Figure 3 Phospholipids in cpoA mutants. Lipids LY333531 were extracted and separated by two dimensional TLC. 1.D and 2.D: first and second dimension (first dimension: CHCl3/MeOH/H20 = 65:25:4;

second dimension: CHCl3/AcOH/MeOH/H20 = 80:14:10:3). Phospholipids were visualized by spraying with Molybdenum Blue spray reagent. PG: phosphatidylgylcerol; CL: cardiolipin. Spots were assigned according to the phosphatidylglycerol standard (see Additional file 1: Figure S1) and Fischer [42]. Pleiotropic phenotype of cpoA mutants The severe changes in membrane lipids in cpoA mutants is consistent with their pleiotropic phenotype described before [1, 7] which included a reduced generation time in liquid medium, decreased susceptibility to beta-lactams, defects in transformability, and a lower amount of PBP1a with less than 20% compared to the parental strain while the pbp1a transcript was unaffected; alterations in other PBPs were not detected. We first verified these properties for the R6ΔcpoA

mutant: the MIC of piperacillin Ipatasertib manufacturer increased from 0.015 μg/ml (R6) to 0.045 μg/ml, the competence for genetic transformation was approximately 20-fold lower and shifted to the early exponential phase compared to R6, and the amount of PBP1a was decreased (not shown). These phenotypes are reminiscent of those displayed by P104/P106 but were more pronounced in R6ΔcpoA, probably a result of the rpsL allele. Several Tryptophan synthase other tests were then performed in order to see whether the altered glycolipid composition affects also cell envelope related properties in general. These included growth at low pH, the requirement for Mg2+, stationary phase autolysis and lysis induced by Triton X100. In all experiments, cpoA mutants showed a clear phenotype distinct from the R6 strain. Growth was severely affected at pH 6 (Figure 4). At pH 6, cpoA mutants showed an increased requirement for Mg2+ (Figure 5). The stationary phase lysis was slightly delayed in all cpoA mutants (Figure 4). Moreover, lysis induced by low concentrations of Triton X100 proceeded significantly more slowly in all cpoA mutants (Figure 6). Figure 4 Growth of cpoA mutants in low pH medium. Strains

were grown in C-medium, and culture density was monitored by nephelometry [NU]. The growth was examined at pH 8 (circles) and pH 6 (squares). A: R6; B: P104; C: P106; D: R6ΔcpoA. Figure 5 Mg 2+ requirement of cpoA mutations. Strains were grown in C-medium pH 6, and culture density was monitored by nephelometry [NU]. The medium contained either 0.195 mg/ml MgCl2 final concentration (filled circles) or 0.39 mg/ml MgCl2 (squares). A: R6; B: P104; C: P106; D: R6ΔcpoA. Figure 6 Triton induced lysis. Cells were grown to OD600 in C-medium. At OD600 = 0.5, Triton (0.01% final concentration) was added. R6: filled GW786034 in vivo circles; R6ΔcpoA: open circles; P106: open triangles; P104: open squares. Susceptibility to non-beta lactam cell wall antibiotics was also tested.