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Molecular Microbiology Jul 2017Nanomachines belonging to the type IV filament (Tff) superfamily serve a variety of cellular functions in prokaryotes, including motility, adhesion, electrical... (Review)
Review
Nanomachines belonging to the type IV filament (Tff) superfamily serve a variety of cellular functions in prokaryotes, including motility, adhesion, electrical conductance, competence and secretion. The type 2 secretion system (T2SS) Tff member assembles a short filament called pseudopilus that promotes the secretion of folded proteins from the periplasm across the outer membrane of Gram-negative bacteria. A combination of structural, biochemical, imaging, computational and in vivo approaches had led to a working model for the assembled nanomachine. High-resolution cryo-electron microscopy and tomography provided the first view of several homologous Tff nanomachines in the cell envelope and revealed the structure of the outer membrane secretin channel, challenging current models of the overall stoichiometry of the T2SS. In addition, recent insights into exoprotein substrate features and interactions with the T2SS have led to new questions about the dynamics of the system and the role of the plasma membrane in substrate presentation. This micro-review will highlight recent advances in the field of type 2 secretion and discuss approaches that can be used to reach a mechanistic understanding of exoprotein recognition, integration into the machine and secretion.
Topics: Amino Acid Sequence; Bacterial Proteins; Bacterial Secretion Systems; Base Sequence; Cryoelectron Microscopy; Gram-Negative Bacteria; Membrane Proteins; Models, Molecular; Periplasm; Protein Binding; Protein Folding; Secretin; Structure-Activity Relationship; Type II Secretion Systems
PubMed: 28486768
DOI: 10.1111/mmi.13704 -
Nature Communications Jul 2021Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan...
Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.
Topics: Amino Acid Motifs; Bacterial Capsules; Catalytic Domain; Cryoelectron Microscopy; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Mass Spectrometry; Membrane Proteins; Models, Molecular; Periplasm; Phosphorylation; Polysaccharides, Bacterial; Protein Conformation, alpha-Helical; Protein-Tyrosine Kinases; Tyrosine
PubMed: 34272394
DOI: 10.1038/s41467-021-24652-1 -
Applied and Environmental Microbiology Jan 2018sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine....
sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of , encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent and in the range of 98 to 111 μM and 69 to 73 μmol · min · mg, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol. Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of spp. Predicted transmembrane domain and signal peptide sequences of were found to be functional in and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.
Topics: Carbaryl; Escherichia coli; Hydrolases; Insecticides; Metabolic Networks and Pathways; Methylamines; Naphthols; Periplasm; Protein Sorting Signals; Pseudomonas; Soil Microbiology
PubMed: 29079626
DOI: 10.1128/AEM.02115-17 -
Biochimica Et Biophysica Acta Aug 2014The Cpx envelope stress response (ESR) has been linked to proteins that are integrated into and secreted across the inner membrane for several decades. Initial studies... (Review)
Review
The Cpx envelope stress response (ESR) has been linked to proteins that are integrated into and secreted across the inner membrane for several decades. Initial studies of the cpx locus linked it to alterations in the protein content of both the inner and outer membrane, together with changes in proton motive driven transport and conjugation. Since the mid 1990s, the predominant view of the Cpx envelope stress response has been that it serves to detect and respond to secreted, misfolded proteins in the periplasm. Recent studies in Escherichia coli and other Gram negative organisms highlight a role for the Cpx ESR in specifically responding to perturbations that occur at the inner membrane (IM). It is clear that Cpx adaptation involves a broad suite of changes that encompass many functions in addition to protein folding. Interestingly, recent studies have refocused attention on Cpx-regulated phenotypes that were initially published over 30years ago, including antibiotic resistance and transport across the IM. In this review I will focus on the insights and models that have arisen from recent studies and that may help explain some of the originally published Cpx phenotypes. Although the molecular nature of the inducing signal for the Cpx ESR remains enigmatic, recently solved structures of signaling proteins are yielding testable models concerning the molecular mechanisms behind signaling. The identification of connections between the Cpx ESR and other stress responses in the cell reveals a complex web of interactions that involves Cpx-regulated expression of other regulators as well as small proteins and sRNAs. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Topics: Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Heat-Shock Proteins; Membrane Proteins; Periplasm; Protein Folding; Protein Kinases; RNA Processing, Post-Transcriptional; Signal Transduction
PubMed: 24184210
DOI: 10.1016/j.bbamcr.2013.10.018 -
Nature Communications Oct 2021The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has...
The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution.
Topics: Binding Sites; Cloning, Molecular; Coliphages; Directed Molecular Evolution; Disulfides; Escherichia coli; Escherichia coli Proteins; Gene Expression; Genetic Vectors; Inteins; Methyltransferases; Models, Molecular; Periplasm; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Disulfide-Isomerases; Protein Interaction Domains and Motifs; Protein Splicing; Receptor, ErbB-2; Recombinant Proteins; Trastuzumab
PubMed: 34645844
DOI: 10.1038/s41467-021-26279-8 -
Nature Sep 2017Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet...
Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.
Topics: ATP-Binding Cassette Transporters; Adenosine Diphosphate; Bacterial Proteins; Biological Transport; Cell Membrane; Cryoelectron Microscopy; Escherichia coli; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Lipopolysaccharides; Models, Molecular; Nanostructures; Periplasm; Protein Binding; Protein Domains
PubMed: 28869968
DOI: 10.1038/nature23649 -
Life Science Alliance Jun 2024The Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized...
The Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized into three regions: a 14-fold symmetric outer membrane cap (OMC) composed of CagY, CagX, CagT, CagM, and Cag3; a 17-fold symmetric periplasmic ring (PR) composed of CagY and CagX; and a stalk with unknown composition. We investigated how CagT, CagM, and a conserved antenna projection (AP) region of CagY contribute to the structural organization of the OMCC. Single-particle cryo-EM analyses showed that complexes purified from Δ or Δ mutants no longer had organized OMCs, but the PRs remained structured. OMCCs purified from a CagY antenna projection mutant (CagYAP) were structurally similar to WT OMCCs, except for the absence of the α-helical antenna projection. These results indicate that CagY and CagX are sufficient for maintaining a stable PR, but the organization of the OMC requires CagY, CagX, CagM, and CagT. Our results highlight an unexpected structural independence of two major subdomains of the Cag T4SS OMCC.
Topics: Helicobacter pylori; Type IV Secretion Systems; Periplasm
PubMed: 38631913
DOI: 10.26508/lsa.202302560 -
PloS One 2022The symport of lactose and H+ is an important physiological process in E. coli, for it is closely related to cellular energy supply. In this paper, we review, extend and...
The symport of lactose and H+ is an important physiological process in E. coli, for it is closely related to cellular energy supply. In this paper, we review, extend and analyse a newly proposed cotransport model that takes the "leakage" phenomenon (uncoupled particle translocation) into account and also satisfies the static head equilibrium condition. Then, we use the model to study the equilibrium properties, including equilibrium solution and the time required to reach equilibrium, of the symport process of E. coli LacY protein, when varying the parameters of the initial state of cotransport system. It can be found that in our extended model, H+ and lactose will reach their equilibrium state separately, and when "leakage" exists, it linearly affects the equilibrium solution, which is a useful property that the original model does not have. We later investigated the effect of the volume of periplasm and cytoplasm on the equilibrium properties. For a certain E. coli cell, as it continues to lose water and contract, the time for cytoplasm pH to be stabilized by symport increases monotonically when the cell survives. Finally, we reproduce the experimental data from a literature to verify the validity of the extension in this symport process. The above phenomena and other findings in this paper may help us to not only further validate or improve the model, but also deepen our understanding of the cotransport process of E. coli LacY protein.
Topics: Biological Transport; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Hydrogen-Ion Concentration; Ion Transport; Kinetics; Lactose; Membrane Transport Proteins; Monosaccharide Transport Proteins; Periplasm; Protons; Reproducibility of Results; Symporters; Thermodynamics
PubMed: 35120164
DOI: 10.1371/journal.pone.0263286 -
Proceedings of the National Academy of... May 2022The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron...
The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron tomography, we report that the P. gingivalis T9SS consists of 18 PorM dimers arranged as a large, caged ring in the periplasm. Near the outer membrane, PorM dimers interact with a PorKN ring complex of ∼52 nm in diameter. PorMKN translocation complexes of a given T9SS adopt distinct conformations energized by the proton motive force, suggestive of different activation states. At the inner membrane, PorM associates with a cytoplasmic complex that exhibits 12-fold symmetry and requires both PorM and PorL for assembly. Activated motors deliver substrates across the outer membrane via one of eight Sov translocons arranged in a ring. The T9SSs are unique among known secretion systems in bacteria and eukaryotes in their assembly as supramolecular machines composed of apparently independently functioning translocation motors and export pores.
Topics: Bacterial Proteins; Bacterial Secretion Systems; Periplasm; Porphyromonas gingivalis; Virulence Factors
PubMed: 35471908
DOI: 10.1073/pnas.2119907119 -
Life Science Alliance Feb 2019Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic...
Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin-fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of the maltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathway the activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.
Topics: Bacterial Outer Membrane Proteins; Cell Membrane Permeability; Cytoplasm; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Proteins; Gene Knockout Techniques; Maltose; Maltose-Binding Proteins; Membrane Transport Proteins; Oligosaccharides; Operon; Periplasm; Periplasmic Binding Proteins; Perylene; Polysaccharides; Porins; Receptors, Virus; Regulon; Trisaccharides
PubMed: 30620010
DOI: 10.26508/lsa.201800242