Expression in periplasmic space of Shewanella oneidensis

A Shewanella expression system has been used for an overproduction of c-type multiheme proteins. The proteins were exported to the periplasmic space for the maturation. Since the periplasmic expression system is attractive, especially for protease-sensitive proteins, an expression vector containing a signal peptide was constructed for expressions in the periplasmic space of Shewanella oneidensis. To evaluate the system, two eukaryotic proteins which originally do not have signal sequences and are difficult to express in Escherichia coli, were selected. The first is human cytochrome c. Properties of the recombinant cytochrome c were identical to those previously reported, indicating the protein is intact. The other was potato calcium-dependent protein kinase. The protein was expressed in periplasmic space. These results indicated that the system is generally applicable for any protein expression including c-type cytochromes, protease-sensitive proteins and those with multi-disulfide bonds because of transportation to the periplasmic space.     

Structure of the periplasmic stress response protein CpxP

CpxP is a novel bacterial periplasmic protein with no homologues of known function. In Gram-negative enteric bacteria, CpxP is thought to interact with the two-component sensor kinase, CpxA, to inhibit induction of the Cpx envelope stress response in the absence of protein misfolding. CpxP has also been shown to facilitate DegP-mediated proteolysis of misfolded proteins. Six mutations that negate the ability of CpxP to function as a signaling protein are localized in or near two conserved LTXXQ motifs that define a class of proteins with similarity to CpxP, Pfam PF07813. To gain insight into how these mutations might affect CpxP signaling and/or proteolytic adaptor functions, the crystal structure of CpxP from Escherichia coli was determined to 2.85-Å resolution. The structure revealed an antiparallel dimer of intertwined α-helices with a highly basic concave surface. Each protomer consists of a long, hooked and bent hairpin fold, with the conserved LTXXQ motifs forming two diverging turns at one end. Biochemical studies demonstrated that CpxP maintains a dimeric state but may undergo a slight structural adjustment in response to the inducing cue, alkaline pH. Three of the six previously characterized cpxP loss-of-function mutations, M59T, Q55P, and Q128H, likely result from a destabilization of the protein fold, whereas the R60Q, D61E, and D61V mutations may alter intermolecular interactions.     

Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli

High level expression of TEM beta-lactamase results in the accumulation of precursor and mature protein in the insoluble fraction of Escherichia coli. The mature polypeptide is sequestered in protein aggregates (inclusion bodies) located within the periplasmic space whereas the insoluble precursor is present in the cytoplasm. With the native beta-lactamase, aggregation is observed when the rate of expression exceeds 2.5% of the total protein synthesis rate. Substitution of the native signal sequence with the outer membrane protein A (OmpA) leader peptide results in extensive aggregation of only the mature protein. Furthermore, for OmpA-beta-lactamase, the accumulation of mature insoluble protein is independent of the rate of protein synthesis. These observations cannot be accounted by the kinetics of export of the OmpA-beta-lactamase and the native precursor, therefore suggesting that the signal sequence affects the conformation of the newly secreted mature polypeptide and in turn, the folding pathway. Previously, we have shown that the aggregation of the mature protein secreted using its own signal sequence can be inhibited by growing the cells in the presence of non-metabolizable sugars such as sucrose (Bowden, G., and Georgiou, G. (1988) Biotechnol. Prog. 4, 97-101). We show here that this phenomenon is not related to osmotic effects, changes in beta-lactamase translation or precursor processing. It follows that the addition of sugars exerts a direct effect on the in vivo pathway of aggregation and folding, in analogy with the well characterized effect of sugars in vitro.     

Lanthanide accumulation in the periplasmic space of Escherichia coli B.

Treatment of growing Escherichia coli B with lanthanide ions [lanthanum(III), terbium(III), and europium(III)] and subsequent aldehyde-OsO4 fixation caused areas of high contrast to appear within the periplasm (the space between inner and outer membrane of the cell envelope). X-ray microanalysis of ultrathin sections of Epon-embedded or acrylic resin-embedded cells revealed the presence of the lanthanide and of phosphorus in the areas, whose contrast greatly exceeded that of other stained structures. Comparatively small amounts of the lanthanide were also present in the outer membrane and in the cytoplasm. The distribution of the periplasmic areas of high contrast was found to be random and not clustered at areas of current or future septum formation. Irregular cell shapes were observed after lanthanide treatment before onset of fixation. In contrast to glutaraldehyde-OsO4 fixation, glutaraldehyde used as the sole fixer caused a scattered distribution of the lanthanide. Cryofixation (slam-freezing) and freeze substitution revealed a lanthanum stain at both the periplasm and the outer part of the outer membrane. Deenergization of the cell membrane by either phage T4 or carbonyl cyanide m-chlorophenylhydrazone abolished the metal accumulation. Furthermore, addition of excess calcium, administered together with the lanthanide solution, diminished the quantity and size of areas of high contrast. Cells grown in media of high NaCl concentration revealed strongly stained areas of periplasmic precipitates, whereas cells grown under low-salt conditions showed very few high-contrast patches in the periplasm. Terbium treatment (during fixation) enhanced the visibility of the sites of inner-outer membrane contact (the membrane adhesion sites) in plasmolized cells, possibly as the result of an accumulation of the metal at the adhesion domains. The data suggest a rapid interaction of the lanthanides with components of the cell envelope, the periplasm, and the energized inner membrane.     

Structure and mechanism of bacterial periplasmic transport systems

Bacterial periplasmic transport systems are complex, multicomponent permeases, present in Gram-negative bacteria. Many such permeases have been analyzed to various levels of detail. A generalized picture has emerged indicating that their overall structure consists of four proteins, one of which is a soluble periplasmic protein that binds the substrate and the other three are membrane bound. The liganded periplasmic protein interacts with the membrane components, which presumably form a complex, and which by a series of conformational changes allow the formation of an entry pathway for the substrate. The two extreme alternatives for such pathway involve either the formation of a nonspecific hydrophilic pore or the development of a ligand-binding site(s) on the membrane-bound complex. One of the membrane-bound components from each system constitutes a family of highly homologous proteins containing sequence domains characteristic of nucleotide-binding sites. Indeed, in several cases, they have been shown to bind ATP, which is thus postulated to be involved in the energy-coupling mechanism. Interestingly, eukaryotic proteins homologous to this family of proteins have been identified (mammalianmdr genes and Drosophilawhite locus), thus indicating that they perform a universal function, presumably related to energy coupling in membrane-related processes. The mechanism of energy coupling in periplasmic permeases is discussed.     

Multilayered distribution of peptidoglycan in the periplasmic space of Escherichia coli

When a staining technique using phosphotungstic acid (PTA) in 10% (w/v) chromic acid was applied to cells of Escherichia coli, the periplasmic space was seen as a dark 15-nm-thick layer of uniform appearance and constant width. Our observations are consistent with peptidoglycan being the main material stained. Isolated sacculi as well as purified peptidoglycan (protein free) were also stained by the same procedure, the thickness of the peptidoglycan being 8.8 +/- 1.8 and 6.6 +/- 1.5 nm, respectively. The increased thickness of the PTA-stained layer in stationary phase cells correlated well with the increased thickness of isolated sacculi or purified peptidoglycan and with the increased amount of peptidoglycan in such cells. Thickness measurements on isolated peptidoglycan were compatible with a two to three layer structure for material from exponential phase cells and with a four to five layer structure for that from stationary phase cells. Furthermore, the results indicated an uneven distribution of peptidoglycan material in the periplasmic space, the peptidoglycan spanning the space from the inner to the outer membrane.     

Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli

The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.     

Compartmentalization of the periplasmic space at division sites in gram-negative bacteria.

Phase-contrast and serial-section electron microscopy were used to study the patterns of localized plasmolysis that occur when cells of Salmonella typhimurium and Escherichia coli are exposed to hypertonic solutions of sucrose. In dividing cells the nascent septum was flanked by localized regions of periseptal plasmolysis. In randomly growing populations, plasmolysis bays that were not associated with septal ingrowth were clustered at the midpoint of the cell and at 1/4 and 3/4 cell lengths. The localized regions of plasmolysis were limited by continuous zones of adhesion that resembled the periseptal annular adhesion zones described previously in lkyD mutants of S. typhimurium (T. J. MacAlister, B. MacDonald, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 80:1372-1376, 1983). When cell division was blocked by growing divC(Ts) cells at elevated temperatures, the localized regions of plasmolysis were clustered along the aseptate filaments at positions that corresponded to sites where septum formation occurred when cell division was permitted to resume by a shift back to the permissive temperature. Taken together the results are consistent with a model in which extended zones of adhesion define localized compartments within the periplasmic space, predominantly located at future sites of cell division.     

Improving the stability of a foreign protein in the periplasmic space of Escherichia coli

An efficient expression/export vector comprising the entire phoS (phosphate binding protein) gene fused to a synthetic gene encoding the human growth hormone releasing factor (mhGRF) has recently been constructed [1]. The hybrid protein (PhoS-mhGRF) was exported to the periplasmic space. However, in this location proteolytic degradation occurred at the C-terminal region. Phenylmethylsulfonyl fluoride (PMSF) increased the stability of the hybrid protein indicating that a serine protease may be involved in the proteolytic cleavage. The correct export and subsequent degradation of the recombinant protein in the periplasmic space were demonstrated in situ using double immunogold labeling on ultrathin sections. Using a phoS-based expression/export vector in the presence of PMSF, 2-4 mg of hybrid protein per liter of culture could be obtained.     

Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli

The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented. The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner. Comparison to the structures of Wolinella succinogenes NrfA and Sulfospirillum deleyianum NrfA, which accept electrons from a membrane-anchored tetra-heme cytochrome (NrfH), reveals notable differences in the protein surface around heme 2, which may be the docking site for the redox partner. The structure shows that four of the NrfA hemes (hemes 2-5) have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand. Analysis of NrfA in solution by magnetic circular dichroism (MCD) suggested that the oxygen ligand arose from water. Electron paramagnetic resonance (EPR) spectra were collected from electrochemically poised NrfA samples. Broad perpendicular mode signals at g similar 10.8 and 3.5, characteristic of weakly spin-coupled S = 5/2, S = 1/2 paramagnets, titrated with E(m) = -107 mV. A possible origin for these are the active site Lys-OH(2) coordinated heme (heme 1) and a nearby bis-His coordinated heme (heme 3). A rhombic heme Fe(III) EPR signal at g(z) = 2.91, g(y) = 2.3, g(x) = 1.5 titrated with E(m) = -37 mV and is likely to arise from bis-His coordinated heme (heme 2) in which the interplanar angle of the imidazole rings is 21.2. The final two bis-His coordinated hemes (hemes 4 and 5) have imidazole interplanar angles of 64.4 and 71.8. Either, or both, of these hemes could give rise to a "Large g max" EPR signal at g(z)() = 3.17 that titrated at potentials between -250 and -400 mV. Previous spectroscopic studies on NrfA from a number of bacterial species are considered in the light of the structure-based spectro-potentiometric analysis presented for the E. coli NrfA.     

Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efflux protein TolC

The trimeric TolC protein of Escherichia coli comprises an outer membrane beta-barrel and a contiguous alpha-helical barrel projecting across the periplasm. This provides a single 140 A long pore for multidrug efflux and protein export. We have previously reported that trivalent cations such as hexammine cobalt can severely inhibit the conductivity of the TolC pore reconstituted in planar lipid bilayers. Here, isothermal calorimetry shows that Co(NH(3))(6)(3+) binds to TolC with an affinity of 20 nM. The crystal structure of the TolC-Co(NH(3))(6)(3+) complex was determined to 2.75 A resolution, and showed no significant difference in the protein when compared with unliganded TolC. An electron density difference map revealed that a single ligand molecule binds at the centre of the periplasmic entrance, the sole constriction of TolC. The octahedral symmetry of the ligand and the three-fold rotational symmetry of the TolC entrance determine a binding site in which the ligand forms hydrogen bonds with the Asp(374) residue of each monomer. When Asp(374) was substituted by alanine, high affinity ligand binding was abolished and inhibition of TolC pore conductivity in lipid bilayers was alleviated. Comparable effects followed independent substitution of the neighbouring Asp(371), indicating that this aspartate ring also contributes to the high affinity ligand binding site. As the electronegative entrance is widely conserved in the TolC family, it may be a useful target for the development of inhibitors against multidrug resistant pathogenic bacteria.     

The OmpL porin does not modulate redox potential in the periplasmic space of Escherichia coli

The Escherichia coli DsbA protein is the major oxidative catalyst in the periplasm. Dartigalongue et al. (EMBO J., 19, 5980-5988, 2000) reported that null mutations in the ompL gene of E.coli fully suppress all phenotypes associated with dsbA mutants, i.e. sensitivity to the reducing agent dithiothreitol (DTT) and the antibiotic benzylpenicillin, lack of motility, reduced alkaline phosphatase activity and mucoidy. They showed that OmpL is a porin and hypothesized that ompL null mutations exert their suppressive effect by preventing efflux of a putative oxidizing-reducing compound into the medium. We have repeated these experiments using two different ompL null alleles in at least three different E.coli K-12 genetic backgrounds and have failed to reproduce any of the ompL suppressive effects noted above. Also, we show that, contrary to earlier results, ompL null mutations alone do not result in partial DTT sensitivity or partial motility, nor do they appreciably affect bacterial growth rates or block propagation of the male-specific bacteriophage M13. Thus, our findings clearly demonstrate that ompL plays no perceptible role in modulating redox potential in the periplasm of E.coli.     

Colicins are not transiently accumulated in the periplasmic space before release from colicinogenic cells

Colicins are released into the spent medium from colicinogenic cells. The pathway of release has been investigated in this study. The localization in producing cells of colicins A, E3 and of cloacin DF13 has been determined at various times after mitomycin C addition: no transient accumulation in the periplasmic space of colicinogenic E. coli K12 strains was detected by electron microscopy for any of the bacteriocins tested. Furthermore, asynchronous induction in individual cells was detected for each bacteriocin tested. These results strongly suggest that colicins, as well as cloacin DF13, do not transit through the periplasmic space before release from colicinogenic cells.     

Auxin-dependent cell wall depositions in the epidermal periplasmic space of graviresponding nodes of Tradescantia fluminensis

Differential growth of the nodal regions of graviresponding Tradescantia fluminensis (Wandering Jew) was analysed with special respect to the extension-restricting epidermal cells of the opposite growing and growth-inhibited organ flanks. Gravicurvature of horizontally gravistimulated isolated nodes depends on auxin (indolyl-3-acetic acid, IAA) and shows a node-specific profile in which the third node below the tip showed the greatest response. Exogenously supplied gibberellic acid induced no gravitropic growth. Vertically oriented isolated nodes supplied with exogenous IAA showed, on an electron microscopical level, conspicuous membrane invaginations with adjacent wall depositions restricted to the outer tangential epidermal cell walls. Their number was more than doubled by exogenously supplied Ca2+, which inhibited IAA-induced growth. No such changes could be detected in water-incubated segments or inner tissues of IAA-supplied segments. Gravistimulated differential growth of nodes of intact shoots and of nodal segments was characterized by changes similar to the ones induced by exogenous IAA, with greatly increased numbers of wall depositions within the epidermal cells of the growth-inhibited upper organ flank. Similar to the gravistimulated wall depositions, an asymmetric distribution pattern of Ca2+ was detected in the epidermal cell walls employing x-ray energy spectrum analysis (EDX). The results indicate that growth of nodes of Tradescantia fluminensis is regulated via IAA-induced secretion and subsequent infiltration of wall components enabling wall extension. The data support the hypothesis that temporary differential growth during gravicurvature of Tradescantia fluminensis is mediated by the antagonistic effect of Ca(2+)-ions on the infiltration of IAA-induced wall-loosening components into the outer, extension-restricting epidermal walls thereby inhibiting growth.     

An elegant means of self-protection in gram-negative bacteria by recognizing and extruding xenobiotics from the periplasmic space

Infection of Pseudomonas aeruginosa in cystic fibrosis patients is a major cause of mortality. This organism shows wide ranging antibiotic resistance that is largely attributable to the expression of xenobiotic efflux pump(s). Here, we show a novel mechanism by which the resistance-nodulation-division-type xenobiotic transporter expels potential hazards and protects the interior of the cells. The xenobiotic transporters MexB and MexY preferentially export beta-lactam and aminoglycoside antibiotics, respectively. When two large extramembrane loops of MexY were replaced by the corresponding loops of MexB, the hybrid protein exhibited beta-lactam selectivity (MexB-type), but failed to recognize aminoglycoside. As the transmembrane segment of MexB was replaced with a corresponding transmembrane segment of MexY, one-by-one for all 12 segments, all the hybrid proteins showed MexB-type antibiotic selectivity. These results clearly demonstrated that the resistance-nodulation-division-type efflux pump in P. aeruginosa selects and transports substrates via the domains that largely protrude over the cytoplasmic membrane. The transmembrane segments were unlikely to have been involved in substrate selectivity. These observations led us to propose a novel mechanism by which the xenobiotic transporters in Gram-negative bacteria select and expel substrates from the periplasmic space before potential hazards penetrate into the cytoplasmic membrane.     

Structure and conservation of the periplasmic targeting factor Tic22 protein from plants and cyanobacteria

Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.