent ZORBAX Rapid Resolution HT column and the MS parameters were fixed as reported previously [17 (link)]. The high resolution electrospray ionization mass spectrometry (ESI-MS) was performed with an Agilent 6,500 Q-TOF LC/MS system operated in ESI-positive mode. The m/z value range to detect the precursor ions was set at m/z 150–400. The precursor ion scan mode targeting at the production ion with m/z 102. Analysis of MS spectra generated and AHL profile was performed as described previously [17 (link)].

HPLC analysis was performed as described earlier [2 (link),6 (link)] with some modifications. PG sacculi were treated with appropriate enzymes for 16h at 37°C, the proteins were heat-inactivated through boiling, and insoluble material was removed by centrifugation (14,000 RPM at RT). Subsequently, the soluble muropeptides were collected and mixed with equal volume of 50 mM sodium borate buffer (pH 9.0). The anomeric carbons of muropeptides were reduced by adding 1 mg of sodium borohydride. Excess borohydride was destroyed by adding 1/20^th volume of orthophosphoric acid and pH was adjusted between 2–4 before loading onto a Zorbax 300 SB RP-C18 (250 X 4.6mm, 5 μm) column. Separation of muropeptides was done by RP-HPLC using Agilent technologies RRLC 1200 system. Samples were injected onto a preheated column at 55°C and binding was allowed at a flow rate of 0.5 ml per minute with a solvent containing 1% acetonitrile and 0.1% trifluoroacetic acid (TFA) for 10 min. A gradient of 1–10% (MepM treated substrates) or 1–15% (untreated PG) acetonitrile containing 0.1% TFA was used for final elution at a flow rate of 0.5 ml per minute. Absorbance was detected at 205 nm.