Expression of the pspA gene stimulates efficient protein export in Escherichia coli

Expression of several mutant forms of outer membrane protein PhoE of Escherichia coli, which are disturbed in normal biogenesis, resulted in high expression of a 26kDa protein. This 26kDa protein fractionated as a peripherally bound inner membrane protein. It appeared to be identical to a previously identified protein (PspA = phage shock protein A) of unknown function that is induced upon infection of E. coli with filamentous phages. PspA was not expressed upon synthesis of mutant PhoE proteins in a secB mutant, nor upon expression of a PhoE mutant that lacks the signal sequence, suggesting that entrance into the export pathway of prePhoE is essential for induction. PspA synthesis was also induced under other conditions that are known to block the export apparatus, i.e. in secA, secD and secF mutants when grown at their non-permissive temperature or upon induction of the synthesis of MalE-LacZ or LamB-LacZ hybrid proteins. The inducing conditions for PspA synthesis suggested a rote for this protein in export. In vivo pulse-chase experiments showed that the translocation of (mutant) prePhoE and of the precursors of other exported proteins was retarded in a pspA mutant strain. Also, in in vitro translocation assays, a role for PspA in protein transport could be demonstrated.     

Filamentous phage assembly: variation on a protein export theme

Biogenesis of both filamentous phage and type-IV pili involves the assembly of many copies of a small, integral inner membrane protein (the phage major coat protein or pilin) into a helical, tubular array that passes through the outer membrane. The occurrence of related proteins required for assembly and export in both systems suggests that there may be similarities at the mechanistic level as well. This report summarizes the properties of filamentous phage and the proteins required for their assembly, with particular emphasis on features they may share with bacterial protein export and pilus biogenesis systems, and it presents evidence that supports the hypothesis that one of the phage proteins functions as an outer membrane export channel.     

PspA can form large scaffolds in Escherichia coli

The phage shock protein A (PspA) of Escherichia coli stabilizes the cytoplasmic membrane under stress conditions. Here we demonstrate that PspA can form hollow spherical or prolate spheroidal particles of about 30-40nm diameter with a scaffold-like arrangement of protein subunits at the surface. The 'PspA-scaffold' is the basic structure that is common to all particles. The PspA-scaffold may be of fundamental importance, as it could allow PspA to stabilize the integrity of membranes through numerous contact points over a large surface area.     

Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway

Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.     

Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin

Dodecamerization and insertion of the outer membrane secretin PulD is entirely determined by the C-terminal half of the polypeptide (PulD-CS). In the absence of its cognate chaperone PulS, PulD-CS and PulD mislocalize to the inner membrane, from which they are extractable with detergents but not urea. Electron microscopy of PulD-CS purified from the inner membrane revealed apparently normal dodecameric complexes. Electron microscopy of PulD-CS and PulD in inner membrane vesicles revealed inserted secretin complexes. Mislocalization of PulD or PulD-CS to this membrane induces the phage shock response, probably as a result of a decreased membrane electrochemical potential. Production of PulD in the absence of the phage shock response protein PspA and PulS caused a substantial drop in membrane potential and was lethal. Thus, PulD-CS and PulD assemble in the inner membrane if they do not associate with PulS. We propose that PulS prevents premature multimerization of PulD and accompanies it through the periplasm to the outer membrane. PulD is the first bacterial outer membrane protein with demonstrated ability to insert efficiently into the inner membrane.     

The first α-helical domain of the vesicle-inducing protein in plastids 1 promotes oligomerization and lipid binding

The vesicle-inducing protein in plastids 1 (Vipp1) is an essential component for thylakoid biogenesis in cyanobacteria and chloroplasts. Vipp1 proteins share significant structural similarity with their evolutionary ancestor PspA (bacterial phage shock protein A), namely a predominantly α-helical structure, the formation of oligomeric high molecular weight complexes (HMW-Cs) and a tight association with membranes. Here, we elucidated domains of Vipp1 from Arabidopsis thaliana involved in homo-oligomerization as well as association with chloroplast inner envelope membranes. We could show that the 21 N-terminal amino acids of Vipp1, which form the first α-helix of the protein, are essential for assembly of the 2 MDa HMW-C but are not needed for formation of smaller subcomplexes. Interestingly, removal of this domain also interferes with association of the Vipp1 protein to the inner envelope. Fourier transform infrared spectroscopy of recombinant Vipp1 further indicates that Escherichia coli lipids bind tightly enough that they can be co-purified with the protein. This feature also depends on the presence of the first helix, which strongly supports an interaction of lipids with the Vipp1 HMW-C but not with smaller subcomplexes. Therefore, Vipp1 oligomerization appears to be a prerequisite for its membrane association. Our results further highlight structural differences between Vipp1 and PspA, which might be important in regard to their different function in thylakoid biogenesis and bacterial stress response, respectively.     

Characterization of the Streptomyces lividans PspA response

Phage shock protein (Psp) is induced by extracytoplasmic stress that may reduce the energy status of the cell. It is encoded in Escherichia coli by the phage shock protein regulon consisting of pspABCDE and by pspF and pspG. The phage shock protein system is highly conserved among a large number of gram-negative bacteria. However, many bacterial genomes contain only a pspA homologue but no homologues of the other genes of the Psp system. This conservation indicates that PspA alone might play an important role in these bacteria. In Streptomyces lividans, a soil-borne gram-positive bacterium, the phage shock protein system consists only of the pspA gene. In this report, we showed that pspA encodes a 28-kDa protein that is present in both the cytoplasmic and the membrane fractions of the S. lividans mycelium. We demonstrated that the pspA gene is strongly induced under stress conditions that attack membrane integrity and that it is essential for growth and survival under most of these conditions. The data reported here clearly show that PspA plays an important role in S. lividans under stress conditions despite the absence of other psp homologues, suggesting that PspA may be more important in most bacteria than previously thought.     

Complex formation of Vipp1 depends on its alpha-helical PspA-like domain

Vipp1 (vesicle-inducing protein in plastids 1) is found in Cyanobacteria and chloroplasts of photosynthetic eukaryotes where it is essential for the formation of the thylakoid membrane. Vipp1 is closely related to the phage shock protein A (PspA), a bacterial protein induced under diverse stress conditions. Vipp1 proteins differ from PspA by an additional C-terminal domain that is required for Vipp1 function in thylakoid biogenesis. We show here that in Cyanobacteria, green algae, and vascular plants, Vipp1 is part of a high molecular mass complex. The complex is formed by multiple copies of Vipp1, and complex formation involves interaction of the central alpha-helical domain that is common to Vipp1 as well as to PspA proteins. In chloroplasts of vascular plants, the Vipp1 complex can be visualized by green fluorescent protein fusion in discrete locations at the inner envelope. Green fluorescent protein fusion analysis furthermore revealed that complex formation is important for proper positioning of Vipp1 at the inner envelope of chloroplasts.     

A trans-envelope protein complex needed for filamentous phage assembly and export

Assembly and export of filamentous phage requires four non-capsid proteins: the outer membrane protein, pIV; the inner membrane proteins, pI and pXI; and a cytoplasmic host factor, thioredoxin. Chemical cross-linking of intact cells demonstrates a trans-membrane complex containing pI and pIV. Formation of the complex protects pI from proteolytic cleavage by an endogenous protease. This protection also requires pXI, which is identical to the C-terminal portion of pI. This indicates that pXI, which is required for phage assembly in its own right, is also part of the complex. This complex forms in the absence of any other phage proteins or the DNA substrate; hence, it represents the first preinitiation step of phage morphogenesis. On the basis of protease protection data, we propose that the preinitiation complex is converted to an initiation complex by binding phage DNA, thioredoxin and the initiating minor coat protein(s).     

Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two-dimensional crystals

TolC is an outer membrane protein required for the export of virulence proteins and toxic compounds without a periplasmic intermediate. We show that TolC is an integral part of the translocator, interacting with inner membrane components, by demonstrating a need for TolC in protein export not only from intact cells but also from sphaeroplasts. To establish the structure of TolC, and thus gain information on how this might be achieved, the protein was purified from the Escherichia coli outer membrane, as a trimer, and crystallized in two-dimensional lattices by reconstitution in phospholipid bilayers. The projection structure at 12 Å resolution showed a threefold symmetric molecule of 58 Å outer diameter, and a single pool of stain filling its centre. Side views parallel to the membrane plane revealed an additional domain outside the membrane. Eighteen membrane-spanning β-strands were predicted for the 51.5 kDa monomer, excluding a 7 kDa C-terminal segment, and this segment was shown to contain a proteinase K-sensitive site that was exposed in reconstituted membranes and sphaeroplasts, but which was protected in intact cells. The combined data suggest that TolC is a trimeric outer membrane protein with each monomer comprising a membrane domain, predicted to be β-barrel, and a C-terminal periplasmic domain. The latter could form part of the bridge to the energized inner membrane component of the translocation complex.     

Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors.

The filamentous phage coat protein pIII has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties. We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of pIII, adjacent to the signal sequence cleavage site. Many functionally defective pIII fusion proteins contained several positively charged amino acids in this region. These residues appear to inhibit proper insertion of pIII into the Escherichia coli inner membrane, blocking the assembly and extrusion of phage particles. Suppressor mutations in the prlA (secY) component of the protein export apparatus dramatically alleviate the phage growth defect caused by the positively charged residues. We conclude that insertion of pIII fusion proteins into the inner membrane can occur by a sec gene-dependent mechanism. The suppressor strains should be useful for increasing the diversity of peptides displayed on pIII in phage libraries.     

Induction and function of the phage shock protein extracytoplasmic stress response in Escherichia coli

The phage shock protein (Psp) F regulon response in Escherichia coli is thought to be induced by impaired inner membrane integrity and an associated decrease in proton motive force (pmf). Mechanisms by which the Psp system detects the stress signal and responds have so far remained undetermined. Here we demonstrate that PspA and PspG directly confront a variety of inducing stimuli by switching the cell to anaerobic respiration and fermentation and by down-regulating motility, thereby subtly adjusting and maintaining energy usage and pmf. Additionally, PspG controls iron usage. We show that the Psp-inducing protein IV secretin stress, in the absence of Psp proteins, decreases the pmf in an ArcB-dependent manner and that ArcB is required for amplifying and transducing the stress signal to the PspF regulon. The requirement of the ArcB signal transduction protein for induction of psp provides clear evidence for a direct link between the physiological redox state of the cell, the electron transport chain, and induction of the Psp response. Under normal growth conditions PspA and PspD control the level of activity of ArcB/ArcA system that senses the redox/metabolic state of the cell, whereas under stress conditions PspA, PspD, and PspG deliver their effector functions at least in part by activating ArcB/ArcA through positive feedback.     

Structure of the filamentous phage pIV multimer by cryo-electron microscopy

The homo-multimeric pIV protein constitutes a channel required for the assembly and export of filamentous phage across the outer membrane of Escherichia coli. We present a 22 A-resolution three-dimensional reconstruction of detergent-solubilized pIV by cryo-electron microscopy associated with image analysis. The structure reveals a barrel-like complex, 13.5 nm in diameter and 24 nm in length, with D14 point-group symmetry, consisting of a dimer of unit multimers. Side views of each unit multimer exhibit three cylindrical domains named the N-ring, the M-ring and the C-ring. Gold labeling of pIV engineered to contain a single cysteine residue near the N or C terminus unambiguously identified the N-terminal region as the N-ring, and the C-terminal region was inferred to make up the C-ring. A large pore, ranging in inner diameter from 6.0 nm to 8.8 nm, runs through the middle of the multimer, but a central domain, the pore gate, blocks it. Moreover, the pore diameter at the N-ring is smaller than the phage particle. We therefore propose that the pIV multimer undergoes a large conformational change during phage transport, with reorganization of the central domain to open the pore, and widening at the N-ring in order to accommodate the 6.5 nm diameter phage particle.