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PubMed1993

Function of periplasmic copper-zinc superoxide dismutase in Caulobacter crescentus.

Caulobacter crescentus is one of a small number of bacterial species that contain a periplasmic copper-zinc superoxide dismutase (CuZnSOD). A C. crescentus mutant, with the CuZnSOD gene interrupted by a promoterless cat gene, was constructed and characterized to analyze CuZnSOD function. Periplasmic SOD does not protect against oxyradical damage in the cytosol or play a major role in maintaining the integrity of the cell envelope. Studies of the effect of sodium citrate on plating efficiency suggest that CuZnSOD protects a periplasmic or membrane function(s) requiring magnesium or calcium.

Cations, Divalent↗
PubMed1994

Inactivation of FhuA at the cell surface of Escherichia coli K-12 by a phage T5 lipoprotein at the periplasmic face of the outer membrane.

Inactivation of phage T5 by lysed cells after phage multiplication is prevented by a phage-encoded lipoprotein (Llp) that inactivates the FhuA outer membrane receptor protein (K. Decker, V. Krauel, A. Meesmann, and K. Heller, Mol. Microbiol. 12:321-332, 1994). Using FhuA derivatives carrying insertions of 4 and 16 amino acid residues and point mutations, we determined whether FhuA inactivation is caused by binding of Llp to FhuA and which regions of FhuA are important for inactivation by Llp. Cells expressing Llp were resistant not only to phage T5 but to all FhuA ligands tested, such as phage phi 80, colicin M, and albomycin, and they were strongly reduced in the uptake of ferrichrome. Most of the FhuA derivatives which were not affected by Llp were, according to a previously published FhuA transmembrane topology model, located in periplasmic turns and in the TonB box close to the periplasm. Since the ligands bind to the cell surface, interaction of FhuA with Llp in the periplasm may induce a FhuA conformation which impairs binding of the ligands. This conclusion was supported by the increase rather than decrease of colicin M sensitivity of two mutants in the presence of Llp. The only Llp-resistant FhuA derivatives with mutations at the cell surface contained insertions of 16 residues in the loop that determines the permeability of the FhuA channel and serves as the principal binding site for all FhuA ligands. This region may be inactivated by steric hindrance in that a portion of Llp penetrates into the channel. Outer membranes prepared with 0.25% Triton X-100 from cells expressing Llp contained inactivated FhuA, suggesting Llp to be an outer membrane protein whose interaction with FhuA was not abolished by Triton X-100. Llp solubilized in 1.1% octylglucoside prevented T5 inactivation by FhuA dissolved in octylglucoside.

Amino Acid Sequence↗
PubMed1995

Inactivation of Serpulina hyodysenteriae flaA1 and flaB1 periplasmic flagellar genes by electroporation-mediated allelic exchange.

Serpulina hyodysenteriae, the etiologic agent of swine dysentery, contains complex periplasmic flagella which are composed of multiple class A and class B polypeptides. To examine the role these proteins play in flagellar synthesis, structure, and function and to develop strains which may provide insight into the importance of motility in the etiology of this pathogen, we constructed specific periplasmic flagellar mutations in S. hyodysenteriae B204. The cloned flaA1 and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and/or kanamycin gene cassettes. Following delivery of these suicide plasmids into S. hyodysenteriae, homologous recombination and allelic exchange at the targeted chromosomal flaA1 and flaB1 genes was verified by PCR, sequence, and Southern analysis. The utility of a chloramphenicol resistance gene cassette for targeted gene disruption was demonstrated and found more amenable than kanamycin as a selective marker in S. hyodysenteriae. Immunoblots of cell lysates of the flagellar mutants with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath or core protein. Both mutations selectively abolished expression of the targeted gene without affecting synthesis of the other flagellar polypeptide. flaA1 and flaB1 mutant strains exhibited altered motility in vitro and were less efficient in movement through a liquid medium. Paradoxically, isogenic strains containing specifically disrupted flaA1 or flaB1 alleles were capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. This is the first report of specific inactivation of a motility-associated gene in spirochetes.

Bacterial Proteins↗
PubMed2000

Cysteine-scanning mutagenesis of the periplasmic loop regions of PomA, a putative channel component of the sodium-driven flagellar motor in Vibrio alginolyticus.

The sodium-driven motor consists of the products of at least four genes, pomA, pomB, motX, and motY, in Vibrio alginolyticus. PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop(1-2)) and at positions 167 to 180 between membrane segments 3 and 4 (loop(3-4)). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembrane segment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also replaced with Cys. Only two mutations, M179C and T186C, conferred a nonmotile phenotype. Many mutations in the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the five successive substitutions from M169C to K173C of loop(3-4) impaired motility. In some mutants that retained substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and N-ethylmaleimide. The profiles of inhibition by the reagents were consistent with the membrane topology predicted from the hydrophobicity profiles. Furthermore, from the profiles of labeling by biotin maleimide, we predicted more directly the membrane topology of loop(3-4). None of the loop(1-2) residues were labeled, suggesting that the environments around the two loops are very different. A few of the mutations were characterized further. The structure and function of the loop regions are discussed.

Amino Acid Sequence↗
PubMed1991

Transmembrane signal transduction and osmoregulation in Escherichia coli. Functional importance of the periplasmic domain of the membrane-located protein kinase, EnvZ.

The EnvZ protein is presumably a membrane-located osmotic sensor which is involved in expression of the ompF and ompC genes in Escherichia coli. Previously, we developed an in vitro method for analyzing the intact form of the EnvZ protein located in isolated cytoplasmic membranes, and demonstrated that this particular form of the EnvZ protein exhibits the ability not only as to OmpR phosphorylation but also OmpR dephosphorylation. In this study, to gain an insight into the structural and functional importance of the putative periplasmic domain of the EnvZ protein, a set of mutant EnvZ proteins, which lack various portions of the periplasmic domain, were characterized in terms of not only their in vivo osmoregulatory phenotypes but also in vitro EnvZ-OmpR phosphotransfer reactions. It was revealed that these deletion mutant EnvZ proteins are normally incorporated into the cytoplasmic membrane. Cells harboring these mutant EnvZ proteins showed a pleiotropic phenotype, namely, OmpF- Mal- LamB- PhoA-, and produced the OmpC protein constitutively irrespective of the medium osmolarity. It was also suggested that all of these mutant EnvZ proteins were defective in their in vitro OmpR dephosphorylation ability, while their OmpR phosphorylation ability remained unaffected. These results imply the functional importance of the periplasmic domain of the EnvZ protein for modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an environmental osmotic stimulus.

Bacterial Outer Membrane Proteins↗
PubMed1990

Iron (III) hydroxamate transport into Escherichia coli. Substrate binding to the periplasmic FhuD protein.

Due to its extreme insolubility, Fe3+ is not transported as a monoatomic ion. In microbes, iron is bound to low molecular weight carriers, designated siderophores. For uptake into cells of Escherichia coli Fe3+ siderophores have to be translocated across two membranes. Transport across the outer membrane is receptor-dependent and energy-coupled; transport across the cytoplasmic membrane seems to follow a periplasmic binding protein-dependent transport mechanism. In support of this notion we demonstrate specific binding of the Fe3+ hydroxamate compounds ferrichrome, aerobactin, and coprogen, which are transported via the Fhu system, to the periplasmic FhuD protein, and no binding of the transport inactive ferrichrome A, ferric citrate, and iron sulfate. About 10(4) ferrichrome molecules were bound to the FhuD protein of cells which overproduced plasmid-encoded FhuD. Binding depended on transport across the outer membrane mediated by the FhuA receptor and the TonB protein. Binding to FhuD was supported by the exclusive resistance of FhuD to proteinase K in the presence of the transport active hydroxamates. The overproduced precursor form of the FhuD protein was not protected by the Fe3+ hydroxamates indicating a conformation different to the mature form. The FhuD protein apparently serves as a periplasmic carrier for Fe3+ hydroxamates with widely different structures.

Bacterial Proteins↗
PubMed1989

Reconstitution of periplasmic transport in inside-out membrane vesicles. Energization by ATP.

The periplasmic histidine permease of Salmonella typhimurium has been reconstituted in inside-out vesicles (IOV) of Escherichia coli by disrupting the cells with a French press in the presence of a high concentration of the periplasmic histidine-binding protein, HisJ. Efflux from IOV, which is equivalent to uptake in whole cells, is induced by ATP. The reconstituted system depends on the presence of the membrane-bound permease proteins, HisQ, HisM, and HisP, and does not function if reconstitution is performed in the presence of a mutant HisJ protein, HisJ5625, that can bind histidine normally but can't interact properly with the membrane complex. Efflux is not induced by the nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate, supporting the contention that ATP hydrolysis is necessary. 8-Azido ATP inactivates IOV, indicating that the ATP effect occurs through the HisP protein, which has previously been shown to be modified by 8-azido ATP (Hobson, A., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 733-7337). The estimated Km of the vesicles for ATP is about 200 microM. Vanadate, an inhibitor of phosphohydrolase enzymes, inhibits ATP-induced efflux. We conclude that ATP is likely to be the proximal energy source for periplasmic permeases.

Adenosine Triphosphate↗
PubMed1984

Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli.

We report the detection in vivo of precursors to the A and the B subunits of the heat-labile enterotoxin (LT) in Escherichia coli. Both pre-LT A (Mr = 29,500) and pre-LT B (Mr = 13,500) are present in the spheroplast fraction of the bacteria after separation of the cells in spheroplasts and periplasm. Two smaller LT A related polypeptides (17 and 23 kDa) were also detected in the spheroplast fraction. Both were degraded with a half-time of about 40 s. Mature subunits (Mr = 27,500 for LT A, and 11,500 for LT B) are released from the spheroplasts soon after processing and occur freely in the periplasm not associated with the cytoplasmic or the outer membranes. Processing occurs mainly post-translationally for both the A and the B subunits. However, they show different kinetics of processing and subsequent segregation into the periplasm. Whereas pre-LT B is processed and released within seconds after chain termination, pre-LT A is processed and released more slowly, and a subfraction of mature LT A may reside in the cytoplasmic membrane for several minutes.

Bacterial Toxins↗
PubMed1982

Renaturation and identification of periplasmic proteins in two-dimensional gels of Escherichia coli.

The locations of the periplasmic proteins of Escherichia coli on standard two-dimensional gel patterns are described. The periplasmic fractions were prepared by osmotic shock of plasmolyzed whole cells and by release during EDTA-lysozyme treatment of whole cells. Within this fraction of proteins, we identify nine binding proteins (leucine-specific, glutamate-aspartate, glutamine, cystine, galactose, maltose, xylose, ribose, and arabinose) in addition to leucine-isoleucine-valine binding protein, which has been previously identified (Bloch, P. L., Phillips, T. A., and Neidhardt, F. C. (1980) J. Bacteriol. 141, 1409-1420). The identifications are based upon genetic criteria, protein induction, and comigration with purified protein. The levels of these proteins are compared in strains K12, B, and HA12 (a derivative of W). A technique was developed for renaturation of the ligand binding sites of periplasmic binding proteins in denaturing two-dimensional gels. This technique was used to demonstrate that leucine-specific and cystine binding proteins both have different isoelectric points in different strains. Renaturation was also used to demonstrate that there are two different charged forms for glutamine binding protein.

Carrier Proteins↗
PubMed1994

The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm.

PreOmpA-bovine pancreatic trypsin inhibitor (BPTI) (Goldenberg, D. P. (1988) Biochemistry 27, 2481-2489) was expressed in Escherichia coli, and the folding pathway of the mature protein in the periplasmic space was analyzed by pulse-chase experiments. Folding intermediates were trapped with iodoacetamide, immunoprecipitated with antisera specific for either the reduced or the native protein, and resolved by electrophoresis. In vivo, native BPTI formed with a half-life of 7 min which is 3-fold faster than the optimal in vitro folding rate in growth media supplemented with low molecular weight disulfides. The measured in vivo half-life includes the time required for translocation and processing by leader peptidase and therefore represents the lower limit for the actual folding rate in the cell. In addition to the native species, two-disulfide intermediates accumulated in the cell at appreciable levels and did not chase to the native species for at least 20 min. We found that the folding of BPTI in E. coli was absolutely dependent on DsbA, a protein which accelerates the formation of disulfide bonds in the periplasm. In a dsbA mutant strain, trace amounts of oxidized BPTI could be detected only in cultures grown under strongly oxidizing conditions. In wild-type cells, the addition of different concentrations of GSH/GSSG or oxidized dithiothreitol did not affect the kinetics of BPTI oxidation by DsbA. Finally, even though the folding of BPTI in vitro decreases by almost 10-fold/unit pH decrease, folding in cells grown at pH 6.0 was only marginally slower than folding in cells grown at neutral pH, despite the fact that the pH of the periplasmic space varies in response to the extracellular fluid.

Animals↗
PubMed1993

Neutrophil activation by nascent FimH subunits of type 1 fimbriae purified from the periplasm of Escherichia coli.

Previous studies of type 1 fimbriae of Escherichia coli have implicated FimH, a minor subunit, as the determinant of its mannose binding property. Structure-function analysis of FimH has not been possible because of the difficulty in obtaining adequate amounts of the subunit from type 1 fimbriae. We have obtained nascent FimH that has not been incorporated into the fimbrial structure from the periplasm of an E. coli strain expressing the cloned fimH gene. Nascently translocated FimH was initially degraded in the periplasm; however, when co-expressed with FimC, a putative fimbrial chaperone, the FimH molecules were stabilized and readily isolated from the periplasmic extract by fractionation on a sodium dodecyl sulfate-polyacrylamide gel followed by electroelution of the FimH band from the gel. The eluted protein was purified to homogeneity by affinity chromatography on a mannose-Sepharose column. Purified FimH displayed the same mannose-inhibitable binding to human neutrophils as type 1 fimbriated bacteria, including triggering an oxidative burst with concomitant release of reactive oxygen metabolites. In addition, inert microspheres coated with FimH, but not those coated with bovine serum albumin, were phagocytosed by neutrophils. These data provide direct evidence that FimH is the determinant on type 1 fimbriae which is responsible for mediating mannose-specific adherence and that isolated FimH is a potent activator of human neutrophils.

Adhesins, Escherichia coli↗
PubMed1996

Production and characterization of biologically active Ala-Ser-(His)6-Ile-Glu-Gly-Arg-human prolactin (tag-hPRL) secreted in the periplasmic space of Escherichia coli.

Human prolactin (hPRL) cDNA was obtained by screening of a pituitary cDNA library with a synthetic 21-mer oligonucleotide and with rat PRL cDNA. For its expression, use was made of a vector, p3SN8, containing tac-promoter-controlled sequences for a bacterial cellulase leader joined to sequences coding for Ala-Ser, a chromatographic affinity site consisting of six histidines and a Factor Xa cleavage site. The hPRL cDNA was inserted at the 3' end of the cleavage-site sequences. Expression in Escherichia coli led to secretion in the periplasmic space of a fully bioactive hPRL variant constituting authentic hPRL with a peptide tag, i.e. Ala-Ser-(His)6-Ile-Glu-Gly-Arg, at its N-terminal. This tag-hPRL could be rapidly and efficiently purified by metal-chelate affinity chromatography. The correct processing and quality of tag-hPRL was monitored by SDS/PAGE, Western-blot analysis, immunoassay and Nb2-lymphoma-cell bioassay. Treatment with Factor Xa for tag removal was only partially successful. Periplasmic secretion of tag-hPRL of the order of 0.7 micrograms/ml per A600 unit and one-step purification indicate feasibility for tag-hPRL production for in vitro diagnostic and research applications. This is the first report describing periplasmic secretion of a bioactive form of hPRL.

Base Sequence↗
PubMed2004

A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA thiol: disulfide isomerase.

Two-dimensional electrophoresis (2-DE) was used to analyze the pleiotropic effects of a deficiency in DsbA, a periplasmic disulfide-bond oxidoreductase, in Salmonella typhi. With this aim, the dsbA gene was cloned and assayed for activity in a dsbA-null mutant of Escherichia coli. A dsbA/chloramphenicol acetylase construct was then used to disrupt the wild-type gene of S. typhi. The resultant dsbA-null mutant of S. typhi, like the E. coli mutant, exhibited a lack of flagellation and of glucose-1-phosphatase activity. Periplasmic extracts from the parental and mutant strains were analyzed by 2-DE using standard denaturing and nondenaturing conditions. Differences in protein expression were more marked in nondenaturing conditions. Ninety-nine protein spots were analyzed by peptide mass fingerprinting, and 65 spots were identified by searching a S. typhi database. Twenty-five spots were exclusively detected in the wild-type strain, 10 were found only in the mutant strain, and 21 were common to both strains. We observed a lack of DsbA, glucose-1-phosphatase and flagellin in the dsbA-null mutant, which explains two of the observed phenotypes. The AI-2 autoinducer-producing protein LuxS, which is involved in quorum-sensing signalling was also absent.

Bacterial Proteins↗
PubMed1999

Selective degradation of unfolded proteins by the self-compartmentalizing HtrA protease, a periplasmic heat shock protein in Escherichia coli.

HtrA, which has a high molecular mass of about 500 kDa, is a periplasmic heat shock protein whose proteolytic activity is essential for the survival of Escherichia coli at high temperatures. To determine the structural organization of HtrA, we have used electron microscopy and chemical cross-linking analysis. The averaged image of HtrA with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity, and its side-on view showed a two-layered structure. Thus, HtrA behaves as a dodecamer consisting of two stacks of hexameric ring. HtrA can degrade thermally unfolded citrate synthase and malate dehydrogenase but cannot when in their native form. HtrA degraded partially unfolded casein more rapidly upon increasing the incubation temperature. However, it hydrolyzed oxidized insulin B-chain, which is fully unfolded, at nearly the same rate at all of the temperatures tested. HtrA also rapidly degraded reduced insulin B-chain generated by treatment of insulin with dithiothreitol but not A-chain or intact insulin. Moreover, HtrA degraded fully unfolded alpha-lactalbumin, of which all four disulfide bonds were reduced, but not the native alpha-lactalbumin and its unfolded intermediates containing two or three disulfide bonds. These results indicate that unfolding of the protein substrates, such as by exposure to high temperatures or reduction of disulfide bonds, is essential for their access into the inner chamber of the double ring-shaped HtrA, where cleavage of peptide bonds may occur. Thus, HtrA with a self-compartmentalizing structure may play an important role in elimination of unfolded proteins in the periplasm of Escherichia coli.

Amino Acid Sequence↗
PubMed2006

nDsbD: a redox interaction hub in the Escherichia coli periplasm.

DsbD is a redox-active protein of the inner Escherichia coli membrane possessing an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain. nDsbD interacts with four different redox proteins involved in the periplasmic disulfide isomerization and in the cytochrome c maturation systems. We review here the studies that led to the structural characterization of all soluble DsbD domains involved and, most importantly, of trapped disulfide intermediate complexes of nDsbD with three of its four redox partners. These results revealed the structural features enabling nDsbD, a 'redox hub' with an immunoglobulin-like fold, to interact efficiently with its different thioredoxin-like partners.

Amino Acid Motifs↗
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

Ligand size is a major determinant of specificity in periplasmic oxyanion-binding proteins: the 1.2 A resolution crystal structure of Azotobacter vinelandii ModA.

BACKGROUND: . Periplasmic receptors constitute a diverse class of binding proteins that differ widely in size, sequence and ligand specificity. Nevertheless, almost all of them display a common beta/alpha folding motif and have similar tertiary structures consisting of two globular domains. The ligand is bound at the bottom of a deep cleft, which lies at the interface between these two domains. The oxyanion-binding proteins are notable in that they can discriminate between very similar ligands. RESULTS: . Azotobacter vinelandii is unusual in that it possesses two periplasmic molybdate-binding proteins. The crystal structure of one of these with bound ligand has been determined at 1.2 A resolution. It superficially resembles the structure of sulphate-binding protein (SBP) from Salmonella typhimurium and uses a similar constellation of hydrogen-bonding interactions to bind its ligand. However, the detailed interactions are distinct from those of SBP and the more closely related molybdate-binding protein of Escherichia coli. CONCLUSIONS: . Despite differences in the residues involved in binding, the volumes of the binding pockets in the A. vinelandii and E. coli molybdate-binding proteins are similar and are significantly larger than that of SBP. We conclude that the discrimination between molybdate and sulphate shown by these binding proteins is largely dependent upon small differences in the sizes of these two oxyanions.

Amino Acid Sequence↗
PubMed1996

The periplasmic TorT protein is required for trimethylamine N-oxide reductase gene induction in Escherichia coli.

Expression of the Escherichia coli torCAD operon, which encodes the trimethylamine N-oxide reductase system, is regulated by the presence of trimethylamine N-oxide through the action of the TorR response regulator. We have identified an additional gene, torT, located just downstream from the torR gene, which is necessary for torCAD structural operon expression. Insertion within the torT gene dramatically reduced the expression of a torA'-'lacZ fusion, while presence of the gene in trans restored the wild-type phenotype. Overproduction of TorR in a torT strain resulted in partial constitutive expression of the torA'-'lacZ fusion, suggesting that TorR acts downstream from TorT. The torT gene codes for a 35.7-kDa periplasmic protein which presents some homology with the periplasmic ribose-binding protein of E. coli. We discuss the possible role of TorT as an inducer-binding protein involved in signal transduction of the tor regulatory pathway.

Amino Acid Sequence↗