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Protein Expression and Purification Jul 2015Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However,...
Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However, expressing recombinant cystatin C is difficult because of low yield and inclusion bodies in Escherichia coli (E. coli). In this study, we cloned HCC gene into pET-22b vector containing PelB leader signal sequence, which could direct the protein to the bacterial periplasm. Large amounts of soluble HCC could be efficiently expressed in the bacterial periplasm at 16°C with 0.1mM IPTG induction. The recombinant HCC was isolated in high purity by cation exchange chromatography and gel filtration chromatography. Furthermore, the HCC was characterized by circular dichroism (CD) and dynamic light scattering (DLS), and displayed biological activity against papain. Here, we provide a method to produce large amounts of soluble mature HCC in E. coli.
Topics: Cystatin C; Escherichia coli; Gene Expression; Humans; Periplasm; Recombinant Proteins
PubMed: 25795130
DOI: 10.1016/j.pep.2015.03.006 -
Journal of the American Chemical Society Apr 2024The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts...
The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring and in real time (i) the hydrolysis of β-lactams by β-lactamases, (ii) the interaction of drugs belonging to the β-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm.
Topics: Anti-Bacterial Agents; Periplasm; Bacteria; beta-Lactams; beta-Lactamases; Magnetic Resonance Spectroscopy
PubMed: 38500259
DOI: 10.1021/jacs.4c00604 -
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 -
Current Topics in Microbiology and... 2015Members of the family Leptospiraceae are thin, spiral, highly motile bacteria that are best visualized by darkfield microscopy. These characteristics are shared with... (Review)
Review
Members of the family Leptospiraceae are thin, spiral, highly motile bacteria that are best visualized by darkfield microscopy. These characteristics are shared with other members of the Order Spirochaetales, but few additional parallels exist among spirochetes. This chapter describes basal features of Leptospira Leptospira that are central to survival and, in the case of pathogenic leptospiral species, intimately linked with pathogenesis, including its morphology, characteristic motility, and unusual metabolism. This chapter also describes the general methodology and critical requirements for in vitro cultivation and storage of Leptospira within a laboratory setting.
Topics: Culture Media; Leptospira; Movement; Periplasm
PubMed: 25388131
DOI: 10.1007/978-3-662-45059-8_3 -
Scientific Reports Mar 2016Periplasmic expression of soluble proteins in Escherichia coli not only offers a much-simplified downstream purification process, but also enhances the probability of...
Periplasmic expression of soluble proteins in Escherichia coli not only offers a much-simplified downstream purification process, but also enhances the probability of obtaining correctly folded and biologically active proteins. Different combinations of signal peptides and target proteins lead to different soluble protein expression levels, ranging from negligible to several grams per litre. Accurate algorithms for rational selection of promising candidates can serve as a powerful tool to complement with current trial-and-error approaches. Accordingly, proteomics studies can be conducted with greater efficiency and cost-effectiveness. Here, we developed a predictor with a two-stage architecture, to predict the real-valued expression level of target protein in the periplasm. The output of the first-stage support vector machine (SVM) classifier determines which second-stage support vector regression (SVR) classifier to be used. When tested on an independent test dataset, the predictor achieved an overall prediction accuracy of 78% and a Pearson's correlation coefficient (PCC) of 0.77. We further illustrate the relative importance of various features with respect to different models. The results indicate that the occurrence of dipeptide glutamine and aspartic acid is the most important feature for the classification model. Finally, we provide access to the implemented predictor through the Periscope webserver, freely accessible at http://lightning.med.monash.edu/periscope/.
Topics: Algorithms; Escherichia coli; Escherichia coli Proteins; Internet; Machine Learning; Periplasm; Regression Analysis; Solubility
PubMed: 26931649
DOI: 10.1038/srep21844 -
Folia Biologica 2014In the cytoplasm of oocytes (ooplasm) located in ovarian follicles with diameters 2000 microm and 2150 microm in Acipenser gueldenstaedtii, and 2000 microm and 2350...
In the cytoplasm of oocytes (ooplasm) located in ovarian follicles with diameters 2000 microm and 2150 microm in Acipenser gueldenstaedtii, and 2000 microm and 2350 microm in A. baerii, periplasm containing a basophilic compartment and endoplasm containing reserve materials was formed. Vesicles involved in polyspermy blocking and in the formation of the embryo were located in the periplasm. These included compact (cCGs), low-electron-dense cortical granules (lCGs), and lamellar bodies. The cCGs were bounded by a membrane, comprised fibrillar material, fibrils and rod-shaped components. The lCGs were membrane-bounded and contained fibrillar material and granular inclusions. Endoplasmic reticulum (ER) and Golgi complexes were involved in the formation of cCG and lCG. The basophilic compartment, ER and Golgi vesicles participated in the formation of lamellar bodies. They comprised numerous membranes and fibrillar material. It is assumed that they transfer membranes and their precursors to the growing furrow during cleavage and release their content to organize the extracellular matrix. The location of compounds in the developing egg envelope of A. gueldenstaedtii was presented and discussed. Ovaries of both investigated species represented the first pubertal stages of development. Such fish should not be used for reproduction.
Topics: Animals; Fishes; Oocytes; Oogenesis; Organelles; Periplasm
PubMed: 25916166
DOI: 10.3409/fb62_4.377 -
Philosophical Transactions of the Royal... Apr 2012Autotransport in Gram-negative bacteria denotes the ability of surface-localized proteins to cross the outer membrane (OM) autonomously. Autotransporters perform this... (Review)
Review
Autotransport in Gram-negative bacteria denotes the ability of surface-localized proteins to cross the outer membrane (OM) autonomously. Autotransporters perform this task with the help of a β-barrel transmembrane domain localized in the OM. Different classes of autotransporters have been investigated in detail in recent years; classical monomeric but also trimeric autotransporters comprise many important bacterial virulence factors. So do the two-partner secretion systems, which are a special case as the transported protein resides on a different polypeptide chain than the transporter. Despite the great interest in these proteins, the exact mechanism of the transport process remains elusive. Moreover, different periplasmic and OM factors have been identified that play a role in the translocation, making the term 'autotransport' debatable. In this review, we compile the wealth of details known on the mechanism of single autotransporters from different classes and organisms, and put them into a bigger perspective. We also discuss recently discovered or rediscovered classes of autotransporters.
Topics: Adhesins, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Secretion Systems; Cell Membrane; Gram-Negative Bacteria; Models, Molecular; Periplasm; Protein Folding; Protein Structure, Tertiary; Protein Transport; Virulence Factors
PubMed: 22411980
DOI: 10.1098/rstb.2011.0208 -
Molecular Microbiology Sep 2015Salmonellae survive and propagate in macrophages to cause serious systemic disease. Periplasmic superoxide dismutase plays a critical role in this survival by combating...
Salmonellae survive and propagate in macrophages to cause serious systemic disease. Periplasmic superoxide dismutase plays a critical role in this survival by combating phagocytic superoxide. Salmonella Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Although both proteins are produced during infection, only SodCI is functional in the macrophage phagosome. We have previously shown that SodCI, relative to SodCII, is both protease resistant and tethered within the periplasm and that either of these properties is sufficient to allow a SodC to protect against phagocytic superoxide. Tethering is defined as remaining cell-associated after osmotic shock or treatment with cationic antimicrobial peptides. Here we show that SodCI non-covalently binds peptidoglycan. SodCI binds to Salmonella and Bacillus peptidoglycan, but not peptidoglycan from Staphylococcus. Moreover, binding can be inhibited by a diaminopimelic acid containing tripeptide, but not a lysine containing tripeptide, showing that the protein recognizes the peptide portion of the peptidoglycan. Replacing nine amino acids in SodCII with the corresponding residues from SodCI confers tethering, partially delineating an apparently novel peptidoglycan binding domain. These changes in sequence increase the affinity of SodCII for peptidoglycan fragments to match that of SodCI and allow the now tethered SodCII to function during infection.
Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Diaminopimelic Acid; Macrophages; Mice, Inbred BALB C; Models, Molecular; Molecular Sequence Data; Osmotic Pressure; Peptidoglycan; Periplasm; Phagosomes; Protein Binding; Salmonella typhimurium; Sequence Alignment; Superoxide Dismutase
PubMed: 25998832
DOI: 10.1111/mmi.13067 -
Molecular Microbiology Jul 2000A set of multidrug efflux systems enables Gram-negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of... (Review)
Review
A set of multidrug efflux systems enables Gram-negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of major efflux pumps of Gram-negative bacteria, which expel from the cells a remarkably broad range of antimicrobial compounds and produce the characteristic intrinsic resistance of these bacteria to antibiotics, detergents, dyes and organic solvents. Each efflux pump consists of three components: the inner membrane transporter, the outer membrane channel and the periplasmic lipoprotein. Similar to the multidrug transporters from eukaryotic cells and Gram-positive bacteria, the inner membrane transporters from Gram-negative bacteria recognize and expel their substrates often from within the phospholipid bilayer. This efflux occurs without drug accumulation in the periplasm, implying that substrates are pumped out across the two membranes directly into the medium. Recent data suggest that the molecular mechanism of the drug extrusion across a two-membrane envelope of Gram-negative bacteria may involve the formation of the membrane adhesion sites between the inner and the outer membranes. The periplasmic components of these pumps are proposed to cause a close membrane apposition as the complexes are assembled for the transport.
Topics: Bacterial Outer Membrane Proteins; Carrier Proteins; Cell Membrane; Drug Resistance, Microbial; Drug Resistance, Multiple; Gram-Negative Bacteria; Ion Channels; Periplasm
PubMed: 10931319
DOI: 10.1046/j.1365-2958.2000.01926.x -
Journal of Bacteriology Dec 2022Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are...
Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, many hypotheses propose distinct periplasmic carriers could be used for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, little support for these models could be found. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower periplasmic levels than PpcA. Mutants containing only PpcC and PpcE showed defects, but these cytochromes were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild-type Fe(III) reduction. PpcA and PpcE from similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme -type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple Δ Δ mutants grew near wild-type rates with normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing and suggest that an as-yet-undiscovered periplasmic mechanism supports electron transfer to electrodes. Many inner and outer membrane cytochromes used by for electron transfer to extracellular acceptors have specific functions. How these are connected by periplasmic carriers remains poorly understood. G. sulfurreducens contains multiple triheme periplasmic cytochromes with unique biochemical properties and expression profiles. It is hypothesized that each could be involved in a different respiratory pathway, depending on redox potential or energy needs. Here, we show that periplasmic cytochromes instead show evidence of being highly promiscuous. Any of 6 triheme cytochromes supported similar growth with soluble or insoluble metals, but none were required when cells utilized electrodes. These findings fail to support many models of electron transfer, and question why these organisms produce such an array of periplasmic cytochromes.
Topics: Geobacter; Periplasm; Ferric Compounds; Electrons; Electron Transport; Cytochromes; Oxidation-Reduction
PubMed: 36383007
DOI: 10.1128/jb.00322-22