Fig 1.
The lack of Lpp-peptidoglycan attachment impairs Rcs activation in response to stress.
A chromosomal PrprA-lacZ fusion was used to monitor Rcs activity. (A) Deletion of ybiS alone (ΔybiS) or of the 3 L,D-transpeptidases (YbiS, ErfK, YcfS) encoded by Escherichia coli (Δldt3) impaired Rcs activity in response to 5 μg/ml A22 or 0.3 μg/ml mecillinam, compared to WT (F(9, 36) = 39.45, P < 0.0001, 2-way ANOVA). No significant difference was observed between nontreated and treated cells from the mutants. (B) Deletion of mdoG constitutively activated Rcs in the WT background (ΔmdoG) but not in cells lacking L,D-transpeptidase activity (Δldt3ΔmdoG). Baseline Rcs activity in ΔmdoG cells is significantly higher than that in Δldt3ΔmdoG cells (F(6, 14) = 68.25, P < 0.0001, 1-way ANOVA). Expressing WT YbiS from a low-copy plasmid (pAM238), but not an inactive mutant (YbiSC186A), restored Rcs activity in the Δldt3ΔmdoG mutant. Rcs activity in Δldt3ΔmdoG cells harbouring pYbiS was significantly higher than that in Δldt3ΔmdoG cells with empty plasmid or pYbiSC186A (F(6, 14) = 68.25, P < 0.0001, 1-way ANOVA). (C) Expressing WT YbiS from a low-copy plasmid (pAM238), but not an inactive mutant (YbiSC186A), also restored Rcs activity in Δldt3 cells in response to mecillinam and A22. Rcs activity in treated cells harbouring pYbiS was significantly higher than that in treated cells with empty plasmid or pYbiSC186A (F(8, 57) = 20.75, P < 0.0001, 2-way ANOVA). All values were normalised by the average β-galactosidase activity of untreated WT cells. Error bars depict standard error of the mean (n = 6). Lpp, Braun’s lipoprotein; Rcs, regulation of capsule synthesis; WT, wild-type.
Fig 2.
Lpp dictates the distance between the IM and the OM in Escherichia coli.
(A) Cryo-EM revealed uniform membrane structures with WT cells, but lppΔK58 cells, in which Lpp can no longer bind peptidoglycan, displayed blebbing of the OM. E. coli strains expressing longer Lpp variants (lpp+14 and lpp+21) did not bleb or exhibit membrane defects, similar to WT (S5 Fig). (B) From cryo-EM projection images, distances between the IM and the OM were measured along the cell axis (while avoiding substantial blebbing regions in cells expressing LppΔK58) and plotted in 1-nm bins. The lppΔK58 mutant strain had a periplasmic (IM-to-OM) distance that was about 3 nm larger than that of the WT strain (P < 0.0001, Kruskal-Wallis) and had a much broader spread of data. The lpp+14 and lpp+21 mutant strains had periplasmic distances that were 3 nm and 4 nm, respectively, larger than WT (P < 0.0001, Kruskal-Wallis), indicating that the IM-to-OM distance varies as a function of the length of Lpp. Avg, average; cryo-EM, cryo-electron microscopy; IM, inner membrane; Lpp, Braun’s lipoprotein; OM, outer membrane; WT, wild-type.
Fig 3.
Increasing the length of Lpp impairs Rcs activity during stress, an effect that can be counteracted by extending the N-terminal linker of RcsF.
β-galactosidase activity was used as a reporter of Rcs activity as in Fig 1. (A) The lengthened Lpp variants Lpp+14 and Lpp+21 impaired the activity of the Rcs system in cells treated with 5 μg/ml A22 or 0.3 μg/ml mecillinam compared to WT (F(4, 33) = 23.75, P < 0.0001, 2-way ANOVA). No significant difference was observed between nontreated and treated mutant cells. (B) Lpp+14 and Lpp+21 also impaired the Rcs response to mdoG deletion compared to WT (F(5, 32) = 62.23, P < 0.0001, 1-way ANOVA). (C, D) Expressing the extended RcsF mutant (RcsF+7) from a low-copy plasmid (pAM238) restored (C) the Rcs response to mdoG deletion and (D) the Rcs response to A22 or mecillinam completely or partially in cells expressing Lpp+14 or Lpp+21, respectively. Rcs activity in ΔmdoGΔrcsF cells harbouring pRcsF+7 was significantly higher than that in ΔmdoGΔrcsF cells with empty plasmid or pRcsF (F(7, 16) = 54.94, P < 0.0001, 1-way ANOVA). Rcs activity in A22- or mecillinam-treated cells harbouring pRcsF+7 was significantly higher than treated cells carrying empty plasmid (pAM238) or pRcsF (F(14, 61) = 22.56, P < 0.0001, 2-way ANOVA). All values were normalised by the average β-galactosidase activity of untreated WT cells. Error bars represent standard error of the mean (n = 6). Lpp, Braun’s lipoprotein; Rcs, regulation of capsule synthesis; WT, wild-type.