Fig 1.
The autotransporter (AT) secretion pathway.
(A) Schematic overview of AT secretion mechanism. The passenger domain remains in a non-native, unfolded conformation during transit across the periplasm. (B) Constructs used in this study. The lengths of the AT variants are drawn to scale and include annotations of promoters and regions encoding annotated domains.
Fig 2.
Expression of prn in an E. coli degP deletion strain is lethal.
(A) Viability of the E. coli degP null strain versus its parent strain was assessed after transforming plasmids that constitutively express AT variants. Viability was measured by counting colony forming units (CFUs); see Methods. Error bars are standard deviations from three biological repetitions. (B) Growth of the E. coli degP null strain versus its parent strain, with or without IPTG-induction of prn expression. Shown are representative growth curves.
Fig 3.
Differential requirement of the DegP protease and chaperone activity during expression of model ATs.
(A) E. coli viability as a function of degP, with or without constitutive expression of prn. To test if the DegP chaperone alone is sufficient to restore viability, a protease deficient version of DegP [16] was produced by co-transforming a plasmid expressing degP S210A. (B) Expression of prn results in production of the precursor and processing of the passenger (samples shown here were collected from cells described in (A). The role of the DegP protease for pertactin accumulation and secretion was assessed by expressing prn when only the DegP chaperone was present, versus the DegP chaperone plus the chromosomal wild type DegP. In the presence of the DegP protease activity, less pertactin precursor accumulates (compare lanes 1 vs. 2). (C) Wild type pet was expressed overnight in E. coli with or without a chromosomal copy of degP (same samples as grey bars in Fig 2A). In the absence of DegP, more full-length precursor accumulates in whole cell lysates (WCL). The yield of secreted Pet in the media after overnight production was not significantly altered with or without DegP. (D) After transforming prnΔβ-barrel in the E. coli degP null strain, very small colonies appeared after prolonged incubation (black triangle). (A, D) Error bars are standard deviations from at least three biological repetitions.
Table 1.
Summary of phenotypes observed upon AT expression in the E. coli degP deletion strain.
Fig 4.
The co-translational DsbA signal sequence, but not the post-translational PhoA signal sequence, bypasses lethality.
(A) Production and secretion of pertactin chimera constructs in the E. coli degP null strain versus its parent. Bacteria were grown to stationary phase and protein production was analyzed by western blotting of whole cell lysates. Successful secretion of the pertactin passenger is measured by appearance of the processed passenger [24,25,29,32,50]. Two non-adjacent portions of the same blot are shown (grey line). (B) Expression of signal sequence chimera constructs does not dramatically affect growth of wild type E. coli. (C) Plasmids to express dsbA(ss):prn and phoA(ss):prn under control of a constitutive promoter were transformed in the E. coli degP null mutant versus its parent as described in Fig 2A. Error bars are standard deviations from at least three biological replicates. (D) Expression of dsbA(ss):prn, dsbA(ss):prnΔβ-barrel and pet does not affect growth in the degP null strain. (B, D) Shown are representative growth curves.
Fig 5.
The Pet signal sequence partially rescues lethality of pertactin in the degP null strain.
(A) The degP null strain and its parent were transformed with plasmids to express pet(ss):prn or prn(ss):pet as described in Fig 2A. Error bars are standard deviations from at least three biological replicates. Filled triangle: After transforming a plasmid to express pet(ss):prn in the degP deletion strain, very small colonies appeared after prolonged incubation. (B) Growth of E. coli with a chromosomal copy of degP is not significantly altered when AT signal sequence chimera variants are produced. (C) Expression of pet and prn(ss):pet does not affect growth in the degP deletion background, whereas expression of pet(ss):prn reduces the growth rate. (D) The chimera pet(ss):prn was expressed and the chimera protein was secreted in E. coli regardless of the presence or absence of DegP, but the secretion yield was reduced in the degP deletion strain. Two non-adjacent portions of the same blot are shown (grey line). (E) Expression of prn(ss):pet resulted in accumulation of the chimera protein at reduced levels in E. coli. Likewise, secretion of the Pet passenger to the medium was reduced, regardless of the presence or absence of DegP. (B, C) Shown are representative growth curves.
Fig 6.
Analysis of proteome-wide changes during pertactin-induced death of E. coli ΔdegP.
(A, B) Expression of prn was induced in the presence (A) or absence (B) of a chromosomal copy of degP and cells were collected at 2 and 5 h post induction for mass spectrometry. For wild type E. coli, two samples were collected for mass spectrometry analysis (2 h and 5 h post prn induction). For the degP deletion strain, a total of four samples were collected (2 h and 5 h post prn induction and uninduced controls at the same time points). To express prn, construct T5p-prn was used and expression was induced in mid-log phase (see also Fig 2B). (C) Workflow to generate dataset for unbiased analysis of the proteome during E. coli death. (D) Quantification of representative periplasmic proteins across all experimental conditions indicates that overall periplasmic proteins decrease during death (last condition, see pink star in (B, C)). (E) Quantification of proteins in the σE stress response pathway reveals a loss of σE during death (last condition, pink star). Note that the σE protein itself along with CutC and YggN are undetectable in the last condition. Error bars correspond to the standard error (∑/mean) for each label-free measurement.
Fig 7.
Model for the interplay between IM translocation, DegP chaperone activity, and the σE stress response.
(A) Co-translational IM translocation results in passenger conformations in the periplasm that are tolerated in the absence of DegP. (B) Post-translational IM translocation results in conditional lethal AT conformations in the periplasm. (C) Lethality can be prevented by interactions with the chaperone DegP. (D) In the absence of DegP, accumulation of pertactin intermediates compromises periplasmic integrity and results in leakage of periplasmic material. This leads to inactivation of the σE stress response. σE inactivation is lethal and causes cell death.