Physiological characteristics of recombinant Escherichia coli cells displaying poly-His peptides

Using the Escherichia coli OmpC protein as an anchoring motif, four different poly-His units (1, 2, 3 and 6 copies of 6-His) were displayed on the seventh loop of the OmpC. Recombinant E. coli strains displaying 1, 3 or 6 copies of poly-His became much more sensitive to SDS (0.1%, w/v) and EDTA (2 mM) compared with control strains. However, recombinant E. coli cells displaying 2 copies of poly-His were resistant to SDS and EDTA; greater than 70% and 90% of cells maintained cell integrity after 60 min treatment with SDS and EDTA, respectively, suggesting its usefulness as a whole cell biosorbent.     

Characterization of Salmonella typhi OmpC and OmpF porins engineered with HIV‐gp41 epitope on the surface loops

Porins form trimers in the outer membrane and help transport nutrients and waste products across the bacterial cell membrane. Porin loops are suitable candidates as display systems due to their high immunogenicity and presentation at the bacterial cell surface. In this study, Salmonella typhi porins (OmpC and OmpF) engineered with the Kennedy peptide from gp41 of HIV were characterised. The chimeric OmpC carrying the Kennedy peptide in loop7 did not trimerise, whereas the chimeric OmpF with the epitope in loop5 formed trimers and also was recognised by the antibodies in the HIV patient serum. The results suggest that chimeric S. typhi OmpF may be taken further as a potential candidate to develop as an epitope display system. Proteins 2017; 85:657–664. © 2016 Wiley Periodicals, Inc.     

Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif.

A novel cell surface display system was developed by employing Escherichia coli outer membrane protein C (OmpC) as an anchoring motif. Polyhistidine peptides consisting of up to 162 amino acids could be successfully displayed on the seventh exposed loop of OmpC. Recombinant cells displaying polyhistidine could adsorb up to 32.0 micromol of Cd(2+) per g (dry weight) of cells.     

Display of Polyhistidine Peptides on the Escherichia coli Cell Surface by Using Outer Membrane Protein C as an Anchoring Motif

A novel cell surface display system was developed by employing Escherichia coli outer membrane protein C (OmpC) as an anchoring motif. Polyhistidine peptides consisting of up to 162 amino acids could be successfully displayed on the seventh exposed loop of OmpC. Recombinant cells displaying polyhistidine could adsorb up to 32.0 μmol of Cd²⁺ per g (dry weight) of cells.     

Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).     

Interaction of anions with the active site of carboxypeptidase A

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Display of avian influenza virus nucleoprotein on <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> cell surface using CTC as a fusion partner

The S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on the cell surface of B. thuringiensis. By fusing np with the anchoring motif of ctc, four recombinant plasmids were constructed. They harbored fusion gene ctc-np, csa-ctc-np (csa representing csaAB operon, very important in anchoring S-layer protein on cell surface), ctc-npp (npp representing the part fragment of np), and csa-ctc-npp, respectively. Five recombinant strains were obtained by transferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. The vegetative cells of five strains were used as agglutinogens for slide agglutination assays. The assays showed recombinant NP proteins successfully displayed on the cell surface of five strains. After immunization of chickens with spores by oral route, all five strains elicited a humoral response to NP and exhibited immunogenicity as indicated by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that one of five strains, CN (bearing csa-ctc-npp), exhibited the highest immunogenicity among five strains, which suggested that the best way of constructing ctc fusion gene was the csa-ctc-npp. The strategy developed in this study suggests the possibility of generating a heat-stable and oral veterinary vaccine with B. thuringiensis surface display system.     

Synthesis, structure and antimicrobial activity of manganese(II) and cobalt(II) complexes of the polyether ionophore antibiotic Sodium Monensin A

Mononuclear neutral manganese(II) and cobalt(II) complexes with the antibiotic Sodium Monensin A (Mon-Na, 1b) were synthesized and characterized. The crystal structures of M(Mon-Na)2Cl2.H2O (M=Mn, 2; M=Co, 3) were determined by X-ray crystallography. The complexes crystallize in monoclinic space group C2 with a tetrahedrally coordinated transition metal attached to oxygen atoms of deprotonated carboxyl groups of two Sodium Monensin molecules and two chloride ions. The sodium ion remains in the cavity of the ligand and cannot be replaced by Mn(II) or Co(II). The complexes were additionally characterized by different spectroscopic techniques (UV-Visible, EPR, FAB-MS). A preferable octahedral environment around the transition metal centers is observed in polar solvents while the complexes retain their tetrahedral structure in non-polar media. The antimicrobial activity of 1b, 2 and 3 was tested against Gram(+) and Gram(-) bacteria.     

High density array screening to identify the genetic requirements for transition metal tolerance in Saccharomyces cerevisiae

Biological systems have developed with a strong dependence on transition metals for accomplishing a number of biochemical reactions. Iron, copper, manganese and zinc are essential for virtually all forms of life with their unique chemistries contributing to a variety of physiological processes including oxygen transport, generation of cellular energy and protein structure and function. Properties of these metals (and to a lesser extent nickel and cobalt) that make them so essential to life also make them extremely cytotoxic in many cases through the formation of damaging oxygen radicals via Fenton chemistry. While life has evolved to exploit the chemistries of transition metals to drive physiological reactions, systems have concomitantly evolved to protect against the damaging effects of these same metals. Saccharomyces cerevisiae is a valuable tool for studying metal homeostasis with many of the genes identified thus far having homologs in higher eukaryotes including humans. Using high density arrays, we have screened a haploid S. cerevisiae deletion set containing 4786 non-essential gene deletions for strains sensitive to each of Fe, Cu, Mn, Ni, Zn and Co and then integrated the six screens using cluster analysis to identify pathways that are unique to individual metals and others with function shared between metals. Genes with no previous implication in metal homeostasis were found to contribute to sensitivity to each metal. Significant overlap was observed between the strains that were sensitive to Mn, Ni, Zn and Co with many of these strains lacking genes for the high affinity Fe transport pathway and genes involved in vacuolar transport and acidification. The results from six genome-wide metal tolerance screens show that there is some commonality between the cellular defenses against the toxicity of Mn, Ni, Zn and Co with Fe and Cu requiring different systems. Additionally, potential new factors been identified that function in tolerance to each of the six metals.     

An assessment of the toxicity of metals to Pseudomonas aeruginosa PU21 (Rip64)

The toxicity of Co(II), Mn(II), Cd(II), and Zn(II) for Pseudomonas aeruginosa PU21, a Hg(II)-hyperresistant strain containing the mercury resistance mer operon, was determined. The metal tolerance of PU21 was strongly influenced by environmental conditions (e.g., existing metal, medium composition). Dose-response analysis on chronic and acute toxicity (e.g., EC(20), median effective dose EC(50), and slope factor B) of divalent cobalt, manganese, cadmium, and zinc cations in LB medium amended with citric acid phosphate buffered saline (CAPBS) suggested a toxicity series of Co > Mn approximately Zn > Cd for EC(50). In contrast, excluding the likely precipitate of Zn(II), the toxicity ranking in phosphate-buffered saline (PBS)-amended LB medium was Co > Cd > Mn. The metal toxicity in PBS, irrespective of metals, was greater than that in CAPBS. This might be attributed to the presence of citric acid in CAPBS as a chelating ligand donating electrons to hold free metals (e.g., Cd(2+), Zn(2+) tetrahedral ML(4) complex). The toxicity assessment established viable operation ranges (ca. 相似文献   

Cobalt Immobilization by Manganese Oxidizing Bacteria from the Indian Ridge System

Co immobilization by two manganese oxidizing isolates from Carlsberg Ridge waters (CR35 and CR48) was compared with that of Mn at same molar concentrations. At a lower concentration of 10 μM, CR35 and CR48 immobilized 22 and 23 fM Co cell(-1) respectively, which was 1.4 to 2 times higher than that of Mn oxidation, while at 10 mM the immobilization was 15-69 times lower than that of Mn. Scanning electron microscope and energy dispersive X-ray analyses of intact bacterial cells grown in 1 mM Co revealed Co peaks showing extracellular binding of the metal. However, it was evident from transmission electron microscope analyses that most of the sequestered Co was bound intracellularly along the cell membrane in both the isolates. Change in morphology was one of the strategies bacteria adopted to counter metal stress. The cells grew larger and thus maintained a lower than normal surface area-volume ratio on exposure to Co to reduce the number of binding sites. An unbalanced growth with increasing Co additions was observed in the isolates. Cells attained a length of 10-18 μm at 10 mM Co which was 11-15 times the original cell length. Extensive cell rupture indicated that Co was harmful at this concentration. It is apparent that biological and optimal requirement of Mn is more than Co. Thus, these differences in the immobilization of the two metals could be driven by the differences in the requirement, cell physiology and the affinities of the isolates for the concentrations of the metals tested.     

Molecular modelling of epitope presentation using membrane protein OmpC

Three-dimensional models of the chimeric S. typhi OmpC protein carrying an epitope from rotavirus VP4 capsid protein on either of two exposed loops (fourth and sixth) were constructed separately, using computer-aided homology modelling. The theoretical model of S. typhi OmpC was used as a template. The monomers were initially energy minimized. The trimers were generated for both the chimeric S. typhi OmpC proteins and the structures were optimized after several cycles of minimization. The surface accessibility calculations for the resulting models show that epitope recognition should be more effective in the fourth loop than in the sixth loop, in accordance with the experimental results on the immunogenic nature of the rotaviral epitope inserted into the two putative loops of S. typhi OmpC.     

Acute and chronic metal exposure impairs locomotion activity in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: a model to study Parkinsonism

The biometals iron (Fe), manganese (Mn) and copper (Cu) have been associated to Parkinson’s disease (PD) and Parkinsonism. In this work, we report for the first time that acute (15 mM for up to 5 days) or chronic (0.5 mM for up to 15 days) Fe, Mn and Cu exposure significantly reduced life span and locomotor activity (i.e. climbing capabilities) in Drosophila melanogaster. It is shown that the concentration of those biometals dramatically increase in Drosophila’s brain acutely or chronically fed with metal. We demonstrate that the metal accumulation in the fly’s head is associated with the neurodegeneration of several dopaminergic neuronal clusters. Interestingly, it is found that the PPL2ab DAergic neuronal cluster was erode by the three metals in acute and chronic metal exposure and the PPL3 DAergic cluster was also erode by the three metals but in acute metal exposure only. Furthermore, we found that the chelator desferoxamine, ethylenediaminetetraacetic acid, and d-penicillamine were able to protect but not rescue D. melanogaster against metal intoxication. Taken together these data suggest that iron, manganese and copper are capable to destroy DAergic neurons in the fly’s brain, thereby impairing their movement capabilities. This work provides for the first time metal-induced Parkinson-like symptoms in D. melanogaster. Understanding therefore the effects of biometals in the Drosophila model may provide insights into the toxic effect of metal ions and more effective therapeutic approaches to Parkinsonism.     

Manganese in biogenic magnetite crystals from magnetotactic bacteria

Magnetotactic bacteria produce either magnetite (Fe₃O₄) or greigite (Fe₃S₄) crystals in cytoplasmic organelles called magnetosomes. Whereas greigite magnetosomes can contain up to 10 atom% copper, magnetite produced by magnetotactic bacteria was considered chemically pure for a long time and this characteristic was used to distinguish between biogenic and abiogenic crystals. Recently, it was shown that magnetosomes containing cobalt could be produced by three strains of Magnetospirillum . Here we show that magnetite crystals produced by uncultured magnetotactic bacteria can incorporate manganese up to 2.8 atom% of the total metal content (Fe+Mn) when manganese chloride is added to microcosms. Thus, chemical purity can no longer be taken as a strict prerequisite to consider magnetite crystals to be of biogenic origin.     

Structure and metal ion binding of the first transmembrane domain of DMT1

DMT1 is an integral membrane protein with 12 putative transmembrane domains. As a divalent metal ion transporter, it plays an important role in metal ion homeostasis from bacteria to human. Loss-function mutations at the conserved motif DPGN located within the first transmembrane domain (TMD1) of DMT1 indicate the significance of TMD1 in the biological function of the protein. In the present work, we study the structure, topology and metal ion binding of DMT1-TMD1 peptide by nuclear magnetic resonance using sodium dodecyl sulfate and dodecylphosphocholine micelles as membrane mimics. We find that the peptide forms an α-helix-extended segment-α-helix configuration in which the motif DPGN locates at the central flexible region. The N-terminal part of the peptide is deeply embedded in micelles, while the motif section and the C-terminal part are close to the surface of micelles. The peptide can bind to Mn2+ and Co2+ ions by the side chains of the negatively charged residues in the motif section and the C-terminal part of TMD1. The crucial role of the central flexible region and the C-terminal part of TMD1 in metal ion capture is confirmed by the binding of the N-terminal part truncated TMD1 to metal ions.     

The impact of length variations in the L2 loop on the structure and thermal stability of non-specific porins: The case of OmpCs from the Yersinia pseudotuberculosis complex

Porins are integral proteins of the outer membranes of gram-negative bacteria. In membranes, they exist as homotrimers and the L2 loops contribute to their stability. Comparison of OmpC porins of the Yersinia pseudotuberculosis complex with other enterobacterial porins demonstrated L2 loop length diversity, which is caused by varying numbers of dipeptide/tripeptide repeats. The OmpC porins are highly homologous to each other, and they can be subdivided into five isoforms based on their L2 loop structure. Optical spectroscopy and SDS-PAGE experiments revealed that particularities of the L2 loops affected the structure and thermal stability of the porins. Thermal denaturation studies showed that porins with shorter loops, compared to porins with longer loops, had more stable tertiary and less stable secondary and quaternary structures. According to our comparative modeling results, the L2 loops differ in their structure by adopting different spatial positions and forming different polar bonds with a neighbor monomer. The replacement of asparagine with arginine at the C-terminus of the L2 loop shifts the loop upwards and causes the loss of contacts with the arginine clusters within the pores. The increase in the length of these loops ensures that they shift down toward the pore and restore their contacts with arginines on the channel wall, as is the case in classical nonspecific porins. Despite the fact that the surface charge density varies considerably among the OmpC porins, the L2 loops form a typical negatively charged region in the center of the trimer.     

The methionyl aminopeptidase from Escherichia coli can function as an iron(II) enzyme.

The identity of the physiologically relevant metal ions for the methionyl aminopeptidase (MetAP) from Escherichia coli was investigated and is suggested to be Fe(II). The metal content of whole cells in the absence and presence of expression of the type I MetAP from E. coli was determined by inductively coupled plasma (ICP) emission analysis. The observed change in whole cell concentrations of cobalt, cadmium, copper, nickel, strontium, titanium, and vanadium upon expression of MetAP was negligible. On the other hand, significant increases in the cellular metal ion concentrations of chromium, zinc, manganese, and iron were observed with the increase in iron concentration being 4.4 and 6.2 times greater than that of manganese and zinc, respectively. Activity assays of freshly lysed BL21(DE3) cells containing the pMetAAP plasmid revealed detectable levels (>2 units/mg) of MetAP activity. Control experiments with BL21(DE3) without the MetAP plasmid showed no detectable enzymatic activity. Since MetAP is active upon expression, these data strongly suggest that cobalt is not the in vivo metal ion for the MetAP from E. coli. The MetAP from E. coli as purified was found to be catalytically inactive (相似文献   

Cobalt(II) Oxidation by the Marine Manganese(II)-Oxidizing Bacillus sp. Strain SG-1

The geochemical cycling of cobalt (Co) has often been considered to be controlled by the scavenging and oxidation of Co(II) on the surface of manganese [Mn(III,IV)] oxides or manganates. Because Mn(II) oxidation in the environment is often catalyzed by bacteria, we have investigated the ability of Mn(II)-oxidizing bacteria to bind and oxidize Co(II) in the absence of Mn(II) to determine whether some Mn(II)-oxidizing bacteria also oxidize Co(II) independently of Mn oxidation. We used the marine Bacillus sp. strain SG-1, which produces mature spores that oxidize Mn(II), apparently due to a protein in their spore coats (R.A. Rosson and K. H. Nealson, J. Bacteriol. 151:1027-1034, 1982; J. P. M. de Vrind et al., Appl. Environ. Microbiol. 52:1096-1100, 1986). A method to measure Co(II) oxidation using radioactive ⁵⁷Co as a tracer and treatments with nonradioactive (cold) Co(II) and ascorbate to discriminate bound Co from oxidized Co was developed. SG-1 spores were found to oxidize Co(II) over a wide range of pH, temperature, and Co(II) concentration. Leucoberbelin blue, a reagent that reacts with Mn(III,IV) oxides forming a blue color, was found to also react with Co(III) oxides and was used to verify the presence of oxidized Co in the absence of added Mn(II). Co(II) oxidation occurred optimally around pH 8 and between 55 and 65°C. SG-1 spores oxidized Co(II) at all Co(II) concentrations tested from the trace levels found in seawater to 100 mM. Co(II) oxidation was found to follow Michaelis-Menten kinetics. An Eadie-Hofstee plot of the data suggests that SG-1 spores have two oxidation systems, a high-affinity-low-rate system (K_m, 3.3 × 10^-8 M; V_max, 1.7 × 10^-15 M · spore^-1 · h^-1) and a low-affinity-high-rate system (K_m, 5.2 × 10^-6 M; V_max, 8.9 × 10^-15 M · spore^-1 · h^-1). SG-1 spores did not oxidize Co(II) in the absence of oxygen, also indicating that oxidation was not due to abiological Co(II) oxidation on the surface of preformed Mn(III,IV) oxides. These results suggest that some microorganisms may directly oxidize Co(II) and such biological activities may exert some control on the behavior of Co in nature. SG-1 spores may also have useful applications in metal removal, recovery, and immobilization processes.     

Naturally high heavy metal concentrations in feathers of the flightless Kagu Rhynochetos jubatus

We assessed heavy metal concentrations in feathers of 38 Kagus Rhynochetos jubatus living on ultramafic soils in New Caledonia. Concentrations of heavy metals in down feathers were similar to concentrations in shafts of primary or secondary feathers, whereas the concentrations in vanes were much higher, indicating that concentrations in down feathers were not due to external contamination but rather to ingestion. Although there was no anthropogenic pollution in our study area, concentrations of iron (Fe), zinc (Zn), manganese (Mn), chromium (Cr), selenium (Se), strontium (Sr) and cobalt (Co) in feathers were 1.2–21 times higher in Kagu than the average in other bird species studied, the majority of those from polluted environments. Kagus may have specific adaptations that enable them to live in environments with naturally high heavy metal concentrations.     

Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity

Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections.