Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Periplasm”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources
PubMed1988

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 (mammalian mdr genes and Drosophila white 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.

Cell Membrane↗
PubMed1989

Analysis and DNA sequence of the osmoregulated treA gene encoding the periplasmic trehalase of Escherichia coli K12.

The treA gene of Escherichia coli K12 codes for a periplasmic trehalase that is induced by growth at high osmolarity. The position of treA within a cloned chromosomal DNA fragment was identified by subcloning of restriction fragments and analysis of the gene product in minicells. The nucleotide sequence of the treA coding region as well as its upstream control region was determined. The treA gene consists of 1695 bp encoding 565 amino acids. The amino-terminus of the mature trehalase was found to begin with the amino acid Glu at position 31 of the open reading frame. The first 30 amino acids resemble a typical signal sequence, consistent with trehalase being a secreted periplasmic enzyme. Two previously isolated phoA fusions to the osmA gene were transferred by homologous recombination on to a treA-containing plasmid and found to be within treA. Analysis of the hybrid genes and their gene products aided the localization of treA and the determination of its direction of transcription within the cloned chromosomal segment. The treA-phoA fusions encoded hybrid proteins which could be found in the periplasm. We found that at high osmolarity the normal pathway for the uptake and utilization of trehalose is blocked. Therefore, the function of the periplasmic trehalase is to provide the cell with the ability to utilize trehalose at high osmolarity by splitting it into glucose molecules that can subsequently be taken up by the phosphotransferase-mediated uptake system.

Amino Acid Sequence↗
PubMed1990

Identification of a periplasmic C-type cytochrome as electron donor to the plasma membrane-bound cytochrome oxidase of the cyanobacterium Nostoc Mac.

Photoautotrophically grown cyanobacterium Nostoc sp. strain Mac (PCC 8009) released up to about 10 nmol of a c-type cytochrome per ml packed cells after treatment with EDTA under conditions that left the plasma membrane absolutely intact as judged from the absence of cytosolic proteins in the supernatant. Spectra of the ascorbate reduced cytochrome revealed peaks at 553, 522 and 416 nm. The protein was purified to an A-553/A-275 ratio of 0.8. Midpoint potential (at pH 7), isoelectric point and apparent molecular weight of the cytochrome were +0.35 V, 8.6, and around 10,500, respectively. The cytochrome proved to be an excellent electron donor to the aa3-type cytochrome oxidase in both plasma and thylakoid membranes isolated and purified from Nostoc Mac. Chemoheterotrophic growth of the cells increased the level of periplasmic cytochrome c up to 10-fold and cytochrome oxidase activity of plasma membranes up to 90-fold. The periplasmic cytochrome also transferred electrons to photosystem I in illuminated thylakoid membranes. We conclude that cyanobacteria contain a periplasmic c-type cytochrome presumably identical to so-called cytochrome c6 or c-553 which has long been known as a photosynthetic (i.e. thylakoid-associated) redox protein in these organisms, and which is capable of donating electrons (from the periplasmic space) to the cytochrome oxidase in the plasma membrane and (from the thylakoid lumen) to both P700 and cytochrome oxidase in the thylakoid membrane.

Cell Membrane↗
PubMed1994

Synthesis of holo Paracoccus denitrificans cytochrome c550 requires targeting to the periplasm whereas that of holo Hydrogenobacter thermophilus cytochrome c552 does not. Implications for c-type cytochrome biogenesis.

Expression from a plasmid of the complete gene, including the codons for the N-terminal periplasmic targeting signal, for cytochrome c550 of Paracoccus denitrificans led to the formation of the holo protein in the periplasms of both P. denitrificans and Escherichia coli. Expression of the gene from which the region coding for the targeting signal had been specifically deleted resulted in formation of apo-protein in the cytoplasms of both organisms. These findings are consistent with haem attachment occurring in the periplasm. In contrast, the formation of holo cytochrome c552 from Hydrogenobacter thermophilus following expression of the gene lacking the periplasmic targeting sequence in either P. denitrificans or E. coli is attributed to spontaneous cytoplasmic attachment of haem to the thermostable protein.

Bacteria, Aerobic↗
PubMed1989

Evidence for partial export of Vitreoscilla hemoglobin into the periplasmic space in Escherichia coli. Implications for protein function.

The Vitreoscilla hemoglobin protein has been implicated in earlier studies to serve a globin-like function under oxygen-limited growth conditions. Evidence is presented using fractionation as well as proteinase K accessibility techniques to prove that a considerable amount of this protein is localized in the periplasmic space of the cell. Genetic evidence points towards the existence of information within the N-terminal domain of the protein that plays a role in the process of protein export. However, this sequence is not cleaved in the process of translocation. Analysis of the primary structure of this region reveals several unusual features. Instead of positively charged residues at its amino terminus, it has a negative charge. The overall hydrophobicity of the central region of this sequence is significantly lower than in typical leader peptides due to the presence of a charged residue. In keeping with the likelihood that such an export signal may not be very efficient, a substantial fraction of the total cellular hemoglobin can also be detected in the cytoplasm. Heme is incorporated in both cytoplasmic and periplasmic globin as indicated by the ability of protein from both fractions to bind carbon monoxide. The secretion of this protein into the periplasm raises questions concerning the physiological significance of its localization. Dimensional analysis of a model based on the facilitated diffusion hypothesis, which was initially proposed to account for the effects of eukaryotic globins on oxygen transport, suggests that periplasmic globin can support an additional oxygen flux to the respiratory apparatus that may be physiologically significant.

Amino Acid Sequence↗
PubMed1991

Periplasmic interaction between two membrane regulatory proteins, ToxR and ToxS, results in signal transduction and transcriptional activation.

ToxR is a transmembrane, DNA-binding protein that can activate transcription of genes encoding cholera toxin (ctxAB). Here we characterize ToxS, a 19 kd transmembrane regulatory protein that interacts with ToxR and stimulates its activity. If a portion of the periplasmic domain of ToxR is deleted, productive interaction with ToxS is abolished. A ToxR-PhoA fusion protein that retains most of the ToxR periplasmic region remains dependent on ToxS for its ToxR activity. ToxS protects this fusion from proteolytic cleavage, suggesting that these two proteins interact within the periplasm. Mutations in a short cytoplasmic domain of ToxR were isolated that disrupt the periplasmic interaction between ToxR and ToxS. This domain is shared by other bacterial transcriptional activators, suggesting that it may play a common role in function of these proteins and in the molecular mechanism of signal transduction.

Alkaline Phosphatase↗
PubMed2001

Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA.

The production of human proinsulin in its disulfide-intact, native form in Escherichia coli requires disulfide bond formation and the periplasmic space is the favourable compartment for oxidative folding. However, the secretory expression of proinsulin is limited by its high susceptibility to proteolysis and by disulfide bond formation, which is rate-limiting for proinsulin folding. In this report we describe a method for the production of high amounts of soluble, native human proinsulin in E. coli. We fused proinsulin to the C-terminus of the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site. As DsbA is the main catalyst of disulfide bond formation in E. coli, we expected increased yields of proinsulin by intra- or intermolecular catalysis of disulfide bond formation. In the context of the fusion protein, proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide bond formation. To increase the yield of DsbA-proinsulin in the periplasm, several parameters were optimised, including host strains and cultivation conditions, and in particular growth medium composition and supplement of low molecular weight additives. We obtained a further, about three-fold increase in the amount of native DsbA-proinsulin by addition of L-arginine or ethanol to the culture medium. The maximum yield of native human proinsulin obtained from the soluble periplasmic fraction after specific cleavage of the fusion protein with trypsin was 9.2 mg g(-1), corresponding to 1.8% of the total cell protein.

Arginine↗
PubMed2000

Periplasmic localization of a GroES homologue in Escherichia coli transformed with groESx cloned from Legionella-like endosymbionts in Amoeba proteus.

Escherichia coli MC4100 transformed with a groE homologous operon cloned from X-bacteria accumulated large amounts of the gene product when cultured at 30 or 37 degrees C. Heat shock for 10-30 min at 42 degrees C or ethanol (5%) shock for 2 h increased GroESx levels to about twice that in E. coli grown at 30 degrees C. The subcellular localization of GroESx in transformed E. coli was determined by several subcellular fractionation methods, by the analysis of extracted proteins in SDS polyacrylamide gels and by assays of marker enzymes. The GroESx protein was detected in both the periplasmic and cytoplasmic extracts and a large amount of the protein was accumulated in the periplasm. The GroEL protein and recombinant beta-galactosidase were exclusively localized in the cytoplasmic fraction, eliminating the possibility that periplasmic GroESx might be due to simple overproduction. N-terminal amino acid sequencing confirmed that the protein resolved on a 2-D gel was GroESx. This work represents the first report of the periplasmic location of GroES homologues in E. coli.

Amoeba↗
PubMedDate not supplied

Facilitating the formation of disulfide bonds in the Escherichia coli periplasm via coexpression of yeast protein disulfide isomerase.

Sacchromyces cerevisiae protein disulfide isomerase (yPDI) was expressed in the E. coli periplasm by using plasmids encoding the OmpA-yPDI-(His)(6) fusion gene under the control of the araBAD, trc, or T7 promoter. The expression levels of yeast PDI under these promoters were compared. Our results showed that yeast PDI expressed into the periplasm could catalyze the formation of disulfide bonds in alkaline phosphatase, restoring the phoA(+) phenotype in dsbA(-) mutants. The yeast PDI was purified from the Escherichia coli periplasm and shown to exhibit catalytic properties comparable to those of the rat enzyme with reduced RNase as substrate. In vivo, coexpression of the yeast PDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) in E. coli by 2-fold, similar to the effect seen previously with the coexpression of the rat enzyme. However yeast PDI was more effective than rat PDI in facilitating the expression of active tissue plasminogen activator (tPA). These results point to differences in the substrate specificity of various PDI enzymes, at least in the context of the E. coli periplasm.

Animals↗
PubMed1996

The N-terminal beta-barrel domain of the Escherichia coli K88 periplasmic chaperone FaeE determines fimbrial subunit recognition and dimerization.

The K88 periplasmic chaperone FaeE is a homodimer, whereas the K99 chaperone FanE is a monomer. The structural requirements for dimerization of the K88 fimbrial periplasmic chaperone and for fimbrial subunit-binding specificity were investigated by analysis of mutant chaperones. FaeE contains a C-terminal extension of 19 amino acid residues when compared to FanE and most other fimbrial chaperones. A C-terminal truncate of the K88 chaperone FaeE was constructed that lacked 19 C-terminal amino acid residues. Expression and complementation experiments revealed that this C-terminal shortened chaperone was still functional in binding the K88 major subunit FaeG and K88 biosynthesis. Two hybrid chaperones were constructed. Each hybrid protein contained one beta-barrel domain of FaeE and the other beta-barrel domain of FanE (Fae/FanE or Fan/FaeE, respectively). Expression and complementation experiments revealed that the Fae/FanE but not the Fan/FaeE hybrid chaperone was functional in the formation of K88 fimbriae. The Fan/FaeE hybrid chaperone was active in the bio-synthesis of K99 fimbriae. The truncated FaeE mutant chaperone and the hybrid Fae/FanE chaperone were able to form stable periplasmic protein complexes with the K88 major fimbrial subunit FaeG. Cross-linking experiments suggested that the C-terminal shortened chaperone and the Fae/FanE hybrid chaperone were homodimers, as is the wild-type K88 chaperone. Altogether, the data suggested that the N-terminal beta-barrel domain of a fimbrial chaperone determines subunit specificity. In the case of the K88 periplasmic chaperone, this N-terminal domain also determines dimerization of the protein.

Amino Acid Sequence↗
PubMed1996

Re-examination of the role of the periplasmic domain of EnvZ in sensing of osmolarity signals in Escherichia coli.

In Escherichia coli, EnvZ senses changes in the osmotic conditions of the growth environment and controls the phosphorylated state of the regulatory protein, OmpR. OmpR-phosphate regulates the expression of the porin genes, ompF and ompC. To investigate the role of the periplasmic domain of EnvZ in sensing of osmolarity signals, portions of this domain were deleted. Cells containing the EnvZ mutant proteins were able to regulate normally the production of OmpF and OmpC in response to changes in osmolarity. The periplasmic domain of EnvZ was also replaced with the non-homologous periplasmic domain of the histidine kinase PhoR of Bacillus subtilis. Osmoregulation of OmpF and OmpC production in cells containing the PhoR-EnvZ hybrid protein was indistinguishable from that in cells containing wild-type EnvZ. Identical results were obtained with an envZ-pta/ack strain, which could not synthesize acetyl phosphate. Thus, acetyl phosphate was not involved in the regulation of ompF and ompC observed in this study. These results indicate that the periplasmic domain of EnvZ is not essential for sensing of osmolarity signals.

Alleles↗
PubMed1995

Biochemical characterization of a Haemophilus influenzae periplasmic iron transport operon.

Bacterial iron transport is critical for growth of pathogens in the host environment, where iron is limited as a form of nonspecific immunity. For Gram-negative bacteria such as Haemophilus influenzae, iron first must be transported across the outer membrane and into the periplasmic space, then from the periplasm to the cytosol. H. influenzae express a periplasmic iron-binding protein encoded by the hitA gene. This gene is organized as the first of a three-gene operon purported to encode a classic high affinity iron acquisition system that includes hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein (hitC). In this study we describe the cloning, overexpression, and purification of the H. influenzae hitA gene product. The function of this protein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2'-dipyridyl, both in vitro and within the periplasmic space of a siderophore-deficient strain of Escherichia coli. Finally, the importance of a functional hitABC operon for iron acquisition is demonstrated by complementation of this siderophore-deficient E. coli to growth on dipyridyl-containing medium. These studies represent a detailed genetic, biochemical, and physiologic description of an active transport system that has evolved to efficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria.

Bacterial Proteins↗
PubMed1996

The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of a periplasmic beta-N-acetylglucosaminidase.

We have described some steps in chitin catabolism by Vibrio furnissii, and proposed that chitin oligosaccharides are hydrolyzed in the periplasmic space to GlcNAc and (GlcNAc)2. Since (GlcNAc)2 is an important inducer in the cascade, it must resist hydrolysis in the periplasm. Known V. furnissii periplasmic hydrolases comprise an endoenzyme (Keyhani, N. O. and Roseman, S. (1996) J. Biol. Chem. 271, 33414-33424), and the beta-N-acetylglucosaminidase, ExoI, reported here. ExoI was isolated from a recombinant strain of Escherichia coli, and hydrolyzes aryl-beta-GlcNAc, aryl-beta-GalNAc, and chitin oligosaccharides. No other beta-GlcNAc glycosides were cleaved. The pH optimum was 7.0 for (GlcNAc)n, n = 3-6, but 5.8 for (GlcNAc)2. At the pH of sea water (8.0-8.3), the enzymatic activity with (GlcNAc)2 is virtually undetectable. These results explain the stability of (GlcNAc)2 in the periplasmic space. The cloned beta-GlcNAcidase gene, exoI, encodes a 69,377-kDa protein (611 amino acids); the predicted N-terminal 20 amino acid residues matched those of the isolated protein. The protein amino acid sequence displays significant homologies to the alpha- and beta-chains of human hexosaminidase despite their marked differences in substrate specificities and pH optima.

Acetylglucosaminidase↗
PubMed1997

Characterization of the Bradyrhizobium japonicum CycY protein, a membrane-anchored periplasmic thioredoxin that may play a role as a reductant in the biogenesis of c-type cytochromes.

A new member of membrane-anchored periplasmic thioredoxin-like proteins was identified in Bradyrhizobium japonicum. It is the product of cycY, the last gene in a cluster of cytochrome c biogenesis genes. Mutational analysis revealed that cycY is essential for the biosynthesis of all c-type cytochromes in this bacterium. The CycY protein was shown to be exported to the periplasm by its N-terminal signal sequence-like domain. Results from Western blot analyses of membrane and soluble fractions indicated that the CycY protein remains bound to the membrane. A soluble version of the protein devoid of its N-terminal membrane anchor (CycY*) was expressed in Escherichia coli and purified to homogeneity from the periplasmic fraction. The protein showed redox reactivity and properties similar to other thioredoxins such as fluorescence quenching in the oxidized form. Its equilibrium constant with glutathione was determined to be 168 mM, from which a standard redox potential of -0.217 V was calculated, suggesting that CycY might act as a reductant in the otherwise oxidative environment of the periplasm. This is in agreement with our hypothesis that CycY is required, directly or indirectly, for the reduction of the heme-binding site cysteines in the CXXCH motif of c-type apocytochromes before heme attachment occurs.

Amino Acid Sequence↗
PubMed1987

The osmotically regulated proU locus of Salmonella typhimurium encodes a periplasmic betaine-binding protein.

The proU locus of Salmonella typhimurium encodes an osmotically induced betaine transport system. We have identified a 31 kDa periplasmic protein, encoded by proU, whose synthesis is induced by osmotic stress. A specific betaine-binding activity with a KD of about 1 microM is also present in the periplasm of osmotically induced cells. This activity is absent in those proU mutants which lack the 31 kDa periplasmic protein. Thus, ProU is a periplasmic binding-protein-dependent transport system.

Bacterial Proteins↗
PubMed1993

Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity.

Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase beta subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the beta subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.

Amino Acid Sequence↗
PubMed1997

The sigma(E) and the Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli.

In Escherichia coli, the heat shock-inducible sigma-factor sigma(E) and the Cpx two-component signal transduction system are both attuned to extracytoplasmic stimuli. For example, sigma(E) activity rises in response to the overproduction of various outer-membrane proteins. Similarly, the activity of the Cpx signal transduction pathway, which consists of an inner-membrane sensor (CpxA) and a cognate response regulator (CpxR), is stimulated by overproduction of the outer-membrane lipoprotein, NlpE. In response to these extracytoplasmic stimuli, sigma(E) and CpxA/CpxR stimulate the transcription of degP, which encodes a periplasmic protease. This suggests that CpxA/CpxR and sigma(E) both mediate protein turnover within the bacterial envelope. Here, we show that CpxA/CpxR and sigma(E) also control the synthesis of periplasmic enzymes that can facilitate protein-folding reactions. Specifically, sigma(E) controls transcription of fkpA, which specifies a periplasmic peptidyl-prolyl cis/trans isomerase. Similarly, the Cpx system controls transcription of the dsbA locus, which encodes a periplasmic enzyme required for efficient disulfide bond formation in several extracytoplasmic proteins. Taken together, these results indicate that sigma(E) and CpxA/CpxR are involved in regulating both protein-turnover and protein-folding activities within the bacterial envelope.

Amino Acid Sequence↗
PubMed1989

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii which Is Distinct from the Periplasmic Form of the Enzyme.

A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system.

Journal Article↗