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At least 343 records · Page 19Linked to original sources
PubMed1998

alpha-Galactoside uptake in Rhizobium meliloti: isolation and characterization of agpA, a gene encoding a periplasmic binding protein required for melibiose and raffinose utilization.

Rhizobium meliloti can occupy at least two distinct ecological niches; it is found in the soil as a free-living saprophyte, and it also lives as a nitrogen-fixing intracellular symbiont in root nodules of alfalfa and related legumes. One approach to understanding how R. meliloti alters its physiology in order to become an integral part of a developing nodule is to identify and characterize genes that are differentially expressed by bacteria living inside nodules. We used a screen to identify genes under the control of the R. meliloti regulatory protein NodD3, SyrM, or SyrA. These regulatory proteins are expressed by bacteria growing inside the root nodule. One gene isolated in this screen was mapped to pSymB and displayed complex regulation. The gene was downregulated by the syrA gene product and also by glucose and succinate. This gene, referred to as agpA, encodes a periplasmic binding protein that is most similar to proteins from the periplasmic oligopeptide binding protein family. It is likely that AgpA binds alpha-galactosides, because alpha-galactosides induce the expression of agpA, and agpA mutants cannot utilize or transport these sugars. Activity of an agpA::TnphoA fusion was downregulated by SyrA. Because syrA is known to be expressed at high levels in intracellular symbiotic R. meliloti and at low levels in the free-living bacteria, we propose that AgpA may belong to the class of gene products whose expression decreases when R. meliloti becomes an intracellular symbiont.

Amino Acid Sequence↗
PubMed1998

MppA, a periplasmic binding protein essential for import of the bacterial cell wall peptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate.

Mutants of a diaminopimelic acid (Dap)-requiring strain of Escherichia coli were isolated which failed to grow on media in which Dap was replaced by the cell wall murein tripeptide, L-alanyl-gamma-D-glutamyl-mesodiaminopimelate. In one such mutant, which is oligopeptide permease (Opp) positive, we have identified a new gene product, designated MppA (murein peptide permease A), that is about 46% identical to OppA, the periplasmic binding protein for Opp. A plasmid carrying the wild-type mppA gene allows the mutant to grow on tripeptide. Two other mutants that failed to grow on tripeptide were resistant to triornithine toxicity, indicating a defect in the opp operon. An E. coli strain whose entire opp operon was deleted but which carried the mppA locus was unable to grow on murein tripeptide unless it was provided with oppBCDF genes in trans. Our data suggest a model whereby the periplasmic MppA binds the murein tripeptide, which is then transported into the cytoplasm via membrane-bound and cytoplasmic OppBCDF. In assessing the affinity of MppA for non-cell wall peptides, we have found that proline auxotrophy can be satisfied with the peptide Pro-Phe-Lys, which utilizes either MppA or OppA in conjunction with OppBCDF for its uptake. Thus, MppA, OppA, and perhaps the third OppA paralog revealed by the E. coli genome sequence may each bind a particular family of peptides but interact with common membrane-associated components for transport of their bound ligands into the cell. As to the physiological function of MppA, the possibility that it may be involved in signal transduction pathway(s) is discussed.

ATP-Binding Cassette Transporters↗
PubMed1999

Structural and functional significance of the FGL sequence of the periplasmic chaperone Caf1M of Yersinia pestis.

The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 beta-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 beta-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.

Amino Acid Sequence↗
PubMed2001

Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity.

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.

Bacterial Proteins↗
PubMed2004

Structural basis for iron binding and release by a novel class of periplasmic iron-binding proteins found in gram-negative pathogens.

We have determined the 1.35- and 1.45-A structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A. M. haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever. The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host. The ferric (Fe(3+)) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion. The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion. Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm.

Animals↗
PubMed2001

Structural and redox properties of the leaderless DsbE (CcmG) protein: both active-site cysteines of the reduced form are involved in its function in the Escherichia coli periplasm.

The thiol/disulfide oxidoreductases play important roles in ensuring the correct formation of disulfide bonds, of which the DsbE protein, also called CcmG, is the one implicated in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli. The soluble, N-terminally truncated DsbE was overexpressed and purified to homogeneity. Here we report the structural and redox properties of the leaderless form (DsbEL-). During the redox reaction, the protein undergoes a structural transformation resulting in a more stable reduced form, but this form shows very low reactivity in thiol/ disulfide exchange of cysteine residues and low activity in accelerating the reduction of insulin. The standard redox potential (E'0) for the active thiol/ disulfide was determined to be -0.186 V; only one of the two cysteines (Cys80) was suggested to be the active residue in the redox reaction. From the aspect of biochemical properties, DsbE can be regarded as a weak reductant in the Escherichia coli periplasm. This implies that the function of DsbE in cytochrome c maturation can be ascribed to its active-site cysteines and the structure of the reduced form.

Binding Sites↗
PubMed1994

Sec-independent translocation of a 100-residue periplasmic N-terminal tail in the E. coli inner membrane protein proW.

The ProW protein, located in the inner membrane of Escherichia coli, has a very unusual topology with a 100-residue-long N-terminal tail protruding into the periplasmic space. We have studied the mechanism of membrane translocation of the periplasmic tail by analysing ProW-PhoA and ProW-Lep fusion proteins, both in wild-type cells and in cells with an impaired sec machinery. Our results show that the translocation efficiency is not affected by treatments that compromise the SecA and SecY functions, but that translocation is completely blocked by dissipation of the proton motive force or by the introduction of extra positively charged residues into the N-terminal tail. This suggests that the sec machinery can act properly only on domains located on the C-terminal side of a translocation signal, and that the N-terminal tail is driven through the membrane by a mechanism that involves the proton motive force.

ATP-Binding Cassette Transporters↗
PubMed2006

The role of periplasmic antioxidant enzymes (superoxide dismutase and thiol peroxidase) of the Shiga toxin-producing Escherichia coli O157:H7 in the formation of biofilms.

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.

Aerobiosis↗
PubMed1999

AcrA is a highly asymmetric protein capable of spanning the periplasm.

AcrA protein is a component of the multi-drug efflux complex AcrAB-TolC of Escherichia coli. Judged by the hypersusceptibility phenotype of acrA mutants, the AcrAB-TolC system pumps out an extraordinarily wide variety of antibiotics, chemotherapeutic agents, detergents and dyes. This complex traverses both the inner and outer membranes of E. coli and catalyzes efflux of the drugs directly into the medium. The coordinated operation of the inner membrane transporter AcrB and outer membrane channel TolC is thought to be mediated by AcrA. The latter is a lipoprotein located in the periplasmic space. We show here that a lipid-deficient derivative of AcrA is functionally active as demonstrated by the complementation of the hypersusceptibility phenotype of the acrA mutant. Purified non-lipidated and intact forms of AcrA were able to restore, with similar efficiency, the activity of AcrA-dependent efflux of erythromycin in Ca2+-sucrose-treated E. coli cells. Using analytical ultracentrifugation and dynamic light scattering techniques we determined hydrodynamic properties of the non-lipidated AcrA and found that AcrA exists in solution as a highly asymmetric monomeric molecule with an axial ratio of 8. This elongated shape of AcrA is compatible with the hypothesis that this protein spans the periplasmic space coordinating the concerted operation of inner and outer membrane components of the complex.

Bacterial Proteins↗
PubMed2000

Extended sugar slide function for the periplasmic coiled coil domain of ScrY.

Several bacterial outer membrane proteins have a periplasmic extension whose structure and function remain elusive. Here, the structure/function relationship of the N-terminal periplasmic domain of the sucrose-specific outer membrane channel ScrY was investigated. Circular dichroism and analytical centrifugation demonstrated that the N-terminal domain formed a parallel, three-stranded coiled coil. When this domain was fused to the maltose-specific channel LamB, permeation of maltooligosaccharides in liposomes increased with increasing sugar chain length whereas wild-type LamB showed the opposite effect. Current fluctuation analysis demonstrated increased off-rates for sugar transport through the fusion protein. Moreover, equilibrium dialysis showed an affinity of sucrose for the isolated N-terminal peptide. Together these results demonstrate a novel function for coiled coil domains, operating as an extended sugar slide.

Alkylation↗
PubMed2000

High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence.

We investigated the ability of signal peptides of eukaryotic origin (human, mouse, and yeast) to efficiently direct model proteins to the Escherichia coli periplasm. These were compared against a well-characterized prokaryotic signal peptide-OmpA. Surprisingly, eukaryotic signal peptides can work very efficiently in E. coli, but require optimization of codon usage by codon-based mutagenesis of the signal peptide coding region. Analysis of the 5' of periplasmic and cytoplasmic E. coli genes shows some codon usage differences.

Amino Acid Sequence↗
PubMed1998

A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens.

During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a DeltafccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens fumarate reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The DeltafccABC mutant of W. succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor.

Amino Acid Sequence↗
PubMed2005

The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity.

The periplasmic loop of MscL, the mechanosensitive channel of large conductance, acts as a spring resisting the opening of the channel. Recently, a high-throughput functional screening of a range of MscL structural mutants indicated that the substitution of residue glutamine (Q) 65 with arginine (R) or leucine (L) leads to a wild-type (WT)-like and a loss-of-function (LOF) phenotype, respectively. We used electron paramagnetic resonance (EPR) spectroscopy, single-channel recording and in vivo experiments to investigate further the effect of R and L mutation of Q65 on the gating mechanism of MscL. Structural analysis of Q65R and Q65L was carried out by coupling the site-directed spin labeling (SDSL) with EPR spectroscopy. A SDSL cysteine mutant of the isoleucine 24 residue (I24C-SL) in the first transmembrane domain, TM1, of MscL served as a reporter residue in EPR experiments. This was due to its strong spin-spin interaction with the neighboring I24C-SL residues in the MscL channel pentamer. The effects of bilayer incorporation of lysophosphatidylcholine on the MscL mutants were also investigated. Functional analysis was carried out using patch-clamp recordings from these mutants and WT MscL reconstituted into artificial liposomes. Although our data are largely in agreement with the high-throughput mutational analysis of Maurer and Dougherty, this study shows that Q65R and Q65L form functional channels and that these mutations lead to partial gain-of-function (GOF) and LOF mutation, respectively. Overall, our study confirms and advances the notion that the periplasmic loop plays a role in setting the channel mechanosensitivity.

Arginine↗
PubMed1988

Control of leucine transport in yeast by periplasmic binding proteins.

The concentrative inward transport of leucine in Saccharomyces carlsbergensis involves two transport systems (S1 and S2); S1 is a system of high affinity and low translocation velocity, and S2 is a system of low affinity and high translocation velocity. The inward transport process of the amino acid is discriminated into two kinetically defined steps: first, binding to periplasmic proteins and second, translocation across the plasmalemma. When cells were incubated with glucose to increase the metabolic energy charge, we observed that JTmax (maximum flux that each system can exhibit for the translocation step) increased for both systems. This increase in JTmax is due to variations in the parameters defining the initial step (Ks (apparent dissociation constant) and N (concentration of binding sites)): for S1, N1 increases and for S2, KS2 diminishes. Dissipation of the electrochemical proton gradient produced an increase of KS1 and a decrease of N2, resulting in a decrease of JTmax in both systems. Instead, osmotic shock decreases N1 and N2, which suggests that periplasmic components were removed, resulting also in a decrease of JTmax in both systems. These results are consistent with the proposition that the total unidirectional flux of the amino acid proceeds by means of a system of multiple components, with the simultaneous operation of two independent transport processes. We propose that the initial interaction of leucine with components of the cellular envelope might be the essential step for the subsequent translocation of the amino acid across the permeability barrier.

Biological Transport, Active↗
PubMed1990

Genetic approach to the role of tryptophan residues in the activities and fluorescence of a bacterial periplasmic maltose-binding protein.

The periplasmic maltose-binding protein (MBP or MalE protein) of Escherichia coli is an essential element in the transport of maltose and maltodextrins and in the chemotaxis towards these sugars. On the basis of previous results suggesting their possible role in the activity and fluorescence of MBP, we have changed independently to alanine each of the eight tryptophan residues as well as asparagine 294, which is conserved among four periplasmic sugar-binding proteins. Five of the tryptophan mutations affected activity. In four cases (substitution of Trp62, Trp230, Trp232 and Trp340), there was a decrease in MBP affinity towards maltose correlated with modifications in transport and chemotaxis. According to the present state of the 2.3 A three-dimensional structure of MBP, all four residues are in the binding site. Residues Trp62 and Trp340 are in the immediate vicinity of the bound substrate and appear to have direct contacts with maltose; this is in agreement with the drastic increases in Kd values (respectively 67 and 300-fold) upon their substitution by alanine residues. The modest increase in Kd (12-fold) observed upon mutation of Trp230 would be compatible with the lesser degree of interaction this residue has with the bound substrate and the idea that it plays an indirect role, presumably by keeping other residues involved directly in binding in their proper orientation. Substitution of Trp232 resulted in a small increase in Kd value (2-fold) in spite of the fact that this residue is the closest to the ligand of the tryptophan residues according to the three-dimensional model. In the fifth case, replacement of Trp158, which is distant from the binding site, strongly reduced the chemotactic response towards maltose without affecting the transport parameters or the sugar-binding activities of the mutant protein. Trp158 may therefore be specifically implicated in the interaction of MBP with the chemotransducer Tar, but this effect is likely to be indirect, since Trp158 is buried in the structure of MBP. Of course, some structural rearrangements could be responsible in part for the effects of these mutations. The remaining four mutations were silent. The corresponding residues (Trp10, Trp94, Trp129 and Asn294) are all distant from the sugar-binding site on the crystallographic model of MBP, which is in agreement with their lack of effect on binding. In addition, our results show that they play no role in the interactions with the other proteins of the maltose transport (MalF, MalG or MalK) or chemotaxis (Tar) systems.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Progress in the identification of interaction sites on the periplasmic maltose binding protein from E coli.

The periplasmic maltose binding protein (MBP) is required for the high affinity transport of maltose and maltodextrins and for chemotaxis towards these sugars. In these functions, MBP interacts with proteins of the cytoplasmic membrane: MalF and MalG for transport, Tar for chemotaxis. A large number of MBP mutations have been isolated by us and other laboratories. We grouped these mutations into classes depending on the interactions affected and we represented the corresponding residues on the 3-D model for MBP so as to further identify the sites of MBP interacting with the MalF-MalG complex and with the Tar protein. MBP (like the other binding proteins) is composed of 2 lobes enclosing a cleft where the substrate binds. The face of the protein opposite the cleft seems to interact neither with MalF-MalG nor with Tar. The other face, corresponding to the cleft, contains sites for interactions with MalF-MalG and Tar. These sites appear to cover both sides of the cleft and may overlap in part. The present definition of the interaction sites suggests further that MBP has different in vivo orientations when it interacts with MalF-MalG or with Tar. This work constitutes an additional step in combining the use of genetic and structural analysis to define the interaction sites on MBP. Because of the structural similarities between periplasmic binding proteins, the regions of interaction defined could be relevant for other members of this family.

ATP-Binding Cassette Transporters↗
PubMed1991

Characterization of an osmoregulated periplasmic glycine betaine-binding protein in Azospirillum brasilense sp7.

Azospirillum brasilense is able to use glycine betaine as a powerful osmoprotectant; the uptake of this compound is strongly stimulated by salt stress, but significantly reduced by cold osmotic shock. Non-denaturing PAGE in the presence of [methyl-14C] glycine betaine and autoradiography demonstrated the presence of one glycine betaine-binding protein (GBBP) in periplasmic shock fluid obtained from high-osmolarity-grown cells. The binding activity was absent in periplasmic fractions from cells grown at low osmolarity. SDS-PAGE analysis showed that the osmotically inducible GBBP has an apparent molecular weight of 32,000. The isoelectric point was between 5.9 and 6.6, as determined by isoelectric focusing. This protein bound glycine betaine with high affinity (KD of 3 microM), but had no affinity for either other betaines (proline betaine, gamma-butyrobetaine, pipecolate betaine, trigonelline, homarine) or related compounds (choline, glycine betaine aldehyde, glycine and proline). Optimum binding activity occurred at pH 7.0 to 7.5, and was not altered whether or not the binding assays were done at low or high osmolarity. Immunoprecipitation and Western blotting showed that immunoadsorbed anti-GBBP antibody from E coli cross-reacted with the GBBP produced by A brasilense cells grown at high osmolarity.

Amino Acids↗
PubMed2004

Siderophore production of a periplasmic transport binding protein kinase gene defective mutant of Magnetospirillum magneticum AMB-1.

A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis. The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed. The transposon was inserted in an open reading frame (ORF) coding for a periplasmic transport binding protein kinase gene homologue. Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon. Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotriacetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore. Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type. These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M. magneticum AMB-1.

Amino Acid Sequence↗