TABLE 1

tolQ, tolR, and tolA mutant salmonellae accumulate PGl and PE molecules within the OM in comparison to the wild typea

m/z of GPL speciesLevel (ng/μl) (avg ± SD) of the individual GPL molecule in the OM fraction of:
WTtolQ mutanttolR mutanttolA mutant
PGl
    71922.43 ± 3.99#34.53 ± 6.41*33.72 ± 6.90*35.26 ± 6.09*
    7331.53 ± 0.28#1.71 ± 0.28*2.14 ± 0.482.08 ± 0.44*
    7473.21 ± 0.70#5.96 ± 1.38*6.55 ± 2.03*6.05 ± 1.35*
    7734.89 ± 0.86#10.16 ± 1.39*11.46 ± 2.92*10.22 ± 1.27*
PE
    688116.76 ± 15.35133.01 ± 14.88*129.50 ± 9.52129.04 ± 14.61
    71435.43 ± 2.7759.42 ± 3.85*48.72 ± 2.84*47.68 ± 1.67*
    71652.91 ± 6.7967.15 ± 7.97*69.50 ± 10.27*66.60 ± 4.83*
    74239.23 ± 6.0869.87 ± 7.16*76.17 ± 11.12*68.12 ± 4.32*
  • a Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify outer membrane glycerophospholipid (OM GPL) molecules. Overnight cultures of wild-type and tolQ, tolR, and tolA mutant salmonellae were back diluted 1:100 in 1 liter of LB broth and incubated at 37°C and 225 rpm for ∼3 h to mid-exponential growth phase. The cells were collected by centrifugation and resuspended in a sucrose solution, which began the osmotic spheroplasting and lysis procedure. The membranes were isolated by discontinuous sucrose density gradient ultracentrifugation (63). Three independent experiments were conducted, and the average values (ng/μl) ± standard deviation (SD) for these biological replicates are depicted. A number sign (#) indicates a statistically significant difference for wild-type S. Typhimurium in the level of the phosphatidylglycerol (PGl) molecule within the OM relative to the level of the PGl molecule within the IM, P < 0.05 (Fig. 3A; Table S3). An asterisk indicates a statistically significant difference in the level of the GPL molecule within the OM for the mutant relative to the level of the molecule within the OM of the wild type, P < 0.05 (Fig. 3B and C). m/z, mass-to-charge ratio; PE, phosphatidylethanolamine.