Isolation, purification, and analysis of LPS

An overnight culture of WT and ΔCbpD was diluted 1:100 and grown in LB medium to the exponential growth phase (OD₆₀₀: 0.8–0.9). Bacteria were washed with PBS, and the pellet was kept at −80 °C until LPS extraction. LPS was extracted from washed and packed cells by the Westphal and Jann (1965) extraction method¹²⁶. Briefly, samples were suspended in water and an equal volume of pre-heated 90% phenol (Sigma), followed by stirring of the reaction mixture at 65 °C for 40 min¹²⁷. Samples were then immediately cooled down to 10 °C and centrifuged (2968 × g, 45 min, 10 °C). The top phenol saturated aqueous layer containing LPS was carefully pipetted out and extensively dialyzed against water to remove phenol. The samples were lyophilized, ultra-centrifuged (120,000 × g, 4 h, 10 °C), and the precipitated LPS was subjected to additional Benzonase nuclease (EMD Millipore) and Proteinase K (Sigma) treatments, followed by the second round of ultra-centrifugation at (120,000 × g, 4 h, 10 °C). The precipitated LPS was used for monosaccharide and fatty acid composition analysis by GC-MS (Agilent Technologies) as a TMS derivative of methyl-glycoside or alditol acetate derivative. Analysis of keto-deoxy-D-manno-8-octanoic acid (Kdo) was done by ultra-performance liquid chromatography with fluorescence detection (RP-UPLC, Acquity, Waters) using a BEH-C18 column (Waters). Briefly, a known amount of LPS was hydrolyzed using 2 M HOAc (80 °C, 3 h), followed by removal of acid, and tagging Kdo with 4, 5 methylenedioxy-1, 2-phenylenediamine dihydrochloride (DMB, Sigma) prior to UPLC analysis. For lipid-A analysis, a known amount of purified LPS was hydrolyzed using 2% HOAc (100 °C, 2 h). Next, the lipid-A was precipitated by centrifugation (5000 × g, 5 min), dried by lyophilization, and dissolved in a 3:1 chloroform: methanol (v/v) mixture containing 1 mM ammonium-formate (Sigma). The samples were analyzed using an LTQ-XL Ion-Trap (Thermo Scientific) mass spectrometer in negative mode.