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D Missiakas

Publications and source records attributed to D Missiakas.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of the Escherichia coli sigma E regulon.

Escherichia coli responds to the accumulation of misfolded proteins by inducing the transcription of heat shock genes. Efinal sigma(E) RNA polymerase controls one of the two heat shock regulons of E. coli. This regulon is activated upon accumulation of misfolded polypeptides in the double membrane envelope of E. coli. final sigma(E) (RpoE) is a member of the extracytoplasmic function subfamily of sigma factors. Here we asked how many genes are activated by Efinal sigma(E) RNA polymerase and what is the identity of these genes. Using two independent genetic approaches, 20 E. coli promoters were identified which activate reporter gene transcription in a final sigma(E)-dependent manner. In all cases examined, a canonical final sigma(E) binding site could be revealed upon mapping transcriptional start sites. 10 identified promoters activated the transcription of previously identified genes with four genes acting directly on the folding of E. coli envelope proteins (dsbC, fkpA, skp, and surA). The remaining promoters transcribed genes that are presumed to encode hitherto unknown extracytoplasmic functions and were named ecf (ecfA-ecfM). Two of these ecf genes were found to be essential for E. coli growth.

Bacterial Proteins↗
PubMed2000

DsbD-catalyzed transport of electrons across the membrane of Escherichia coli.

Dsb proteins catalyze folding and oxidation of polypeptides in the periplasm of Escherichia coli. DsbC reduces wrongly paired disulfides by transferring electrons from its catalytic dithiol motif (98)CGYC. Genetic evidence suggests that recycling of this motif requires at least three proteins, the cytoplasmic thioredoxin reductase (TrxB) and thioredoxin (TrxA) as well as the DsbD membrane protein. We demonstrate here that electrons are transferred directly from thioredoxin to DsbD and from DsbD to DsbC. Three cysteine pairs within DsbD undergo reversible disulfide rearrangements. Our results suggest a novel mechanism for electron transport across membranes whereby electrons are transferred sequentially from cysteine pairs arranged in a thioredoxin-like motif (CXXC) to a cognate reactive disulfide.

Cell Membrane↗
PubMed2000

RseB binding to the periplasmic domain of RseA modulates the RseA:sigmaE interaction in the cytoplasm and the availability of sigmaE.RNA polymerase.

The Escherichia coli sigmaE regulon has evolved to sense the presence of misfolded proteins in the bacterial envelope. Expression of periplasmic chaperones and folding catalysts is under the control of sigmaE RNA polymerase. The N-terminal domain of RseA sequesters sigmaE in the cytoplasmic membrane, preventing its association with core RNA polymerase. The C-terminal domain of RseA interacts with RseB, a periplasmic protein. The relative concentration of sigmaE:RseA:RseB is 2:5:1 and this ratio remains unaltered upon heat shock induction of the sigmaE regulon. Purification from crude cellular extracts yields cytoplasmic, soluble sigmaE RNA polymerase as well as membrane sequestered sigmaE.RseA and sigmaE.RseA.RseB. RseB binding to the C-terminal domain of RseA increases the affinity of RseA for sigmaE by 2- to 3-fold (Kd 50-100 nM). RseB binds also to the misfolded aggregates of MalE31, a variant of maltose binding protein that forms inclusion bodies in the periplasm. We discuss a model whereby the RseB-RiseA interaction represents a measure for misfolded polypeptides in the bacterial envelope, modulating the assembly of sigmaE RNA polymerase and the cellular heat shock response.

ATP-Binding Cassette Transporters↗
PubMed2000

Transfer of electrons across the cytoplasmic membrane by DsbD, a membrane protein involved in thiol-disulphide exchange and protein folding in the bacterial periplasm.

Reduction of non-native protein disulphides in the periplasm of Escherichia coli is catalysed by three enzymes, DsbC, DsbG and DsbE, each of which harbours a catalytic Cys-X-X-Cys dithiol motif. This dithiol motif requires continuous reduction for activity. Genetic evidence suggests that the source of periplasmic reducing power resides within the cytoplasm, provided by thioredoxin (trxA) and thioredoxin reductase (trxB). Cytoplasmic electrons donated by thioredoxin are thought to be transferred into the periplasm via the DsbD membrane protein. To understand the molecular nature of electron transfer, we have analysed the membrane topology of DsbD. DsbD is exported by an N-terminal signal peptide. The N- and C-terminal domains are positioned in the periplasmic space, connected by eight transmembrane segments. Electron transfer was shown to require five cysteine sulphydryl of DsbD. Trans complementation of mutant DsbD molecules revealed intermolecular electron transfer. We discuss a model whereby the membrane-embedded disulphides of DsbD accept electrons from cytoplasmic thioredoxin and transfer them to the C-terminal periplasmic dithiol motif of DsbD.

Amino Acid Sequence↗
PubMed1998

The functional properties of DsbG, a thiol-disulfide oxidoreductase from the periplasm of Escherichia coli.

Genetic studies have recently identified DsbG, a new member of the dsb group of redox proteins, which catalyze protein disulfide bond formation in the periplasm of Escherichia coli. We now demonstrate that DsbG functions primarily as an oxidant during protein disulfide bond formation, which is consistent with the low stability of its active site disulfide bond. There are indications, however, that the substrate range of DsbG may be narrower than the other periplasmic oxidative enzymes, DsbA and DsbC. Our observations further elaborate the pathway of disulfide bond formation in E. coli.

Aprotinin↗
PubMed1998

The extracytoplasmic function sigma factors: role and regulation.

Alternative sigma factors provide a means of regulating gene expression in response to various extracellular changes. One such class of sigma factors appears to control a variety of functions, including expression of heat-shock genes in Escherichia coli, biosynthesis of alginates and carotenoids in Pseudomonas aeruginosa and Myxococcus xanthus, respectively, iron uptake in E. coli and Pseudomonas spp., nickel and cobalt efflux in Alcaligenes europhus, plant pathogenicity in Pseudomonas syringae and synthesis of outer membrane proteins in Photobacterium sp. strain SS9. Most of these activities deal with extracytoplasmic functions, and such sigmas have been designated as ECF sigma factors. They have also been characterized in Mycobacteria as well as gram-positive bacteria such as Streptomyces coelicolor and Bacillus subtilus and the archaea Sulpholobus acidocaldarius. ECF factors belong to a subfamily of the sigma 70 class, based on their sequence conservation and function across bacterial species. The promoter consensus sequences recognized by the ECF factors are also highly conserved. In most of the cases, the activity of these factors is modulated by a cognate inner membrane protein that has been shown, both in E. coli and in P. aeruginosa, to act as an anti-sigma activity. This inner membrane protein is presumed to serve as a sensor and signalling molecule, allowing an adaptive response to specific environmental change. Presumably, an on-and-off switch of the anti-sigma activity leads to the release of the sigma factor and thereby to the co-ordinate transcription of the specific regulon it governs.

Gene Expression Regulation, Bacterial↗
PubMed1997

Signal transduction pathways in response to protein misfolding in the extracytoplasmic compartments of E. coli: role of two new phosphoprotein phosphatases PrpA and PrpB.

It is now well established that the sigmaE regulon of Escherichia coli is induced by misfolding of proteins in the periplasm and the outer membrane. htrA belongs to this regulon and encodes a periplasmic protease involved in the degradation of misfolded proteins. htrA transcription is also under the positive control of a two component signal transduction system CpxR CpxA. Closer examination of the putative signal transduction pathway modulating htrA transcription has led us to the identification of two new genes. Biochemical and genetic evidence shows that these two genes encode two phosphoprotein phosphatases, designated PrpA and PrpB. These are the first examples of typical serine/threonine and tyrosine phosphatases described in E. coli. PrpA and PrpB are involved in signaling protein misfolding via the CpxR CpxA transducing system. In addition, both PrpA and PrpB modulate the phosphorylated status of some other phosphoproteins in E. coli. Finally, we show that PrpA is a heat shock protein.

Amino Acid Sequence↗
PubMed1997

Protein misfolding in the cell envelope of Escherichia coli: new signaling pathways.

Depending on their cellular localization, misfolded proteins in Escherichia coli trigger two different heat-shock responses. Cytoplasmic proteins induce the 'classical' heat-shock regulon transcribed by the E sigma 32 polymerase. By contrast, misfolding of proteins in the cell envelope induces the newly described E sigma E-dependent regulon. This implies that there is an inducible transduction machinery in the inner membrane. The response to protein misfolding in the cell envelope is a finely tuned system regulated by a cascade of phosphorylation and dephosphorylation reactions.

Bacterial Proteins↗
PubMed1997

Modulation of the Escherichia coli sigmaE (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins.

The sigma(E) (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities. The sigma(E) regulon is induced upon misfolding of proteins in the periplasm or the outer membrane. Similar to other alternative sigma factors, the activity of sigma(E) is tightly regulated in E. coli. We have previously shown that sigma(E) is positively autoregulated at the transcriptional level. DNA sequencing, coupled with transcriptional analyses, have shown that sigma(E) is encoded by the first gene of a four-gene operon. The second gene of this operon, rseA, encodes an anti-sigma(E) activity. This was demonstrated at both the genetic and biochemical levels. For example, mutations in rseA constitutively increase sigma(E) activity. Consistent with this, overproduction of RseA leads to an inhibitory effect on sigma(E) activity. Topological analysis of RseA suggests the existence of one transmembrane domain, with the N-terminal part localized in the cytoplasm. Overproduction of this N-terminal domain alone was shown to inhibit sigma(E) activity. These observations were confirmed in vitro, because either purified RseA or only its purified N-terminal domain inhibited transcription from Esigma(E)-dependent promoters. Furthermore, RseA and sigma(E) co-purify, and can be co-immunoprecipitated, and chemically cross-linked. The sigma(E) activity is further modulated by the products of the remaining genes in this operon, rseB and rseC. RseB is a periplasmic protein, which negatively regulates sigma(E) activity and specifically interacts with the C-terminal periplasmic domain of RseA. In contrast, RseC is an inner membrane protein that positively modulates sigma(E) activity. Most of these protein-protein interactions were verified in vivo using the yeast two-hybrid system.

Bacterial Proteins↗
PubMed1997

A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins.

We have identified and functionally characterized a new Escherichia coli gene, dsbG, whose product is involved in disulphide bond formation in the periplasm. The dsbG gene was cloned from a multicopy plasmid library lacking the dsbB redox protein-encoding gene. Multicopy dsbG-carrying clones were selected, since they allowed E. coli to grow at lethal concentrations of dithiothreitol. In a complementary genetic approach, point mutations were independently obtained and mapped to the dsbG gene. Such mutations led simultaneously to a dithiothreitol-sensitive phenotype and an increased sigmaE-dependent heat shock response, which reflects the presence of misfolded proteins in the extracytoplasm. In agreement with these observations, dsbG mutants were shown to accumulate reduced forms of a variety of disulphide bond-containing proteins in the periplasm. This DsbG defect could be rescued by addition to the growth medium of either oxidized dithiothreitol or cystine, or by overexpression of the dsbA or dsbB genes. DsbG is synthesized as a precursor form of 27.5 kDa and processed to a 25.7kDa mature species located in the periplasm. DsbG was overproduced, purified to homogeneity and shown to have redox properties of thiol-disulphide oxidoreductases in vitro. Replacement of the first Cys residue of the predicted active site, Phe-(Xaa)4-Cys-Pro-Tyr-Cys by Ala, completely inactivated DsbG protein function. Taken together, all our results demonstrate that DsbG acts in vivo as an efficient thiol-disulphide oxidase. In addition, dsbG is the first member of the dsb family for which null mutations are conditionally lethal and can be propagated only if supplemented with oxidants in the growth medium. We propose that the main role of DsbG is to maintain the proper redox balance between the DsbA/DsbB and DsbC systems.

Alkaline Phosphatase↗
PubMed1997

Making and breaking disulfide bonds.

It is now well established that protein folding requires the assistance of folding helpers in vivo. The formation or isomerization of disulfide bonds in proteins is a slow process requiring catalysis. In nascent polypeptide chains the cysteine residues are in the thiol form. The formation of the disulfide bonds usually occurs simultaneously with the folding of the polypeptide, which means in the endoplasmic reticulum of eukaryotes or in the periplasm of Gram-negative bacteria. In prokaryotes, the existence of redox proteins involved in the formation of disulfide bonds containing proteins has recently been revealed in the periplasm. The discovery of these redox proteins through various genetic approaches will be summarized, as well as the most recent insights regarding their biochemical and biological activities.

Bacterial Proteins↗
PubMed1996

Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.

Heat shock response in Escherichia coli is autoregulated. Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures. A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin. We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin. One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon. HslV/ClpQ shares a very high degree of homology with members of the beta-type subunit, constituting the catalytic core of the 20S proteasome. HslU/ClpY is 50% identical to the ClpX protein of E. coli, which is known to present large polypeptides to its partner, the ATP-independent proteolytic enzyme ClpP. We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides. Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP.

ATP-Dependent Proteases↗
PubMed1996

Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains in Escherichia coli K12.

The assembly of anaerobically induced electron transfer chains in Escherichia coli strains defective in periplasmic disulphide bond formation was investigated. Strains deficient in DsbA, DsbB or DipZ (DsbD) were unable to catalyse formate-dependent nitrite reduction (Nrf activity) or synthesize any of the known c-type cytochromes. The Nrf+ activity and cytochrome c content of mutants defective in DsbC, DsbE or DsbF were similar to those of the parental, wild-type strain. Neither DsbC expressed from a multicopy plasmid nor a second mutation in dipZ (dsbD) was able to compensate for a dsbA mutation by restoring nitrite reductase activity and cytochrome c synthesis. In contrast, only the dsbB and dipZ (dsbD) strains were defective in periplasmic nitrate reductase activity, suggesting that DsbB might fulfil an additional role in anaerobic electron transport. Mutants defective in dipZ (dsbD) were only slightly more sensitive to Cu++ ions at concentrations above 5 mM than the parental strain, but strains defective in DsbA, DsbB, DsbC, DsbE or DsbF were unaffected. These results are consistent with our earlier proposals that DsbA, DsbB and DipZ (DsbD) are part of the same pathway for ensuring that haem groups are attached to the correct pairs of cysteine residues of apocytochromes c in the E. coli periplasm. However, neither DsbE nor DsbF are essential for the reduction of DipZ (DsbD).

Anaerobiosis↗
PubMed1996

Crystallization of DsbC, the disulfide bond isomerase of Escherichia coli.

DsbC is a 2 x 23 kDa soluble dimeric protein molecule involved in protein disulfide bond formation in the E. coli periplasm, primarily catalyzing disulfide bond rearrangements. Crystals of both the native and selenomethione protein suitable for structure determination were obtained using the hanging-drop vapour-diffusion method. The best crystals were obtained using 18-22% (v/v) polyethylene glycol 550 monomethyl ether in 100 mM Tris-HCl (pH 8.9). Seeding methods were used to produce large crystals diffracting to 2 A resolution, and the detergent n-octyl-beta-glucoside was used to improve crystal quality. Significant variation in cell dimensions and crystal order was observed. Cell dimensions obtained for frozen crystals were in the range a = 58.8 (0.3), b = 78.9 (0.5), c = 95.2 (5.0) A. The lattice is orthorhombic and systematic absences indicate that the space group is P2(1)2(1)2(1).

Journal Article↗
PubMed1996

Probing the structural role of an alpha beta loop of maltose-binding protein by mutagenesis: heat-shock induction by loop variants of the maltose-binding protein that form periplasmic inclusion bodies.

The maltose-binding protein (MBP) of Escherichia coli is the periplasmic receptor of the maltose transport system. Previous studies have identified amino acid substitutions in an alpha/beta loop of the structure of MBP that are critical for the in vivo folding. To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malE were mutagenized. The maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivo folding pathway of the protein. By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies. Heat-shock induction with production of misfolded loop variants was examined using both lon-lacZ and htrA-lacZ fusions. We found that the extent of formation of inclusion bodies in the periplasm of E. coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock sigma factor, sigma 24.

ATP-Binding Cassette Transporters↗
PubMed1996

New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH.

A global search for extracytoplasmic folding catalysts in Escherichia coli was undertaken using different genetic systems that produce unstable or misfolded proteins in the periplasm. The extent of misfolding was monitored by the increased activity of the sigma E regulon that is specifically induced by misfolded proteins in the periplasm. Using multicopy libraries, we cloned two genes, surA and fkpA, that decreased the sigma E-dependent response constitutively induced by misfolded proteins. According to their sequences and their biochemical activities, SurA and FkpA belong to two different peptidyl prolyl isomerase (PPI) families. Interestingly, surA was also selected as a multicopy suppressor of a defined htrM (rfaD) null mutation. Such mutants produce a defective lipopolysaccharide that is unable to protect outer membrane proteins from degradation during folding. The SurA multicopy suppression effect in htrM (rfaD) mutant bacteria was directly associated with its ability to catalyse the folding of outer membrane proteins immediately after export. Finally, Tn10 insertions were isolated, which led to an increased activity of the sigma E regulon. Such insertions were mapped to the dsb genes encoding catalysts of the protein disulphide isomerase (PDI) family, as well as to the surA, fkpA and ompH/skp genes. We propose that these three proteins (SurA, FkpA and OmpH/Skp) play an active role either as folding catalysts or as chaperones in extracytoplasmic compartments.

Bacterial Outer Membrane Proteins↗
PubMed1995

Identification and characterization of a new disulfide isomerase-like protein (DsbD) in Escherichia coli.

Previous studies have established that DsbA and DsbC, periplasmic proteins of Escherichia coli, are two key players involved in disulfide bond formation. A search for extragenic mutations able to compensate for the lack of dsbA function in vivo led us to the identification of a new gene, designated dsbD. Lack of DsbD protein leads to some, but not all, of the phenotypic defects observed with other dsb mutations, such as hypersensitivity to dithiothreitol and to benzylpenicillin. In addition, unlike the rest of the dsb genes, dsbD is essential for bacterial growth at temperatures above 42 degrees C. Cloning of the wild-type gene and sequencing and overexpression of the protein show that dsbD is part of an operon and encodes an inner membrane protein. A 138 amino acid subdomain of the protein was purified and shown to possess an oxido-reductase activity in vitro. Expressing this subdomain in the periplasmic space helped restore the phenotypic defects associated with a dsbD null mutation. Interestingly, this domain shares 45% identity with the portion of the eukaryotic protein disulfide isomerase carrying the active site. We further show that in dsbD mutant bacteria the dithiol active sites of DsbA and DsbC proteins are mostly oxidized, as compared with wild-type bacteria. Our results argue that DsbD generates a reducing source in the periplasm, which is required for maintaining proper redox conditions. The finding that overexpression of DsbD leads to a Dsb- phenotype, very similar to that exhibited by dsbA null mutants, is in good agreement with such a model.

Amino Acid Sequence↗