Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications

Abstract

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling.We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution 31 P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase.

On the role of nucleotides and lipids in the polymerization of the actin homolog MreB from a Gram-positive bacterium

“…QCM-D was previously applied to study, for example, the binding affinity of the division proteins MinD and MinE of E. coli to SLBs ( Renner and Weibel, 2012 ). E. coli and B. subtilis cytoplasmic membranes are mainly composed of phospholipids, with the negatively charged phosphatidylglycerol (PG) and the zwitterionic phosphatidylethanolamine (PE) being the dominant species ( Bernat et al, 2016 ; Bishop et al, 1967 ; den Kamp et al, 1969 ; Laydevant et al, 2022 ; Nickels et al, 2017 ; Seydlová and Svobodová, 2008 ; Sohlenkamp and Geiger, 2016 ). Although lipid proportions vary widely depending on the strains and growth conditions, PE is largely dominant in E. coli while PG is more dominant in B. subtilis , indicating that phospholipids are more negatively charged in Gram-positive membranes.…”

Polymerization cycle of actin homolog MreB from a Gram-positive bacterium

“…QCM-D was previously applied to study, for example, the binding affinity of the division proteins MinD and MinE of E. coli to SLBs (Renner & Weibel, 2012). E. coli and B. subtilis membranes are mainly composed of phospholipids, with the anionic phosphatidylglycerol (PG) and the zwitterionic phosphatidylethanolamine (PE) being the dominant species (Bernat et al , 2016; Bishop et al , 1967; den Kamp et al , 1969; Laydevant et al , 2022; Nickels et al , 2017; Seydlova & Svobodova, 2008; Sohlenkamp & Geiger, 2016). Although lipid proportions vary widely depending on the strains and growth conditions, PE is largely dominant in E. coli while PG is more dominant in B. subtilis , indicating that phospholipids are more negatively charged in G+ membranes.…”

In situ solid-state NMR study of antimicrobial peptide interactions with erythrocyte membranes

“…The lipid profile was determined following the procedure of Laydevant et al (37). Lipid extraction was performed in triplicate using the Folch protocol (38), and freeze-dried sample weights were measured using XP205 Mettler Toledo Analytical Balance (up to 0.01 mg readability).…”

“…1D 31 Samples used for solution NMR analyses were then recovered and prepared for FA analysis by GCMS. Briefly, samples were filtered then transesterification was carried out in 2 mL of H 2 SO 4 (2% in methanol) and 0.8 mL of toluene for 10 min at 100°C.The GC-MS analyses were carried out as described by Laydevant et al (37) using a polar HP-5MS column (30 m length  250 µm diameter  0.25 µm film thickness). The injection volume was 1 µL and the oven temperature was programmed to heat at 140 °C for 5 min followed by a 4°C/min ramp up to 300°C, prior to electron ionization and detection with a quadrupole MS. Data acquisition and processing were performed with the Chemstation software.…”

“…31 P resonances of the main phospholipids (PE, PC, SM and PS) were identified by comparison with the chemical shifts of standards and the literature(37). Lipid proportions were determined by integrating those resonances and are reported in Table…”