Properties of the phage-shock-protein (Psp) regulatory complex that govern signal transduction and induction of the Psp response in Escherichia coli

Abstract

The phage-shock-protein (Psp) response maintains the proton-motive force (pmf) under extracytoplasmic stress conditions that impair the inner membrane (IM) in bacterial cells. In Escherichia coli transcription of the pspABCDE and pspG genes requires activation of σ(54)-RNA polymerase by the enhancer-binding protein PspF. A regulatory network comprising PspF-A-C-B-ArcB controls psp expression. One key regulatory point is the negative control of PspF imposed by its binding to PspA. It has been proposed that under stress conditions, the IM-bound sensors PspB and PspC receive and transduce the signal(s) to PspA via protein-protein interactions, resulting in the release of the PspA-PspF inhibitory complex and the consequent induction of psp. In this work we demonstrate that PspB self-associates and interacts with PspC via putative IM regions. We present evidence suggesting that PspC has two topologies and that conserved residue G48 and the putative leucine zipper motif are determinants required for PspA interaction and signal transduction upon stress. We also establish that PspC directly interacts with the effector PspG, and show that PspG self-associates. These results are discussed in the context of formation and function of the Psp regulatory complex.

Fig. 1.

Topologies of Psp proteins. (a) Schematic representation of PspA, PspB, PspC and PspG topologies according to Elderkin et al. (2005), Jones et al. (2003), Kleerebezem et al. (1996) and Engl et al. (2009), respectively. Per, periplasm; IM, inner membrane; Cyt, cytoplasm; N, N-terminus of the protein; C, C-terminus of the protein. (b) Representation of the predicted conserved domain organization of PspC: the cytoplasmic extrusion domain (Cyt, residues 1–39), the transmembrane portion (TM, residues 40–64), the periplasmic extrusion domain (Per, residues 65–119) and the conserved PspC domain (residues 1–68, light grey). The putative leucine zipper motif (LeuZ; residues 77–98) is as indicated. The numbering refers to E. coli PspC.

Fig. 2.

Co-purification of PspA, PspB and PspG with PspC_6His. PspC_6His co-expressed with PspB (pRD047His) was purified using metal-affinity chromatography (see Methods). As a control non-tagged PspC co-expressed with PspB (pRD047) was also purified. Peak fractions from both purifications (corresponding to fractions 2–4), were visualized using Western blotting and (a) anti-PspC, (b) anti-PspB, (c) anti-PspA or (d) anti-PspG antibodies as indicated. In (a) the positions of monomeric PspC (PspC; arrow) and an additional slower-migrating anti-PspC-reactive (double asterisk) species are as indicated. PspB (b), PspA (and non-specific band, asterisk) (c) and PspG (d) co-purified with PspC_6His fractions (2–4) but not with PspC (non-tagged). (e) PspC_6His co-expressed with PspB was also purified from a ΔpspA strain and the peak fractions were visualized using Western blotting and anti-PspG antibodies. The positions of molecular mass marker proteins (kDa) are indicated.

Fig. 3.

PspG–GFP forms higher oligomers in vivo. The number of molecules in fluorescent PspG–GFP complexes was estimated using ImageJ software. The intensity of a single pixel in a fluorescent PspG–GFP (pGJ7) cluster expressed in E. coli MG1655 ΔpspG cells (MVA40) was measured and compared to GFP fluorescence in E. coli MG1655 cell lysates harbouring pDSW209 (GFP alone). (a) The mean fluorescence intensity of 50 PspG–GFP complexes within living cells was on average at least three times higher than that of GFP spots, suggesting that PspG can form at least a dimer/trimer in vivo. (b) The frequency distribution among the 50 complexes analysed illustrates that PspG–GFP self-assembles into a single major distinct oligomeric class.

Fig. 4.

Oligomerization state of PspC. Western blots (using anti-PspC) illustrating PspC expression from the chromosome in either WT (MG1655) or ΔpspA (MG1655ΔpspA) cells (in the absence or presence of pIV). The positions of species that specifically cross-react with anti-PspC are highlighted as monomeric PspC (13.5 kDa; arrow) and a putative dimer (double asterisk). The positions of the marker proteins (kDa) are indicated. Below: the relative expression levels of monomeric PspC (labelled as PspC monomer) and the putative PspC dimer (double asterisk) were quantified within each strain tested, and the results expressed as a percentage of the induced corresponding protein band (+pIV; lanes 2 and 4, 100 %). ‘Proteins’ refers to the loading control. Importantly, these results demonstrate that in the absence of PspA (lanes 3 and 4), the relative expression levels of monomeric PspC are clearly highly elevated compared to WT in the presence of pIV, whereas the putative dimer expression level remains relatively unchanged, suggesting that this band corresponds to an unspecific anti-PspC cross-reacting species.

Fig. 5.

psp expression in the presence of overexpressed PspBC. (a) Induction of chromosomal Φ(pspA–lacZ) in a ΔpspC strain (MVA13) by overexpression of PspB (pAJM1), PspC (pAJM2) or PspBC (pAJM3) (using 0.02 % Ara). (b) Overexpression of PspC decreases pmf while co-expression with PspB counteracts this effect. Δψ was determined in a ΔpspF strain (MG1655ΔpspF) overexpressing PspB, PspC or PspBC. (c) Overexpression of PspBC directly induces psp. PspBC (pAJM3) or PspB^LeuZmC (pGJ49; PspB^LeuZm does not transduce the psp-inducing signal) were co-expressed in either ΔpspBC (MVA45) or ΔpspBCΔarcB (MVA83; ΔarcB diminishes psp induction) cells (using 0.02 % Ara). PspBC were co-expressed in a ΔpspBC strain (since the arcB mutation reduces induction by pIV; using 0.02 % Ara) in the presence of pIV. Vector, pBAD18-cm. As a control, psp expression was determined in the absence of PspA (ΔpspA, MVA27; to prevent negative regulation).

Fig. 6.

psp expression by the PspC fragments. (a) Full-length PspC is required for signal transduction upon pIV-dependent psp-inducing stress. Induction of the chromosomal Φ(pspA–lacZ) fusion in a ΔpspC strain (MVA13) expressing a low level of PspC (1–119, pAJM2) or PspC fragments (1–68, pAJM7; 40–68, pAJM5; 40–119, pAJM8) (using 0.001 % Ara) in the absence or presence of pIV (pGJ4) (see Methods). (b) The TM-periplasmic region of PspC (PspC_40–119) is sufficient for PspB-independent induction of psp. Induction of the chromosomal Φ(pspA–lacZ) fusion in a ΔpspC strain (MVA13) overexpressing PspC (1–119, pAJM2) or PspC fragments on its own (as in a) or with PspB [PspBC (pAJM3) or PspBC fragments (1–68, pAJM12; 40–68, pAJM10; 40–119, pAJM13)] (using 0.02 % Ara) (see Methods). (c) Overexpression of PspC_1–68 decreases pmf while co-expression with PspB counteracts this effect in a ΔpspF strain (MG1655ΔpspF).

Fig. 7.

PspC determinants involved in signal transduction and induction of psp. (a) The PspC LeuZ and residue G48 are required for pIV-induced psp expression. Induction of the chromosomal Φ(pspA–lacZ) fusion in a ΔpspC strain (MVA13) expressing low-level PspBC (pAJM3) or PspBC mutants (PspC mutants: G48A, pGJ54; G74A, pGJ55; LeuZm, pGJ57) (using 0.001 % Ara) in the absence or presence of pIV (pGJ4) (see Methods). As a control, pIV-dependent induction of psp in WT cells (MVA4) is presented. (b) High-level co-expression of PspBC^G48A, PspBC^LeuZm, (pGJ60) and (pGJ61) mutants failed to directly induce psp expression. Induction of the chromosomal Φ(pspA–lacZ) fusion in a ΔpspC strain (MVA13) by overexpression of PspBC or PspBC mutants (using 0.02 % Ara). (c) The PspC periplasmic region may exist in two topologies: schematic illustrating the potential topologies of PspC (A, B or C) and PspC_40–119 (the periplasmic region containing the LeuZ; D).