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PubMed2002

Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases.

NapC is a tetra-haem member of a family of bacterial membrane-anchored multi-haem c -type cytochromes implicated in electron transfer between membrane quinols and periplasmic enzymes. The water-soluble tetra-haem fragment of Paracoccus pantotrophus NapC has been expressed as a periplasmic protein (NapC(sol)) in Paracoccus denitrificans, P. pantotrophus and Escherichia coli. Site-specific mutagenesis of NapC(sol), combined with spectroscopic studies, suggests that each haem iron centre has bis -histidinyl co-ordination. Four proximal ligands arise from each of four Cys-Xaa-Xaa-Cys-His haem-binding motifs; candidates for the four distal ligands are His(81), His(99), His(174) and His(194). NapC(H81A), NapC(H99A), NapC(H174A) and NapC(H194A) mutants (with alanine substituted for each of the four candidate residues) have all been purified from E. coli. In each case, one of the haems has become high-spin, as judged by the presence of a broad absorption band between 620 nm and 650 nm for the oxidized cytochrome; this feature is absent for wild-type protein and presumably arises because of the absence of the distal histidine ligand from one of the haems. NapC(H81A) and NapC(H174A) are less well expressed in E. coli than NapC(H99A) and NapC(H194A) and cannot be detected when expressed in P. denitrificans or P. pantotrophus. In vitro and in vivo complementation studies demonstrate that the soluble periplasmic NapC can mediate electron transfer from quinols to the periplasmic nitrate reductase. This capacity was retained in vitro with the NapC(H99A) and NapC(H194A) mutants but was lost in vivo. A model for the structural organization of NapC(sol) into two domains, each containing a di-haem pair, is proposed. In this model, each haem pair obtains one distal haem ligand from its own domain and a second from the other domain. The suggestion of two domains is supported by observations that the 24 kDa NapC(sol) cleaves to yield a 12 kDa haem-staining band. Determination of the cleavage site showed it was between two equally sized di-haem domains predicted from sequence analysis.

Amino Acid Sequence↗
PubMed1999

Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins.

Using a cross-linking approach, we have analyzed the function of Skp, a presumed molecular chaperone of the periplasmic space of Escherichia coli, during the biogenesis of an outer membrane protein (OmpA). Following its transmembrane translocation, OmpA interacts with Skp in close vicinity to the plasma membrane. In vitro, Skp was also found to bind strongly and specifically to pOmpA nascent chains after their release from the ribosome suggesting the ability of Skp to recognize early folding intermediates of outer membrane proteins. Pulse labeling of OmpA in spheroplasts prepared from an skp null mutant revealed a specific requirement of Skp for the release of newly translocated outer membrane proteins from the plasma membrane. Deltaskp mutant cells are viable and show only slight changes in the physiology of their outer membranes. In contrast, double mutants deficient both in Skp and the periplasmic protease DegP (HtrA) do not grow at 37 degrees C in rich medium. We show that in the absence of an active DegP, a lack of Skp leads to the accumulation of protein aggregates in the periplasm. Collectively, our data demonstrate that Skp is a molecular chaperone involved in generating and maintaining the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of Gram-negative bacteria.

Bacterial Outer Membrane Proteins↗
PubMed2003

Roles of DegP in prevention of protein misfolding in the periplasm upon overexpression of penicillin acylase in Escherichia coli.

Enhancement of the production of soluble recombinant penicillin acylase in Escherichia coli via coexpression of a periplasmic protease/chaperone, DegP, was demonstrated. Coexpression of DegP resulted in a shift of in vivo penicillin acylase (PAC) synthesis flux from the nonproductive pathway to the productive one when pac was overexpressed. The number of inclusion bodies, which consist primarily of protein aggregates of PAC precursors in the periplasm, was highly reduced, and the specific PAC activity was highly increased. DegP was a heat shock protein induced in response to pac overexpression, suggesting that the protein could possibly suppress the physiological toxicity caused by pac overexpression. Coexpression of DegP(S210A), a DegP mutant without protease activity but retaining chaperone activity, could not suppress the physiological toxicity, suggesting that DegP protease activity was primarily responsible for the suppression, possibly by degradation of abnormal proteins when pac was overexpressed. However, a shortage of periplasmic protease activity was not the only reason for the deterioration in culture performance upon pac overexpression because coexpression of a DegP-homologous periplasmic protease, DegQ or DegS, could not suppress the physiological toxicity. The chaperone activity of DegP is proposed to be another possible factor contributing to the suppression.

Enzyme Precursors↗
PubMed2025

Repair of oxidized methionine residues in the chaperone Spy maintains periplasmic proteostasis under chlorite stress in Escherichia coli.

The bacterial cell envelope is exposed to various stresses, including oxidative stress caused by different types of oxidants, such as reactive oxygen species (ROS) and reactive chlorine species (RCS). In Escherichia coli, the reduction of chlorate into chlorite, a toxic RCS compound, induces the expression of the MsrPQ system, which repairs periplasmic proteins oxidized at methionine residues (methionine sulfoxide, Met-O). In this study, using a proteomic-based approach, we show that chlorite stress also triggers the overproduction of the periplasmic molecular chaperone Spheroplast Protein Y (Spy). This response is mediated by the activation of the BaeSR two-component system. Furthermore, both in vivo and in vitro evidence reveal that Spy's susceptibility to oxidation is critical for its chaperone activity. We demonstrate that the MsrPQ repair system ensures Spy's functionality by reducing its Met-O, thereby safeguarding its role in periplasmic protein homeostasis. Overall, this work reveals Spy as a key target of chlorite-induced oxidative damage and underscores the essential role of MsrPQ in preserving periplasmic protein quality control.

Methionine↗
PubMed2002

A strategy for in vivo screening of subtilisin E reaction specificity in E. coli periplasm.

We developed a protocol for efficient expression of the functional serine protease, subtilisin E, in Escherichia coli periplasm that permits direct in vivo measurement of the enzyme's catalytic activity. Activity assays and SDS-PAGE/Western blot analysis showed that the levels of expressed subtilisin varied and were correlated with both the culture conditions and the induction procedures. The highest level of subtilisin expression was achieved at 0.10-0.15% (w/v) of arabinose as inducer and a temperature of 20-22 degrees C, and was ca. eightfold higher as compared to the expression level at 30 degrees C. Cultivation of bacterial cells to a steady state of balanced growth before induction was required for uniform subtilisin expression in cell cultures growing in wells of microtiter plates. Amidase and esterase cell-based kinetic assays on microtiter plates were developed based on the direct measurement of subtilisin activity in vivo. Intact E. coli cells displaying wild-type, dimethylformamide-resistant, and temperature-resistant subtilisins were assayed on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-acetyl-Phe-p-nitrophenyl ester for their amidase and esterase activity, respectively. Additionally, the periplasmic fractions were isolated from the three E. coli strains expressing the respective subtilisins and tested for amidase activity. The amidase activity of the three subtilisins was ca. 15-fold higher than the esterolytic activity when measured in both the intact cells and in the periplasmic fractions. The strategy combining periplasmic expression of subtilisins with two cell-based kinetic assays permits rapid screening of subtilisin mutant libraries for desired activities.

Amidohydrolases↗
PubMed1999

Isolation of salt-induced periplasmic proteins from Synechocystis sp. strain PCC 6803.

Periplasmic proteins were obtained from control cells and salt-adapted cells of the cyanobacterium Synechocystis sp. PCC 6803 using the method of cold osmotic shock. Two of these proteins (PP 1, apparent mol. mass 27.6 kDa, and PP 3, apparent mol. mass 39.9 kDa) were accumulated in high amounts in the periplasm of salt-adapted cells, while the major periplasmic protein (PP 2, apparent mol. mass 36.0 kDa) was accumulated independently from salt. After isolation from gels and partial sequencing, the proteins could be assigned to proteins deduced from the complete genome sequence of Synechocystis. Neither salt-induced periplasmic proteins (PP 1, Slr0924 and PP 3, Slr1485) exhibited sequence similarity to proteins of known function from databases. The major protein (PP 2-Slr0513) showed significant sequence similarities to iron-binding proteins. All proteins included typical leader sequences at their N-terminus.

Adaptation, Physiological↗
PubMed2002

The periplasmic space in Thermus thermophilus: evidence from a regulation-defective S-layer mutant overexpressing an alkaline phosphatase.

The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed in a mutant (HB8(Delta)UTR1) defective in the regulation of its S-layer (surface crystalline layer). This mutant forms round multicellular bodies (MBs) surrounded by a common envelope as the culture approaches the stationary phase. Confocal microscopy revealed that the origin of the MBs is the progressive detachment of the external layers coupled with the accumulation of NH(2)-containing material between the external envelopes and the peptidoglycan. A specific pattern of proteins was found as soluble components of the intercellular space of the MBs by a single fractionation procedure, suggesting that they are periplasmic-like components. To demonstrate this, we cloned a gene ( phoA) from T. thermophilus HB8 encoding a signal peptide-wearing alkaline phosphatase (AP), and engineered it to be overexpressed in the mutant from a shuttle vector. Most of the AP activity (>80%) was found as a soluble component of the MBs' intercellular fraction. All these data indicate that Thermus thermophilus actually has a periplasmic space which is functionally similar to that of Proteobacteria. The potential application of the HB8(Delta)UTR1 mutant for the overexpression of periplasmic thermophilic proteins is discussed.

Alkaline Phosphatase↗
PubMed2005

The membrane anchors of the heme chaperone CcmE and the periplasmic thioredoxin CcmG are functionally important.

The cytochrome c maturation system of Escherichia coli contains two monotopic membrane proteins with periplasmic, functional domains, the heme chaperone CcmE and the thioredoxin CcmG. We show in a domain swap experiment that the membrane anchors of these proteins can be exchanged without drastic loss of function in cytochrome c maturation. By contrast, the soluble periplasmic forms produced with a cleavable OmpA signal sequence have low biological activity. Both the chimerical CcmE (CcmG'-'E) and the soluble periplasmic CcmE produce low levels of holo-CcmE and thus are impaired in their heme receiving capacity. Also, both forms of CcmE can be co-precipitated with CcmC, thus restricting the site of interaction of CcmE with CcmC to the C-terminal periplasmic domain. However, the low level of holo-CcmE formed in the chimera is transferred efficiently to cytochrome c, indicating that heme delivery from CcmE does not involve the membrane anchor.

Bacterial Outer Membrane Proteins↗
PubMed2005

The periplasmic binding protein of a tripartite tricarboxylate transporter is involved in signal transduction.

A new type of solute importer has been identified recently in various bacterial genera and called the tripartite tricarboxylate transporter (TTT). TTTs consist of two cytoplasmic membrane proteins and a periplasmic solute-binding protein. In the whooping cough agent Bordetella pertussis, a TTT system that has been called BctCBA mediates the uptake of citrate, with BctA and BctB being the membrane components and BctC, the periplasmic protein. Here, we describe that the expression of the bctCBA operon is induced by the presence of citrate in the milieu. The signalling cascade involves both BctC and the signal transduction two-component system BctDE, encoded by an operon adjacent to bctCBA. Furthermore, two-hybrid analyses and affinity chromatography experiments indicated that citrate-liganded BctC interacts with the periplasmic domain of the sensor protein, BctE. Thus, BctC is part of the signalling cascade leading to upregulation of the transporter operon in the presence of its solute, a new function for periplasmic binding proteins of TT transporters.

Bacterial Proteins↗
PubMedDate not supplied

Analysis of the Escherichia coli Tol-Pal and TonB systems by periplasmic production of Tol, TonB, colicin, or phage capsid soluble domains.

The aim of this review is to describe an in vivo assay of the interactions taking place in the Tol-Pal or TonB-ExbB-ExbD envelope complexes in the periplasm of Escherichia coli and between them and colicins or g3p protein of filamentous bacteriophages. Domains of colicins or periplasmic soluble domains of Tol or TonB proteins can be artificially addressed to the periplasm of bacteria by fusing them to a signal sequence from an exported protein. These domains interact specifically in the periplasm with the Tol or TonB complexes and disturb their function, which can be directly detected by the appearance of specific tol or tonB phenotypes. This technique can be used to detect new interactions, to characterize them biochemically and to map them or to induce tol or tonB phenotypes to study the functions of these two complexes.

Bacterial Proteins↗
PubMed2003

Localisation of Helicobacter pylori catalase in both the periplasm and cytoplasm, and its dependence on the twin-arginine target protein, KapA, for activity.

Helicobacter pylori induces a severe inflammatory response in the gastric mucosa. It is able to withstand the inflammatory response by producing proteins such as KatA and KapA. The C-terminus of KatA possesses a unique tetra-lysine motif not found in other catalases or other known protein sequences. Mutants deficient in this motif were constructed by site-directed mutagenesis. Cytoplasmic and periplasmic catalase activities were measured for the parental strain, a truncated KatA mutant (deficient in the unique C-terminal tetra-lysine motif) and a previously constructed KapA-deficient mutant (confirming previous observations regarding the possible periplasmic localisation of KatA). No differences were observed in the cytoplasmic catalase activities, however, the KapA-deficient mutant had approximately 5.5 times less catalase activity in the periplasmic extract when compared to the periplasmic preparations of either parental strain or KatA truncated mutant. N-terminal sequencing of KatA revealed no cleaved N-terminal signal peptide, indicating Sec-independent transport. These findings support previous reports that there is some form of interaction between KatA and KapA of H. pylori, an interaction which still needs to be characterised.

Amino Acid Sequence↗
PubMed2005

Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization.

Escherichia coli EnvZ is a membrane sensor histidine kinase that plays a pivotal role in cell adaptation to changes in extracellular osmolarity. Although the cytoplasmic histidine kinase domain of EnvZ has been extensively studied, both biochemically and structurally, little is known about the structure of its periplasmic domain, which has been implicated in the mechanism underlying its osmosensing function. In the present study, we report the biochemical and biophysical characterization of the periplasmic region of EnvZ (Ala38-Arg162). This region was found to form a dimer in solution, and to consist of two well-defined domains: an N-terminal a-helical domain and a C-terminal core domain (Glu83-Arg162) containing both a-helical and b-sheet secondary structures. Our pull-down assays and analytical ultracentrifugation analysis revealed that dimerization of the periplasmic region is highly sensitive to the presence of CHAPS, but relatively insensitive to salt concentration, thus suggesting the significance of hydrophobic interactions between the homodimeric subunits. Periplasmic homodimerization is mediated predominantly by the C-terminal core domain, while a regulatory function may be attributed mainly to the N-terminal a-helical domain, whose mutations have been shown previously to produce a high-osmolarity phenotype.

Amino Acid Sequence↗
PubMed1992

A periplasmic protein disulfide oxidoreductase is required for transformation of Haemophilus influenzae Rd.

The mutated gene in JG16, a Haemophilus influenzae strain deficient in competence-induced DNA binding and uptake, was cloned and the wild-type allele was sequenced. The gene was shown by Northern analysis to be constitutively expressed on a 1.7-kilobase transcript. The gene product was identified as a 20.6-kDa protein targeted to the periplasm. The protein contains the sequence Cys-Pro-His-Cys (CPHC) and is highly similar to two other periplasmic CPHC motif-containing proteins: DsbA, an Escherichia coli protein (45% identity, 87% homology) and TcpG, a Vibrio cholerae protein (32% identity, 74% homology). Both DsbA and TcpG promote disulfide bond formation in periplasmic proteins, are required for pilus biogenesis, and, like thioredoxin, are capable of reducing insulin in vitro. The Haemophilus protein was shown to complement an E. coli mutation in DsbA and was named Por (periplasmic oxidoreductase). In JG16 the competence-dependent redistribution of inner membrane proteins did not occur. These findings suggest that Por is required for the correct assembly and/or folding of one or more disulfide-containing cell envelope protein involved either in competence development or in the DNA-binding and -uptake machinery.

Amino Acid Sequence↗
PubMed2001

The early interaction of the outer membrane protein phoe with the periplasmic chaperone Skp occurs at the cytoplasmic membrane.

Spheroplasts were used to study the early interactions of newly synthesized outer membrane protein PhoE with periplasmic proteins employing a protein cross-linking approach. Newly translocated PhoE protein could be cross-linked to the periplasmic chaperone Skp at the periplasmic side of the inner membrane. To study the timing of this interaction, a PhoE-dihydrofolate reductase hybrid protein was constructed that formed translocation intermediates, which had the PhoE moiety present in the periplasm and the dihydrofolate reductase moiety tightly folded in the cytoplasm. The hybrid protein was found to cross-link to Skp, indicating that PhoE closely interacts with the chaperone when the protein is still in a transmembrane orientation in the translocase. Removal of N-terminal parts of PhoE protein affected Skp binding in a cumulative manner, consistent with the presence of two Skp-binding sites in that region. In contrast, deletion of C-terminal parts resulted in variable interactions with Skp, suggesting that interaction of Skp with the N-terminal region is influenced by parts of the C terminus of PhoE protein. Both the soluble as well as the membrane-associated Skp protein were found to interact with PhoE. The latter form is proposed to be involved in the initial interaction with the N-terminal regions of the outer membrane protein.

Binding Sites↗
PubMed2001

Overproduction of bacterial protein disulfide isomerase (DsbC) and its modulator (DsbD) markedly enhances periplasmic production of human nerve growth factor in Escherichia coli.

Production of eukaryotic proteins with multiple disulfide bonds in the Escherichia coli periplasm often encounters difficulty in obtaining soluble products with native structure. Human nerve growth factor beta (NGF) contains three disulfide bonds between nonconsecutive cysteine residues and forms insoluble aggregates when expressed in E. coli. We now report that overexpression of Dsb proteins known to catalyze formation and isomerization of disulfide bonds can substantially enhance periplasmic production of NGF. A set of pACYC184-based plasmids that permit dsb expression under the araB promoter were introduced into cells carrying a compatible plasmid that expresses NGF. The efficiency of periplasmic production of NGF fused to the OmpT signal peptide was strikingly improved by coexpression of DsbCD or DsbABCD proteins (up to 80% of total NGF produced). Coexpression of DsbAB was hardly effective, whereas that of DsbAC increased the total yield but not the periplasmic expression. These results suggest synergistic roles of DsbC and DsbD in disulfide isomerization that appear to become limiting upon NGF production. Furthermore, recombinant NGF produced with excess DsbCD (or DsbABCD) was biologically active judged by the neurite outgrowth assay using rat PC12 cells.

Animals↗
PubMed2003

Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles.

Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5alpha, HB101, and MC4100 transformed with plasmid-encoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5alpha and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.

Bacterial Outer Membrane Proteins↗
PubMed2005

A bacterial glutathione transporter (Escherichia coli CydDC) exports reductant to the periplasm.

Glutathione (GSH), a major biological antioxidant, maintains redox balance in prokaryotes and eukaryotic cells and forms exportable conjugates with compounds of pharmacological and agronomic importance. However, no GSH transporter has been characterized in a prokaryote. We show here that a heterodimeric ATP-binding cassette-type transporter, CydDC, mediates GSH transport across the Escherichia coli cytoplasmic membrane. In everted membrane vesicles, GSH is imported via an ATP-driven, protonophore-insensitive, orthovanadate-sensitive mechanism, equating with export to the periplasm in intact cells. GSH transport and cytochrome bd quinol oxidase assembly are abolished in the cydD1 mutant. Glutathione disulfide (GSSG) was not transported in either Cyd(+) or Cyd(-) strains. Exogenous GSH restores defective swarming motility and benzylpenicillin sensitivity in a cydD mutant and also benzylpenicillin sensitivity in a gshA mutant defective in GSH synthesis. Overexpression of the cydDC operon in dsbD mutants defective in disulfide bond formation restores dithiothreitol tolerance and periplasmic cytochrome b assembly, revealing redundant pathways for reductant export to the periplasm. These results identify the first prokaryotic GSH transporter and indicate a key role for GSH in periplasmic redox homeostasis.

ATP-Binding Cassette Transporters↗
PubMed1995

Escherichia coli periplasmic protein FepB binds ferrienterobactin.

Most high-affinity systems for iron uptake in Gram-negative bacteria are thought to employ periplasmic-binding-protein-dependent transport. In Escherichia coli, FepB is a periplasmic protein required for uptake of iron complexed to its endogenously-synthesized siderophore enterobactin (Ent). Direct evidence that ferrienterobactin (FeEnt) binds to FepB is lacking because high background binding by FeEnt prevents use of the usual binding protein assays. Here the membrane localization vehicle LppOmpA [Francisco, J.A., Earhart, C.F. & Georgiou, G. (1992). Proc Natl Acad Sci USA 89, 2713-2717] was employed to place FepB in the E. coli outer membrane. Plasmid pTX700 was constructed and shown to encode, under lac operator control, the 'tribrid' protein LppOmpAFepB; the carboxy-terminal FepB portion lacks at most two amino acids of mature FepB. After short induction periods, most of the tribrid was in the outer membrane. A number of LppOmpAFepB species could be detected; some were degradation products and some may be related to the multiplicity of FepB forms previously observed in minicells and maxicells. Outer membrane harbouring the tribrid and lacking FepA, the normal outer membrane receptor for FeEnt, bound approximately four times more FeEnt than outer membrane from uninduced cells, from cells lacking pTX700 and from cells expressing only an LppOmpA 'dibrid'. Similarly, whole UT5600(fepA)/pTX700 cells induced for tribrid synthesis bound FeEnt and this binding was not affected by energy poisons. The results demonstrated that FepB can bind FeEnt, thereby definitely placing FeEnt transport in the periplasmic permease category of transport systems, and that the LppOmpA localization vehicle can be used with periplasmic binding proteins.

Azides↗