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BioTechniques Jan 2022High-yield expression of quality antibody fragments is indispensable for research and diagnosis. Most recombinant antibody fragments are expressed in using liquid...
High-yield expression of quality antibody fragments is indispensable for research and diagnosis. Most recombinant antibody fragments are expressed in using liquid cultures; however, their yields and quality are often poor. Here the authors expressed a single-chain variable fragment in cultivated on the wet surface of a solid support. Compared with a liquid culture, the authors obtained 2.5-times more single-chain variable fragments with membrane-cultivated . This method has two important advantages: it enables high yields of periplasmic single-chain variable fragments compared with liquid culture and offers simple and rapid expression and extraction.
Topics: Escherichia coli; Escherichia coli Proteins; Periplasm; Recombinant Proteins; Single-Chain Antibodies
PubMed: 34841891
DOI: 10.2144/btn-2021-0093 -
Trends in Microbiology May 2021Copper (Cu) plays a key role at the host-pathogen interface as both an essential element and a toxic element. Intracellular strains of pathogenic Salmonella have...
Copper (Cu) plays a key role at the host-pathogen interface as both an essential element and a toxic element. Intracellular strains of pathogenic Salmonella have acquired the periplasmic Cu chaperone, CueP, and the thiol oxidoreductases complex Scs, while losing the ancestral Cu-detoxification Cus system. Coregulation of these species-specific factors link Cu with redox stress and allows Salmonella to counteract Cu toxicity during infection.
Topics: Animals; Bacterial Proteins; Cell Membrane; Copper; Host-Pathogen Interactions; Humans; Oxidation-Reduction; Periplasm; Salmonella typhimurium; Virulence
PubMed: 33516594
DOI: 10.1016/j.tim.2021.01.004 -
Scientific Reports Oct 2022Bovine enterokinase light chain (EK) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However,...
Bovine enterokinase light chain (EK) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EK gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by k/K, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased k and T values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site.
Topics: Cattle; Animals; Enteropeptidase; Escherichia coli; Inclusion Bodies; Periplasm; Biological Products; Recombinant Proteins
PubMed: 36271247
DOI: 10.1038/s41598-022-22574-6 -
MBio Sep 2015In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the...
UNLABELLED
In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the periplasm. Central to these systems, the Cu(+) ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the periplasm. The fate of copper in the periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The ΔcopI mutant is more susceptible to copper than the Cu(+) ATPase copA mutant. CopI is induced by copper, localized in the periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes (cbb3 oxidase and photosystem) in both ΔcopI and copA-null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in ΔcopI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c-type cytochromes would contribute also to the copper toxicity in the periplasm of the wild-type cells when they are exposed to high copper concentrations.
IMPORTANCE
Copper is an essential cation required as a cofactor in enzymes involved in vital processes such as respiration, photosynthesis, free radical scavenging, and pathogenesis. However, copper is highly toxic and has been implicated in disorders in all organisms, including humans, because it can catalyze the production of toxic reactive oxygen species and targets various biosynthesis pathways. Identifying copper targets, provides insights into copper toxicity and homeostatic mechanisms for copper tolerance. In this work, we describe for the first time a direct effect of excess copper on cytochrome c assembly. We show that excess copper specifically affects periplasmic and membrane cytochromes c, thus suggesting that the copper toxicity targets c-type cytochrome biogenesis.
Topics: Betaproteobacteria; Copper; Cytochromes c; Gene Deletion; Molecular Sequence Data; Periplasm; Periplasmic Proteins; Protein Multimerization; Sequence Analysis, DNA
PubMed: 26396241
DOI: 10.1128/mBio.01007-15 -
Protein Science : a Publication of the... Jul 2022Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are...
Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion-conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.
Topics: Biological Transport; Copper; Homeostasis; Humans; Membrane Proteins; Periplasm
PubMed: 35762724
DOI: 10.1002/pro.4364 -
Structure (London, England : 1993) May 2021The periplasm of Gram-negative bacteria is a complex, highly crowded molecular environment. Little is known about how antibiotics move across the periplasm and the...
The periplasm of Gram-negative bacteria is a complex, highly crowded molecular environment. Little is known about how antibiotics move across the periplasm and the interactions they experience. Here, atomistic molecular dynamics simulations are used to study the antibiotic polymyxin B1 within models of the periplasm, which are crowded to different extents. We show that PMB1 is likely to be able to "hitchhike" within the periplasm by binding to lipoprotein carriers-a previously unreported passive transport route. The simulations reveal that PMB1 forms both transient and long-lived interactions with proteins, osmolytes, lipids of the outer membrane, and the cell wall, and is rarely uncomplexed when in the periplasm. Furthermore, it can interfere in the conformational dynamics of native proteins. These are important considerations for interpreting its mechanism of action and are likely to also hold for other antibiotics that rely on diffusion to cross the periplasm.
Topics: Anti-Bacterial Agents; Bacterial Outer Membrane; Escherichia coli; Escherichia coli Proteins; Molecular Dynamics Simulation; Periplasm; Periplasmic Binding Proteins; Polymyxins
PubMed: 33577754
DOI: 10.1016/j.str.2021.01.009 -
Proceedings of the National Academy of... Jul 2014Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is...
Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de-N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB's N- and C-terminal domains forms a cleft required for the binding and de-N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C-terminal domain that would allow for a processive mechanism. PgaB's C-terminal domain (PgaB310-672) directly binds PNAG oligomers with dissociation constants of ∼1-3 mM, and the structures of PgaB310-672 in complex with β-1,6-(GlcNAc)6, GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de-N-acetylated PNAG (dPNAG). Furthermore, PgaB310-672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB42-655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB310-672 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.
Topics: Amidohydrolases; Biofilms; Biological Transport, Active; Escherichia coli; Escherichia coli Proteins; Molecular Docking Simulation; Periplasm; Polysaccharides, Bacterial; Protein Structure, Secondary; Protein Structure, Tertiary; beta-Glucans
PubMed: 24994902
DOI: 10.1073/pnas.1406388111 -
Physical Review Letters Dec 2011Spirochetes are a unique group of motile bacteria that are distinguished by their helical or flat-wave shapes and the location of their flagella, which reside within the...
Spirochetes are a unique group of motile bacteria that are distinguished by their helical or flat-wave shapes and the location of their flagella, which reside within the tiny space between the bacterial cell wall and the outer membrane (the periplasm). In Borrelia burgdorferi, rotation of the flagella produces cellular undulations that drive swimming. How these shape changes arise due to the forces and torques that act between the flagella and the cell body is unknown. It is possible that resistive forces come from friction or from fluid drag, depending on whether or not the flagella are in contact with the cell wall. Here, we consider both of these cases. By analyzing the motion of an elastic flagellum rotating in the periplasmic space, we show that the flagella are most likely separated from the bacterial cell wall by a lubricating layer of fluid. This analysis then provides drag coefficients for rotation and sliding of a flagellum within the periplasm.
Topics: Biomechanical Phenomena; Borrelia burgdorferi; Elasticity; Flagella; Periplasm; Rotation; Torque
PubMed: 22243185
DOI: 10.1103/PhysRevLett.107.268101 -
Applied and Environmental Microbiology Dec 2018Extracellular electron transfer (EET) is a strategy for respiration in which electrons generated from metabolism are moved outside the cell to a terminal electron...
Extracellular electron transfer (EET) is a strategy for respiration in which electrons generated from metabolism are moved outside the cell to a terminal electron acceptor, such as iron or manganese oxide. EET has primarily been studied in two model systems, and Metal reduction has also been reported in numerous microorganisms, including spp., which are ubiquitous found in aquatic ecosystems, with some species capable of pathogenesis in humans and fish. Genomic comparisons of spp. revealed a potential outer membrane conduit homologous to MtrCAB. While the ability to respire metals and mineral oxides is not widespread in the genus , 90% of the sequenced isolates contain MtrCAB homologs. ATCC 7966 mutants lacking are unable to reduce metals. Expression of in an mutant lacking homologous components restored metal reduction. Although the outer membrane conduits for metal reduction were similar, homologs of the inner membrane and periplasmic EET components CymA, FccA, and CctA were not found in We characterized a cluster of genes predicted to encode components related to a formate-dependent nitrite reductase (NrfBCD), here named NetBCD (for rf-like lectron ransfer), and a predicted diheme periplasmic cytochrome, PdsA (eriplasmic iheme huttle). We present genetic evidence that proteins encoded by this cluster facilitate electron transfer from the cytoplasmic membrane across the periplasm to the MtrCAB conduit and function independently from an authentic NrfABCD system. mutants lacking and were unable to reduce metals, while heterologous expression of these genes could restore metal reduction in an mutant background. EET may therefore allow and other species of to persist and thrive in iron- or manganese-rich oxygen-limited environments. Metal-reducing microorganisms are used for electricity production, bioremediation of toxic compounds, wastewater treatment, and production of valuable compounds. Despite numerous microorganisms being reported to reduce metals, the molecular mechanism has primarily been studied in two model systems, and We have characterized the mechanism of extracellular electron transfer in , which uses the well-studied system, MtrCAB, to move electrons across the outer membrane; however, most spp. appear to use a novel mechanism to transfer electrons from the inner membrane through the periplasm and to the outer membrane. The conserved use of MtrCAB in spp. and spp. for metal reduction and conserved genomic architecture of metal reduction genes in spp. may serve as genomic markers for identifying metal-reducing microorganisms from genomic or transcriptomic sequencing. Understanding the variety of pathways used to reduce metals can allow for optimization and more efficient design of microorganisms used for practical applications.
Topics: Aeromonas hydrophila; Bacterial Proteins; Cell Membrane; Electron Transport; Electrons; Gene Expression Regulation, Bacterial; Manganese Compounds; Metals; Multigene Family; Oxidation-Reduction; Oxides; Periplasm; Phylogeny; Shewanella
PubMed: 30266730
DOI: 10.1128/AEM.02134-18 -
The Biochemical Journal Jan 2019Two-component systems (TCSs) are essential for bacteria to sense, respond, and adapt to changing environments, such as elevation of Cu(I)/Ag(I) ions in the periplasm. In...
Two-component systems (TCSs) are essential for bacteria to sense, respond, and adapt to changing environments, such as elevation of Cu(I)/Ag(I) ions in the periplasm. In , the CusS-CusR TCS up-regulates the genes under increased periplasmic Cu(I)/Ag(I) concentrations to help maintain metal ion homeostasis. The CusS histidine kinase is a homodimeric integral membrane protein that binds to periplasmic Cu(I)/Ag(I) and transduces a signal to its cytoplasmic kinase domain. However, the mechanism of how metal binding in the periplasm activates autophosphorylation in the cytoplasm is unknown. Here, we report that only one of the two metal ion-binding sites in CusS enhances dimerization of the sensor domain. Utilizing nanodisc technology to study full-length CusS, we show that metal-induced dimerization in the sensor domain triggers kinase activity in the cytoplasmic domain. We also investigated autophosphorylation in the cytoplasmic domain of CusS and phosphotransfer between CusS and CusR. analyses show that CusS autophosphorylates its conserved H271 residue at the N1 position of the histidine imidazole. The phosphoryl group is removed by the response regulator CusR in a reaction that requires a conserved aspartate at position 51. Functional analyses of CusS and CusR variants with mutations in the autophosphorylation or phosphoacceptor residues suggest that the phosphotransfer event is essential for metal resistance in Biochemical analysis shows that the CusS dimer autophosphorylates using a mechanism. Our results support a signal transduction model in which rotation and bending movements in the cytoplasmic domain maintain the mode of autophosphorylation.
Topics: Copper; Dimerization; Escherichia coli; Escherichia coli Proteins; Histidine Kinase; Periplasm; Phosphorylation; Protein Domains; Protein Structure, Quaternary; Signal Transduction; Silver; Trans-Activators
PubMed: 30530842
DOI: 10.1042/BCJ20180577