FIG 6.
Phosphatidylglycerol is the substrate for the synthesis of ECAPG. (A) PG is not essential in strains where pressure on lipoprotein maturation is relieved (ΔrcsF Δlpp). Growth curves were performed with PgsA depletion strains in this background alone (blue), with wzzE and waaL deletions (orange), or an isogenic strain with a wecA deletion (green) to investigate growth phenotypes caused by loss of ECA and disruptions to peptidoglycan biosynthesis caused by accumulation of ECA precursor. Cultures were diluted 1:10,000 and treated with arabinose to induce expression from the PBAD promoter (solid lines) or glucose to repress expression (hashed lines). Depletion of PgsA in the ΔrcsF Δlpp background allowed full growth. Cells without ECA (ΔwecA-wzzE ΔwaaL) stopped growing when PG was depleted and then showed a slow decrease in OD600. However, cells making only ECAPG (ΔwzzE ΔwaaL) grew to stationary phase and then quickly lysed. The data are means of four biological replicates ± the SEM. (B) CL is not essential. Cultures for growth curves were grown as in panel A. Depletion of ClsA in strains with deletions of clsA, clsB, and clsC did not cause lysis either alone or when combined with ΔwzzE ΔwaaL. The data are means of three biological replicates ± the SEM. (C) ECALPS levels were assayed by WGA staining of strains with deletions in the indicated genes. Where indicated, cultures were grown with 50 mM Mg2+ to allow survival of strains lacking PE (pss93). Strains lacking CL and PE had small but significant decreases in ECALPS levels. The strain lacking PG had a large decrease in ECALPS; however, ECALPS levels remained detectable. The data are the mean of three biological replicates ± the SEM. *, P < 0.05 (by paired t test); **, P < 0.005 (by paired t test). (D) The activity of Pwec was assayed as in Fig. 3. The data are shown as the fold of the relative luminescence to the wild type and are means of six biological replicates ± the SEM. No significant changes determined by t test were observed for contiguous data points. (E) ECAPG levels in strains without CL (ΔclsABC) or without PE (pss93) were assayed in the ΔwzzE ΔwaaL (ECAPG only) strain background. Cultures for lanes 5 and 6 were grown with 50 mM Mg2+. The ΔwecA strain serves as a negative control. Both strains lacking CL and strains lacking PE retained production of ECAPG. The images are representative of two independent experiments. (F) Since depletion of PG causes lysis in an ECAPG only background, the effect of the ΔpgsA mutation was assayed in a ΔwaaL background where only ECAPG can be observed by immunoblotting. Immunoblot analysis was performed to assay ECAPG levels in strains without CL (ΔclsABC) or without PG (ΔrcsF Δlpp ΔpgsA). The ΔwecA strain serves as a negative control, indicating nonspecific bands. Deletion of waaL in the presence of ECACYC causes inhibition of ECAPG synthesis (56), leading to lower ECA levels overall. ECAPG is observed in the strain without CL. However, only a low-abundance, low-molecular-weight band (arrow) is observed in the strain without PG. The images are representative of three independent experiments.