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Sample records for bacteriophage t4 lysozyme

  1. Domain Motions in Bacteriophage T4 Lysozyme: A Comparison Between Molecular Dynamics

    E-print Network

    van Aalten, Daan

    Domain Motions in Bacteriophage T4 Lysozyme: A Comparison Between Molecular Dynamics) simula- tions of bacteriophage T4 lysozyme in solvent with X-ray data is presented. Essential dynam- ics lysozyme, inferred from its X-ray structure,1,2 is more than twenty years old.3 Although bacteriophage T4

  2. Control of Bacteriophage T4 Tail Lysozyme Activity During the Infection Process

    SciTech Connect

    Kanamaru, Shuji; Ishiwata, Yasutaka; Suzuki, Toshiharu; Rossmann, Michael G.; Arisaka, Fumio

    2010-07-19

    Bacteriophage T4 has an efficient mechanism for injecting the host Escherichia coli cell with genomic DNA. Its gene product 5 (gp5) has a needle-like structure attached to the end of a tube through which the DNA passes on its way out of the head and into the host. The gp5 needle punctures the outer cell membrane and then digests the peptidoglycan cell wall in the periplasmic space. gp5 is normally post-translationally cleaved between residues 351 and 352. The function of this process in controlling the lysozyme activity of gp5 has now been investigated. When gp5 is over-expressed in E. coli, two mutants (S351H and S351A) showed a reduction of cleavage products and five other mutants (S351L, S351K, S351Y, S351Q, and S351T) showed no cleavage. Furthermore, in a complementation assay at 20 C, the mutants that had no cleavage of gp5 produced a reduced number of plaques compared to wild-type T4. The crystal structure of the non-cleavage phenotype mutant of gp5, S351L, complexed with gene product 27, showed that the 18 residues in the vicinity of the potential cleavage site (disordered in the wild-type structure) had visible electron density. The polypeptide around the potential cleavage site is exposed, thus allowing access for an E. coli protease. The lysozyme activity is inhibited in the wild-type structure by a loop from the adjacent gp5 monomer that binds into the substrate-binding site. The same inhibition is apparent in the mutant structure, showing that the lysozyme is inhibited before gp5 is cleaved and, presumably, the lysozyme is activated only after gp5 has penetrated the outer membrane.

  3. T4 LYSOZYME AND ATTACIN GENES ENHANCE RESISTANCE OF TRANSGENIC 'GALAXY' APPLE AGAINST ERWINIA AMYLOVORA (BURR.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genes encoding T4 lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of 'Galaxy' apple, enha...

  4. Structure and function of bacteriophage T4

    PubMed Central

    Yap, Moh Lan; Rossmann, Michael G

    2014-01-01

    Bacteriophage T4 is the most well-studied member of Myoviridae, the most complex family of tailed phages. T4 assembly is divided into three independent pathways: the head, the tail and the long tail fibers. The prolate head encapsidates a 172 kbp concatemeric dsDNA genome. The 925 Å-long tail is surrounded by the contractile sheath and ends with a hexagonal baseplate. Six long tail fibers are attached to the baseplate’s periphery and are the host cell’s recognition sensors. The sheath and the baseplate undergo large conformational changes during infection. X-ray crystallography and cryo-electron microscopy have provided structural information on protein–protein and protein–nucleic acid interactions that regulate conformational changes during assembly and infection of Escherichia coli cells. PMID:25517898

  5. An improved 96-well turbidity assay for T4 lysozyme activity

    PubMed Central

    Toro, Tasha B.; Nguyen, Thao P.; Watt, Terry J.

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: • Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays; • Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and • Incorporates a simplified expression and purification protocol for T4 lysozyme. PMID:26150996

  6. RAPID COMMUNICATION The Structure of Isometric Capsids of Bacteriophage T4

    E-print Network

    Baker, Timothy S.

    RAPID COMMUNICATION The Structure of Isometric Capsids of Bacteriophage T4 Norman H. Olson,* Mari-dimensional structure of DNA-filled, bacteriophage T4 isometric capsids has been determined by means of cryoelectron: bacteriophage T4; cryoelectron microscopy; three-dimensional image reconstruction. Introduction. Bacteriophage T

  7. The Molecular Architecture of the Bacteriophage T4 Neck

    PubMed Central

    Fokine, Andrei; Zhang, Zhihong; Kanamaru, Shuji; Bowman, Valorie D.; Aksyuk, Anastasia A.; Arisaka, Fumio; Rao, Venigalla B.; Rossmann, Michael G.

    2013-01-01

    A hexamer of the bacteriophage T4 tail terminator protein, gp15, attaches to the top of the phage tail stabilizing the contractile sheath and forming the interface for binding of the independently assembled head. Here we report the crystal structure of the gp15 hexamer, describe its interactions in T4 virions that have either an extended tail or a contracted tail, and discuss its structural relationship to other phage proteins. The neck of T4 virions is decorated by the “collar” and “whiskers”, made of fibritin molecules. Fibritin acts as a chaperone helping to attach the long tail fibers to the virus during the assembly process. The collar and whiskers are environment-sensing devices, regulating the retraction of the long tail fibers under unfavorable conditions, thus preventing infection. Cryo-electron microscopy analysis suggests that twelve fibritin molecules attach to the phage neck with six molecules forming the collar and six molecules forming the whiskers. PMID:23434847

  8. Specificity of Interactions among the DNApackaging Machine Components of T4related Bacteriophages

    E-print Network

    Huang, Ching-Tsan

    Bacteriophages Song Gao and Venigalla B. Rao THE JOURNAL OF BIOLOGICAL CHEMISTRY 286, 39443956(2010) Shih bacteriophagemolecular motorgenomic DNA DNADNA T4related bacteriophage packaging components (gp17gp16;2 T4 related BacteriophagesDNA packing machine gp16gp17gp20gp17large terminasedomain domain

  9. The effect of alpha particles on bacteriophage T4Br+.

    PubMed

    Leont'eva, G A; Akoev, I G; Grigor'ev, A E

    1983-01-01

    It is generally accepted that heavy charged particles play an important part in generating the secondary flux of nuclear particles formed by the interaction of space hadrons with nuclei. It is assumed that these particles are responsible for the high biological efficiency of space hadrons in causing cellular damage by their strong interactions. To examine this assumption we investigated the effects of 5.3 MeV alpha particles on bacteriophage T4. This energy provides a LET value of 88.6 KeV/micrometer lying in the range of the highest biological efficiency. PMID:11542756

  10. Genetic complementation by cloned bacteriophage T4 late genes.

    PubMed Central

    Jacobs, K A; Albright, L M; Shibata, D K; Geiduschek, E P

    1981-01-01

    Bacteriophage T4 containing nonsense mutations in late genes was found to be genetically complemented by four conjugate T4 genes (7, 11, 23, or 24) located on plasmid or phage vectors. Complementation was at a very low level unless the infecting phage carried a denB mutation (which abolishes T4 DNA endonuclease IV activity). In most experiments, the infecting phage also had a denA mutation, which abolishes T4 DNA endonuclease II activity. Mutations in the alc/unf gene (which allow dCMP-containing T4 late genes to be expressed) further increased complementation efficiency. Most of the alc/unf mutant phage strains used for these experiments were constructed to incorporate a gene 56 mutation, which blocks dCTP breakdown and allows replication to generate dCMP-containing T4 DNA. Effects of the alc/unf:56 mutant combination on complementation efficiency varied among the different T4 late genes. Despite regions of homology, ranging from 2 to 14 kilobase pairs, between cloned T4 genes and infecting genomes, the rate of formation of recombinants after T4 den:alc phage infection was generally low (higher for two mutants in gene 23, lower for mutants in gene 7 and 11). More significantly, when gene 23 complementation had to be preceded by recombination, the complementation efficiency was drastically reduced. We conclude that high complementation efficiency of cloned T4 late genes need not depend on prior complete breakage-reunion events which transpose those genes from the resident plasmid to a late promoter on the infecting T4 genome. The presence of the intact gene 23 on plasmids reduced the yield of T4 phage. The magnitude of this negative complementation effect varied in different plasmids; in the extreme case (plasmid pLA3), an almost 10-fold reduction of yield was observed. The cells can thus be said to have been made partly nonpermissive for this lytic virus by incorporating a part of the viral genome. Images PMID:6456362

  11. Nanomechanical Characterization of the Triple b-Helix Domain in the Cell Puncture Needle of Bacteriophage T4 Virus

    E-print Network

    Müftü, Sinan

    of Bacteriophage T4 Virus SINAN KETEN,1 J. FERNANDO RODRIGUEZ ALVARADO,2 SINAN MU¨ FTU¨ ,3 and MARKUS J. BUEHLER 1 of the bacteriophage T4 virus. We characterize the compressive mechanical strength of this protein nanotube using full

  12. Functional heterogeneity as reflected by topological parameters in a classical protein molecular model: t4 phage lysozyme.

    PubMed

    Caruso, Lisa Beatrice; Giuliani, Alessandro; Colosimo, Alfredo

    2016-01-01

    A systematic comparison with the Wild-Type (WT) of one-point mutants of bacteriophage T4 lysozyme was carried out using as difference markers the topological parameters of the protein contact networks corresponding to each crystallographic structure. The investigation concerned changes at the resolution level of single residue along the protein sequence. The results were correlated with (reported) changes in functional properties and (observed) changes in the information provided by the energy dissipation algorithm of the "Turbine" software simulation tool. The critical factor leading to significant difference among mutants and WT is in most cases associated to the sensitivity towards mutation of relatively short windows in the amino acidic sequence not necessarily contiguous to the active site. PMID:26412794

  13. Investigation of bacteriophage T4 by atomic force microscopy

    PubMed Central

    Kuznetsov, Yuri G; Chang, Sheng-Chieh

    2011-01-01

    Bacteriophage T4 was visualized using atomic force microscopy (AFM). The images were consistent with, and complementary to electron microscopy images. Head heights of dried particles containing DNA were about 75 nm in length and 60 nm in width, or about 100 nm and 85 nm respectively when scanned in fluid. The diameter of hydrated tail assemblies was 28 nm and their lengths about 130 nm. Seven to eight pronounced, right-handed helical turns with a pitch of 15 nm were evident on the tail assemblies. At the distal end of the tail was a knob shaped mass, presumably the baseplate. The opposite end, where the tail assembly joins the head, was tapered and connected to the portal complex, which was also visible. Phage that had ejected their DNA revealed the internal injection tube of the tail assembly. Heads disrupted by osmotic shock yielded boluses of closely packed DNA that unraveled slowly to expose threads composed of multiple twisted strands of nucleic acid. Assembly errors resulted in the appearance of several percent of the phage exhibiting two rather than one tail assemblies that were consistently oriented at about 72° to one another. No pattern of capsomeres was visible on native T4 heads. A mutant that is negative for the surface proteins hoc and soc, however, clearly revealed the icosahedral arrangement of ring shaped capsomeres on the surface. The hexameric rings have an outside diameter of about 14 nm, a pronounced central depression, and a center-to-center distance of 15 nm. Phage collapsed on cell surfaces appeared to be dissolving, possibly into the cell membrane. PMID:22164350

  14. ON THE THERMODYNAMICS AND KINETICS OF THE COOPERATIVE BINDING OF BACTERIOPHAGE T4-

    E-print Network

    Kowalczykowski, Stephen C.

    ON THE THERMODYNAMICS AND KINETICS OF THE COOPERATIVE BINDING OF BACTERIOPHAGE T4- CODED GENE 32 of thermodynamic, and preliminary kinetic, studies on the molecular details and specificity of interaction of phage T4-coded gene 32-protein (GP32) with nucleic acid lattices. It is shown that the binding ofGP32

  15. Single-Molecule Measurements of T4 Lysozyme using Carbon Nanotube Electronic Circuits

    NASA Astrophysics Data System (ADS)

    Sims, Patrick Craig

    Because of their unique electronic and chemical properties, single-walled carbon nanotubes (SWNTs) are attractive candidates for label-free, single-molecule sensing and detection applications. In this work, a field-effect transistor (FET) architecture comprised of an individual SWNT is used to transduce the conformational motion of a single T4 lysozyme protein, conjugated to the SWNT side wall, into a corresponding electrical current signal. The SWNTs are grown using chemical vapor deposition, and metal electrical contacts are formed using electron beam evaporation. Using N-(1-Pyrene)maleimide, the protein is conjugated to the SWNT side wall. After conjugation, the sensing area of the device is submerged in an electrolyte solution, and the source-drain current is measured while applying an electrolyte-gate. Analysis of the signal provided single-molecule resolution of the dynamical activity of lysozyme as it hydrolyzes macromolecular peptidoglycan, a component of bacterial cell walls. This analysis revealed seven different independent time scales that govern the activity of lysozyme, the pH dependence of these time scales, and a lower limit on the number rate-limiting steps in lysozyme's hinge opening and closing motions. Furthermore, the signals elucidated differences in how lysozyme traverses and catalyzes structurally varying peptidoglycan constructs.

  16. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection

    PubMed Central

    Jin, Qingwen; Chen, Hong; Wang, Xingxia; Zhao, Liandong; Xu, Qingchen; Wang, Huijuan; Li, Guanyu; Yang, Xiaofan; Ma, Hongming; Wu, Haoquan; Ji, Xiaohui

    2015-01-01

    Background Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. Methods We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. Results Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. Conclusions Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents. PMID:26154172

  17. Deciphering Lysis and its Regulation in Bacteriophage T4 

    E-print Network

    Moussa, Samir

    2012-10-19

    Like all phages, T4 requires a holin (T) to effect lysis. The lysis event depends on the temporally regulated action of T, which accumulates in the inner membrane (IM) until, at an allele-specific time, it triggers to ...

  18. Control of Gene Function in Bacteriophage T4

    PubMed Central

    Sauerbier, Walter; Schweiger, Manfred; Herrlich, Peter

    1971-01-01

    Synthesis of early T4 protein, which is normally shut off at 10 min after infection, continues until lysis when host cells have been preinfected with T3 sam+. In host cells preinfected with T3 sam?, synthesis of early enzymes is shut off as normal. Thus, S-adenosylmethionine is required for the turnoff of early T4 functions (at least when host cells have been preinfected with T3). PMID:4943682

  19. Encapsulation of T4 bacteriophage in electrospun poly(ethylene oxide)/cellulose diacetate fibers.

    PubMed

    Korehei, Reza; Kadla, John F

    2014-01-16

    Phage therapy is a potentially beneficial approach to food preservation and storage. Sustained delivery of bacteriophage can prevent bacterial growth on contaminated food surfaces. Using coaxial electrospinning bacteriophage can be encapsulated in electrospun fibers with high viability. The resulting bio-based electrospun fibers may have potential as a food packaging material. In the present work, T4 bacteriophage (T4 phage) was incorporated into core/shell electrospun fibers made from poly(ethylene oxide) (PEO), cellulose diacetate (CDA), and their blends. Fibers prepared using PEO as the shell polymer showed an immediate burst release of T4 phage upon submersion in buffer. The blending of CDA with PEO significantly decreased the rate of phage release, with no released T4 phage being detected from the solely CDA fibers. Increasing the PEO molecular weight increased the electrospun fiber diameter and viscosity of the releasing medium, which resulted in a relatively slower T4 phage release profile. SEM analyses of the electrospun fiber morphologies were in good agreement with the T4 phage release profiles. Depending on the PEO/CDA ratio, the post-release electrospun fiber morphologies varied from discontinuous fibers to minimally swollen fibers. From these results it is suggested that the T4 phage release mechanism is through solvent activation/polymer dissolution in the case of the PEO fibers and/or by diffusion control from the PEO/CDA blend fibers. PMID:24188849

  20. Bacteriophage T4 whiskers: a rudimentary environment-sensing device.

    PubMed Central

    Conley, M P; Wood, W B

    1975-01-01

    The 400 A filaments or "whiskers," which extend outward from the collar region of the phage, control retraction and extension of the tail fibers in response to certain environmental conditions. The tail fibers of normal phage retract in the absence of a required adsorption cofactor, at low pH, at low ionic strength, at low temperature, and at high concentrations of polyethylene glycol. The tail fibers of mutant whiskerless (wac) phage still retract under the first two conditions, but not the last three. Antibodies to whiskers neutralize T4, probably by fixing tail fibers in the retracted configuration. Phage with retracted tail fibers adsorb poorly to host bacterial cells, and their adsorption rate increases as the fibers become extended. These results suggest that one function of the whiskers is to retract the tail fibers and thereby prevent adsorption to host cells under certain conditions that might be unfavorable for production of phage progeny following infection. PMID:242007

  1. Role of cavities and hydration in the pressure unfolding of T4 lysozyme

    PubMed Central

    Nucci, Nathaniel V.; Fuglestad, Brian; Athanasoula, Evangelia A.; Wand, A. Joshua

    2014-01-01

    It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A–benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins. PMID:25201963

  2. Probing the folded state and mechanical unfolding pathways of T4 lysozyme using all-atom and coarse-grained molecular simulation

    NASA Astrophysics Data System (ADS)

    Zheng, Wenjun; Glenn, Paul

    2015-01-01

    The Bacteriophage T4 Lysozyme (T4L) is a prototype modular protein comprised of an N-terminal and a C-domain domain, which was extensively studied to understand the folding/unfolding mechanism of modular proteins. To offer detailed structural and dynamic insights to the folded-state stability and the mechanical unfolding behaviors of T4L, we have performed extensive equilibrium and steered molecular dynamics simulations of both the wild-type (WT) and a circular permutation (CP) variant of T4L using all-atom and coarse-grained force fields. Our all-atom and coarse-grained simulations of the folded state have consistently found greater stability of the C-domain than the N-domain in isolation, which is in agreement with past thermostatic studies of T4L. While the all-atom simulation cannot fully explain the mechanical unfolding behaviors of the WT and the CP variant observed in an optical tweezers study, the coarse-grained simulations based on the Go model or a modified elastic network model (mENM) are in qualitative agreement with the experimental finding of greater unfolding cooperativity in the WT than the CP variant. Interestingly, the two coarse-grained models predict different structural mechanisms for the observed change in cooperativity between the WT and the CP variant—while the Go model predicts minor modification of the unfolding pathways by circular permutation (i.e., preserving the general order that the N-domain unfolds before the C-domain), the mENM predicts a dramatic change in unfolding pathways (e.g., different order of N/C-domain unfolding in the WT and the CP variant). Based on our simulations, we have analyzed the limitations of and the key differences between these models and offered testable predictions for future experiments to resolve the structural mechanism for cooperative folding/unfolding of T4L.

  3. Probing the folded state and mechanical unfolding pathways of T4 lysozyme using all-atom and coarse-grained molecular simulation

    SciTech Connect

    Zheng, Wenjun Glenn, Paul

    2015-01-21

    The Bacteriophage T4 Lysozyme (T4L) is a prototype modular protein comprised of an N-terminal and a C-domain domain, which was extensively studied to understand the folding/unfolding mechanism of modular proteins. To offer detailed structural and dynamic insights to the folded-state stability and the mechanical unfolding behaviors of T4L, we have performed extensive equilibrium and steered molecular dynamics simulations of both the wild-type (WT) and a circular permutation (CP) variant of T4L using all-atom and coarse-grained force fields. Our all-atom and coarse-grained simulations of the folded state have consistently found greater stability of the C-domain than the N-domain in isolation, which is in agreement with past thermostatic studies of T4L. While the all-atom simulation cannot fully explain the mechanical unfolding behaviors of the WT and the CP variant observed in an optical tweezers study, the coarse-grained simulations based on the Go model or a modified elastic network model (mENM) are in qualitative agreement with the experimental finding of greater unfolding cooperativity in the WT than the CP variant. Interestingly, the two coarse-grained models predict different structural mechanisms for the observed change in cooperativity between the WT and the CP variant—while the Go model predicts minor modification of the unfolding pathways by circular permutation (i.e., preserving the general order that the N-domain unfolds before the C-domain), the mENM predicts a dramatic change in unfolding pathways (e.g., different order of N/C-domain unfolding in the WT and the CP variant). Based on our simulations, we have analyzed the limitations of and the key differences between these models and offered testable predictions for future experiments to resolve the structural mechanism for cooperative folding/unfolding of T4L.

  4. Preparation of isolated biomolecules for SFM observations: T4 bacteriophage as a test sample.

    PubMed Central

    Droz, E; Taborelli, M; Wells, T N; Descouts, P

    1993-01-01

    The T4 bacteriophage has been used to investigate protocols for the preparation of samples for scanning force microscopy in air, in order to obtaining reproducible images. The resolution of images and the distribution of bacteriophages on the substrate depends on the buffer type, its concentration, the surface treatment of substrate, and the method of deposition. The best imaging conditions for the phages require dilution in a volatile buffer at low ionic strength and adsorption onto hydrophilic surfaces. When imaging with the scanning force microscopy the quality of the images is influenced by the vertical and lateral forces applied on the sample and by the tip geometry. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 6 PMID:8241398

  5. Identification of P48 and P54 as components of bacteriophage T4 baseplates.

    PubMed Central

    Berget, P B; Warner, H R

    1975-01-01

    The involvement of two bacteriophage T4 gene products in the initiation of T4 tail tube and sheath polymerization on mature baseplates has been studied by radioautography of acrylamide gels of various partially completed tail structures. The products of genes 48 and 54 (P48[the nomenclature P48 refers to the protein product of bacteriophage T4 gene 48] and P54), which are known to be required for the synthesis of mature baseplates, have been shown to be structural components of the baseplate. These gene products have molecular weights of 42,000 and 33,000, respectively. The addition of P54 to the baseplate not only permits the polymerization of the core protein, P19, onto the baseplate, but also caused the disappearance of a polypeptide of molecular weight about 15,000 from the supernatant fraction of infected cells. Another gene product, P27, has been identified in the crude extracts of infected cells. This gene product, which is required for the synthesis of baseplate structures, has the same mobility as one of the unidentified structural polypeptides of the baseplate and is therefore probably also a baseplate component. Images PMID:1202250

  6. Theoretical studies on solvation contribution to the thermodynamic stability of mutants of lysozyme T4.

    PubMed

    Deep, Shashank; Ahluwalia, J C

    2003-06-01

    Atomic solvation parameters (ASPs) are widely used to estimate the solvation contribution to the thermodynamic stability of proteins as well as the free energy of association for protein-ligand complexes. In view of discrepancies in the results of free energies of solvation of folding for various proteins obtained using different atomic solvation parameter sets, systematic studies have been carried out for the calculation of accessible surface area and the changes in free energy of solvation of folding (deltaG(s,f)) for mutants of lysozyme T4 where threonine 157 is replaced by amino acids: cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, asparagine, arginine, serine and valine. The deviations of the calculated results from the experimental results are discussed to highlight the discrepancies in the atomic solvation parameter sets and possible reasons for them. The results are also discussed to throw light on the effect of chain free energy and hydrogen bonding on the stability of mutants. The octanol to water-based ASP sets 'Sch1' and 'EM' perform better than the vacuum to water-based ASP sets. The vacuum to water-based ASP sets 'Sch3' and 'WE' can be used to predict the stability of mutants if a proper method to calculate the hydrogen bond contribution to overall stability is in place. PMID:12874374

  7. Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins

    SciTech Connect

    Fokine, Andrei; Bowman, Valorie D.; Battisti, Anthony J.; Li Qin; Chipman, Paul R.; Rao, Venigalla B.; Rossmann, Michael G.

    2007-10-25

    The bacteriophage T4 capsid contains two accessory surface proteins, the small outer capsid protein (Soc, 870 copies) and the highly antigenic outer capsid protein (Hoc, 155 copies). As these are dispensable for capsid formation, they can be used for displaying proteins and macromolecular complexes on the T4 capsid surface. Anthrax toxin components were attached to the T4 capsid as a fusion protein of the N-terminal domain of the anthrax lethal factor (LFn) with Soc. The LFn-Soc fusion protein was complexed in vitro with Hoc{sup -}Soc{sup -}T4 phage. Subsequently, cleaved anthrax protective antigen heptamers (PA63){sub 7} were attached to the exposed LFn domains. A cryo-electron microscopy study of the decorated T4 particles shows the complex of PA63 heptamers with LFn-Soc on the phage surface. Although the cryo-electron microscopy reconstruction is unable to differentiate on its own between different proposed models of the anthrax toxin, the density is consistent with a model that had predicted the orientation and position of three LFn molecules bound to one PA63 heptamer.

  8. Bacteriophage Tail Components IV. Pteroyl Polyglutamate Synthesis in T4D-Infected Escherichia coli B

    PubMed Central

    Kozloff, Lloyd M.; Lute, Murl

    1973-01-01

    The nature of pteroyl polyglutamates in uninfected and T4D bacteriophage-infected Escherichia coli B has been examined. 3H-p-aminobenzoic acid has been used to label the folate compounds and gel permeation chromatography on glass beads to separate the folate compound by molecular size. It has been found that, although the major folate compound in uninfected bacteria is pteroyl triglutamate, E. coli B cells also contain folate compounds having as many as six glutamate residues. Infection with T4D stimulated the addition of glutamate residues to the lower-molecular-weight host pteroyl compounds, resulting in the conversion of the host compounds into the hexaglutamate form. This viral-induced conversion is chloramphenicol sensitive and appears to be due to a late phage gene product. The phage gene responsible for this conversion has not been identified. In cells infected with a T4D mutant defective in gene 28, there was an apparent production of the large pteroyl polyglutamates equivalent in size to pte(glu)9-12. These high-molecular-weight forms were converted into pte(glu)6 by incubation with bacterial extracts made after infection with T4D 28+. Apparently, the product of T4D gene 28+ is capable of specifically cleaving the high-molecular-weight polyglutamates to the form necessary for phage tail assembly. PMID:4575283

  9. A New Epistasis Group for the Repair of DNA Damage in Bacteriophage T4: Replication Repair

    PubMed Central

    Wachsman, Joseph T.; Drake, John W.

    1987-01-01

    The gene 32 mutation amA453 sensitizes bacteriophage T4 to the lethal effects of ultraviolet (UV) irradiation, methyl methanesulfonate and angelicin-mediated photodynamic irradiation when treated particles are plated on amber-suppressing host cells. The increased UV sensitivity caused by amA453 is additive to that caused by mutations in both the T4 excision repair (denV) and recombination repair (uvsWXY) systems, suggesting the operation of a third kind of repair system. The mutation uvs79, with many similarities to amA453 but mapping in gene 41, is largely epistatic to amA453. The mutation mms1, also with many similarities to amA453, maps close to amA453 within gene 32 and is largely epistatic to uvs79. Neither amA453 nor uvs79 affect the ratio of UV-induced mutational to lethal hits, nor does amA453 affect spontaneous or UV-enhanced recombination frequencies. Gene 32 encodes the major T4 ssDNA-binding protein (the scaffolding of DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication. We conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before or after DNA replication. PMID:3552872

  10. Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure.

    PubMed

    Lerch, Michael T; López, Carlos J; Yang, Zhongyu; Kreitman, Margaux J; Horwitz, Joseph; Hubbell, Wayne L

    2015-05-12

    Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306-E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron-electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ? 3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111-114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2-3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape. PMID:25918400

  11. Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure

    PubMed Central

    Lerch, Michael T.; López, Carlos J.; Yang, Zhongyu; Kreitman, Margaux J.; Horwitz, Joseph; Hubbell, Wayne L.

    2015-01-01

    Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306–E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron–electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ?3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111–114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2–3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape. PMID:25918400

  12. Placing Single-Molecule T4 Lysozyme Enzymes on a Bacterial Cell Surface: Toward Probing Single-Molecule Enzymatic Reaction in Living Cells

    SciTech Connect

    Hu, Dehong; Lu, H PETER.

    2004-07-01

    TheT4 lysozyme enzymatic hydrolyzation reaction of bacterial cell walls is an important biological process, and single-molecule enzymatic reaction dynamics had been studied under physiological condition using purified E. Coli cell walls as substrates. Here, we report progress toward characterizing the T4 lysozyme enzymatic reaction on a living bacterial cell wall using a combined single-molecule placement and spectroscopy. Placing a dye-labeled single T4 lysozyme molecule on a targeted cell wall by using a hydrodynamic micro-injection approach, we monitored single-molecule rotational motions during binding, attachment to, and dissociation from the cell wall by tracing single-molecule fluorescence intensity time trajectories and polarization. The single-molecule attachment duration of the T4 lysozyme to the cell wall during enzymatic reactions was typically shorter than photobleaching time under physiological conditions.

  13. Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs.

    PubMed

    Durand, Sylvain; Richard, Graziella; Bontems, François; Uzan, Marc

    2012-05-01

    The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine-Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5'-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5'-monophosphate termini, to attack mRNAs from the 5'-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5' terminus for mRNA stability and depicts a pathway of mRNA degradation with 5'- to 3'-polarity in cells devoid of 5'-3' exonucleases. It also ascribes a role for T4 PNK during normal phage development. PMID:22499790

  14. Atomic force microscopy images of T4 bacteriophages on silicon substrates

    SciTech Connect

    Kolbe, W.F.; Ogletree, D.F.; Salmeron, M.B.

    1991-08-01

    A new atomic force microscope incorporating microfabricated cantilevers and employing laser beam deflection for force detection has been constructed and is being applied to studied of biological material. In this study, T4 bacteriophage virus particles were deposited from solution onto electronic grade flat silicon wafers and imaged in air with the microscope. Microliter droplets of the solution were deposited and either allowed to dry or removed with blotting paper. The images show both isolated viruses and aggregates of various sizes. The external structure as well as strands believed to be DNA streaming out of the virus could be observed. The construction of the microscope and its performance are also described. 19 refs., 4 figs.

  15. Evidence for an electrostatic mechanism of force generation by the bacteriophage T4 DNA packaging motor

    NASA Astrophysics Data System (ADS)

    Migliori, Amy D.; Keller, Nicholas; Alam, Tanfis I.; Mahalingam, Marthandan; Rao, Venigalla B.; Arya, Gaurav; Smith, Douglas E.

    2014-06-01

    How viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free-energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle.

  16. The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria

    SciTech Connect

    Aksyuk, Anastasia A.; Leiman, Petr G.; Kurochkina, Lidia P.; Shneider, Mikhail M.; Kostyuchenko, Victor A.; Mesyanzhinov, Vadim V.; Rossmann, Michael G.

    2009-07-22

    The contractile tail of bacteriophage T4 is a molecular machine that facilitates very high viral infection efficiency. Its major component is a tail sheath, which contracts during infection to less than half of its initial length. The sheath consists of 138 copies of the tail sheath protein, gene product (gp) 18, which surrounds the central non-contractile tail tube. The contraction of the sheath drives the tail tube through the outer membrane, creating a channel for the viral genome delivery. A crystal structure of about three quarters of gp18 has been determined and was fitted into cryo-electron microscopy reconstructions of the tail sheath before and after contraction. It was shown that during contraction, gp18 subunits slide over each other with no apparent change in their structure.

  17. A bacteriophage T4 in vitro system to clone long DNA molecules. Final report, June 1, 1990--January 31, 1996

    SciTech Connect

    Rao, V.B.

    1997-09-01

    A summary is presented of the following objectives: development of a bacteriophage T4 in vitro system, and techniques to clone long segments of foreign DNA; development of a giant prohead DNA packaging system that could potentially be used to clone even a megabase size DNA; and development of techniques to rapidly map the cloned DNA inserts.

  18. Crystallization of the carboxy-terminal region of the bacteriophage T4 proximal long tail fibre protein gp34

    SciTech Connect

    Granell, Meritxell; Namura, Mikiyoshi; Alvira, Sara; Garcia-Doval, Carmela; Singh, Abhimanyu K.; Gutsche, Irina; Raaij, Mark J. van Kanamaru, Shuji

    2014-06-19

    The crystallization of three C-terminal fragments of the bacteriophage T4 protein gp34 is reported. Diffraction data have been obtained for three native crystal forms and two selenomethionine derivatives, one of which contained high-quality anomalous signal.

  19. Protein structural plasticity exemplified by insertion and deletion mutants in T4 lysozyme.

    PubMed Central

    Vetter, I. R.; Baase, W. A.; Heinz, D. W.; Xiong, J. P.; Snow, S.; Matthews, B. W.

    1996-01-01

    To further investigate the ways in which proteins respond to changes in the length of the polypeptide chain, a series of 32 insertions and five deletions were made within nine different alpha-helices of T4 lysozyme. In most cases, the inserted amino acid was a single alanine, although in some instances up to four residues, not necessarily alanine, were used. Different insertions destabilized the protein by different amounts, ranging from approximately 1 to 6 kcal/mol. In one case, no protein could be obtained. An "extension" mutant in which the carboxy terminus of the molecule was extended by four alanines increased stability by 0.3 kcal/mol. For the deletions, the loss in stability ranged from approximately 3 to 5 kcal/mol. The structures of six insertion mutants, as well as one deletion mutant and the extension mutant, were determined, three in crystal forms nonisomorphous with wild type. In all cases, including previously described insertion mutants within a single alpha-helix, there appears to be a strong tendency to preserve the helix by translocating residues so that the effects of the insertion are propagated into a bend or loop at one end or the other of the helix. In three mutants, even the hydrophobic core was disrupted so as to permit the preservation of the alpha-helix containing the insertion. Translocation (or "register shift") was also observed for the deletion mutant, in this case a loop at the end of the helix being shortened. In general, when translocation occurs, the reduction in stability is only moderate, averaging 2.5 kcal/mol. Only in the most extreme cases does "bulging" or "looping-out" occur within the body of an alpha-helix, in which case the destabilization is substantial, averaging 4.9 kcal/mol. Looping-out can occur for insertions close to the end of a helix, in which case the destabilization is less severe, averaging 2.6 kcal/mol. Mutant A73-[AAA] as well as mutants R119-[A] and V131-[A], include shifts in the backbone of 3-6 A, extending over 20 residues or more. As a result, residues 114-142, which form a "cap" on the carboxy-terminal domain, undergo substantial reorganizations such that the interface between this "cap" and the rest of the protein is altered substantially. In the case of mutant A73-[AAA], two nearby alpha-helices, which form a bend of approximately 105 degrees in the wild-type structure, reorganize in the mutant structure to form a single, essentially straight helix. These structural responses to mutation demonstrate the plasticity of protein structures and illustrate ways in which their three-dimensional structures might changes during evolution. PMID:8976549

  20. HIGH-PRESSURE X-RAY CRYSTALLOGRAPHY AND CORE HYDROPHOBICITY OF T4 LYSOZYMES

    E-print Network

    Gruner, Sol M.

    separation between oil-like non-polar and charged or po- lar amino acid residues drives the formation unfolding is a different process from thermally or chemically driven unfolding. Separately, little is known no impact on the pressure response over the wild-type lysozyme. Instead, four water molecules cooperatively

  1. Structure-function analysis of the DNA translocating portal of the bacteriophage T4 packaging machine.

    PubMed

    Padilla-Sanchez, Victor; Gao, Song; Kim, Hyung Rae; Kihara, Daisuke; Sun, Lei; Rossmann, Michael G; Rao, Venigalla B

    2014-03-01

    Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages ?29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging. PMID:24126213

  2. Analysis of the cooperative thermal unfolding of the td intron of bacteriophage T4.

    PubMed Central

    Brion, P; Michel, F; Schroeder, R; Westhof, E

    1999-01-01

    The thermal stability of folded transcripts of the td intron of bacteriophage T4 that carried up to three base substitutions was investigated by temperature gradient gel electrophoresis (TGGE) and UV melting. The unfolding of this autocatalytic group I intron is endothermic and entropically driven. Although the effects of mutations in base pairs follow in most cases the expected order G-C>A-U>G.U>A.C, the extent of global destabilization varies strongly according to the helix in which substitutions are located. Effects are more pronounced in the P7 helix which forms, together with the P3 helix, the central pseudoknot of group I introns. The stability of the tertiary fold was also monitored as a function of ionic concentration and of the nature of the ion. At low ionic strength, the stabilizing effect of divalent ions is independent of the nature of the ion. However, with increasing ionic concentration, stabilization is most pronounced for Mg2+and less for Mn2+with Ca2+having intermediate effects. Ammonium ions stabilize folding with a similar slope, but at concentrations about 400 times higher than divalent ions. The apparent enthalpic change associated with the tertiary structure thermal unfolding increases strongly with increasing concentrations of divalent ions. A similar increase is observed with the monovalent ammonium ions. However, in the presence of NH4+ions, the apparent enthalpy peaks at 2.0 M and decreases beyond. PMID:10352179

  3. T4-Related Bacteriophage LIMEstone Isolates for the Control of Soft Rot on Potato Caused by ‘Dickeya solani’

    PubMed Central

    Adriaenssens, Evelien M.; Van Vaerenbergh, Johan; Vandenheuvel, Dieter; Dunon, Vincent; Ceyssens, Pieter-Jan; De Proft, Maurice; Kropinski, Andrew M.; Noben, Jean-Paul; Maes, Martine; Lavigne, Rob

    2012-01-01

    The bacterium ‘Dickeya solani’, an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with ‘Dickeya solani’, the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics. PMID:22413005

  4. Diversity of the major capsid genes (g23) of T4-like bacteriophages in the eutrophic Lake Kotokel in East Siberia, Russia.

    PubMed

    Butina, Tatyana V; Belykh, Olga I; Potapov, Sergey A; Sorokovikova, Ekaterina G

    2013-07-01

    Numerous studies revealed high diversity of T4-like bacteriophages in various environments, but so far, little is known about T4-like virus diversity in freshwater bodies, particularly in eutrophic lakes. The present study was aimed at elucidating molecular diversity of T4-like bacteriophages in eutrophic Lake Kotokel located near Lake Baikal by partial sequencing of the major capsid genes (g23) of T4-like bacteriophages. The majority of g23 fragments from Lake Kotokel were most similar to those from freshwater lakes and paddy fields. Despite the proximity and direct water connection between Lake Kotokel and Lake Baikal, g23 sequence assemblages from two lakes were different. UniFrac analysis showed that uncultured T4-like viruses from Lake Kotokel tended to cluster with those from the distant lake of the same trophic status. This fact suggested that the trophic conditions affected the formation of viral populations, particularly of T4-like viruses, in freshwater environments. PMID:23539063

  5. Structure of the 3.3 MDa, in Vitro Assembled, Hubless Bacteriophage T4 Baseplate

    PubMed Central

    Yap, Moh Lan; Klose, Thomas; Plevka, Pavel; Aksyuk, Anastasia; Zhang, Xinzheng; Arisaka, Fumio; Rossmann, Michael G.

    2014-01-01

    The bacteriophage T4 baseplate is the control center of the virus, where the recognition of an E. coli host by the long tail fibers is translated into a signal to initiate infection. The short tail fibers unfold from the baseplate for firm attachment to the host, followed by shrinkage of the tail sheath that causes the tail tube to enter and cross the periplasmic space ending with injection of the genome into the host. During this process, the 6.5 MDa baseplate changes its structure from a “dome” shape to a “star” shape. An in vitro assembled hubless baseplate has been crystalized. It consists of six copies of the recombinantly expressed trimeric gene product (gp) 10, monomeric gp7, dimeric gp8, dimeric gp6 and monomeric gp53. The diffraction pattern extends, at most, to 4.0 Å resolution. The known partial structures of gp10, gp8, and gp6 and their relative position in the baseplate derived from earlier electron microscopy studies were used for molecular replacement. An electron density map has been calculated based on molecular replacement, single isomorphous replacement with anomalous dispersion data and 2-fold non-crystallographic symmetry averaging between two baseplate wedges in the crystallographic asymmetric unit. The current electron density map indicates that there are structural changes in the gp6, gp8, and gp10 oligomers compared to their structures when separately crystallized. Additional density is also visible corresponding to gp7, gp53 and the unknown parts of gp10 and gp6. PMID:24998893

  6. Orthogonal Spin Labeling and Gd(III)-Nitroxide Distance Measurements on Bacteriophage T4-Lysozyme

    PubMed Central

    Garbuio, Luca; Bordignon, Enrica; Brooks, Evan K.; Hubbell, Wayne L.; Jeschke, Gunnar; Yulikov, Maxim

    2013-01-01

    We present the first example of chemo-selective site-specific spin labeling of a monomeric protein with two spectroscopically orthogonal spin labels: a Gadolinium (III) chelate complex and a nitroxide radical. A detailed analysis of the performance of two commercially available Gd(III) ligands in the Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) experiment is reported. A modification of the flip angle of the pump pulse in the Gd(III)-nitroxide DEER experiment is proposed to optimize sensitivity. PMID:23442004

  7. Analysis of the effectiveness of proline substitutions and glycine replacements in increasing the stability of phage T4 lysozyme.

    PubMed

    Nicholson, H; Tronrud, D E; Becktel, W J; Matthews, B W

    1992-11-01

    It was previously shown that the two replacements Gly 77-->Ala (G77A) and Ala 82-->Pro (A82P) increase the thermostability of phage T4 lysozyme at pH 6.5. Such replacements are presumed to restrict the degrees of freedom of the unfolded protein and so decrease the entropy of unfolding [B. W. Matthews, H. Nicholson, and W. J. Becktel (1987) Proceedings of the National Academy of Science USA Vol. 84, pp. 6663-6667]. To further test this approach, three additional replacements--G113A, K60P and A93P--have been constructed. On the basis of model building, each of these three replacements was judged to be less than optimal because it would tend to introduce unfavorable van der Waals contacts with neighboring parts of the protein. The presence of such contacts was verified for G113A and K60P by conformational adjustments seen in the crystal structures of these mutant proteins. In the case of G113A there are backbone conformational changes of 0.5-1.0 A in the short alpha-helix, 108-113, that includes the site of substitution. In the case of K60P the pyrrolidine ring shows evidence of strain. The thermal stability of each of the three variants at both pH 2.0 and pH 6.5 was found to be very close to that of wild-type lysozyme. The results suggest that the procedure used to predict sites for both Xaa-->Pro and Gly-->Ala is, in principle, correct. At the same time, the increase in stability expected from substitutions of this type is modest, and can easily be offset by strain associated with introduction of the alanine or proline. This means that the criteria used to select substitutions that will increase thermostability have to be stringent at least. In the case of T4 lysozyme this severely limits the number of sites. The analysis reveals a significant discrepancy between the conformational energy surface predicted for the residue preceding a proline and the conformations observed in crystal structures. PMID:1457724

  8. Cryo-EM structure of the bacteriophage T4 portal protein assembly at near-atomic resolution

    NASA Astrophysics Data System (ADS)

    Sun, Lei; Zhang, Xinzheng; Gao, Song; Rao, Prashant A.; Padilla-Sanchez, Victor; Chen, Zhenguo; Sun, Siyang; Xiang, Ye; Subramaniam, Sriram; Rao, Venigalla B.; Rossmann, Michael G.

    2015-07-01

    The structure and assembly of bacteriophage T4 has been extensively studied. However, the detailed structure of the portal protein remained unknown. Here we report the structure of the bacteriophage T4 portal assembly, gene product 20 (gp20), determined by cryo-electron microscopy (cryo-EM) to 3.6 Å resolution. In addition, analysis of a 10 Å resolution cryo-EM map of an empty prolate T4 head shows how the dodecameric portal assembly interacts with the capsid protein gp23 at the special pentameric vertex. The gp20 structure also verifies that the portal assembly is required for initiating head assembly, for attachment of the packaging motor, and for participation in DNA packaging. Comparison of the Myoviridae T4 portal structure with the known portal structures of ?29, SPP1 and P22, representing Podo- and Siphoviridae, shows that the portal structure probably dates back to a time when self-replicating microorganisms were being established on Earth.

  9. Cryo-EM structure of the bacteriophage T4 portal protein assembly at near-atomic resolution

    PubMed Central

    Sun, Lei; Zhang, Xinzheng; Gao, Song; Rao, Prashant A.; Padilla-Sanchez, Victor; Chen, Zhenguo; Sun, Siyang; Xiang, Ye; Subramaniam, Sriram; Rao, Venigalla B.; Rossmann, Michael G.

    2015-01-01

    The structure and assembly of bacteriophage T4 has been extensively studied. However, the detailed structure of the portal protein remained unknown. Here we report the structure of the bacteriophage T4 portal assembly, gene product 20 (gp20), determined by cryo-electron microscopy (cryo-EM) to 3.6?Å resolution. In addition, analysis of a 10?Å resolution cryo-EM map of an empty prolate T4 head shows how the dodecameric portal assembly interacts with the capsid protein gp23 at the special pentameric vertex. The gp20 structure also verifies that the portal assembly is required for initiating head assembly, for attachment of the packaging motor, and for participation in DNA packaging. Comparison of the Myoviridae T4 portal structure with the known portal structures of ?29, SPP1 and P22, representing Podo- and Siphoviridae, shows that the portal structure probably dates back to a time when self-replicating microorganisms were being established on Earth. PMID:26144253

  10. Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases.

    PubMed

    Gordon, L K; Haseltine, W A

    1980-12-25

    A comparison was made of the activity of the UV-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultravilet light-irradiated DNA substrates of defined sequence. The two enzymes cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same, suggesting that the scission of DNA by T4 endonuclease V occurs via the combined actin of a pyrimidine dimer specific DNA glycosylase and an apyrimidinic-apurinic (AP) endonuclease as was recently shown for the M. luteus enzyme. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA. PMID:6254991

  11. Genetic effects of cosmic radiation on bacteriophage T4Br+ (on materials of biological experiment "Soyuz-Apollo").

    PubMed

    Yurov, S S; Akoev, I G; Akhmadieva, A K; Livanova, I A; Leont'eva, G A; Marennyi, A M; Popov, V I

    1979-01-01

    During the experiment "Spore-ring Forming Fungi Biorhythm" of the Apollo-Soyuz test project the Rhythm-1 apparatus contained a dried film culture of bacteriophage T4Br+, growing cultures of Actinomyces and plastic nuclear particle detectors. The following were studied: the frequency of induction of r mutations in the bacteriophage film per 2 X 10(4) surviving particles, the spectrum of mutant types obtained (rI, rII, rIII), and the possible molecular mechanisms for the occurrence of rII mutants with due regard to the registered tracks of heavy nuclear particles. The studies showed that the local radiation due to heavy nuclear particle tracks plays a major role in space radiation damage. PMID:12008698

  12. The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase

    SciTech Connect

    Cheng, X.; Pflugrath, J.W.; Zhang, X.; Studier F.W.

    1994-04-26

    The lysozyme of bacteriophage T7 is a bifunctional protein that cuts amide bonds in the bacterial cell wall and binds to and inhibits transcription by T7 RNA polymerase. The structure of a mutant T7 lysozyme has been determined by x-ray crystallography and refined at 2.2-{angstrom} resolution. The protein folds into an {alpha}/{beta}-sheet structure that has a prominent cleft. A zinc atom is located in the cleft, bound directly to three amino acids and, through a water molecule, to a fourth. Zinc is required for amidase activity but not for inhibition of T7 RNA polymerase. Alignment of the zinc ligands of T7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amidase and these zinc proteases. Mutational analysis identified presumed catalytic residues for amidase activity within the cleft and a surface that appears to be the site of binding to T7 RNA polymerase. Binding of T7 RNA polymerase inhibits amidase activity.

  13. Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

    SciTech Connect

    Fokine, Andrei; Islam, Mohammad Z.; Zhang, Zhihong; Bowman, Valorie D.; Rao, Venigalla B.; Rossmann, Michael G.

    2011-09-16

    The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.

  14. Complete Genome Sequences of T4-Like Bacteriophages RB3, RB5, RB6, RB7, RB9, RB10, RB27, RB33, RB55, RB59, and RB68

    E-print Network

    Esvelt, Kevin M.

    T4-like bacteriophages have been explored for phage therapy and are model organisms for phage genomics and evolution. Here, we describe the sequencing of 11 T4-like phages. We found a high nucleotide similarity among the ...

  15. Molecular genetic analysis of a prokaryotic transcriptional coactivator: functional domains of the bacteriophage T4 gene 33 protein.

    PubMed Central

    Winkelman, J W; Kassavetis, G A; Geiduschek, E P

    1994-01-01

    The bacteriophage T4 gene 33 encodes a small, acidic RNA polymerase-binding protein that mediates enhancement of transcriptional initiation at T4 late promoters by the T4 DNA replication accessory proteins. A set of nested deletions in the gene 33 open reading frame was constructed by oligonucleotide site-directed mutagenesis. The resulting variant gene 33 proteins were radiolabeled during overexpression employing a T7 RNA polymerase-based system and substantially purified. Each variant was analyzed for three properties of gp33: RNA polymerase binding activity, ability to mediate enhancer-dependent transcriptional activation, and repression of unenhanced transcription. Two separate regions of gp33 were required to form stable complexes with RNA polymerase, whereas the extreme carboxyl terminus of gp33 was essential for mediating late gene activation. Variant gene 33 proteins lacking the carboxyl terminus nevertheless repressed nonenhanced transcription, demonstrating that the functional domains required for transcriptional activation and repression of unenhanced transcription are separable. The possible roles of gp33 in mediating late gene expression are discussed in the light of the identification of these functional domains. Images PMID:8106327

  16. Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

    PubMed Central

    Vafabakhsh, Reza; Kondabagil, Kiran; Earnest, Tyler; Lee, Kyung Suk; Zhang, Zhihong; Dai, Li; Dahmen, Karin A.; Rao, Venigalla B.; Ha, Taekjip

    2014-01-01

    Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly. PMID:25288726

  17. Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA.

    PubMed Central

    Wovcha, M G; Chiu, C S; Tomich, P K; Greenberg, G R

    1976-01-01

    Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis. PMID:789911

  18. Bacteriophage T4 lysis and lysis inhibition: molecular basis of an ancient story 

    E-print Network

    Tran, Tram Anh Thi

    2009-05-15

    T4 requires two proteins: holin, T (lesion formation and lysis timing) and endolysin, E (cell wall degradation) to lyse the host at the end of its life cycle. E is a cytoplasmic protein that sequestered away from its substrate, but the inner...

  19. Structure and Biophysical Properties of a Triple-Stranded Beta-Helix Comprising the Central Spike of Bacteriophage T4

    PubMed Central

    Buth, Sergey A.; Menin, Laure; Shneider, Mikhail M.; Engel, Jürgen; Boudko, Sergei P.; Leiman, Petr G.

    2015-01-01

    Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender ?-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded ?-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem. Here, we report structural and biophysical characterization of T4 gp5 ?-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the ?-helix is small. It is located near the C-terminal end of the ?-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the ?-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete ?-helix. PMID:26295253

  20. An ATP hydrolysis sensor in the DNA packaging motor from bacteriophage T4 suggests an inchworm-type translocation mechanism.

    PubMed

    Draper, Bonnie; Rao, Venigalla B

    2007-05-25

    Tailed bacteriophages and large eukaryotic viruses employ powerful molecular motors to translocate dsDNA into a preassembled capsid shell. The phage T4 motor is composed of a dodecameric portal and small and large terminase subunits assembled at the special head-tail connector vertex of the prohead. The motor pumps DNA through the portal channel, utilizing ATP hydrolysis energy provided by an ATPase present in the large terminase subunit. We report that the ATPase motors of terminases, helicases, translocating restriction enzymes, and protein translocases possess a common coupling motif (C-motif). Mutations in the phage T4 terminase C-motif lead to loss of stimulated ATPase and DNA translocation activities. Surprisingly, the mutants can catalyze at least one ATP hydrolysis event but are unable to turn over and reset the motor. This is the first report of a catalytic block in translocating ATPase motor after ATP hydrolysis occurred. We suggest that the C-motif is an ATP hydrolysis sensor, linking product release to mechanical motion. A novel terminase-driven mechanism is proposed for translocation of dsDNA in viruses. PMID:17428497

  1. Structure and Biophysical Properties of a Triple-Stranded Beta-Helix Comprising the Central Spike of Bacteriophage T4.

    PubMed

    Buth, Sergey A; Menin, Laure; Shneider, Mikhail M; Engel, Jürgen; Boudko, Sergei P; Leiman, Petr G

    2015-08-01

    Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender ?-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded ?-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem. Here, we report structural and biophysical characterization of T4 gp5 ?-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the ?-helix is small. It is located near the C-terminal end of the ?-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the ?-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete ?-helix. PMID:26295253

  2. Experimental electromagnetic effects on the model organism Escherichia coli and the bacteriophage T4

    NASA Astrophysics Data System (ADS)

    Lisiewski, Darlene Mildred

    This experimentally-based work was designed to answer the research question as to whether the phenomenon of nuclear magnetic resonance (NMR) can produce observable effects upon the bacterial virus activity of T4, with such activity demonstrated through the infection of its host bacterium Escherichia coli. The biological samples were placed for three hours within a coil antenna assembly propagating oscillating fields of radio frequency electromagnetic energy generated at the frequency of 5.6 MHz, and set at right angles within a magnetic field of 1450 gauss (recognizing such conditions are not set for the maximum effective resonance for hydrogen nuclei). The laboratory technique of plaque formation was the basis upon which the statistically tested data were compiled. Exposure of the bacterium alone exhibited an increase in viral activity over the control group (40--68% higher numbers of plaque formation), while exposure of T4 alone saw a decrease (approximately 23%) in infection rates. Depending on the protocol, placement of both T4 and E. coli into the coil assembly saw a decrease of either approximately 50% or 42% in infection rates. Future research must address identification of the effects being observed.

  3. Enhanced pyrimidine dimer repair in cultured murine epithelial cells transfected with the denV gene of bacteriophage T4.

    PubMed

    Kusewitt, D F; Budge, C L; Ley, R D

    1994-04-01

    The patch size for excision repair of ultraviolet radiation (UV)-induced pyrimidine dimers was determined in cultured murine epithelial cells with normal and enhanced pyrimidine dimer repair capabilities. Cells with enhanced pyrimidine dimer repair were produced by transfecting 308 cells with the denV gene of bacteriophage T4; this gene encodes the enzyme endonuclease V. Pyrimidine dimer repair following exposure to UV from an FS-40 sunlamp was determined by micrococcal dimer-specific nuclease digestion and alkaline sucrose ultracentrifugation. Patch size ws estimated based on the photolytic lability of bromodeoxyuridine-substituted DNA. Excision repair of UV-induced pyrimidine dimers in denV-transfected 308 cells was enhanced two- to threefold. Production of mRNA from the denV gene in cell lines with enhanced repair was confirmed by RNA blotting. In control cells, the patch size for excision repair of DNA photoproducts was estimated to be 34 nucleotides per photoproduct removed; in denV-transfected cells, a smaller average patch size of 10-16 nucleotides per photoproduct removed was calculated. Thus, endonuclease V activity appears to alter not only the extent, but also the nature of excision repair in UV-exposed mammalian epithelial cells. PMID:8151125

  4. The Structure of Gene Product 6 of Bacteriophage T4, the Hinge-Pin of the Baseplate

    SciTech Connect

    Aksyuk, Anastasia A.; Leiman, Petr G.; Shneider, Mikhail M.; Mesyanzhinov, Vadim V.; Rossmann, Michael G.

    2009-07-21

    The baseplate of bacteriophage T4 is a multicomponent protein complex, which controls phage attachment to the host. It assembles from six wedges and a central hub. During infection the baseplate undergoes a large conformational change from a dome-shaped to a flat, star-shaped structure. We report the crystal structure of the C-terminal half of gene product (gp) 6 and investigate its motion with respect to the other proteins during the baseplate rearrangement. Six gp6 dimers interdigitate, forming a ring that maintains the integrity of the baseplate in both conformations. One baseplate wedge contains an N-terminal dimer of gp6, whereas neighboring wedges are tied together through the C-terminal dimer of gp6. The dimeric interactions are preserved throughout the rearrangement of the baseplate. However, the hinge angle between the N- and C-terminal parts of gp6 changes by {approx}15{sup o}, accounting for a 10 {angstrom} radial increase in the diameter of the gp6 ring.

  5. Analyzing indirect secondary electron contrast of unstained bacteriophage T4 based on SEM images and Monte Carlo simulations

    SciTech Connect

    Ogura, Toshihiko

    2009-03-06

    The indirect secondary electron contrast (ISEC) condition of the scanning electron microscopy (SEM) produces high contrast detection with minimal damage of unstained biological samples mounted under a thin carbon film. The high contrast image is created by a secondary electron signal produced under the carbon film by a low acceleration voltage. Here, we show that ISEC condition is clearly able to detect unstained bacteriophage T4 under a thin carbon film (10-15 nm) by using high-resolution field emission (FE) SEM. The results show that FE-SEM provides higher resolution than thermionic emission SEM. Furthermore, we investigated the scattered electron area within the carbon film under ISEC conditions using Monte Carlo simulation. The simulations indicated that the image resolution difference is related to the scattering width in the carbon film and the electron beam spot size. Using ISEC conditions on unstained virus samples would produce low electronic damage, because the electron beam does not directly irradiate the sample. In addition to the routine analysis, this method can be utilized for structural analysis of various biological samples like viruses, bacteria, and protein complexes.

  6. Cloning and identification of bacteriophage T4 gene 2 product gp2 and action of gp2 on infecting DNA in vivo.

    PubMed Central

    Lipinska, B; Rao, A S; Bolten, B M; Balakrishnan, R; Goldberg, E B

    1989-01-01

    We sequenced bacteriophage T4 genes 2 and 3 and the putative C-terminal portion of gene 50. They were found to have appropriate open reading frames directed counterclockwise on the T4 map. Mutations in genes 2 and 64 were shown to be in the same open reading frame, which we now call gene 2. This gene codes for a protein of 27,068 daltons. The open reading frame corresponding to gene 3 codes for a protein of 20,634 daltons. Appropriate bands on polyacrylamide gels were identified at 30 and 20 kilodaltons, respectively. We found that the product of the cloned gene 2 can protect T4 DNA double-stranded ends from exonuclease V action. Images PMID:2644202

  7. Involvement of the Escherichia coli endoribonucleases G and E in the secondary processing of RegB-cleaved transcripts of bacteriophage T4.

    PubMed

    Zajanckauskaite, Aurelija; Truncaite, Lidija; Strazdaite-Zieliene, Zivile; Nivinskas, Rimas

    2008-06-01

    Sequence-specific endoribonuclease RegB of bacteriophage T4 cleaves early phage mRNAs and facilitates the transition between early and subsequent phases of T4 gene expression. The great majority of RegB targets have been identified in the intergenic regions of T4 transcripts, frequently in the Shine-Dalgarno sequences. Here we show that localization of RegB targets is not restricted to intergenic regions of mRNA. We detected 30 intragenic RegB sites in T4 transcripts that are differently susceptible to cleavage. Four RegB-processed mRNAs were previously shown to undergo further processing at so-called "secondary sites". We have found three additional transcripts carrying clear targets for both RegB and another endoribonuclease. We show that secondary cuts within RegB-processed T4 mRNAs are generated mainly by Escherichia coli RNase G, but that in some cases RNase E can recognize the same targets. Using plasmid-phage systems we demonstrate that T4 infection favours cleavage by the host endoribonucleases at these sites. PMID:18395239

  8. In Vitro Ligation of Oligodeoxynucleotides Containing C8-Oxidized Purine Lesions using Bacteriophage T4 DNA Ligase†

    PubMed Central

    Zhao, Xiaobei; Muller, James G.; Halasyam, Mohan; David, Sheila S.; Burrows, Cynthia J.

    2008-01-01

    Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3? end of the nick compared to the 5? end and to lesions located in the intact template strand. In general, substrates containing the OG·A mismatch were more readily ligated than OG·C. Ligations of duplexes containing the OA·T base pair (OA=8-oxo-7,8- dihydroadenosine) that could adopt an anti-anti conformation proceeded in high efficiencies. An OI·Acontaining duplex (OI = 8-oxo-7,8-dihydroinosine) behaved similarly to OG·A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G although the overall ligation efficiencies were much lower than most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90–100-fold for OG·C, OI·C, OI·A and OA·T containing duplexes compared to G·C. Substrates containing Gh·A, Gh·G, Sp·A and Sp·G base pairs showed Km values 20–70-fold higher than G·C while the Km value for OG·A was 5 times lower than G·C. PMID:17323928

  9. Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22

    SciTech Connect

    Hartman, P.E.; Hartman, Z.; Citardi, M.J.

    1988-05-01

    Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C). T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+. Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H/sub 2/O/sub 2/ or some product derived therefrom was predominant in causing inactivation of plaque formation. Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected. A similar effect was found for the polyamine, spermidine. In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection. L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man. L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals.

  10. ISOLATION AND CHARACTERIZATION OF T4 BACTERIOPHAGE GP17 TERMINASE: A LARGE SUBUNIT MULTIMER WITH ENHANCED ATPASE ACTIVITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phage T4 terminase is a two-subunit enzyme that binds to prohead portal protein and cuts and packages concatemeric DNA. To characterize the T4 terminase large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a chitin-binding fusion protein. Following cleavage and release of gp17 from ch...

  11. Isolation and characterization of EMS induced splicing defective point mutations within the intron of the nrdB gene of bacteriophage T4.

    PubMed

    Khan, A U; Lal, S K; Ahmad, M

    1998-01-01

    The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598-base-pair self-splicing intron which is closely related to other group I introns of T4 and eukaryotes. The screening, isolation, and mapping of 31 nrdB intron mutations were conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dyhydrofolate reductase or thymidylate synthase. These intron mutations cluster towards the ends, mainly the 3' end, and show a defect in self-splicing. These mutations map in regions of conserved structural elements, thus supporting secondary structure predictions. A distinct pattern of clustering is observed with the highest number of mutations mapping within three of the smaller regions (A, C, and D) of the nrdB intron and no mutations mapping in the largest (B) region. The highest density of mutations mapped in the smallest region (C) of the intron, containing only 96 bases, thus showing a distinct pattern of clustering within the catalytic core. PMID:9439601

  12. Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the ?-subunit flap domain of Escherichia coli RNA polymerase

    PubMed Central

    Twist, Kelly-Anne F.; Campbell, Elizabeth A.; Deighan, Padraig; Nechaev, Sergei; Jain, Vikas; Geiduschek, E. Peter; Hochschild, Ann; Darst, Seth A.

    2011-01-01

    Activated transcription of the bacteriophage T4 late genes, which is coupled to concurrent DNA replication, is accomplished by an initiation complex containing the host RNA polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the DNA-mounted sliding-clamp processivity factor of the phage T4 replisome (gp45, the activator). We have determined the 3.0 ?-resolution X-ray crystal structure of gp33 complexed with its RNA polymerase binding determinant, the ?-flap domain. Like domain 4 of the promoter specificity ? factor (?4), gp33 interacts with RNA polymerase primarily by clamping onto the helix at the tip of the ?-flap domain. Nevertheless, gp33 and ?4 are not structurally related. The gp33/?-flap structure, combined with biochemical, biophysical, and structural information, allows us to generate a structural model of the T4 late promoter initiation complex. The model predicts protein/protein interactions within the complex that explain the presence of conserved patches of surface-exposed residues on gp33, and provides a structural framework for interpreting and designing future experiments to functionally characterize the complex. PMID:22135460

  13. Interaction of Escherichia coli B and B/4 and Bacteriophage T4D with Berea Sandstone Rock in Relation to Enhanced Oil Recovery

    PubMed Central

    Chang, Philip L.; Yen, Teh Fu

    1984-01-01

    Much research and development is needed to recover oil reserves presently unattainable, and microbially enhanced oil recovery is a technology that may be used for this purpose. To address the problem of bacterial contamination in an oil field injection well region, we connected each end of a Teflon-sleeved Berea sandstone rock to a flask containing nutrient medium. By inoculating one flask with Escherichia coli B, we could observe bacterial growth in the uninoculated flask resulting from the transport and establishment of cells across the rock. Differences in bacterial populations occurred depending on whether bacteriophage T4D was first adsorbed to the rock. The results of these experiments indicate that the inhibition of bacterial establishment within a rock matrix is possible via lytic interaction. Some nonlytic effects are also implied by experiments with B/4 cells, which are T4D-resistant mutants of E. coli B. A 10 to 40% retention of T4 by the rock occurred when it was loaded with 105 to 106 PFU. We also describe a lysogenic system for possible use in microbially enhanced oil recovery techniques. PMID:16346492

  14. Interaction of Escherichia coli B and B/4 and Bacteriophage T4D with Berea Sandstone Rock in Relation to Enhanced Oil Recovery.

    PubMed

    Chang, P L; Yen, T F

    1984-03-01

    Much research and development is needed to recover oil reserves presently unattainable, and microbially enhanced oil recovery is a technology that may be used for this purpose. To address the problem of bacterial contamination in an oil field injection well region, we connected each end of a Teflon-sleeved Berea sandstone rock to a flask containing nutrient medium. By inoculating one flask with Escherichia coli B, we could observe bacterial growth in the uninoculated flask resulting from the transport and establishment of cells across the rock. Differences in bacterial populations occurred depending on whether bacteriophage T4D was first adsorbed to the rock. The results of these experiments indicate that the inhibition of bacterial establishment within a rock matrix is possible via lytic interaction. Some nonlytic effects are also implied by experiments with B/4 cells, which are T4D-resistant mutants of E. coli B. A 10 to 40% retention of T4 by the rock occurred when it was loaded with 10 to 10 PFU. We also describe a lysogenic system for possible use in microbially enhanced oil recovery techniques. PMID:16346492

  15. Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme

    NASA Astrophysics Data System (ADS)

    Georgieva, Elka R.; Roy, Aritro S.; Grigoryants, Vladimir M.; Borbat, Petr P.; Earle, Keith A.; Scholes, Charles P.; Freed, Jack H.

    2012-03-01

    Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (?100 ?s) samples to those from commonly shock-frozen (slow freeze, 1 s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions.

  16. The MotA transcription factor from bacteriophage T4 contains a novel DNA-binding domain : the 'double wing' motif.

    SciTech Connect

    Li, N.; Sickmier, E. A.; Zhang, R.; Joachimiak, A.; White, S. W.; Biosciences Division; St. Jude Children's Research Hospital; Univ. of Tennessee Health Science Center; Corixa Inc.

    2002-01-01

    MotA is a transcription factor from bacteriophage T4 that helps adapt the host Escherichia coli transcription apparatus to T4 middle promoters. We have determined the crystal structure of the C-terminal DNA-binding domain of MotA (MotCF) to 1.6 A resolution using multiwavelength, anomalous diffraction methods. The structure reveals a novel DNA-binding alpha/beta motif that contains an exposed beta-sheet surface that mediates interactions with the DNA. Independent biochemical experiments have shown that MotCF binds to one surface of a single turn of DNA through interactions in adjacent major and minor grooves. We present a model of the interaction in which beta-ribbons at opposite corners of the six-stranded beta-sheet penetrate the DNA grooves, and call the motif a 'double wing' to emphasize similarities to the 'winged-helix' motif. The model is consistent with data on how MotA functions at middle promoters, and provides an explanation for why MotA can form non-specific multimers on DNA.

  17. Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding

    PubMed Central

    Jose, Davis; Weitzel, Steven E.; Baase, Walter A.; von Hippel, Peter H.

    2015-01-01

    Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these ‘macromolecular machines’. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3?-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2–3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3?-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and ‘DNA map’) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex. PMID:26275775

  18. A single-molecule view of the assembly pathway, subunit stoichiometry and unwinding activity of the bacteriophage T4 primosome (helicase-primase) complex

    PubMed Central

    Lee, Wonbae; Jose, Davis; Phelps, Carey; Marcus, Andrew H.; von Hippel, Peter H.

    2013-01-01

    Single molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase-primase (primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks ‘internally’ labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time-dependence of the smFRET signals was monitored during the unwinding process and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the primosome and the assembly pathway required to form functional and fully active primosome-DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)6 complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabilized the resulting primosome complex at the fork and resulted in fully active and processive primosome helicases with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit ratios substantially reduced the primosome unwinding activity. The use of alternative assembly pathways resulted in loss of helicase activity and formation of metastable DNA-protein aggregates, which were easily detected in our smFRET experiments as intense light-scattering foci. These single molecule experiments provide a detailed real time visualization of the assembly pathway and duplex DNA unwinding activity of the T4 primosome and are consistent with more indirect equilibrium and steady state results obtained in bulk solution studies. PMID:23578280

  19. Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function

    PubMed Central

    Kondabagil, Kiran; Dai, Li; Vafabakhsh, Reza; Ha, Taekjip; Draper, Bonnie; Rao, Venigalla B.

    2015-01-01

    The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000 bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH? nucleophile. The ATPase Walker B motif, thus, may be redefined as “?-strand (4–6 hydrophobic-rich amino acids)–DE-hydrophobic/nonpolar amino acid”. PMID:25443668

  20. Portal-large terminase interactions of the bacteriophage T4 DNA packaging machine implicate a molecular lever mechanism for coupling ATPase to DNA translocation.

    PubMed

    Hegde, Shylaja; Padilla-Sanchez, Victor; Draper, Bonnie; Rao, Venigalla B

    2012-04-01

    DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation. PMID:22345478

  1. A novel approach for isolation and mapping of intron mutations in a ribonucleotide reductase encoding gene (nrdB) of bacteriophage T4 using the white halo plaque phenotype.

    PubMed

    Lal, S K; Hall, D H

    1993-10-29

    The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 base pair self splicing intron which is closely related to other group I introns of T4 and eukaryotes. The screening, isolation and mapping of the nrbB intron mutations was conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dhydrofolate reductase or thymidylate synthase. We have isolated 159 hydroxylamine-induced nrdB mutants, determined which mutations are in nrdB by marker rescue with clones of the nrdB gene and have mapped these mutations by marker rescue using subclones of the nrdB intron. Thirty out of the 159 nrdB mutations are in or near the intron. These mutations cluster towards the ends, mainly the 3' end. We have performed deletion mapping to further map mutations in the 3' end of the intron. The mutations map in regions of conserved structural elements, thus supporting secondary structure predictions similar to those of the well studied td intron in the T4 gene coding for thymidylate synthase. PMID:8240371

  2. In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

    SciTech Connect

    Shivachandra, Sathish B.; Rao, Mangala; Janosi, Laszlo; Sathaliyawala, Taheri; Matyas, Gary R.; Alving, Carl R.; Leppla, Stephen H.; Rao, Venigalla B. . E-mail: rao@cua.edu

    2006-02-05

    An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc {sup -} phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.

  3. Effects of Uvsx, Uvsy and DNA Topoisomerase on the Formation of Tandem Duplications of the Rii Gene in Bacteriophage T4

    PubMed Central

    Kumagai, M.; Yamashita, T.; Honda, M.; Ikeda, H.

    1993-01-01

    We have characterized tandem duplications in the rII regions of phage T4. The rII deletion r1589 blocks only the function of the rIIA cistron, although it extends into the B cistron. Another rII deletion, r1236, blocks the function of the rIIB cistron and overlaps r1589. When a cross is made between r1589 and r1236, true rII(+) progeny cannot form. Instead, anomalous phenotypically rII(+) phages are detected carrying an rII region from each parent. Analyses of nucleotide sequences of the recombination junctions indicate that recombination takes place between short regions of homology (from 2 to 10 bp). Open reading frames of the recombinants deduced from the nucleotide sequences reveal that they contain a normal rIIA cistron and one of a variety of fused, duplicated rIIB cistrons. The T4 uvsX and uvsY genes, which participate in homologous recombination, are involved in this duplication formation. T4 DNA topoisomerase is encoded by genes 39, 52 and 60. Mutations in 52 and 60 reduced the frequency of such duplications, but mutations in gene 39 and some in gene 52 did not. Hence, the effects of topoisomerase mutations are allele-specific. Models are proposed in which these proteins are involved in tandem duplication. PMID:7916730

  4. Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

    PubMed Central

    Jose, Davis; Weitzel, Steven E.; Baase, Walter A.; Michael, Miya M.; von Hippel, Peter H.

    2015-01-01

    We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5?-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex. PMID:26275774

  5. Biochemical and Genetic Characterization of Bacteriophage Holins 

    E-print Network

    To, Kam Ho

    2013-11-06

    , there are also the following enzymatic classes: glycosidase or “true lysozyme” (e.g., T4 gpe lysozyme and P22 gp19 lysozyme), amidase (e.g., T7 gp3.5 lysozyme and 11 murein hydrolase), and endopeptidase (e.g., 11 murein hydrolase) (5). There is no dependence... ....................................................................................... 139 Bacterial strains, culture growth, and reagents ...................................... 139 Construction and phenotypic analysis of Mu ?(gp19 gp20) .................. 139 Bioinformatic identification of lambda holin homologues in phage genomes...

  6. Single Molecule Recordings of Lysozyme Activity

    PubMed Central

    Choi, Yongki; Weiss, Gregory A.

    2013-01-01

    Single molecule bioelectronic circuits provide an opportunity to study chemical kinetics and kinetic variability with bond-by-bond resolution. To demonstrate this approach, we examined the catalytic activity of T4 lysozyme processing peptidoglycan substrates. Monitoring a single lysozyme molecule through changes in a circuit’s conductance helped elucidate unexplored and previously invisible aspects of lysozyme’s catalytic mechanism and demonstrated lysozyme to be a processive enzyme governed by 9 independent time constants. The variation of each time constant with pH or substrate crosslinking provided different insights into catalytic activity and dynamic disorder. Overall, ten lysozyme variants were synthesized and tested in single molecule circuits to dissect the transduction of chemical activity into electronic signals. Measurements show that a single amino acid with the appropriate properties is sufficient for good signal generation, proving that the single molecule circuit technique can be easily extended to other proteins. PMID:23752924

  7. Structural analysis of bacteriophage-encoded peptidoglycan hydrolase domain KMV36C: crystallization and preliminary X-ray diffraction

    SciTech Connect

    Van Hecke, Kristof; Briers, Yves; Derua, Rita; Waelkens, Etienne; Lavigne, Rob; Van Meervelt, Luc

    2008-04-01

    Crystallization and X-ray data collection of the C-terminus of gp36 from bacteriophage ?KMV (KMV36C) are reported. The C-terminus of gp36 of bacteriophage ?KMV (KMV36C) functions as a particle-associated muramidase, presumably as part of the injection needle of the ?KMV genome during infection. Crystals of KMV36C were obtained by hanging-drop vapour diffusion and diffracted to a resolution of 1.6 Å. The crystals belong to the cubic space group P432, with unit-cell parameters a = b = c = 102.52 Å. KMV36C shows 30% sequence identity to T4 lysozyme (PDB code)

  8. Lysozyme Crystal

    NASA Technical Reports Server (NTRS)

    2004-01-01

    To the crystallographer, this may not be a diamond but it is just as priceless. A Lysozyme crystal grown in orbit looks great under a microscope, but the real test is X-ray crystallography. The colors are caused by polarizing filters. Proteins can form crystals generated by rows and columns of molecules that form up like soldiers on a parade ground. Shining X-rays through a crystal will produce a pattern of dots that can be decoded to reveal the arrangement of the atoms in the molecules making up the crystal. Like the troops in formation, uniformity and order are everything in X-ray crystallography. X-rays have much shorter wavelengths than visible light, so the best looking crystals under the microscope won't necessarily pass muster under the X-rays. In order to have crystals to use for X-ray diffraction studies, crystals need to be fairly large and well ordered. Scientists also need lots of crystals since exposure to air, the process of X-raying them, and other factors destroy them. Growing protein crystals in space has yielded striking results. Lysozyme's structure is well known and it has become a standard in many crystallization studies on Earth and in space.

  9. Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme.

    PubMed Central

    Jucovic, M.; Poteete, A. R.

    1998-01-01

    Model-free approaches (random mutagenesis, DNA shuffling) in combination with more "rational," three-dimensional information-guided randomization have been used for directed evolution of lysozyme activity in a defective T4 lysozyme mutant. A specialized lysozyme cloning vector phage, derived from phage lambda, depends upon T4 lysozyme function for its ability to form plaques. The substitution W138P in T4 lysozyme totally abolishes its plaque-forming ability. Compensating mutations in W138P T4 lysozyme after sequential random mutagenesis of the whole gene as well as after targeted randomization of residues in the vicinity of Trp138 were selected. In a second stage, these mutations were randomly recombined by the recombinatorial PCR method of DNA shuffling. Shuffled and selected W138P T4 lysozyme variants provide the hybrid lambda phage with sufficient lysozyme activity to produce normal-size plaques, even at elevated temperature (42 degrees C). The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis. The best evolved W138P T4 lysozymes, however, accumulated mutations originating from both randomly mutagenized as well as target-randomized variants. PMID:9792108

  10. StructureFunction Analysis of the DNA Translocating Portal of the Bacteriophage

    E-print Network

    Kihara, Daisuke

    Structure­Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging.jmb.2013.10.011 Edited by J. Johnson Abstract Tailed bacteriophages and herpesviruses consist bacteriophages and herpesviruses use powerful molecular machines to package their gen- omes into a head

  11. Lytic bacteriophages

    PubMed Central

    Sharma, Manan

    2013-01-01

    Foodborne illnesses resulting from the consumption of produce commodities contaminated with enteric pathogens continue to be a significant public health issue. Lytic bacteriophages may provide an effective and natural intervention to reduce bacterial pathogens on fresh and fresh-cut produce commodities. The use of multi-phage cocktails specific for a single pathogen has been most frequently assessed on produce commodities to minimize the development of bacteriophage insensitive mutants (BIM) in target pathogen populations. Regulatory approval for the use of several lytic phage products specific for bacterial pathogens such as Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in foods and on food processing surfaces has been granted by various agencies in the US and other countries, possibly allowing for the more widespread use of bacteriophages in the decontamination of fresh and minimally processed produce. Research studies have shown lytic bacteriophages specific for E. coli O157:H7, Salmonella spp. and Listeria monocytogenes have been effective in reducing pathogen populations on leafy greens, sprouts and tomatoes. PMID:24228223

  12. Sorption of MS2 Bacteriophage to Layered Double Hydroxides: Effects of Reaction Time, pH, and Competing Anions

    E-print Network

    Sparks, Donald L.

    Sorption of MS2 Bacteriophage to Layered Double Hydroxides: Effects of Reaction Time, p,to investigate the potential of layered double hydroxides (LDHs) to remove bacteriophage MS2 from contaminated waters. All four of 70% of bacteriophages (T4 and MS2) were removed the LDHs evaluated in this study had

  13. T4 test

    MedlinePLUS

    ... in which the thyroid produces too much hormone) Hypothyroidism (underactive thyroid, in which the thyroid does not ... normal level of T4 may be due to: Hypothyroidism (including Hashimoto disease and other disorders involving an ...

  14. Template reporter bacteriophage platform and multiple bacterial detection assays based thereon

    NASA Technical Reports Server (NTRS)

    Goodridge, Lawrence (Inventor)

    2007-01-01

    The invention is a method for the development of assays for the simultaneous detection of multiple bacteria. A bacteria of interest is selected. A host bacteria containing plasmid DNA from a T even bacteriophage that infects the bacteria of interest is infected with T4 reporter bacteriophage. After infection, the progeny bacteriophage are plating onto the bacteria of interest. The invention also includes single-tube, fast and sensitive assays which utilize the novel method.

  15. Lysozyme mediated calcium carbonate mineralization.

    PubMed

    Wang, Xiaoqiang; Sun, Hailing; Xia, Yongqing; Chen, Cuixia; Xu, Hai; Shan, Honghong; Lu, Jian R

    2009-04-01

    Lysozyme, a major component of egg white proteins, has been speculated to participate in the calcification of avian eggshells. However, its detailed role during the eggshell formation is not well understood. In this work, the influence of lysozyme on the precipitation of CaCO(3) has been investigated using a combined study of FTIR, XRD, and SEM. The precipitation was produced from (NH(4))(2)CO(3) vapor diffusion into CaCl(2) aqueous solution using a specially built chamber. In the absence of lysozyme, hexagonal platelets of vaterite and their spherical aggregates dominated the precipitates during the first 3-12 h crystallization period studied, with the (001) crystal face well expressed in the hexagonal direction. In contrast, calcite was favored to precipitate in the presence of lysozyme during the same period and the effect was found to be proportional to lysozyme concentration. Furthermore, the (110) face of calcite was expressed in addition to the common (104) face, and the morphological modification was also lysozyme concentration dependent. We attributed these phenomena to the selective adsorption of ammonium ions and lysozyme onto different crystal faces. Our findings have clearly revealed the concentration and face dependent role of lysozyme in CaCO(3) precipitation. This, together with the abundance of lysozyme in the uterine fluid, implies its direct contribution to the hierarchical structures of calcite during the initial stage of eggshell formation. PMID:19167007

  16. THE CELL LYSIS ACTIVITY OF THE STREPTOCOCCUS AGALACTIAE BACTERIOPHAGE B30 ENDOLYSIN RELIES ON THE CHAP ENDOPEPTIDASE DOMAIN.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 443 amino acid Streptococcus agalactiae bacteriophage B30 endolysin gene contains a CHAP endopeptidase domain, an Acm lysozyme-like glycosidase, and a C-terminal SH3b cell wall binding domain. Although both hydrolase domains are enzymatically functional, it is unknown the degree to which each c...

  17. Microneedle-mediated transdermal bacteriophage delivery

    PubMed Central

    Ryan, Elizabeth; Garland, Martin J.; Singh, Thakur Raghu Raj; Bambury, Eoin; O’Dea, John; Migalska, Katarzyna; Gorman, Sean P.; McCarthy, Helen O.; Gilmore, Brendan F.; Donnelly, Ryan F.

    2012-01-01

    Interest in bacteriophages as therapeutic agents has recently been reawakened. Parenteral delivery is the most routinely-employed method of administration. However, injection of phages has numerous disadvantages, such as the requirement of a health professional for administration and the possibility of cross-contamination. Transdermal delivery offers one potential means of overcoming many of these problems. The present study utilized a novel poly (carbonate) (PC) hollow microneedle (MN) device for the transdermal delivery of Escherichia coli-specific T4 bacteriophages both in vitro and in vivo. MN successfully achieved bacteriophage delivery in vitro across dermatomed and full thickness skin. A concentration of 2.67 × 106 PFU/ml (plaque forming units per ml) was detected in the receiver compartment when delivered across dermatomed skin and 4.0 × 103 PFU/ml was detected in the receiver compartment when delivered across full thickness skin. An in vivo study resulted in 4.13 × 103 PFU/ml being detected in blood 30 min following initial MN-mediated phage administration. Clearance occurred rapidly, with phages being completely cleared from the systemic circulation within 24 h, which was expected in the absence of infection. We have shown here that MN-mediated delivery allows successful systemic phage absorption. Accordingly, bacteriophage-based therapeutics may now have an alternative route for systemic delivery. Once fully-investigated, this could lead to more widespread investigation of these interesting therapeutic viruses. PMID:22750416

  18. BACTERIOPHAGE: BIOLOGY AND GENETICS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage are viruses that infect bacteria. Bacteriophage are very small and made up of a protein coat with an inner core containing their genetic material. They infect bacterium, by attaching to the bacterial cell and injecting their nucleic acids into the bacteria. The phages then use the bac...

  19. Bacteriophages Infecting Propionibacterium acnes

    PubMed Central

    2013-01-01

    Viruses specifically infecting bacteria, or bacteriophages, are the most common biological entity in the biosphere. As such, they greatly influence bacteria, both in terms of enhancing their virulence and in terms of killing them. Since the first identification of bacteriophages in the beginning of the 20th century, researchers have been fascinated by these microorganisms and their ability to eradicate bacteria. In this review, we will cover the history of the Propionibacterium acnes bacteriophage research and point out how bacteriophage research has been an important part of the research on P. acnes itself. We will further discuss recent findings from phage genome sequencing and the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs). Finally, the potential to use P. acnes bacteriophages as a therapeutic strategy to combat P. acnes-associated diseases will be discussed. PMID:23691509

  20. Structure of the Small Outer Capsid Protein, Soc: A Clamp for Stabilizing Capsids of T4-like Phages

    SciTech Connect

    Qin, Li; Fokine, Andrei; O'Donnell, Erin; Rao, Venigalla B.; Rossmann, Michael G.

    2010-07-22

    Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a 'glue' between neighboring hexameric capsomers, forming a 'cage' that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 {angstrom} resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.

  1. Sweetness characterization of recombinant human lysozyme.

    PubMed

    Matano, Mami; Nakajima, Kana; Kashiwagi, Yutaka; Udaka, Shigezo; Maehashi, Kenji

    2015-10-01

    Lysozyme, a bacteriolytic enzyme, is widely distributed in nature and is a component of the innate immune system. It is established that chicken egg lysozyme elicits sweetness. However, the sweetness of human milk lysozyme, which is vital for combating microbial infections of the gastrointestinal tract of breast-fed infants, has not been characterized. This study aimed to assess the elicitation of sweetness using recombinant mammalian lysozymes expressed in Pichia pastoris. Recombinant human lysozyme (h-LZ) and other mammalian lysozymes of mouse, dog, cat and bovine milk elicited similar sweetness as determined using a sensory test, whereas bovine stomach lysozyme (bs-LZ) did not. Assays of cell cultures showed that h-LZ activated the human sweet taste receptor hT1R2/hT1R3, whereas bs-LZ did not. Point mutations confirmed that the sweetness of h-LZ was independent of enzyme activity and substrate-binding sites, although acidic amino acid residues of bs-LZ played a significant role in diminishing sweetness. Therefore, we conclude that elicitation of sweetness is a ubiquitous function among all lysozymes including mammalian lysozymes. These findings may provide novel insights into the biological implications of T1R2/T1R3-activation by mammalian lysozyme in the oral cavity and gastrointestinal tract. However, the function of lysozyme within species lacking the functional sweet taste receptor gene, such as cat, is currently unknown. PMID:26027787

  2. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

  3. BACTERIOPHAGE THERAPY AND CAMPYLOBACTER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The book chapter reports efforts to exploit Campylobacter-specific bacteriophages to reduce the numbers of Campylobacter jejuni and C. coli colonizing poultry and contaminating poultry meat products. Controlling campylobacters in poultry represents one of the greatest challenges to the agriculture a...

  4. Chlamydial plasmids and bacteriophages.

    PubMed

    Pawlikowska-Warych, Ma?gorzata; ?liwa-Dominiak, Joanna; Deptu?a, Wies?aw

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia. PMID:25654356

  5. Problem-Solving Test: RNA and Protein Synthesis in Bacteriophage-Infected "E. coli" Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

    The classic experiment presented in this problem-solving test was designed to identify the template molecules of translation by analyzing the synthesis of phage proteins in "Escherichia coli" cells infected with bacteriophage T4. The work described in this test led to one of the most seminal discoveries of early molecular biology: it dealt a…

  6. Fluorescence Studies of Lysozyme Nucleation

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Smith, Lori

    1998-01-01

    Fluorescence is one of the most powerful tools available for the study of macromolecules. For example, fluorescence can be used to study self association through methods such as anisotropy (the rotational rate of the molecule in solution), quenching (the accessibility of a bound probe to the bulk solution), and resonance energy transfer (measurement of the distance between two species). Fluorescence can also be used to study the local environment of the probe molecules, and the changes in that environment which accompany crystal nucleation and growth. However fluorescent techniques have been very much underutilized in macromolecular growth studies. One major advantage is that the fluorescent species generally must be at low concentration, typically ca 10-5 to 10-6 M. Thus one can study a very wide range of solution conditions, ranging from very high to very low protein concentration, he latter of which are not readily accessible to scattering techniques. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme (CEWL). Fluorescent probes have been attached to two different sites, ASP 101 and the N-terrninal amine, with a sought for use in different lines of study. Preliminary resonance energy transfer studies have been -carried out using pyrene acetic acid (Ex 340 mn, Em 376 nm) lysozyme as a donor and cascade blue (Ex 377 run, Em 423 nm) labeled lysozyme as an acceptor. The emission of both the pyrene and cascade blue probes was followed as a function of the salt protein concentrations. The data show an increase in cascade blue and a concomitant decrease in the pyrene fluorescence as either the salt or protein concentrations are increased, suggesting that the two species are approaching each other close enough for resonance energy transfer to occur. This data can be analyzed to measure the distance between the probe molecules and, knowing their locations on the protein molecule their distances from and orientations with respect to each other. The results of these and other studies will be discussed.

  7. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F. William (Stony Brook, NY); Rosenberg, Alan H. (Setauket, NY)

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

  8. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F.W.; Rosenberg, A.H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

  9. Structural and Mechanistic Studies of Pesticin, a Bacterial Homolog of Phage Lysozymes*

    PubMed Central

    Patzer, Silke I.; Albrecht, Reinhard; Braun, Volkmar; Zeth, Kornelius

    2012-01-01

    Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme. PMID:22593569

  10. PROTEOLYTIC REMOVAL OF THE CARBOXYL TERMINUS OF THE T4 GENE 32 HELIX-DESTABILIZING PROTEIN ALTERS THE T4 IN VITRO REPLICATION COMPLEX

    SciTech Connect

    Burke, R.L.; Alberts, B.M.; Hosoda, J.

    1980-07-01

    The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. (1) Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32*I protein for this synthesis. (2) Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. (3) Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. (4) The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3{prime}-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities.

  11. Phenotypic characterization and genomic analysis of the Shigella sonnei bacteriophage SP18.

    PubMed

    Kim, Kyoung-Ho; Chang, Ho-Won; Nam, Young-Do; Roh, Seong Woon; Bae, Jin-Woo

    2010-04-01

    A novel bacteriophage that infects Shigella sonnei was isolated from the Gap River in Korea, and its phenotypic and genomic characteristics were investigated. The virus, called SP18, showed morphology characteristic of the family Myoviridae, and phylogenetic analysis of major capsid gene (gp23) sequences classified it as a T4-like phage. Based on host spectrum analysis, it is lytic to S. sonnei, but not to Shigella flexneri, Shigella boydii or members of the genera Escherichia and Salmonella. Pyrosequencing of the SP18 bacteriophage genome revealed a 170-kb length sequence. In total, 286 ORFs and 3 tRNA genes were identified, and 259 ORFs showed similarity (BLASTP e-value<0.001) to genes of other bacteriophages. The results from comparative genomic analysis indicated that the enterophage JS98, isolated from human stool, is the closest relative of SP18. Based on phylogenetic analysis of gp23 protein-coding sequences, dot plot comparison and BLASTP analysis of genomes, SP18 and JS98 appear to be closely related to T4-even phages. However, several insertions, deletions, and duplications indicate differences between SP18 and JS98. Comparison of duplicated gp24 genes and the soc gene showed that duplication events are responsible for the differentiation and evolution of T4-like bacteriophages. PMID:20437154

  12. Lysozyme

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity experiments with data from laboratory experiments to study the equilibrium rate of hanging drop experiments in microgravity.

  13. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F. William (Stony Brook, NY); Dubendorff, John W. (Sound Beach, NY)

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  14. Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages

    DOEpatents

    Studier, F. William (Stony Brook, NY); Dubendorff, John W. (Sound Beach, NY)

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  15. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F.W.; Dubendorff, J.W.

    1998-10-20

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  16. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F.W.; Dubendorff, J.W.

    1998-11-03

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  17. The bacteriophage f29 portal motor can package DNA

    E-print Network

    Croquette, Vincent

    ................................................................. The bacteriophage f29 portal molecular motors, which couple rotation to DNA translocation6 . Previously, bacteriophage DNA packaging has

  18. DNA unwinding and inhibition of T4 DNA ligase by anthracyclines.

    PubMed Central

    Montecucco, A; Pedrali-Noy, G; Spadari, S; Zanolin, E; Ciarrocchi, G

    1988-01-01

    The ability to alter DNA tertiary structure of ten anthracycline derivatives whose antitumor potency is known was studied by an assay that makes use of nicked circular DNA and bacteriophage T4 DNA ligase. This assay allows the detection of tertiary structure alterations caused by DNA binding of both intercalating and non-intercalating drugs. The determination of these events can be obtained at different temperatures in the range of activity of DNA ligase. The results indicate that anthracyclines alter the DNA tertiary structure but this property does not correlate with their cytotoxic or antitumor activities. An additional interesting finding was that several anthracyclines inhibit T4 DNA ligase. The inhibition can be complete and is a cubic function of drug concentration. The inhibition of DNA ligase does not correlate with the ability of anthracyclines to alter the tertiary structure of DNA but is dependent from the presence of an amino group on the sugar ring. Images PMID:3287337

  19. BACTERIOPHAGE T4 MULTIPLICATION IN A GLUCOSE-LIMITED ESCHERICHIA COLI BIOFILM. (R825503)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  20. Acetylated Lysozyme as Impurity in Lysozyme Crystals: Constant Distribution Coefficient

    NASA Technical Reports Server (NTRS)

    Thomas, B. R.; Chernov, A. A.

    2000-01-01

    Hen egg white lysozyme (HEWL) was acetylated to modify molecular charge keeping the molecular size and weight nearly constant. Two derivatives, A and B, more and less acetylated, respectively, were obtained, separated, purified and added to the solution from which crystals of tetragonal HEWL crystals were grown. Amounts of the A or B impurities added were 0.76, 0.38 and 0.1 milligram per millimeter while HEWL concentration were 20, 30 and 40 milligram per milliliter. The crystals grown in 18 experiments for each impurity were dissolved and quantities of A or B additives in these crystals were analyzed by cation exchange high performance liquid chromatography. All the data for each set of 18 samples with the different impurity and regular HEWL concentrations is well described by one distribution coefficient K = 2.15 plus or minus 0.13 for A and K = 3.42 plus or minus 0.25 for B. The observed independence of the distribution coefficient on both the impurity concentration and supersaturation is explained by the dilution model described in this paper. It shows that impurity adsorption and incorporation rate is proportional to the impurity concentration and that the growth rate is proportional to the crystallizing protein in solution. With the kinetic coefficient for crystallization, beta = 5.10(exp -7) centimeters per second, the frequency at which an impurity molecule near the growing interface irreversibly joins a molecular site on the crystal was found to be 3 1 per second, much higher than the average frequency for crystal molecules. For best quality protein crystals it is better to have low microheterogeneous protein impurity concentration and high supers aturation.

  1. Widespread genetic exchange among terrestrial bacteriophages

    E-print Network

    Turner, Paul

    Widespread genetic exchange among terrestrial bacteriophages Olin K. Silander*§ , Daniel M October 27, 2005 (received for review April 15, 2005) Bacteriophages are the most numerous entities Microbes are the most numerous entities in the biosphere, and viruses that infect bacteria (bacteriophages

  2. Inactivation of Bacteriophages via Photosensitization of Fullerol

    E-print Network

    Alvarez, Pedro J.

    Inactivation of Bacteriophages via Photosensitization of Fullerol Nanoparticles A P P A L A R A J U bacteriophage inactivation rate nearly doubled due to the presence of singlet oxygen and increased by 125% due that generate superoxide and singlet oxygen, respectively (Figure 1) (12). The MS2 bacteriophage was chosen

  3. Dispersing biofilms with engineered enzymatic bacteriophage

    E-print Network

    Collins, James J.

    Dispersing biofilms with engineered enzymatic bacteriophage Timothy K. Lu* and James J. Collins engineered bacteriophage to express a biofilm-degrading en- zyme during infection to simultaneously attack substances. We show that the efficacy of biofilm removal by this two-pronged enzymatic bacteriophage strategy

  4. Coacervates of lysozyme and ?-casein.

    PubMed

    Anema, Skelte G; de Kruif, C G Kees

    2013-05-15

    Complexes are formed when positively charged lysozyme (LYZ) is mixed with negatively charged caseins. Adding ?-casein (BCN) to LYZ leads to flocculation even at low addition levels. Titrating LYZ into BCN shows that complexes are formed up to a critical composition (x=[LYZ]/([LYZ]+[BCN]). The formation of these complex coacervates increases asymptotically toward the molar charge equivalent ratio (xcrit), where the size of the complexes also seems to grow asymptotically. At xcrit, insoluble precipitates of charge-neutral complexes are formed. The precipitates can be re-dispersed by adding NaCl. The value of xcrit shifts to higher values on the LYZ side with increasing salt concentration and pH. Increasing the pH, de-protonates the BCN and protonates the LYZ, and therefore, charge neutrality will shift toward the LYZ side. xcrit increases linearly from 0.2 at no salt to 0.5 at 0.5M NaCl. It ends abruptly at a salt concentration of 0.5M after which a clear mixed solution remains. Away from the charge equivalent ratio, it seems that the buildup of charges limits the complex size. A simple scaling law to predict the size of the complex is proposed. By assuming that surface charge density is constant or can reach only a maximum value, it follows that scattering intensity is proportional to |(1-x/xcrit)|(-3) where x is the mole fraction of one protein and xcrit the value of the mole fraction at the charge equivalent ratio. Both scattering intensity and particle size seem to obey this simple assumption. For BCN-LYZ, the buildup occurs only at the LYZside in contrast to lactoferrin which forms stable complexes on either side of xcrit. The reason that the complexes are formed at the BCN side only may be due to the small size of LYZ, which induces a bending energy in the BCN on adsorption. PMID:23511012

  5. Growth kinetics of tetragonal lysozyme crystals

    NASA Technical Reports Server (NTRS)

    Pusey, M.; Naumann, R.

    1986-01-01

    A method for immobilizing protein crystals in small volumes to determine growth rates on various faces is applied to study the growth kinetics of the (100) face of tetragonal hen-egg white lysozyme crystals at different degrees of bulk saturation. In normal gravity, transport is found to be dominated by convection for crystal sizes larger than a few microns, while in a microgravity environment, transport is diffusion-limited for sizes up to a few mm. It is found that convection can be significant even in microgravity for crystals approaching cm sizes, and that lysozyme growth is limited by surface kinetics in normal gravity.

  6. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  7. Recombinant goose-type lysozyme in channel catfish: Lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  8. Morphogenesis of the T4 tail and tail fibers

    PubMed Central

    2010-01-01

    Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study. PMID:21129200

  9. Virulence reduction in bacteriophage resistant bacteria

    PubMed Central

    León, Marcela; Bastías, Roberto

    2015-01-01

    Bacteriophages can influence the abundance, diversity, and evolution of bacterial communities. Several bacteriophages have been reported to add virulence factors to their host and to increase bacterial virulence. However, lytic bacteriophages can also exert a selective pressure allowing the proliferation of strains with reduced virulence. This reduction can be explained because bacteriophages use structures present on the bacterial surface as receptors, which can be virulence factors in different bacterial species. Therefore, strains with modifications in these receptors will be resistant to bacteriophage infection and may also exhibit reduced virulence. This mini-review summarizes the reports on bacteriophage-resistant strains with reductions in virulence, and it discusses the potential consequences in phage therapy and in the use of bacteriophages to select attenuated strains for vaccines. PMID:25954266

  10. Lysogenic bacteriophage isolated from acidophilium

    DOEpatents

    Ward, Thomas W. (Idaho Falls, ID); Bruhn, Debby F. (Idaho Falls, ID); Bulmer, Deborah K. (Idaho Falls, ID)

    1992-01-01

    A bacteriophage identified as .phi.Ac1 capable of infecting acidophilic heterotropic bacteria (such as Acidiphilium sp.) and processes for genetically engineering acidophilic bacteria for biomining or sulfur removal from coal are disclosed. The bacteriophage is capable of growth in cells existing at pH at or below 3.0. Lytic forms of the phage introduced into areas experiencing acid drainage kill the bacteria causing such drainage. Lysogenic forms of the phase having genes for selective removal of metallic or nonmetallic elements can be introduced into acidophilic bacteria to effect removal of the desired element form ore or coal.

  11. Comparative analysis of microsatellites and compound microsatellites in T4-like viruses.

    PubMed

    Zhou, Lan; Deng, Liang; Fu, Yongzhuo; Wu, Xiaolong; Zhao, Xiangyan; Chen, Yubao; Li, Mingfu; Tan, Zhongyang

    2016-01-10

    Microsatellites or simple sequence repeats (SSRs) are known to present ubiquitously in genomes of eukaryotes and prokaryotes, as well as viruses. A comprehensive analysis of microsatellites and compound microsatellites (CM) was performed for 67 T4-like bacteriophage genomes. We found that the number of repeats was generally proportional to the size of the genome. CM were more abundant in genic regions, while their relative abundance was higher in intergenic regions. Meanwhile, the number of CM rapidly decreased with the increase of complexity but gradually increased with higher dMAX (maximum distance between any two adjacent microsatellites). (A)n/(T)n, (AT)n/(TA)n and (AAG)n were the most abundant repeats of mono-, di- and trinucleotide microsatellites, respectively. The number of microsatellites in reference sequences was significantly lower than that in corresponding random sequences. This result was mainly attributed to mono- and dinucleotide repeats which hardly exceeded 6bp in T4-like viruses. These observations may be helpful to understand the distribution of microsatellites and viral genetic diversity in T4-like viruses. PMID:26410414

  12. Enumeration of bacteriophage particles

    PubMed Central

    Rashid, Mohammed H; Carter, Chandi; Pasternack, Gary; Rajanna, Chythanya; Revazishvili, Tamara; Dean, Timothy; Senecal, Andre; Sulakvelidze, Alexander

    2011-01-01

    Bacteriophages are increasingly being utilized and considered for various practical applications, ranging from decontaminating foods and inanimate surfaces to human therapy; therefore, it is important to determine their concentrations quickly and reliably. Traditional plaque assay (PA) is the current “gold standard” for quantitating phage titers. However, it requires at least 18 h before results are obtained, and they may be significantly influenced by various factors. Therefore, two alternative assays based on the quantitative real-time polymerase chain reaction (QPCR) and NanoSight Limited (NS) technologies were recently proposed for enumerating phage particles. The present study compared the three approaches' abilities to quantitate Listeria monocytogenes-, Escherichia coli O157:H7- and Yersinia pestis-specific lytic phages quickly and reproducibly. The average coefficient of variation (CVS) of the PA method including all three phages was 0.15. The reproducibility of the PA method decreased dramatically when multiple investigators performed the assays, and mean differences of as much as 0.33 log were observed. The QPC R method required costly equipment and the synthesis of phage-specific oligonucleotide primers, but it determined phage concentrations faster (within about 4 h) and more precisely than did PA (CVS = 0.13). NS technology required costly equipment, was less precise (CVS = 0.28) than the PA and QPCR methods, and only worked when the phages were suspended in clear medium. However, it provided results within 5 min. After the overall correlation is established with the PA method, either of the two assays may be useful for quickly and reproducibly determining phage concentrations. PMID:22334864

  13. Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage [psi]29 tail

    SciTech Connect

    Xiang, Ye; Morais, Marc C.; Cohen, Daniel N.; Bowman, Valorie D.; Anderson, Dwight L.; Rossmann, Michael G.

    2009-08-28

    The small bacteriophage {phi}29 must penetrate the {approx}250-{angstrom} thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage {phi}29 is noncontractile and {approx}380 {angstrom} long. A 1.8-{angstrom} resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal end of the {phi}29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13{sup -} mutants with the {phi}29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.

  14. Crystal Structure of the Phage T4 Recombinase UvsX and Its Functional Interaction with the T4 SF2 Helicase UvsW

    SciTech Connect

    Gajewski, Stefan; Webb, Michael R.; Galkin, Vitold; Egelman, Edward H.; Kreuzer, Kenneth N.; White, Stephen W.

    2012-07-11

    Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resemble those of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed electron microscopy reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms: a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates.

  15. Bacteriophage therapy in animal production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concerns over the consequences of bacterial resistance to antibiotics with the use of antibiotics in animal production have led to an increase in research on alternatives to antibiotics. Bacteriophages kill bacteria, are natural, safe, plentiful, self replicating, self limiting, can be used to spec...

  16. Bacteriophage endolysins as novel antimicrobials

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can...

  17. The antimicrobial peptide lysozyme is induced after multiple trauma.

    PubMed

    Klüter, Tim; Fitschen-Oestern, Stefanie; Lippross, Sebastian; Weuster, Matthias; Mentlein, Rolf; Steubesand, Nadine; Neunaber, Claudia; Hildebrand, Frank; Pufe, Thomas; Tohidnezhad, Mersedeh; Beyer, Andreas; Seekamp, Andreas; Varoga, Deike

    2014-01-01

    The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction. PMID:25258475

  18. The Antimicrobial Peptide Lysozyme Is Induced after Multiple Trauma

    PubMed Central

    Klüter, Tim; Fitschen-Oestern, Stefanie; Lippross, Sebastian; Weuster, Matthias; Pufe, Thomas; Tohidnezhad, Mersedeh; Beyer, Andreas; Seekamp, Andreas; Varoga, Deike

    2014-01-01

    The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction. PMID:25258475

  19. Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability

    PubMed Central

    Oliveira Silva, Catarina; Petersen, Steffen B.; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa

    2015-01-01

    The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N–formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 ?W and 0.3 ?W. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation. PMID:26656259

  20. Subdiffusive motion of bacteriophage in mucosal surfaces increases the frequency of bacterial encounters

    PubMed Central

    Barr, Jeremy J.; Auro, Rita; Sam-Soon, Nicholas; Kassegne, Sam; Peters, Gregory; Bonilla, Natasha; Hatay, Mark; Mourtada, Sarah; Bailey, Barbara; Youle, Merry; Felts, Ben; Baljon, Arlette; Nulton, Jim; Salamon, Peter; Rohwer, Forest

    2015-01-01

    Bacteriophages (phages) defend mucosal surfaces against bacterial infections. However, their complex interactions with their bacterial hosts and with the mucus-covered epithelium remain mostly unexplored. Our previous work demonstrated that T4 phage with Hoc proteins exposed on their capsid adhered to mucin glycoproteins and protected mucus-producing tissue culture cells in vitro. On this basis, we proposed our bacteriophage adherence to mucus (BAM) model of immunity. Here, to test this model, we developed a microfluidic device (chip) that emulates a mucosal surface experiencing constant fluid flow and mucin secretion dynamics. Using mucus-producing human cells and Escherichia coli in the chip, we observed similar accumulation and persistence of mucus-adherent T4 phage and nonadherent T4?hoc phage in the mucus. Nevertheless, T4 phage reduced bacterial colonization of the epithelium >4,000-fold compared with T4?hoc phage. This suggests that phage adherence to mucus increases encounters with bacterial hosts by some other mechanism. Phages are traditionally thought to be completely dependent on normal diffusion, driven by random Brownian motion, for host contact. We demonstrated that T4 phage particles displayed subdiffusive motion in mucus, whereas T4?hoc particles displayed normal diffusion. Experiments and modeling indicate that subdiffusive motion increases phage–host encounters when bacterial concentration is low. By concentrating phages in an optimal mucus zone, subdiffusion increases their host encounters and antimicrobial action. Our revised BAM model proposes that the fundamental mechanism of mucosal immunity is subdiffusion resulting from adherence to mucus. These findings suggest intriguing possibilities for engineering phages to manipulate and personalize the mucosal microbiome. PMID:26483471

  1. Classification of Myoviridae bacteriophages using protein sequence similarity

    PubMed Central

    2009-01-01

    Background We advocate unifying classical and genomic classification of bacteriophages by integration of proteomic data and physicochemical parameters. Our previous application of this approach to the entirely sequenced members of the Podoviridae fully supported the current phage classification of the International Committee on Taxonomy of Viruses (ICTV). It appears that horizontal gene transfer generally does not totally obliterate evolutionary relationships between phages. Results CoreGenes/CoreExtractor proteome comparison techniques applied to 102 Myoviridae suggest the establishment of three subfamilies (Peduovirinae, Teequatrovirinae, the Spounavirinae) and eight new independent genera (Bcep781, BcepMu, FelixO1, HAP1, Bzx1, PB1, phiCD119, and phiKZ-like viruses). The Peduovirinae subfamily, derived from the P2-related phages, is composed of two distinct genera: the "P2-like viruses", and the "HP1-like viruses". At present, the more complex Teequatrovirinae subfamily has two genera, the "T4-like" and "KVP40-like viruses". In the genus "T4-like viruses" proper, four groups sharing >70% proteins are distinguished: T4-type, 44RR-type, RB43-type, and RB49-type viruses. The Spounavirinae contain the "SPO1-"and "Twort-like viruses." Conclusion The hierarchical clustering of these groupings provide biologically significant subdivisions, which are consistent with our previous analysis of the Podoviridae. PMID:19857251

  2. Dynamics of Lysozyme in Trehalose solutions

    NASA Astrophysics Data System (ADS)

    Ghatty, Pavan; Uberbacher, Edward C.

    2008-03-01

    Anhydrobiosis in Tardigrades and Nematodes has been a topic of constant interest and intrigue in the scientific community. An increase in the concentration of Trehalose has been attributed to the ability of some organisms to survive extreme conditions of temperature, pressure and pH. Although there exist many experimental studies attributing this effect to Trehalose, the molecular details governing the interaction between Trehalose and proteins remains unclear. We have conducted a 20ns study of Lysozyme in varying concentrations of Trehalose in water. Strong and weak hydrogen bonds and hydrophobic interactions between water, Trehalose and protein seem to dictate the interactions in the system. We have observed a hydrogen bonded network of Trehalose around the protein entrapping a layer of water between itself and protein. Lysozyme remains in a near-native conformation throughout the simulation giving hints on the ability of Trehalose in preserving the structure of protiens.

  3. Elasticity and Strength of Biomacromolecular Crystals: Lysozyme

    NASA Technical Reports Server (NTRS)

    Holmes, A. M.; Witherow, W. K.; Chen, L. Q.; Chernov, A. A.

    2003-01-01

    The static Young modulus, E = 0.1 to 0.5 GPa, the crystal critical strength (sigma(sub c)) and its ratio to E,sigma(sub c)/E is approximately 10(exp 3), were measured for the first time for non cross-linked lysozyme crystals in solution. By using a triple point bending apparatus, we also demonstrated that the crystals were purely elastic. Softness of protein crystals built of hard macromolecules (26 GPa for lysozyme) is explained by the large size of the macromolecules as compared to the range of intermolecular forces and by the weakness of intermolecular bonds as compared to the peptide bond strength. The relatively large reported dynamic elastic moduli (approximately 8 GPa) from resonance light scattering should come from averaging over the moduli of intracrystalline water and intra- and intermolecular bonding.

  4. The Structure of the Phage T4 DNA Packaging Motor Suggests a Mechanism Dependent on Electrostatic Forces

    SciTech Connect

    Sun, Siyang; Kondabagil, Kiran; Draper, Bonnie; Alam, Tanfis I.; Bowman, Valorie D.; Zhang, Zhihong; Hegde, Shylaja; Fokine, Andrei; Rossmann, Michael G.; Rao, Venigalla B.

    2009-06-30

    Viral genomes are packaged into procapsids by powerful molecular motors. We report the crystal structure of the DNA packaging motor protein, gene product 17 (gp17), in bacteriophage T4. The structure consists of an N-terminal ATPase domain, which provides energy for compacting DNA, and a C-terminal nuclease domain, which terminates packaging. We show that another function of the C-terminal domain is to translocate the genome into the procapsid. The two domains are in close contact in the crystal structure, representing a tensed state. A cryo-electron microscopy reconstruction of the T4 procapsid complexed with gp17 shows that the packaging motor is a pentamer and that the domains within each monomer are spatially separated, representing a relaxed state. These structures suggest a mechanism, supported by mutational and other data, in which electrostatic forces drive the DNA packaging by alternating between tensed and relaxed states. Similar mechanisms may occur in other molecular motors.

  5. Structural and Thermodynamic Characterization of T4 Lysozyme Mutants and the Contribution of Internal Cavities to Pressure Denaturation

    E-print Network

    Gruner, Sol M.

    of Internal Cavities to Pressure Denaturation Nozomi Ando, Buz Barstow,§ Walter A. Baase,| Andrew Fields-ray scattering (SAXS) and tryptophan fluorescence spectroscopy, we have identified multiple compact denatured that the mechanism of pressure denaturation is the penetration of water into the protein rather than the transfer

  6. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1490 Lysozyme (muramidase) test system. (a)...

  7. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1490 Lysozyme (muramidase) test system. (a)...

  8. Probing the toxic mechanism of Ag? with lysozyme.

    PubMed

    Wu, Qianqian; Zhang, Hao; Sun, Tao; Zhang, Bin; Liu, Rutao

    2015-12-01

    Silver (Ag) is widely used in human activities, which provides possibilities to distribute in organisms and tissues, resulting in harmful effects on human health. In this work, lysozyme was chosen as the target molecule to study the mechanism of toxic interactions between Ag(+) and protein using fluorescence emission spectra, synchronous fluorescence spectra, UV-vis absorption spectra, circular dichroism (CD) spectra, isothermal titration calorimetry (ITC), and enzyme activity assay. The results of fluorescence emission and synchronous fluorescence showed that there were interactions between Ag(+) and lysozyme by eliminating the inner filter effect (IFE). Data from UV-vis spectra indicated that the frame structure of lysozyme became looser with Ag(+) existent, while the micro-environment of aromatic amino acid residues did not show any significant alteration. CD results suggested that the secondary structure of lysozyme presented a decrease in ?-helix contents with the increasing amount of Ag(+). ITC results showed Ag(+) can spontaneously bind with lysozyme through hydrogen bonding and van der Waals forces with one binding site (Ka=1.93×10(6)). The lysozyme activity was inhibited by Ag(+) according to the enzyme activity assay, revealing that Ag(+) bound to lysozyme at the active site which resulted in inhibition of lysozyme activity. This work showed that Ag(+) can cause damages to the structure and function of lysozyme. PMID:26125992

  9. Removal of Endotoxins from Bacteriophage Preparations by Extraction with Organic Solvents

    PubMed Central

    Szermer-Olearnik, Bo?ena; Boraty?ski, Janusz

    2015-01-01

    Lipopolysaccharide (LPS, endotoxin, pyrogen) constitutes a very troubling contaminant of crude phage lysates produced in Gram-negative bacteria. Toxicity of LPS depends on the strong innate immunity response including the cytokines. Therefore, its removal is important for bacteriophage applications. In this paper, we present a procedure for extractive removal of endotoxin from bacteriophage preparations with water immiscible solvents (1-octanol or 1-butanol). During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin (expressed as endotoxin units, EU) in the aqueous bacteriophage-containing fraction determined by limulus amebocyte lysate or EndoLISA assay were exceptionally low. While the initial endotoxin levels in the crude phage lysates ranged between 103 and 105 EU/ml the average level after organic extraction remaining in the aqueous fraction was 5.3 EU/ml. These values when related to phage titers decreased from 103-105 EU/109 PFU (plaque forming units) down to an average of 2.8 EU/109 PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli) and F8 (P. aeruginosa). PMID:25811193

  10. Lysozyme pattern formation in evaporating droplets

    NASA Astrophysics Data System (ADS)

    Gorr, Heather Meloy

    Liquid droplets containing suspended particles deposited on a solid, flat surface generally form ring-like structures due to the redistribution of solute during evaporation (the "coffee ring effect"). The forms of the deposited patterns depend on complex interactions between solute(s), solvent, and substrate in a rapidly changing, far from equilibrium system. Solute self-organization during evaporation of colloidal sessile droplets has attracted the attention of researchers over the past few decades due to a variety of technological applications. Recently, pattern formation during evaporation of various biofluids has been studied due to potential applications in medical screening and diagnosis. Due to the complexity of 'real' biological fluids and other multicomponent systems, a comprehensive understanding of pattern formation during droplet evaporation of these fluids is lacking. In this PhD dissertation, the morphology of the patterns remaining after evaporation of droplets of a simplified model biological fluid (aqueous lysozyme solutions + NaCl) are examined by atomic force microscopy (AFM) and optical microscopy. Lysozyme is a globular protein found in high concentration, for example, in human tears and saliva. The drop diameters, D, studied range from the micro- to the macro- scale (1 microm -- 2 mm). In this work, the effect of evaporation conditions, solution chemistry, and heat transfer within the droplet on pattern formation is examined. In micro-scale deposits of aqueous lysozyme solutions (1 microm < D < 50 microm), the protein motion and the resulting dried residue morphology are highly influenced by the decreased evaporation time of the drop. The effect of electrolytes on pattern formation is also investigated by adding varying concentrations NaCl to the lysozyme solutions. Finally, a novel pattern recognition program is described and implemented which classifies deposit images by their solution chemistries. The results presented in this PhD dissertation provide insight into the evaporative behavior and pattern formation in droplets of simplified model biological fluids (aqueous lysozyme + NaCl). The patterns that form depend sensitively on the evaporation conditions, characteristic time and length scales, and the physiochemical properties of the solutions. The patterns are unique, dependent on solution chemistry, and may therefore act as a "fingerprint" in identifying fluid properties.

  11. Bacteriophage Transduction in Staphylococcus aureus.

    PubMed

    Olson, Michael E

    2016-01-01

    The genetic manipulation of Staphylococcus aureus for molecular experimentation is a valuable tool for assessing gene function and virulence. Genetic variability between strains coupled with difficult laboratory techniques for strain construction is a frequent roadblock in S. aureus research. Bacteriophage transduction greatly increases the speed and ease of S. aureus studies by allowing movement of chromosomal markers and plasmids between strains. This technique enables the S. aureus research community to focus investigations on clinically relevant isolates. PMID:25646608

  12. Genetic Dissection of T4 Lysis

    PubMed Central

    Moussa, Samir H.; Lawler, Jessica L.

    2014-01-01

    t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an Nin-Cout topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; Nout-Cin) of the coliphage lambda and S68 (2 TMDs; Nin-Cin) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation. PMID:24706740

  13. Filamentous Bacteriophage Promote Biofilm Assembly and Function.

    PubMed

    Secor, Patrick R; Sweere, Johanna M; Michaels, Lia A; Malkovskiy, Andrey V; Lazzareschi, Daniel; Katznelson, Ethan; Rajadas, Jayakumar; Birnbaum, Michael E; Arrigoni, Allison; Braun, Kathleen R; Evanko, Stephen P; Stevens, David A; Kaminsky, Werner; Singh, Pradeep K; Parks, William C; Bollyky, Paul L

    2015-11-11

    Biofilms-communities of bacteria encased in a polymer-rich matrix-confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies. PMID:26567508

  14. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lysozyme (muramidase) test system. 862.1490 Section 862.1490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1490 Lysozyme (muramidase)...

  15. Continuing adventures in lysozyme crystal growth. [in earth laboratory experiments

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.

    1992-01-01

    Results obtained on the crystal nucleation and growth of lysozyme are presented. Special attention is given to the anion-protein binding, the nucleation kinetics, the mechanisms of aggregation, and the conditions that promote or inhibit lysozyme (110)-face growth rate. The emerging theory that is currently being used for data interpretation and for designing new experimental approaches is outlined.

  16. Immobilization of lysozyme on cotton fabrics; synthesis, characterication, and activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antimicrobial activity of lysozyme derives from the hydrolysis of the bacterial cell wall polysaccharide at the glycosidic bond that links N-acetyl-glucosamine and N-acetyl-muramic acid. Maintaining the activity of lysozyme while bound to a cellulose substrate is a goal toward developing enzyme...

  17. Tetragonal Lysozyme, From Monomer to Crystal

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    The data now leads us to a comprehensive model for the process by which tetragonal lysozyme crystals are nucleated and subsequently grow. Lysozyme is typically desolubilized by addition of ionic salts. The salt anions bind to basic and other sites on the protein and promote protein-protein interactions, i.e., initiate the nucleation self assembly process. Formation of protein-protein interactions occurs at the expense of the protein-anion interactions, with the anions being released to the solution. The association follows a defined pattern, forming the "head to side" interactions of the crystal 4(3) helix. The presence of the high salt also promotes hydrophobic interactions between the protein molecules, further tightening their interaction. The solute assembly process persists after crystal nucleation, and the 4(3) helical structures form the subsequent growth units. AFM measurements show that the growth units follow the dimensions of these helices, and that those on the surface are more compact about the c-axis than in the bulk crystal, with adjacent helices riot being in contact. This further supports the role of hydrophobic interactions, as the surface is still in contact with the bulk solution. Once buried within the crystal the protein:salt ratio radically changes and the hydrophobic interactions relax to those measured crystallographically. Thus the crystal growth process recapitulates the initial stages of the nucleation process, and the two seamlessly merge. Experimental evidence, based upon face growth rate, AFM, and fluorescence energy transfer data, for a postulated model of the nucleation of tetragonal lysozyme crystals and how it transitions into crystal growth will be presented.

  18. Rationalising lysozyme amyloidosis: insights from the structure and solution dynamics of T70N lysozyme.

    PubMed

    Johnson, Russell J K; Christodoulou, John; Dumoulin, Mireille; Caddy, Gemma L; Alcocer, Marcos J C; Murtagh, Gareth J; Kumita, Janet R; Larsson, Göran; Robinson, Carol V; Archer, David B; Luisi, Ben; Dobson, Christopher M

    2005-09-30

    T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo. PMID:16126226

  19. Structure of the Bacteriophage [phi]KZ Lytic Transglycosylase gp144

    SciTech Connect

    Fokine, Andrei; Miroshnikov, Konstantin A.; Shneider, Mikhail M.; Mesyanzhinov, Vadim V.; Rossmann, Michael G.

    2008-04-02

    Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage {phi}KZ has been determined to 2.5-{angstrom} resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. {phi}KZ gp144 is a 260-residue {alpha}-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G d-Ala-d-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu{sup 115} in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the {phi}KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine){sub 4} has been determined to 2.6-{angstrom} resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

  20. Determination of monomer concentrations in crystallizing lysozyme solutions

    NASA Technical Reports Server (NTRS)

    Wilson, L. J.; Pusey, Marc L.

    1992-01-01

    We have developed a non-optical technique for the study of aggregation in lysozyme and other protein solutions. By monitoring the rate at which lysozyme traverses a semipermeable membrane it was possible to quantitate the degree of aggregation in supersaturated solutions. Using this technique, we have measured the concentration of monomers and larger aggregates in under- and oversaturated lysozyme solutions, and in the presence of crystals, at pH 4.0 and 3 percent NaCl (0.1M NaAc). Comparison of these concentration profiles with (110) face growth rate data supports the theory that tetragonal lysozyme crystals grow by addition of preformed aggregates and not by monomer addition. The data suggest that a considerable population of aggregates larger than dimers are present at lysozyme concentrations above 22 mg/ml. Determination of dimer concentrations, and equilibrium constants for subsequent aggregation levels, are currently underway.

  1. Lysozyme, intradermal diffusion of india ink and peritoneal capillary permeability in mice.

    PubMed

    Bianchi, C; Goi, A; Ronchini, A

    1982-04-01

    Hen egg white lysozyme injected intracutaneously into mice inhibits the intradermal diffusion of India Ink. The effect of lysozyme persists in presence of hyaluronidase. Lysozyme injected i.p. into mice inhibits acetic acid (i.p.) induced leakage of Pontamine Sky Blue into the abdominal cavity. The data suggest that lysozyme might be tried for applications outside the microbiological field. PMID:7079584

  2. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium1

    PubMed Central

    Seal, Bruce S.

    2014-01-01

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently used antibiotics. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a significant role in human foodborne disease as well as nonfoodborne human, animal, and avian diseases. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated diseases in poultry. To address these issues, new antimicrobial agents, putative lysins encoded by the genomes of bacteriophages, are being identified in our laboratory. Poultry intestinal material, soil, sewage, and poultry processing drainage water were screened for virulent bacteriophages that could lyse C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long noncontractile tails, members of the family Siphoviridae, and with short noncontractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded N-acetylmuramoyl-l-alanine amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain that has not been previously reported in viral genomes. Putative phage lysin genes (ply) were cloned and expressed in Escherichia coli. The recombinant lysins were amidases capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays, but did not lyse any clostridia beyond the species. Consequently, bacteriophage gene products could eventually be used to target bacterial pathogens, such as C. perfringens via a species-specific strategy, to control animal and human diseases without having deleterious effects on beneficial probiotic bacteria. PMID:23300321

  3. The Effects of Acetate Buffer Concentration on Lysozyme Solubility

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Pusey, Marc L.

    1996-01-01

    The micro-solubility column technique was employed to systematically investigate the effects of buffer concentration on tetragonal lysozyme solubility. While keeping the NaCl concentrations constant at 2%, 3%, 4%, 5% and 7%, and the pH at 4.0, we have studied the solubility of tetragonal lysozyme over an acetate buffer concentration range of 0.01M to 0.5M as a function of temperature. The lysozyme solubility decreased with increasing acetate concentration from 0.01M to 0.1M. This decrease may simply be due to the net increase in solvent ionic strength. Increasing the acetate concentration beyond 0.1M resulted in an increase in the lysozyme solubility, which reached a peak at - 0.3M acetate concentration. This increase was believed to be due to the increased binding of acetate to the anionic binding sites of lysozyme, preventing their occupation by chloride. In keeping with the previously observed reversal of the Hoffmeister series for effectiveness of anions in crystallizing lysozyme, acetate would be a less effective precipitant than chloride. Further increasing the acetate concentration beyond 0.3M resulted in a subsequent gradual decrease in the lysozyme solubility at all NaCl concentrations.

  4. Stable multilayer thin films composed of gold nanoparticles and lysozyme

    NASA Astrophysics Data System (ADS)

    Su, Yan-lei; Li, Chao

    2008-01-01

    It needs appropriately attractive forces to construct multilayer thin films by layer-by-layer (LBL) assembly technique. It is feasible to prepare multilayer thin films on glass slides with negatively charged gold nanoparticles and positively charged lysozyme through the electrostatic LBL assembly technique. The gold nanoparticles/lysozyme multilayer thin films are highly stable; immersion in 0.1 M HCl, NaOH, and surfactant sodium dodecyl sulfate aqueous solutions cannot destroy the films. The highly stable gold nanoparticles/lysozyme multilayer thin films have potential application in long-term antibacterial coating.

  5. Reentrant condensation of lysozyme: Implications for studying dynamics of lysozyme in aqueous solutions of lithium chloride

    SciTech Connect

    Mamontov, Eugene; O'Neill, Hugh Michael

    2014-01-01

    Recent studies have outlined the use of eutectic solution of lithium chloride in water to study microscopic dynamics of lysozyme in an aqueous solvent that is remarkably similar to pure water in many respects, yet allows experiments over a wide temperature range without the solvent crystallization. The eutectic point in (H2O)R(LiCl) system corresponds to R 7.3, and it is of interest to investigate whether less concentrated aqueous solutions of LiCl could be employed in low-temperature studies of a solvated protein. We have investigated a range of concentrations of lysozyme and LiCl in aqueous solutions to identify systems that do not show phase separation and avoid solvent crystallization on cooling down. Compared to the lysozyme concentration in solution, the concentration of LiCl in the aqueous solvent plays the major role in determining systems suitable for low-temperature studies. We have observed interesting and rich phase behavior reminiscent of reentrant condensation of proteins.

  6. Lytic Clostridium perfringens Bacteriophage 39-O Genomic

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Screening for bacteriophages lytic for Clostridium perfringens was completed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Following limit dilution cloning and three rounds of plaque purification lytic phage preparations ...

  7. Arthrobacter globiformis and its bacteriophage in soil

    NASA Technical Reports Server (NTRS)

    Casida, L. E., Jr.; Liu, K.-C.

    1974-01-01

    An attempt was made to correlate bacteriophages for Arthrobacter globiformis with soils containing that bacterium. The phages were not detected unless the soil was nutritionally amended (with glucose or sucrose) and incubated for several days. Phage was continuously produced after amendment without the addition of host Arthrobacter. These results indicate that the bacteriophage is present in a masked state and that the bacteria are present in an insensitive form which becomes sensitive after addition of nutrient.

  8. Taking Bacteriophage Therapy Seriously: A Moral Argument

    PubMed Central

    Verbeken, Gilbert; Huys, Isabelle; Jennes, Serge; Chanishvili, Nina; Górski, Andrzej; De Vos, Daniel

    2014-01-01

    The excessive and improper use of antibiotics has led to an increasing incidence of bacterial resistance. In Europe the yearly number of infections caused by multidrug resistant bacteria is more than 400.000, each year resulting in 25.000 attributable deaths. Few new antibiotics are in the pipeline of the pharmaceutical industry. Early in the 20th century, bacteriophages were described as entities that can control bacterial populations. Although bacteriophage therapy was developed and practiced in Europe and the former Soviet republics, the use of bacteriophages in clinical setting was neglected in Western Europe since the introduction of traditional antibiotics. Given the worldwide antibiotic crisis there is now a growing interest in making bacteriophage therapy available for use in modern western medicine. Despite the growing interest, access to bacteriophage therapy remains highly problematic. In this paper, we argue that the current state of affairs is morally unacceptable and that all stakeholders (pharmaceutical industry, competent authorities, lawmakers, regulators, and politicians) have the moral duty and the shared responsibility towards making bacteriophage therapy urgently available for all patients in need. PMID:24868534

  9. Dynamical changes in hydration water accompanying lysozyme thermal denaturation

    NASA Astrophysics Data System (ADS)

    Mallamace, Francesco; Corsaro, Carmelo; Mallamace, Domenico; Cicero, Nicola; Vasi, Sebastiano; Dugo, Giacomo; Stanley, H. Eugene

    2015-10-01

    We study the dynamics of the first hydration shell of lysozyme to determine the role of hydration water that accompanies lysozyme thermal denaturation. We use nuclear magnetic resonance spectroscopy to investigate both the translational and rotational contributions. Data on proton self-diffusion and reorentational correlation time indicate that the kinetics of the lysozyme folding/unfolding process is controlled by the dynamics of the water molecules in the first hydration shell. When the hydration water dynamics change, because of the weakening of the hydrogen bond network, the three-dimensional structure of the lysozyme is lost and denaturation is triggered. Our data indicates that at temperatures above approximately 315 K, water behaves as a simple liquid and is no longer a good solvent.

  10. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...muramidase) in serum, plasma, leukocytes, and urine. Lysozyme measurements are used in the diagnosis and treatment of monocytic leukemia and kidney disease. (b) Classification. Class I (general controls). The device is exempt from the premarket...

  11. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...muramidase) in serum, plasma, leukocytes, and urine. Lysozyme measurements are used in the diagnosis and treatment of monocytic leukemia and kidney disease. (b) Classification. Class I (general controls). The device is exempt from the premarket...

  12. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...muramidase) in serum, plasma, leukocytes, and urine. Lysozyme measurements are used in the diagnosis and treatment of monocytic leukemia and kidney disease. (b) Classification. Class I (general controls). The device is exempt from the premarket...

  13. Preliminary crystallographic examination of a novel fungal lysozyme from Chalaropsis

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; He, Xiao-Min; Lyne, James E.; Stubbs, Gerald; Hash, John H.

    1990-01-01

    The lysozyme from the fungus of the Chalaropsis species has been crystallized. This lysozyme displays no sequence homology with avian, phage, or mammalian lysozymes, however, preliminary studies indicate significant sequence homology with the bacterial lysozyme from Streptomyces. Both enzymes are unusual in possessing beta-1,4-N-acetylmuramidase and beta-1,4-N,6-O-diacetylmuramidase activity. The crystals grow from solutions of ammonium sulfate during growth periods from several months to a year. The space group is P2(1)2(1)2(1) with a = 34.0 A, b = 42.6 A, c = 122.1 A. Preliminary data indicate that there is 1 molecule/asymmetric unit.

  14. Location of Bromide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

    Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

    1998-01-01

    Anions have been shown to play a dominant role in the crystallization of chicken egg white lysozyme from salt solutions. Previous studies employing X-ray crystallography had found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystal grown in bromide and chloride solutions. Five possible anion binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four of these sites corresponded to four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed.

  15. Characterization of tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa

    SciTech Connect

    Kurochkina, Lidia P.; Sachkova, Maria Yu.; Sykilinda, Nina N.; Mesyanzhinov, Vadim V.

    2009-12-20

    The tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa encoded by gene 29 was identified and its expression system was developed. Localization of the protein on the virion was confirmed by immunoelectron microscopy. Properties of gene product (gp) 29 were studied by electron microscopy, immunoblotting and limited trypsinolysis. Recombinant gp29 assembles into the regular tubular structures (polysheaths) of variable length. Trypsin digestion of gp29 within polysheaths or extended sheath of virion results in specific cleavage of the peptide bond between Arg135 and Asp136. However, this cleavage does not affect polymeric structure of polysheaths, sheaths and viral infectivity. Digestion by trypsin of the C-truncated gp29 mutant, lacking the ability to self-assemble, results in formation of a stable protease-resistant fragment. Although there is no sequence homology of phiKZ proteins to proteins of other bacteriophages, some characteristic biochemical properties of gp29 revealed similarities to the tail sheath protein of bacteriophage T4.

  16. Improved activity in acidic media of immobilized lysozyme.

    PubMed

    Zacchigna, M; Di Luca, G; Lassiani, L; Varnavas, A; Pitotti, A; Boccù, E

    1999-03-01

    Polyethylene glycols (PEGs) of various chain length were used to crosslink lysozyme onto an insoluble support such as oxirane. A very high degree of modification and no inactivation of lysozyme were obtained with PEG 20000, but enzymatic activity increased up to 20 times at pH 3.0, at which point the activity of the native enzyme was lower when using Leuconostok oenus as a macromolecular substrate. PMID:15304727

  17. On the problem of phase transitions in lysozyme crystals

    E-print Network

    Yu. Vasylkiv; Yu. Nastishin; R. Vlokh

    2007-06-17

    We present experimental evidence of the fact that lysozyme crystals, which are grown from their mother solution and exist in it, dissolve on heating above T=307 K. We argue that the anomaly in the light scattering recently observed at the temperature T=307 K and identified in the reference [Svanidze A. V. et al. 2006. JETP Lett. 84: 551] as a structural crystalline phase transition in the single lysozyme crystals, in fact, corresponds to a temperature limit of the crystal existence.

  18. Destroying activity of magnetoferritin on lysozyme amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Kopcansky, Peter; Siposova, Katarina; Melnikova, Lucia; Bednarikova, Zuzana; Timko, Milan; Mitroova, Zuzana; Antosova, Andrea; Garamus, Vasil M.; Petrenko, Viktor I.; Avdeev, Mikhail V.; Gazova, Zuzana

    2015-03-01

    Presence of protein amyloid aggregates (oligomers, protofilaments, fibrils) is associated with many diseases as diabetes mellitus or Alzheimer's disease. The interaction between lysozyme amyloid fibrils and magnetoferritin loaded with different amount of iron atoms (168 or 532 atoms) has been investigated by small-angle X-rays scattering and thioflavin T fluorescence measurements. Results suggest that magnetoferritin caused an iron atom-concentration dependent reduction of lysozyme fibril size.

  19. Strong and Selective Adsorption of Lysozyme on Graphene Oxide

    PubMed Central

    2015-01-01

    Biosensing methods and devices using graphene oxide (GO) have recently been explored for detection and quantification of specific biomolecules from body fluid samples, such as saliva, milk, urine, and serum. For a practical diagnostics application, any sensing system must show an absence of nonselective detection of abundant proteins in the fluid matrix. Because lysozyme is an abundant protein in these body fluids (e.g., around 21.4 and 7 ?g/mL of lysozyme is found in human milk and saliva from healthy individuals, and more than 15 or even 100 ?g/mL in patients suffering from leukemia, renal disease, and sarcoidosis), it may interfere with detections and quantification if it has strong interaction with GO. Therefore, one fundamental question that needs to be addressed before any development of GO based diagnostics method is how GO interacts with lysozyme. In this study, GO has demonstrated a strong interaction with lysozyme. This interaction is so strong that we are able to subsequently eliminate and separate lysozyme from aqueous solution onto the surface of GO. Furthermore, the strong electrostatic interaction also renders the selective adsorption of lysozyme on GO from a mixture of binary and ternary proteins. This selectivity is confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), fluorescence spectroscopy, and UV–vis absorption spectroscopy. PMID:24684375

  20. Penetration and fusion of phospholipid vesicles by lysozyme

    SciTech Connect

    Kim, J.; Kim, H.

    1989-10-01

    The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-(({sup 125}I)iodophenyl)diazirine (({sup 125}I)TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent ({sup 125}I)TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.

  1. Visible Light Sensitized Inactivation of MS-2 Bacteriophage by a

    E-print Network

    Alvarez, Pedro J.

    Visible Light Sensitized Inactivation of MS-2 Bacteriophage by a Cationic Amine-Functionalized C60 bacteriophage), double stranded DNA virus with (PRD1) and without (T7) internal lipid membrane. Tegos et al. (7

  2. Interplay between the mechanics of bacteriophage fibers and the strength of virus-host links

    NASA Astrophysics Data System (ADS)

    Ares, P.; Garcia-Doval, C.; Llauró, A.; Gómez-Herrero, J.; van Raaij, M. J.; de Pablo, P. J.

    2014-05-01

    Viral fibers play a central role in many virus infection mechanisms since they recognize the corresponding host and establish a mechanical link to its surface. Specifically, bacteriophages have to anchor to bacteria through the fibers surrounding the tail before starting the viral DNA translocation into the host. The protein gene product (gp) 37 from bacteriophage T4 long tail fibers forms a fibrous parallel homotrimer located at the distal end of the long tail fibers. Biochemical data indicate that, at least, three of these fibers are required for initial host cell interaction but do not reveal why three and no other numbers are required. By using atomic force microscopy, we obtained high-resolution images of gp37 fibers adsorbed on a mica substrate in buffer conditions and probed their local mechanical properties. Our experiments of radial indentation at the nanometer scale provided a radial stiffness of ˜0.08 N/m and a breaking force of ˜120 pN. In addition, we performed finite element analysis and determined a Young's modulus of ˜20 MPa. From these mechanical parameters, we hypothesize that three viral fibers provide enough mechanical strength to prevent a T4 virus from being detached from the bacteria by the viral particle Brownian motion, delivering a biophysical justification for the previous biochemical data.

  3. Real time observations of single bacteriophage DNA ejections in vitro.

    E-print Network

    Phillips, Rob

    Real time observations of single bacteriophage DNA ejections in vitro. Paul Grayson, Lin Han, chemical, and structural features of bacteriophage genome release have been the subject of much recent in this study are probably generic features of DNA translocation in bacteriophages and have implications

  4. The Tripartite Associations between Bacteriophage, Wolbachia, and Arthropods

    E-print Network

    Bordenstein, Seth

    The Tripartite Associations between Bacteriophage, Wolbachia, and Arthropods Seth R. Bordenstein with incompatibility strength. The recent discovery of a temperate bacteriophage (WO-B) of Wolbachia containing ankyrin-encoding genes and virulence factors has led to intensifying debate that bacteriophage WO-B induces CI. However

  5. Bacteriophage Assembly, O 1981 Alan R. Liss, lnc.,

    E-print Network

    Harrison, Stephen C.

    Bacteriophage Assembly, O 1981 Alan R. Liss, lnc., pages 3-18 150 Filth Avenue, New York, NY 100i1 Harvard University Cambridqe, Mass . 02138 Subunits that form assembfies fike bacteriophage heads or taifs experiments on a variety of bacteriophage assembly pathways (thoroughly reviewed by Klng and Casjens, 1975

  6. 2006 Nature Publishing Group Structure of epsilon15 bacteriophage reveals

    E-print Network

    Jiang, Wen

    #12;© 2006 Nature Publishing Group Structure of epsilon15 bacteriophage reveals genome organization bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedralDNA) bacteriophages are vectors for gene transfer among enteric bacteria, including important human pathogens7

  7. Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: lateral reorganization of LPS monolayer, model of the Escherichia coli outer membrane.

    PubMed

    Derde, Melanie; Nau, Françoise; Lechevalier, Valérie; Guérin-Dubiard, Catherine; Paboeuf, Gilles; Jan, Sophie; Baron, Florence; Gautier, Michel; Vié, Véronique

    2015-01-01

    Lysozyme is mainly described active against Gram-positive bacteria, but is also efficient against some Gram-negative species. Especially, it was recently demonstrated that lysozyme disrupts Escherichia coli membranes. Moreover, dry-heating changes the physicochemical properties of the protein and increases the membrane activity of lysozyme. In order to elucidate the mode of insertion of lysozyme into the bacterial membrane, the interaction between lysozyme and a LPS monolayer mimicking the E. coli outer membrane has been investigated by tensiometry, ellipsometry, Brewster angle microscopy and atomic force microscopy. It was thus established that lysozyme has a high affinity for the LPS monolayer, and is able to insert into the latter as long as polysaccharide moieties are present, causing reorganization of the LPS monolayer. Dry-heating increases the lysozyme affinity for the LPS monolayer and its insertion capacity; the resulting reorganization of the LPS monolayer is different and more drastic than with the native protein. PMID:25450345

  8. Class I self-splicing introns are found in the T-even bacteriophage family

    SciTech Connect

    Chu, F.K.; Maley, F.; Maley, G.F.

    1987-05-01

    The thymidylate synthase gene (td) and ribonucleotide reductase B2 subunit gene (nrdB) EMBO both of bacteriophage T4 in origin, are procaryotic intron-containing protein-encoding genes. To screen for other procaryotic introns, southern hybridization analysis of several procaryotic genomes was carried out, using T4 phage td DNA restriction fragments and synthetic oligodeoxynucleotides defining strategic td exon and intron regions. Furthermore, the labeling pattern of total RNA with (..cap alpha..-/sup 32/P)GTP, a typical reaction of self-splicing RNAs (class I), was examined. Experimental data implicate multiple self-splicing introns only in the T-even phages: five (1, 0.9, 0.83, 0.75 and 0.6 kb) in T4 and three (1, 0.9 and 0.75 kb) each in T2 and T6 phages. Northern hybridization analysis of total RNA extracted from T-even phage-infected cells confirms that the 1 kb RNA from each phage is in fact the excised intron segment from the precursor RNA transcribed from an intron-containing td gene in each case. This RNA cyclizes to form a contiguous circular molecule. The 0.6 kb RNA is most likely the T4 phage nrdB intron which seems to be absent from the corresponding gene in T2 and T6. The remaining RNA species are candidates for other self-splicing introns in these phages.

  9. A new lysozyme from the eastern oyster, Crassostrea virginica, and a possible evolutionary pathway for i-type lysozymes in bivalves from host defense to digestion

    PubMed Central

    2010-01-01

    Background Lysozymes are enzymes that lyse bacterial cell walls, an activity widely used for host defense but also modified in some instances for digestion. The biochemical and evolutionary changes between these different functional forms has been well-studied in the c-type lysozymes of vertebrates, but less so in the i-type lysozymes prevalent in most invertebrate animals. Some bivalve molluscs possess both defensive and digestive lysozymes. Results We report a third lysozyme from the oyster Crassostrea virginica, cv-lysozyme 3. The chemical properties of cv-lysozyme 3 (including molecular weight, isoelectric point, basic amino acid residue number, and predicted protease cutting sites) suggest it represents a transitional form between lysozymes used for digestion and immunity. The cv-lysozyme 3 protein inhibited the growth of bacteria (consistent with a defensive function), but semi-quantitative RT-PCR suggested the gene was expressed mainly in digestive glands. Purified cv-lysozyme 3 expressed maximum muramidase activity within a range of pH (7.0 and 8.0) and ionic strength (I = 0.005-0.01) unfavorable for either cv-lysozyme 1 or cv-lysozyme 2 activities. The topology of a phylogenetic analysis of cv-lysozyme 3 cDNA (full length 663 bp, encoding an open reading frame of 187 amino acids) is also consistent with a transitional condition, as cv-lysozyme 3 falls at the base of a monophyletic clade of bivalve lysozymes identified from digestive glands. Rates of nonsynonymous substitution are significantly high at the base of this clade, consistent with an episode of positive selection associated with the functional transition from defense to digestion. Conclusion The pattern of molecular evolution accompanying the shift from defensive to digestive function in the i-type lysozymes of bivalves parallels those seen for c-type lysozymes in mammals and suggests that the lysozyme paralogs that enhance the range of physiological conditions for lysozyme activity may provide stepping stones between defensive and digestive forms. PMID:20633278

  10. Bacteriophage protein-protein interactions.

    PubMed

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian; Uetz, Peter

    2012-01-01

    Bacteriophages T7, ?, P22, and P2/P4 (from Escherichia coli), as well as ?29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages ? and T7. For example, the ?55 proteins encoded by the T7 genome are connected by ?43 interactions with another ?15 between the phage and its host. The chapter compiles published interactions for the well-studied phages ? (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ?29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage ? and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  11. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    PubMed Central

    Costa, Liliana; Faustino, Maria Amparo F.; Neves, Maria Graça P. M. S.; Cunha, Ângela; Almeida, Adelaide

    2012-01-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. PMID:22852040

  12. Effects of purification on the crystallization of lysozyme

    NASA Astrophysics Data System (ADS)

    Ewing, Felecia L.; Forsythe, Elizabeth L.; van der Woerd, Mark; Pusey, Marc L.

    1996-03-01

    We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20°C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal ? orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

  13. Effects of Purification on the Crystallization of Lysozyme

    NASA Technical Reports Server (NTRS)

    Ewing, Felecia L.; Forsythe, Elizabeth L.; Van Der Woerd, Mark; Pusey, Marc L.

    1996-01-01

    We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20 C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal- orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

  14. Experimental and computational studies on the DNA translocation mechanism of the T4 viral packaging motor

    NASA Astrophysics Data System (ADS)

    Migliori, Amy; Arya, Gaurav; Smith, Douglas E.

    2012-10-01

    Bacteriophage T4 is a double stranded DNA virus that infects E.coli by injecting the viral genome through the cellular wall of a host cell. The T4 genome must be ejected from the viral capsid with sufficient force to ensure infection. To generate high ejection forces, the genome is packaged to high density within the viral capsid. A DNA translocation motor, in which the protein gp17 hydrolyzes ATP and binds to the DNA, is responsible for translocating the genome into the capsid during viral maturation of T4. This motor generates forces in excess of 60 pN and packages DNA at rates exceeding 2000 base pairs/second (bp/s)1. Understanding these small yet powerful motors is important, as they have many potential applications. Though much is known about the activity of these motors from bulk and single molecule biophysical techniques, little is known about their detailed molecular mechanism. Recently, two structures of gp17 have been obtained: a high-resolution X-ray crystallographic structure showing a monomeric compacted form of the enzyme, and a cryo-electron microscopic structure of the extended form of gp17 in complex with actively packaging prohead complexes. Comparison of these two structures indicates several key differences, and a model has been proposed to explain the translocation action of the motor2. Key to this model are a set of residues forming ion pairs across two domains of the gp17 molecule that are proposed to be involved in force generation by causing the collapse of the extended form of gp17. Using a dual optical trap to measure the rates of DNA packaging and the generated forces, we present preliminary mutational data showing that these several of these ion pairs are important to motor function. We have also performed preliminary free energy calculations on the extended and collapsed state of gp17, to confirm that these interdomain ion pairs have large contributions to the change in free energy that occurs upon the collapse of gp17 during the proposed ratcheting mechanism.

  15. Formation of amyloid fibrils from fully reduced hen egg white lysozyme

    E-print Network

    Luhua, Lai

    Formation of amyloid fibrils from fully reduced hen egg white lysozyme AONENG CAO, DAOYING HU hen egg white lysozyme (HEWL), which is a good model of random coil structure, has been converted. 2001; Kallberg et al. 2001). Hen egg white lysozyme (HEWL), an extensively studied small protein

  16. Structureenergy relations in hen egg white lysozyme observed during refolding from a quenched unfolded statew

    E-print Network

    Structure­energy relations in hen egg white lysozyme observed during refolding from a quenched by rapidly quenching thermally unfolded hen egg white lysozyme in a glassy matrix, followed by reheating possible tech- nique to quench fast-folding proteins.9 Hen egg white lysozyme was chosen as the model

  17. Salt induced reduction of lysozyme adsorption at charged interfaces

    NASA Astrophysics Data System (ADS)

    Göhring, Holger; Paulus, Michael; Salmen, Paul; Wirkert, Florian; Kruse, Theresa; Degen, Patrick; Stuhr, Susan; Rehage, Heinz; Tolan, Metin

    2015-06-01

    A study of lysozyme adsorption below a behenic acid membrane and at the solid-liquid interface between aqueous lysozyme solution and a silicon wafer in the presence of sodium chloride is presented. The salt concentration was varied between 1 mmol L-1 and 1000 mmol L-1. X-ray reflectivity data show a clear dependence of the protein adsorption on the salt concentration. Increasing salt concentrations result in a decreased protein adsorption at the interface until a complete suppression at high concentrations is reached. This effect can be attributed to a reduced attractive electrostatic interaction between the positively charged proteins and negatively charged surfaces by charge screening. The measurements at the solid-liquid interfaces show a transition from unoriented order of lysozyme in the adsorbed film to an oriented order with the short protein axis perpendicular to the solid-liquid interface with rising salt concentration.

  18. Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood

    PubMed Central

    Majewska, Joanna; Beta, Weronika; Lecion, Dorota; Hodyra-Stefaniak, Katarzyna; K?opot, Anna; Ka?mierczak, Zuzanna; Miernikiewicz, Paulina; Piotrowicz, Agnieszka; Ciekot, Jaros?aw; Owczarek, Barbara; Kopciuch, Agnieszka; Wojtyna, Karolina; Harhala, Marek; M?kosa, Mateusz; D?browska, Krystyna

    2015-01-01

    A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA) were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79); the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus) presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design. PMID:26308042

  19. Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood.

    PubMed

    Majewska, Joanna; Beta, Weronika; Lecion, Dorota; Hodyra-Stefaniak, Katarzyna; K?opot, Anna; Ka?mierczak, Zuzanna; Miernikiewicz, Paulina; Piotrowicz, Agnieszka; Ciekot, Jaros?aw; Owczarek, Barbara; Kopciuch, Agnieszka; Wojtyna, Karolina; Harhala, Marek; M?kosa, Mateusz; D?browska, Krystyna

    2015-08-01

    A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA) were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79); the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus) presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design. PMID:26308042

  20. Bacteriophage lysis: mechanism and regulation.

    PubMed Central

    Young, R

    1992-01-01

    Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E. Images PMID:1406491

  1. Bacteriophage control of foodborne bacteriat.

    PubMed

    Greer, G Gordon

    2005-05-01

    Bacteriophages are measurable components of the natural microflora in the food production continuum from the farm to the retail outlet. Phages are remarkably stable in these environments and are readily recovered from soil, sewage, water, farm and processing plant effluents, feces, and retail foods. Purified high-titer phage lysates have been used for the species-specific control of bacteria during the pre- and postharvest phases of food production and storage. For example, the inhibition of the phytopathogens Erwinia amylovara and Xanthomonas campestris has reduced the incidence of diseases such as fire blight in apples and bacterial spot of tomato and peaches. Research on preslaughter treatment of food animals has demonstrated phage control of salmonellosis in chickens, enteropathogenic Escherichia coli infections in calves, piglets, and lambs, and E. coli O157:H7 shedding by beef cattle. Phages have also been applied to control the growth of pathogens such as Listeria monocytogenes, Salmonella, and Campylobacter jejuni in a variety of refrigerated foods such as fruit, dairy products, poultry, and red meats. Phage control of spoilage bacteria (e.g., Pseudomonas spp. and Brochothrix thermosphacta) in raw chilled meats can result in a significant extension of storage life. Phage biocontrol strategies for food preservation have the advantages of being self-perpetuating, highly discriminatory, natural, and cost-effective. Some of the drawbacks of biopreservation with phages are a limited host range, the requirement for threshold numbers of the bacterial targets, phage-resistant mutants, and the potential for the transduction of undesirable characteristics from one bacterial strain to another. Most research to date has involved experimentally infected plants and animals or artificially inoculated foods. This technology must be transferred to the field and to commercial environments to assess the possibility of controlling natural contaminants under more realistic production and processing conditions. PMID:15895751

  2. The solubility of hen egg-white lysozyme

    NASA Technical Reports Server (NTRS)

    Howard, Sandra B.; Twigg, Pamela J.; Baird, James K.; Meehan, Edward J.

    1988-01-01

    The equilibrium solubility of chicken egg-white lysozyme in the presence of crystalline solid state was determined as a function of NaCl concentration, pH, and temperature. The solubility curves obtained represent a region of the lysozyme phase diagram. This diagram makes it possible to determine the supersaturation of a given set of conditions or to achieve identical supersaturations by different combinations of parameters. The temperature dependence of the solubility permits the evaluation of Delta-H of crystallization. The data indicate a negative heat of crystallization for the tetragonal crystal form but a positive heat of crystallization for the high-temperature orthorhombic form.

  3. A stochastic model for bacteriophage therapies

    E-print Network

    Bardina, Xavier; Rovira, Carles; Tindel, Samy

    2011-01-01

    In this article, we analyze a system modeling bacteriophage treatments for infections in a noisy context. In the small noise regime, we show that after a reasonable amount of time the system is close to a sane equilibrium (which is a relevant biologic information) with high probability. Mathematically speaking, our study hinges on concentration techniques for delayed stochastic differential equations.

  4. Bacteriophage ecology in commercial sauerkraut fermentations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ecology of bacteriophages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 independent phage isolates, including ...

  5. ADSORPTION OF BACTERIOPHAGES ON CLAY MINERALS

    EPA Science Inventory

    Theability to predict the fate of microorganisms in soil is dependent on an understanding of the process of their sorption on soil and subsurface materials. Presently, we have focused on studying the thermodynamics of sorption of bacteriophages (T-2, MS-2, and

  6. Use of wide-host-range bacteriophages to reduce Salmonella on poultry products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages used to treat infections are typically amplified in a pathogenic host. However, this practice introduces the risk of administering any remaining bacteriophage-resistant pathogen during bacteriophage application if separate techniques are less than perfect. In this study, bacteriopha...

  7. Temperature Affects the Tripartite Interactions between Bacteriophage WO, Wolbachia, and Cytoplasmic

    E-print Network

    Bordenstein, Seth

    Temperature Affects the Tripartite Interactions between Bacteriophage WO, Wolbachia influence the symbiosis. For example, Wolbachia in arthropods harbor a widespread temperate bacteriophage that temperatures at the extreme edges of an insect's habitable range alter bacteriophage WO inducibility

  8. Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM

    E-print Network

    Jiang, Wen

    Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo important contribu- tions to modern structural biology. Bacteriophages, the most diverse and abundant previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution

  9. STUDIES ON THE PURIFICATION OF BACTERIOPHAGE.

    PubMed

    Kalmanson, G; Bronfenbrenner, J

    1939-11-20

    A simple method of concentrating and purifying bacteriophage has been described. The procedure consisted essentially in collecting the active agent on a reinforced collodion membrane of a porosity that would just retain all the active agent and permit extraneous material to pass through. Advantage was taken of the fact that B. coli will proliferate and regenerate bacteriophage in a completely diffusible synthetic medium with ammonia as the only source of nitrogen, which permitted the purification of the bacteriophage by copious washing. The material thus obtained was concentrated by suction and after thorough washing possessed all the activity of the original filtrate. It was labile, losing its activity in a few days on standing, and was quickly and completely inactivated upon drying. This material contained approximately 15 per cent of nitrogen and with 2 or 3 mg. samples of inactive dry residue it was possible to obtain positive protein color tests. The concentrated and purified bacteriophage has about 10(-14) mg. of nitrogen, or 6 x 10(-17) gm. of protein per unit of lytic activity. Assuming that each unit of activity represents a molecule, the calculated maximum average molecular weight would be approximately 36,000,000, and on the assumption of a spherical shape of particles and a density of 1.3, the calculated radius would be about 22 millimicra. By measurement of the diffusion rate, the average radius of particle of the fraction of the purified bacteriophage which diffuses most readily through a porous plate was found to be of the order of magnitude of 9 millimicra, or of a calculated molecular weight of 2,250,000. Furthermore, when this purified bacteriophage was fractionated by forcing it through a thin collodion membrane, which permits the passage of only the smaller particles, it was possible to demonstrate in the ultrafiltrate active particles of about 2 millimicra in radius, and of a calculated molecular weight of 25,000. It was of interest to apply this method of purification to a staphylococcus bacteriophage. Since this organism does not readily grow in synthetic medium, a diffusate of yeast extract medium was employed. The better of two preparations contained about 10(-12) mg. of nitrogen per unit of lytic activity. Although this is about one hundred times the amount of nitrogen found in an active unit of B. coli bacteriophage, nevertheless, the diffusion rate experiments gave results which paralleled those obtained with the coliphage. The diffusible particles of the crude staphylococcus bacteriophage had a radius of about 7 millimicra, and a calculated molecular weight of about 1,000,000, while the particles of the same phage which appeared in the ultrafiltrate through a thin collodion membrane had a radius of about 2.4 millimicra and a calculated molecular weight of about 45,000. It appears, therefore, that the active principle is distributed as particles of widely different sizes. However, since the smaller particles have all the properties of bacteriophage, the larger particles probably do not represent free molecules, but either are aggregates, or more likely, inactive colloids to which the active agent is adsorbed. The protein isolated, which bears the phage activity, is capable of stimulating the production of antilytic antibodies on parenteral injection into rabbits or guinea pigs. It retains its specific antigenicity when inactivated by formalin, but not when inactivated by drying. PMID:19873149

  10. Mapping the solid-state properties of crystalline lysozyme during pharmaceutical unit-operations.

    PubMed

    Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T

    2015-10-10

    Bulk crystallisation of protein therapeutic molecules towards their controlled drug delivery is of interest to the biopharmaceutical industry. The complexity of biotherapeutic molecules is likely to lead to complex material properties of crystals in the solid state and to complex transitions. This complexity is explored using batch crystallised lysozyme as a model. The effects of drying and milling on the solid-state transformations of lysozyme crystals were monitored using differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), FT-Raman, and enzymatic assay. XRPD was used to characterise crystallinity and these data supported those of crystalline lysozyme which gave a distinctive DSC thermogram. The apparent denaturation temperature (Tm) of the amorphous lysozyme was ?201 °C, while the Tm of the crystalline form was ?187 °C. Raman spectra supported a more ?-helix rich structure of crystalline lysozyme. This structure is consistent with reduced cooperative unit sizes compared to the amorphous lysozyme and is consistent with a reduction in the Tm of the crystalline form. Evidence was obtained that milling also induced denaturation in the solid-state, with the denatured lysozyme showing no thermal transition. The denaturation of the crystalline lysozyme occurred mainly through its amorphous form. Interestingly, the mechanical denaturation of lysozyme did not affect its biological activity on dissolution. Lysozyme crystals on drying did not become amorphous, while milling-time played a crucial role in the crystalline-amorphous-denatured transformations of lysozyme crystals. DSC is shown to be a key tool to monitor quantitatively these transformations. PMID:26068908

  11. Electrostatic self-assembly between biological polymers & macroions: Interactions of F-actin & DNA with lysozyme

    NASA Astrophysics Data System (ADS)

    Sanders, Lori K.; Matthews, Brian W.; Wong, Gerard C. L.

    2005-03-01

    The pathological self-assembly of polyelectrolytes such as DNA and F-actin with cationic antimicrobial proteins such as lysozyme may have significant clinical consequences in Cystic Fibrosis (CF) lung infections. Wild-type lysozyme is a compact, cationic, globular protein which carries a net charge of +9e at neutral pH. Our Small Angle X-ray Scattering (SAXS) experiments on F-actin-lysozyme complexes indicate that the wild-type lysozyme close packs into 1-D columns between hexagonally organized F-actin filaments. We will present SAXS results of the interactions of F-actin and DNA with genetically engineered lysozyme mutants that carry a reduced charge of +5e. We have also used fluorescence microscopy to investigate the morphologies and sizes of such bundles induced with divalent cations, wild-type lysozyme, and mutant lysozymes.

  12. Purification, Crystallization and Preliminary X-ray Diffraction Analysis of the Phage T4 Vertex Protein Gp24 and its Mutant Forms

    SciTech Connect

    Boeshans,K.; Liu, F.; Peng, G.; Idler, W.; Jang, S.; Marekov, L.; Black, L.; Ahvazi, B.

    2006-01-01

    The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6 {angstrom} resolution compared to wild-type gp24 at 3.80 {angstrom} resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.

  13. Dye functionalized cryogel columns for reversible lysozyme adsorption.

    PubMed

    Uygun, Murat; Akduman, Begüm; Uygun, Deniz Akta?; Akgöl, Sinan; Denizli, Adil

    2015-01-01

    In this study, poly (methyl methacrylate-glycidyl methacrylate) [poly(MMA-GMA)] cryogels were prepared by radical cryocopolymerization of MMA with GMA as a functional comonomer. Reactive Green 19 dye was then attached to the cryogel by nucleophilic substitution reaction, and this dye-attached cryogel column was used for lysozyme adsorption. Characterization of the cryogel was performed by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, Brunauer-Emmett-Teller, and energy dispersive X-ray analysis. Pore size of the cryogels was 15-30??m and pores were interconnected structure. Attached amount of Reactive Green 19 to cryogel support was calculated as 106.25??mol/g cryogel. Lysozyme adsorption studies were carried out by using a continuous system. It was found that the maximum amount of lysozyme adsorption (32?mg/g cryogel) obtained from experimental results was found to be approximately same with the calculated Langmuir adsorption capacity (33?mg/g cryogel). Desorption of adsorbed lysozyme was carried out by using 1.5?M NaCl in pH 4.5 acetate buffer, and desorption yield was found to be 97.4%. Cryogels were very stable, and it was found that there was no remarkable reduction in the adsorption capacity at the end of ten adsorption-desorption cycles. As a result, Reactive Green 19-attached cryogels have great advantages such as easy preparation, rapid adsorption, and desorption, being economic and allowing the direct separation of proteins. PMID:25555198

  14. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lysozyme (muramidase) test system. 862.1490 Section 862.1490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  15. Locations of Bromide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

    Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

    1998-01-01

    Anions have been shown to play a dominant role in the crystallization of chicken egg-white lysozyme from salt solutions. Previous studies employing X-ray crystallography have found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystals grown in bromide and chloride solutions. Five possible anion-binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four further sites were found which corresponded to the four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion-binding sites in lysozyme remain unchanged even when different anions and different crystal forms of lysozyme are employed.

  16. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lysozyme (muramidase) test system. 862.1490 Section 862.1490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  17. Science Study Aids 6: Lysozyme - The Cooperative Enzyme.

    ERIC Educational Resources Information Center

    Boeschen, John; Alderton, Gordon

    This publication is the sixth of a series of seven supplementary investigative materials for use in secondary science classes providing up-to-date research-related investigations. This unit is structured for grade levels 10 through 12. It is concerned with the crystallization of an enzyme, lysozyme, from egg white. The first part of this guide…

  18. Bacteriophages were discovered indepen-dently in 1915 by Frederick Twort, a British

    E-print Network

    Cai, Long

    Bacteriophages were discovered indepen- dently in 1915 by Frederick Twort, a British pathologist1 the term `bacteriophage' -- literally meaning bacteria-eater. Phages are obligate intracellular para- sites

  19. Chicken-type lysozyme in channel catfish: expression analysis, lysozyme activity, and efficacy as immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with...

  20. Chicken-type lysozyme in channel catfish: Expression analysis, lysozyme activity and efficacy as immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with...

  1. Functional relationship between bacteriophages G4 and phi X174.

    PubMed Central

    Borrias, W E; Hagenaar, M; Van Den Brekel, R; Kühlemeijer, C; Weisbeek, P J

    1979-01-01

    Mutants of bacteriophage G4 were isolated and characterized, and their mutations were mapped. They constitute six different genes, namely, A, B, E, F, G, and H. The functional relationship with bacteriophage phi X174 was determined by complementation experiments using amber mutants of phi X and amber mutants of G4. Bacteriophage phi X was able to use the products of G4 genes E, F, G, and H. In bacteriophage G4, however, only the phi X gene H product was functional. Images PMID:480475

  2. Generation of recombinant destabilase-lysozyme from medicinal leeches in three different expression systems.

    PubMed

    Manuvera, Valentin A; Kurdyumov, Alexey S; Filonova, Kseniya A; Lazarev, Vassili N

    2015-12-01

    Destabilase-lysozyme (mlDL) is an enzyme secreted by the salivary gland cells of medicinal leeches. Destabilase-lysozyme possesses lysozyme and isopeptidase activities. We generated recombinant destabilase-lysozyme isoform 2 in three expression systems, i.e., in the bacteria Escherichia coli, in the yeast Pichia pastoris, and in the human cell line Expi293F. In E. coli, we generated both polypeptide in inclusion bodies that was later undergone to the refolding and soluble protein that had been fused with the chaperone SlyD. The chaperone was later cleaved by a specific TEV-protease. In cultures of the yeast P. pastoris and the human cell line Expi293F, the soluble form of destabilase-lysozyme was accumulated in the culture media. For the generated enzymes, we determined the lysozyme, isopeptidase and fibrinolytic activities and tested their general antimicrobial effects. The comparisons of the enzymes generated in the different expression systems revealed that all of the destabilase-lysozymes obtained in the soluble forms possessed equal levels of lysozyme, isopeptidase and fibrinolytic activities that exceeded several to ten times the levels of the same activities of the destabilase-lysozyme renaturated from the inclusion bodies. A similar pattern of the differences in the levels of the general antimicrobial effects was observed for the destabilase-lysozymes generated in the soluble form and as inclusion bodies. PMID:26277552

  3. Kinetically trapped structure in the renaturation of reduced oxindolealanine 62 lysozyme.

    PubMed

    Ueda, T; Abe, Y; Ohkuri, T; Kawano, K; Terada, Y; Imoto, T

    1995-12-12

    The refolded products of reduced native lysozyme and reduced OX62 lysozyme, in which Trp62 is converted to oxindolealanine (OX62) during the renaturation of sulfhydryl-disulfide interchange reactions at pH 8 and 37 degrees C, were investigated. On gel-chromatography eluted with 10% aqueous acetic acid containing 4 M urea, two peaks appeared in the refolded product of reduced OX62 lysozyme while a single peak appeared in the refolded product of reduced native lysozyme. From the analyses of the activity and primary and the tertiary structures of the derivative, the structure of the derivative from reduced native lysozyme was confirmed to be identical to that of the untreated one. On the other hand, the refolded product from reduced OX62 lysozyme had the same primary structure but a different tertiary structure compared to the untreated one. The tertiary structure of the refolded product from the reduced OX62 lysozyme was changed to that of the untreated one by the denaturation-renaturation treatment under nonreduced conditions. However, the refolded species was barely changed to that of the untreated one by incubation under physiological conditions. Therefore, the refolded product from reduced OX62 lysozyme was suggested to be a metastable and kinetically trapped product in the renaturation process of reduced OX62 lysozyme. In addition, an interaction involving the folding process of reduced lysozyme was discussed on the basis of the NMR analyses of the metastable structure. PMID:8519775

  4. Call for a dedicated European legal framework for bacteriophage therapy.

    PubMed

    Verbeken, Gilbert; Pirnay, Jean-Paul; Lavigne, Rob; Jennes, Serge; De Vos, Daniel; Casteels, Minne; Huys, Isabelle

    2014-04-01

    The worldwide emergence of antibiotic resistances and the drying up of the antibiotic pipeline have spurred a search for alternative or complementary antibacterial therapies. Bacteriophages are bacterial viruses that have been used for almost a century to combat bacterial infections, particularly in Poland and the former Soviet Union. The antibiotic crisis has triggered a renewed clinical and agricultural interest in bacteriophages. This, combined with new scientific insights, has pushed bacteriophages to the forefront of the search for new approaches to fighting bacterial infections. But before bacteriophage therapy can be introduced into clinical practice in the European Union, several challenges must be overcome. One of these is the conceptualization and classification of bacteriophage therapy itself and the extent to which it constitutes a human medicinal product regulated under the European Human Code for Medicines (Directive 2001/83/EC). Can therapeutic products containing natural bacteriophages be categorized under the current European regulatory framework, or should this framework be adapted? Various actors in the field have discussed the need for an adapted (or entirely new) regulatory framework for the reintroduction of bacteriophage therapy in Europe. This led to the identification of several characteristics specific to natural bacteriophages that should be taken into consideration by regulators when evaluating bacteriophage therapy. One important consideration is whether bacteriophage therapy development occurs on an industrial scale or a hospital-based, patient-specific scale. More suitable regulatory standards may create opportunities to improve insights into this promising therapeutic approach. In light of this, we argue for the creation of a new, dedicated European regulatory framework for bacteriophage therapy. PMID:24500660

  5. Inactivation of E. coli, B. subtilis spores, and MS2, T4, and T7 phage using UV/H2O2 advanced oxidation.

    PubMed

    Mamane, Hadas; Shemer, Hilla; Linden, Karl G

    2007-07-31

    The goal of this study was to evaluate the potential of an advanced oxidation process (AOP) for microbiocidal and virucidal inactivation. The viruses chosen for this study were bacteriophage MS2, T4, and T7. In addition, Bacillus subtilis spores and Escherichia coli were studied. By using H(2)O(2) in the presence of filtered ultraviolet (UV) irradiation (UV/H(2)O(2)) to generate wavelengths above 295nm, the direct UV photolysis disinfection mechanism was minimized, while disinfection by H(2)O(2) was also negligible. Virus T4 and E. coli in phosphate buffered saline (PBS) were sensitive to >295nm filtered UV irradiation (without H(2)O(2)), while MS2 was very resistant. Addition of H(2)O(2) at 25mg/l in the presence of filtered UV irradiation over a 15min reaction time did not result in any additional disinfection of virus T4, while an additional one log inactivation for T7 and 2.5 logs for MS2 were obtained. With E. coli, only a slight additional effect was observed when H(2)O(2) was added. B. subtilis spores did not show any inactivation at any of the conditions used in this study. The OH radical exposure (CT value) was calculated to present the relationship between the hydroxyl radical dose and microbial inactivation. PMID:17532124

  6. Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: optical absorbance and fluorescence studies.

    PubMed Central

    Cherepanov, A V; de Vries, S

    2001-01-01

    The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the K(d) between 0.1 and 20 microM. Nucleotide binding results in a decrease of absorbance at 260 nm due to pi-stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the pi-stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K(d) for the noncovalent site is approximately 1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity. PMID:11721015

  7. Dynamical Properties on the Thermal Denaturation of Lysozyme-Trehalose Solution

    NASA Astrophysics Data System (ADS)

    Sasanuma, Keita; Seshimo, Yuichi; Hashimoto, Eiji; Ike, Yuji; Kojima, Seiji

    2008-02-01

    We studied the dynamics of lysozyme solution and the bioprotective effect of trehalose. Thermodynamics and elastic properties related to the thermal denaturation were investigated by Modulated-temperature DSC (MDSC) and Brillouin scattering. By MDSC measurements, it is found that the thermal stability of lysozyme depends on the trehalose concentration, and trehalose suppresses the denaturation induced by pH change. The sound velocity of lysozyme-trehalose solution is studied as a function of trehalose concentration. With increasing trehalose concentration, the number density of tetrahedral structure of water molecules decreases. Furthermore, we reveal the interaction between lysozyme and trehalose increases, especially around room temperature. We suggerst that trehalose molecules tend to associate lysozyme by preferential hydration, and the trehalose-induced bioprotection phenomenon may result from the mechanical suppression of lysozyme unfolding.

  8. Trehalose Bioprotective Effects in Lysozyme Aqueous Solution Studied by Brillouin Scattering and Calorimetric Measurements

    NASA Astrophysics Data System (ADS)

    Sasanuma, Keita; Seshimo, Yuichi; Hashimoto, Eiji; Ike, Yuji; Kojima, Seiji

    2008-05-01

    The bioprotective effect of trehalose in lysozyme aqueous solutions has been investigated by Brillouin scattering and modulated-temperature differential scanning calorimetry (MDSC). MDSC experiments show that the isothermal kinetics of thermally irreversible denaturation can be described by the Arrhenius equation. By the addition of trehalose, the irreversible denaturation of lysozyme is suppressed, and its activation energy is half that of the denaturation without trehalose. The sound velocity of lysozyme-trehalose-water ternary solutions obviously depends on the trehalose concentration. With increasing trehalose concentration, the sound velocity becomes higher because the hydration of trehalose reduces the hydrogen bonds between water molecules. Moreover, hydration around lysozyme molecules increases the sound velocity further. Trehalose molecules tend to aggregate with lysozyme molecules at high trehalose concentrations. The bioprotective effect of trehalose probably originates from the mechanical suppression of conformational fluctuations of lysozyme molecules.

  9. Changes in Galleria mellonella lysozyme level and activity during Pseudomonas aeruginosa infection.

    PubMed

    Andrejko, M; Mizerska-Dudka, M; Jakubowicz, T

    2008-01-01

    The level of lysozyme in fat body, hemocytes and cell-free hemolymph from Galleria mellonella larvae infected with Pseudomonas aeruginosa was determined and evaluated. In the samples of fat body and hemocytes, an increase in lysozyme content was detected 1 d after infection and then a significant decrease was observed after a prolonged infection time. In the case of cell-free hemolymph, an increase in the lysozyme level was noticeable during the first 30 h post injection and stayed at a similar level for 42 h. The smaller decrease of the lysozyme level after 42 h might be associated with the development of bacteremia of P. aeruginosa in insects. In addition, the gradual increase in the content of lysozyme correlated with the increase of its activity in the hemolymph of the infected larvae as a response to injection with P. aeruginosa. The G. mellonella lysozyme appeared to be insensitive to extracellular proteinases produced in vivo by P. aeruginosa. PMID:18500634

  10. Tetragonal Chicken Egg White Lysozyme Solubility in Sodium Chloride Solutions

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Judge, Russell A.; Pusey, Marc L.

    1998-01-01

    The solubility of chicken egg white lysozyme, crystallized in the tetragonal form was measured in sodium chloride solutions from 1.6 to 30.7 C, using a miniature column solubility apparatus. Sodium chloride solution concentrations ranged from 1 to 7% (w/v). The solutions were buffered with 0.1 M sodium acetate buffer with the solubility being measured at pH values in 0.2 pH unit increments in the range pH 4.0 to 5.4, with data also included at pH 4.5. Lysozyme solubility was found to increase with increases in temperature and decreasing salt concentration. Solution pH has a varied and unpredictable effect on solubility.

  11. Protein crystal growth - Growth kinetics for tetragonal lysozyme crystals

    NASA Technical Reports Server (NTRS)

    Pusey, M. L.; Snyder, R. S.; Naumann, R.

    1986-01-01

    Results are reported from theoretical and experimental studies of the growth rate of lysozyme as a function of diffusion in earth-gravity conditions. The investigations were carried out to form a comparison database for future studies of protein crystal growth in the microgravity environment of space. A diffusion-convection model is presented for predicting crystal growth rates in the presence of solutal concentration gradients. Techniques used to grow and monitor the growth of hen egg white lysozyme are detailed. The model calculations and experiment data are employed to discuss the effects of transport and interfacial kinetics in the growth of the crystals, which gradually diminished the free energy in the growth solution. Density gradient-driven convection, caused by presence of the gravity field, was a limiting factor in the growth rate.

  12. Actin - Lysozyme Interactions in Model Cystic Fibrosis Sputum

    NASA Astrophysics Data System (ADS)

    Sanders, Lori; Slimmer, Scott; Angelini, Thomas; Wong, Gerard C. L.

    2003-03-01

    Cystic fibrosis sputum is a complex fluid consisting of mucin (a glycoprotein), lysozyme (a cationic polypeptide), water, salt, as well as a high concentration of a number of anionic biological polyelectrolytes such as DNA and F-actin. The interactions governing these components are poorly understood, but may have important clinical consequences. For example, the formation of these biological polyelectrolytes into ordered gel phases may contribute significantly to the observed high viscosity of CF sputum. In this work, a number of model systems containing actin, lysozyme, and KCl were created to simulate CF sputum in vitro. These model systems were studied using small angle x-ray scattering and confocal fluorescence microscopy. Preliminary results will be presented. This work was supported by NSF DMR-0071761, the Beckman Young Investigator Program, and the Cystic Fibrosis Foundation.

  13. Locations of Halide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

    Lim, Kap; Adimurthy, Ganapathi; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

    1998-01-01

    Anions play an important role in the crystallization of lysozyme, and are known to bind to the crystalline protein. Previous studies employing X-ray crystallography had found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. Studies using other approaches have reported more chloride ion binding sites, but their locations were not known. Knowing the precise location of these anions is also useful in determining the correct electrostatic fields surrounding the protein. In the first part of this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from the lysozyme crystals grown in bromide and chloride solutions under identical conditions. The anion locations were then obtained from standard crystallographic methods and five possible anion binding sites were found in this manner. The sole chloride ion binding site found in previous studies was confirmed. The remaining four sites were new ones for tetragonal lysozyme crystals. However, three of these new sites and the previously found one corresponded to the four unique binding sites found for nitrate ions in monoclinic crystals. This suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed. It is unlikely that there are many more anions in the tetragonal lysozyme crystal structure. Assuming osmotic equilibrium it can be shown that there are at most three more anions in the crystal channels. Some of the new anion binding sites found in this study were, as expected, in pockets containing basic residues. However, some of them were near neutral, but polar, residues. Thus, the study also showed the importance of uncharged, but polar groups, on the protein surface in determining its electrostatic field. This was important for the second part of this study where the electrostatic field surrounding the protein was accurately determined. This was achieved by solving the linearized version of the Poisson-Boltzmann equation for the protein in solution. The solution was computed employing the commercial code Delphi which uses a finite difference technique. This has recently become available as a module in the general protein visualization code Insight II. Partial charges were assigned to the polar groups of lysozyme for the calculations done here. The calculations showed the complexity of the electrostatic field surrounding the protein. Although most of the region near the protein surface had a positive field strength, the active site cleft was negatively charged and this was projected a considerable distance. This might explain the occurrence of "head-to-side" interactions in the formation of lysozyme aggregates in solution. Pockets of high positive field strength were also found in the vicinity of the anion locations obtained from the crystallographic part of this study, confirming the validity of these calculations. This study clearly shows not only the importance of determining the counterion locations in protein crystals and the electrostatic fields surrounding the protein, but also the advantage of performing them together.

  14. Critical Evaluation of Bacteriophage to Prevent and Treat Colibacillosis in Poultry.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage are viruses that infect and kill bacteria. Bacteriophage do not infect animal and plant cells making them a potentially safe alternative to antibiotics. We have conducted research on the efficacy of bacteriophage to both prevent and treat colibacillosis in poultry. Bacteriophage lyt...

  15. Bacteriophage Flux in Endosymbionts (Wolbachia): Infection Frequency, Lateral Transfer, and Recombination Rates

    E-print Network

    Bordenstein, Seth

    Bacteriophage Flux in Endosymbionts (Wolbachia): Infection Frequency, Lateral Transfer in the free-living microbial world--bacteriophages. This study yields three results that show bacteriophages endosymbiont of invertebrates, Wolbachia. First, we show that bacteriophage WO is more widespread in Wolbachia

  16. Bacteriophage in polar inland waters Christin Sawstrom John Lisle Alexandre M. Anesio

    E-print Network

    Priscu, John C.

    REVIEW Bacteriophage in polar inland waters Christin Sa¨wstro¨m Æ John Lisle Æ Alexandre M. Anesio / Published online: 10 January 2008 Ó Springer 2007 Abstract Bacteriophages are found wherever microbial life of bacteriophage ecology have been undertaken at temperate latitudes. Data on bacteriophages in polar inland waters

  17. Complete Genome Sequence of Vibrio vulnificus Bacteriophage SSP002

    PubMed Central

    Lee, Hyun Sung; Choi, Slae

    2012-01-01

    Vibrio vulnificus phages are abundant in coastal marine environments, shellfish, clams, and oysters. SSP002, a V. vulnificus-specific bacteriophage, was isolated from oysters from the west coast of South Korea. In this study, the complete genome of SSP002 was sequenced and analyzed for the first time among the V. vulnificus-specific bacteriophages. PMID:22733877

  18. Complete genome sequence of Vibrio vulnificus bacteriophage SSP002.

    PubMed

    Lee, Hyun Sung; Choi, Slae; Choi, Sang Ho

    2012-07-01

    Vibrio vulnificus phages are abundant in coastal marine environments, shellfish, clams, and oysters. SSP002, a V. vulnificus-specific bacteriophage, was isolated from oysters from the west coast of South Korea. In this study, the complete genome of SSP002 was sequenced and analyzed for the first time among the V. vulnificus-specific bacteriophages. PMID:22733877

  19. Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy

    E-print Network

    Collins, James J.

    Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy Timothy K. Lua, there is a pressing need for new antibacterial therapies that can be readily designed and implemented. In this work, we engineered bacteriophage to overexpress proteins and attack gene networks that are not directly

  20. Remarkable diversity of Salmonella bacteriophages in swine and poultry.

    PubMed

    Cortés, Pilar; Spricigo, Denis A; Bardina, Carlota; Llagostera, Montserrat

    2015-01-01

    The diversity of 55 Salmonella-specific bacteriophages isolated from 191 fecal samples of poultry and swine from farms located in diverse geographic areas of Spain was determined using lysis profiling, DNA restriction and random amplification of polymorphic DNA (RAPD-PCR). Among them, lysis profiling and RAPD-PCR exhibited 100% typeability and DNA restriction 96%, with discriminatory power of 0.978 (± 0.016), 0.938 (± 0.028) and 0.982 (± 0.013), respectively. The highest concordance (0.974) was that between RAPD-PCR and lysis profiling. None of the bacteriophages isolated from poultry and swine shared any DNA restriction or RAPD-PCR patterns and only two lysis profiles were common to bacteriophages isolated from poultry and swine. The major part of the lysis and RAPD-PCR profiles from the bacteriophages isolated from poultry included only one or two bacteriophages, while those obtained from swine contained more than two bacteriophages. Overall, our results provide evidence of the remarkable diversity exhibited by bacteriophages of Salmonella in farm animals. Moreover, they also show that RAPD-PCR may also be suitable for the pre-screening of the diversity of Salmonella bacteriophages for further use in biocontrol and therapeutic strategies. PMID:25670704

  1. Potential of Bacteriophage to Prevent and Treat Poultry Diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage are viruses plentiful in nature that kill bacteria, and represent a safe alternative to antibiotics. Bacteriophage lytic to Escherichia coli were isolated from municipal waste water treatment and poultry processing plants. This E. coli isolate is pathogenic to poultry, causing a sev...

  2. Immune Interference of Bacteriophage Efficacy When Treating Colibacillosis In Poultry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was conducted to determine if prior exposure of broiler chickens with bacteriophage would limit the ability of the same bacteriophage to treat colibacillosis. There were 5 treatments with 3 replicate pens of 20 birds per pen. The treatments consisted of 1) control; 2) birds treated with ba...

  3. Biochemical characterization of lysozymes present in egg white of selected species of anatid birds.

    PubMed

    D'Surney, S J; deKloet, S R

    1985-01-01

    The isolation of lysozyme from the egg white of several representative species of waterfowl is described. The purified lysozymes were analyzed to determine the type and molecular weight of each enzyme. All enzymes found in duck egg whites were found to be of the c-type. In contrast all true geese, and the mute swan species as well as the northern blackneck screamer contain lysozyme g in their egg white. PMID:4042624

  4. Biochemical characterization of lysozymes present in egg white of selected species of anatid birds.

    PubMed

    D'Surney, S J; deKloet, S R

    1985-01-01

    The isolation of lysozyme from the egg white of several representative species of waterfowl is described. The purified lysozymes were analyzed in order to determine the type and molecular weight of each enzyme. All enzymes found in duck egg whites were found to be of the c-type. In contrast all true geese, the Mute Swan as well as the Northern Blackneck Screamer contain lysozyme g in their egg white. PMID:4085215

  5. The effects of bacteriophage and nanoparticles on microbial processes

    NASA Astrophysics Data System (ADS)

    Moody, Austin L.

    There are approximately 1031 tailed phages in the biosphere, making them the most abundant organism. Bacteriophages are viruses that infect bacteria. Due to the large diversity and abundance, no two bacteriophages that have been isolated are genetically the same. Phage products have potential in disease therapy to solve bacteria-related problems, such as infections resulting from resistant strains of Staphylococcus aureus. A bacteriophage capable of infecting methicillin-resistant S. aureus (MRSA) was isolated from bovine hair. The bacteriophage, named JB phage, was characterized using purification, amplification, cesium chloride banding, scanning electron microscopy, and transmission electron microscopy. JB phage and nanoparticles were used in various in vitro and in vivo models to test their effects on microbial processes. Scanning and transmission electron microscopy studies revealed strong interactions between JB phage and nanoparticles, which resulted in increased bacteriophage infectivity. JB phage and nanoparticle cocktails were used as a therapeutic to treat skin and systemic infections in mice caused by MRSA.

  6. Lysozyme as a recognition element for monitoring of bacterial population.

    PubMed

    Zheng, Laibao; Wan, Yi; Yu, Liangmin; Zhang, Dun

    2016-01-01

    Bacterial infections remain a significant challenge in biomedicine and environment safety. Increasing worldwide demand for point-of-care techniques and increasing concern on their safe development and use, require a simple and sensitive bioanalysis for pathogen detection. However, this goal is not yet achieved. A design for fluorescein isothiocyanate-labeled lysozyme (FITC-LYZ), which provides quantitative binding information for gram-positive bacteria, Micrococcus luteus, and detects pathogen concentration, is presented. The functional lysozyme is used not only as the pathogenic detection platform, but also as a tracking reagent for microbial population in antibacterial tests. A nonlinear relationship between the system response and the logarithm of the bacterial concentration was observed in the range of 1.2×10(2)-1.2×10(5)cfumL(-1). The system has a potential for further applications and provides a facile and simple method for detection of pathogenic bacteria. Meanwhile, the fluorescein isothiocyanate -labeled lysozyme is also employed as the tracking agent for antibacterial dynamic assay, which show a similar dynamic curve compared with UV-vis test. PMID:26695267

  7. Relationship Between Equilibrium Forms of Lysozyme Crystals and Precipitant Anions

    NASA Technical Reports Server (NTRS)

    Nadarajah, Arunan

    1996-01-01

    Molecular forces, such as electrostatic, hydrophobic, van der Waals and steric forces, are known to be important in determining protein interactions. These forces are affected by the solution conditions and changing the pH, temperature or the ionic strength of the solution can sharply affect protein interactions. Several investigations of protein crystallization have shown that this process is also strongly dependent on solution conditions. As the ionic strength of the solution is increased, the initially soluble protein may either crystallize or form an amorphous precipitate at high ionic strengths. Studies done on the model protein hen egg white lysozyme have shown that different crystal forms can be easily and reproducibly obtained, depending primarily on the anion used to desolubilize the protein. In this study we employ pyranine to probe the effect of various anions on the water structure. Additionally, lysozyme crystallization was carried out at these conditions and the crystal form was determined by X-ray crystallography. The goal of the study was to understand the physico-chemical basis for the effect of changing the anion concentration on the equilibrium form of lysozyme crystals. It will also verify the hypothesis that the anions, by altering the bulk water structure in the crystallizing solutions, alter the surface energy of the between the crystal faces and the solution and, consequently, the equilibrium form of the crystals.

  8. ON THE REPLICATION OF INCOMPLETE CHROMOSOMES OF PHAGE T4*

    PubMed Central

    Mosig, G.; Werner, R.

    1969-01-01

    Some small particles of phage T4 contain incomplete chromosomes measuring two thirds of the length of normal T4 chromosomes. They cannot produce progeny phage after single infection because their incomplete chromosomes lack random segments of the genetic map. We have investigated the replication of DNA following single infection with small particles. Our results indicate that approximately two thirds of the incomplete chromosomes can initiate DNA replication and apparently extend the process from a genetically fixed origin to one end of the molecule. However, few if any of the incomplete chromosomes can initiate a second round of replication. The incomplete chromosomes which do not replicate also do not attach to bacterial membranes. PMID:5261046

  9. Estimation of the initial equilibrium constants in the formation of tetragonal lysozyme nuclei

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.

    1991-01-01

    Results are presented from a study of the equilibria, kinetic rates, and the aggregation pathway which leads from a lysozyme monomer crystal to a tetragonal crystal, using dialyzed and recrystallized commercial hen eggwhite lysozyme. Relative light scattering intensity measurements were used to estimate the initial equilibrium constants for undersaturated lysozyme solutions in the tetragonal regime. The K1 value was estimated to be (1-3) x 10 exp 4 L/mol. Estimates of subsequent equilibrium constants depend on the crystal aggregation model chosen or determined. Experimental data suggest that tetragonal lysozyme crystal grows by addition of aggregates preformed in the bulk solution, rather than by monomer addition.

  10. Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica).

    PubMed

    Xue, Qing-Gang; Schey, Kevin L; Volety, Aswani K; Chu, Fu-Lin E; La Peyre, Jerome F

    2004-09-01

    Lysozyme was purified from the plasma of eastern oysters (Crassostrea virginica) using a combination of ion exchange and gel filtration chromatographies. The molecular mass of purified lysozyme was estimated at 18.4 kDa by SDS-PAGE, and its isoelectric point was greater than 10. Mass spectrometric analysis of the purified enzyme revealed a high-sequence homology with i-type lysozymes. No similarity was found however between the N-terminal sequence of oyster plasma lysozyme and N-terminal sequences of other i-type lysozymes, suggesting that the N-terminal sequences of the i-type lysozymes may vary to a greater extent between species than reported in earlier studies. The optimal ionic strength, pH, cation concentrations, sea salt concentrations, and temperature for activity of the purified lysozyme were determined, as well as its temperature and pH stability. Purified oyster plasma lysozyme inhibited the growth of Gram-positive bacteria (e.g., Lactococcus garvieae, Enterococcus sp.) and Gram-negative bacteria (e.g., Escherichia coli, Vibrio vulnificus). This is a first report of a lysozyme purified from an oyster species and from the plasma of a bivalve mollusc. PMID:15364284

  11. Management of stage T3 and T4 glottic carcinomas

    SciTech Connect

    Yuen, A.; Medina, J.E.; Goepfert, H.; Fletcher, G.

    1984-10-01

    Between 1959 and 1979, 242 patients with T3 and T4 lesions of the vocal cords were treated at our institution. Treatment consisted of total laryngectomy in all patients. Different modalities of regional node dissections were performed on 187 patients. In addition, 50 patients received irradiation with cobalt-60 postoperatively for specific features of the disease. In the group of 192 patients whose treatment consisted of surgery alone, 28 (14 percent) had recurrence in the neck and 10 (5 percent) had stomal recurrence. Of the patients treated with combined therapy, three (6 percent) had ipsilateral neck recurrences and one (2 percent) had stomal recurrence. For lesions staged N0, failure rates above the clavicles were 16 percent and 31 percent for patients with T3 and T4 lesions, respectively, in the group treated by surgery alone, 9 percent and 6 percent for patients with T3 and T4 lesions, respectively, in the combined therapy group. The rate of failure above the clavicles for lesions staged N+ was 32 percent in the group treated with surgery alone and 8 percent in the combined therapy group. In this study, a correlation was made between the failure rates above the clavicles and different clinical and histologic characteristics of the tumor, surgical findings, and the different modalities of cervical node dissection used. From analysis of the data, recommendations have been made for the selective treatment of patients with advanced glottic carcinomas.

  12. Crystallization and Preliminary X-ray Analysis of Bacteriophasge T4 UvsY Recombination Mediator Protein

    SciTech Connect

    Xu,H.; Beernink, H.; Rould, M.; Morrical, S.

    2006-01-01

    Bacteriophage T4 UvsY protein is considered to be the prototype of recombination mediator proteins, a class of proteins which assist in the loading of recombinases onto DNA. Wild-type and Se-substituted UvsY protein have been expressed and purified and crystallized by hanging-drop vapor diffusion. The crystals diffract to 2.4 {angstrom} using in-house facilities and to 2.2 {angstrom} at NSLS, Brookhaven National Laboratory. The crystals belong to space group P422, P4{sub 2}22, P42{sub 1}2 or P4{sub 2}2{sub 1}2, the ambiguity arising from pseudo-centering, with unit-cell parameters a = b = 76.93, c = 269.8 {angstrom}. Previous biophysical characterization of UvsY indicates that it exists primarily as a hexamer in solution. Along with the absence of a crystallographic threefold, this suggests that the asymmetric unit of these crystals is likely to contain either three monomers, giving a solvent content of 71%, or six monomers, giving a solvent content of 41%.

  13. M13 Bacteriophage Based Protein Sensors

    NASA Astrophysics Data System (ADS)

    Lee, Ju Hun

    Despite significant progress in biotechnology and biosensing, early detection and disease diagnosis remains a critical issue for improving patient survival rates and well-being. Many of the typical detection schemes currently used possess issues such as low sensitivity and accuracy and are also time consuming to run and expensive. In addition, multiplexed detection remains difficult to achieve. Therefore, developing advanced approaches for reliable, simple, quantitative analysis of multiple markers in solution that also are highly sensitive are still in demand. In recent years, much of the research has primarily focused on improving two key components of biosensors: the bio-recognition agent (bio-receptor) and the transducer. Particular bio-receptors that have been used include antibodies, aptamers, molecular imprinted polymers, and small affinity peptides. In terms of transducing agents, nanomaterials have been considered as attractive candidates due to their inherent nanoscale size, durability and unique chemical and physical properties. The key focus of this thesis is the design of a protein detection and identification system that is based on chemically engineered M13 bacteriophage coupled with nanomaterials. The first chapter provides an introduction of biosensors and M13 bacteriophage in general, where the advantages of each are provided. In chapter 2, an efficient and enzyme-free sensor is demonstrated from modified M13 bacteriophage to generate highly sensitive colorimetric signals from gold nanocrystals. In chapter 3, DNA conjugated M13 were used to enable facile and rapid detection of antigens in solution that also provides modalities for identification. Lastly, high DNA loadings per phage was achieved via hydrozone chemistry and these were applied in conjunction with Raman active DNA-gold/silver core/shell nanoparticles toward highly sensitive SERS sensing.

  14. Manipulation of lysozyme phase behavior by additives as function of conformational stability.

    PubMed

    Galm, Lara; Morgenstern, Josefine; Hubbuch, Jürgen

    2015-10-15

    Undesired protein aggregation in general and non-native protein aggregation in particular need to be inhibited during bio-pharmaceutical processing to ensure patient safety and to maintain product activity. In this work the potency of different additives, namely glycerol, PEG 1000, and glycine, to prevent lysozyme aggregation and selectively manipulate lysozyme phase behavior was investigated. The results revealed a strong pH dependency of the additive impact on lysozyme phase behavior, lysozyme solubility, crystal size and morphology. This work aims to link this pH dependent impact to a protein-specific parameter, the conformational stability of lysozyme. At pH 3 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1M glycine stabilized or destabilized lysozymes' native conformation resulting in a modified size of the crystallization area without influencing lysozyme solubility, crystal size and morphology. Addition of 1M glycine even promoted non-native aggregation at pH 3 whereas addition of PEG 1000 completely inhibited non-native aggregation. At pH 5 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1M glycine did not influence lysozymes' native conformation, but strongly influenced the position of the crystallization area, lysozyme solubility, crystal size and morphology. The observed pH dependent impact of the additives could be linked to a differing lysozyme conformational stability in the binary systems without additives at pH 3 and pH 5. However, in any case lysozyme phase behavior could selectively be manipulated by addition of glycerol, PEG 1000 and glycine. Furthermore, at pH 5 crystal size and morphology could selectively be manipulated. PMID:26302861

  15. Genetic Recombination in Bacteriophage ?X174

    PubMed Central

    Benbow, Robert M.; Zuccarelli, Anthony J.; Davis, Gloria C.; Sinsheimer, Robert L.

    1974-01-01

    Genetic recombination in bacteriophage ?X174 usually takes place early in the infection process and involves two parental replicative form (double-stranded) DNA molecules. The host recA protein is required; none of the nine known ?X174 cistron products is essential. The products of a single recombination event are nonreciprocal and asymmetric. Typically, only one of the parental genotypes and one recombinant genotype are recovered from a single cell. An alternative, less efficient recombination mechanism which requires an active ?X174 cistron A protein is observed in the absence of the host recA gene product. PMID:4595301

  16. The amino acid sequence of Lady Amherst's pheasant (Chrysolophus amherstiae) and golden pheasant (Chrysolophus pictus) egg-white lysozymes.

    PubMed

    Araki, T; Kuramoto, M; Torikata, T

    1990-09-01

    The amino acids of Lady Amherst's pheasant and golden pheasant egg-white lysozymes have been sequenced. The carboxymethylated lysozymes were digested with trypsin followed by sequencing of the tryptic peptides. Lady Amherst's pheasant lysozyme proved to consist of 129 amino acid residues, and a relative molecular mass of 14,423 Da was calculated. This lysozyme had 6 amino acids substitutions when compared with hen egg-white lysozyme: Phe3 to Tyr, His15 to Leu, Gln41 to His, Asn77 to His, Gln 121 to Asn, and a newly found substitution of Ile124 to Thr. The amino acid sequence of golden pheasant lysozyme was identical to that of Lady Amherst's phesant lysozyme. The phylogenetic tree constructured by the comparison of amino acid sequences of phasianoid birds lysozymes revealed a minimum genetic distance between these pheasants and the turkey-peafowl group. PMID:1368578

  17. Bacteriophage-Mediated Dispersal of Campylobacter jejuni Biofilms ?

    PubMed Central

    Siringan, Patcharin; Connerton, Phillippa L.; Payne, Robert J. H.; Connerton, Ian F.

    2011-01-01

    Bacteria in their natural environments frequently exist as mixed surface-associated communities, protected by extracellular material, termed biofilms. Biofilms formed by the human pathogen Campylobacter jejuni may arise in the gastrointestinal tract of animals but also in water pipes and other industrial situations, leading to their possible transmission into the human food chain either directly or via farm animals. Bacteriophages are natural predators of bacteria that usually kill their prey by cell lysis and have potential application for the biocontrol and dispersal of target bacteria in biofilms. The effects of virulent Campylobacter specific-bacteriophages CP8 and CP30 on C. jejuni biofilms formed on glass by strains NCTC 11168 and PT14 at 37°C under microaerobic conditions were investigated. Independent bacteriophage treatments (n ? 3) led to 1 to 3 log10 CFU/cm2 reductions in the viable count 24 h postinfection compared with control levels. In contrast, bacteriophages applied under these conditions effected a reduction of less than 1 log10 CFU/ml in planktonic cells. Resistance to bacteriophage in bacteria surviving bacteriophage treatment of C. jejuni NCTC 11168 biofilms was 84% and 90% for CP8 and CP30, respectively, whereas bacteriophage resistance was not found in similarly recovered C. jejuni PT14 cells. Dispersal of the biofilm matrix by bacteriophage was demonstrated by crystal violet staining and transmission electron microscopy. Bacteriophage may play an important role in the control of attachment and biofilm formation by Campylobacter in situations where biofilms occur in nature, and they have the potential for application in industrial situations leading to improvements in food safety. PMID:21441325

  18. T4 DNA condensation in water-alcohol media

    E-print Network

    M. O. Gallyamov; O. A. Pyshkina; V. G. Sergeyev; I. V. Yaminsky

    2011-07-21

    The process of compaction of high molecular weight DNA T4 is investigated directly in a AFM liquid cell. The AFM-images of globules formed by DNA molecules in the result of compaction in water-alcohol environments at high izopropanol concentration (80%) are received; it is found that at intermediate concentration of izopropanol (40-50%) the DNA molecules form partially compacted formations in which the separate coils of macromolecules twist in toroidal structures. It is shown using the technique of deconvolution of the AFM-images that the globule include only one closely packed DNA molecule. The model of DNA packing is proposed on the basis of AFM experiment.

  19. Lysozyme as an alternative to antibiotics improves performance in nursery pigs during an indirect immune challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this study was to determine the effect of lysozyme and antibiotics on growth performance and immune response during an indirect immune challenge. Two replicates of 600 pigs each were weaned from the sow at 2...

  20. Osmotic Pressure in Escherichia coli as Rendered Detectable by Lysozyme Attack

    PubMed Central

    Scheie, Paul

    1973-01-01

    The enhanced susceptibility of plasmolyzed Escherichia coli to lysozyme attack was used to estimate the internal osmotic pressure of these cells under various conditions. Differences were detected between strains, culture media, stages in the growth cycle, and the osmotically active material used to produce plasmolysis. Lysozyme also was found to attack unplasmolyzed cells at 0 C and between 50 and 70 C. PMID:4574692

  1. Thermodynamic Exploration of Eosin-Lysozyme Binding: A Physical Chemistry and Biochemistry Laboratory Experiment

    ERIC Educational Resources Information Center

    Huisman, Andrew J.; Hartsell, Lydia R.; Krueger, Brent P.; Pikaart, Michael J.

    2010-01-01

    We developed a modular pair of experiments for use in the undergraduate physical chemistry and biochemistry laboratories. Both experiments examine the thermodynamics of the binding of a small molecule, eosin Y, to the protein lysozyme. The assay for binding is the quenching of lysozyme fluorescence by eosin through resonant energy transfer. In…

  2. [The lysozyme-antilysozyme functional system in hydrobionts and its role in forming aqueous biocenoses].

    PubMed

    Solovykh, G N; Nemtseva, N V

    1994-01-01

    Lysozyme activity has been evaluated in hydrobionts of different trophic levels: algae, zooplankton and germs. The existence of the functional system "Lysozyme of hydrobionts--antilysozyme of germs", functioning as a regulating element in the formation of aqueous microbiocenosis, has been substantiated on the basis of data provided by laboratory experiments and investigations carried out in natural water pools. PMID:7856360

  3. Isothermal Titration Calorimetry and Macromolecular Visualization for the Interaction of Lysozyme and Its Inhibitors

    ERIC Educational Resources Information Center

    Wei, Chin-Chuan; Jensen, Drake; Boyle, Tiffany; O'Brien, Leah C.; De Meo, Cristina; Shabestary, Nahid; Eder, Douglas J.

    2015-01-01

    To provide a research-like experience to upper-division undergraduate students in a biochemistry teaching laboratory, isothermal titration calorimetry (ITC) is employed to determine the binding constants of lysozyme and its inhibitors, N-acetyl glucosamine trimer (NAG[subscript 3]) and monomer (NAG). The extremely weak binding of lysozyme/NAG is…

  4. Genetic control of the humoral immune response to avian egg white lysozymes in the chicken

    SciTech Connect

    Flanagan, M.P.

    1987-01-01

    Chickens from two closely related sublines, GHs-B6 and GHs-B13, differing serologically at the major histocompatibility complex, were significantly different in their humoral response to three avian egg white lysozymes. Specific antisera levels were measured by radioimmunoassay using /sup 125/I-labeled lysozymes. Antibodies elicited in response to these lysozymes are assumed to be directed against sites on these lysozymes where their amino acid sequence differs from that of the recipient G. domesticus egg white lysozyme (HEL). GHs-B6 birds produced a high level of antibody in response to immunization of turkey (TEL), pheasant (PhL) and guinea hen (GHL) lysozymes. GHs-B13 birds produced no detectable antibody to TEL, were intermediate in their response to PhL and equaled the antibody production of GHs-B6 birds in response to GHL. Antisera to each lysozyme were examined for crossreactivity with all other lysozymes by use of a competitive binding assay.

  5. Midgut lysozymes of Lucilia sericata - new antimicrobials involved in maggot debridement therapy.

    PubMed

    Valachova, I; Takac, P; Majtan, J

    2014-12-01

    Larvae of Lucilia sericata are used for maggot debridement therapy (MDT) because of their ability to remove necrotic tissue and eradicate bacterial pathogens of infected wounds. So far, very few antibacterial factors have been fully characterized (eg lucifensin). Using a molecular approach, some other putative antimicrobial compounds, including three novel lysozymes, have been previously identified and predicted to be involved in MDT. Nevertheless, data on lysozymes tissue origin and their functions have never been elucidated. Therefore, the aim of this study was to investigate the expression of three lysozymes in L.?sericata and confirm their antibacterial effects within MDT. Moreover, we characterized the eradication process of bacteria within the digestive system of maggots and determined the role of lysozymes in this process. We found that three lysozymes are expressed in specific sections of the L.?sericata midgut. Recombinant lysozymes displayed comparable antibacterial activity against Micrococcus luteus. Furthermore, the majority of Gram-positive bacteria were destroyed in vivo within the particular section of the L.?sericata midgut where lysozymes are produced. Larval ingestion and subsequent eradication of wound pathogens during their passage through the intestine of maggots are due to, at least in part, antibacterial action of three midgut lysozymes. PMID:25098233

  6. Lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology in nursery pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this experiment was to determine if lysozyme in nursery diets improved growth performance and gastrointestinal health of pigs weaned from the sow at 24 d of age. Two replicates of 96 pigs (192 total 96 males,...

  7. Collective Dynamics of Lysozyme in Water: Terahertz Absorption Spectroscopy and Comparison with Theory

    E-print Network

    Xu, Jing

    confirm the existence of dense, overlapping normal modes in the terahertz frequency range. Our observed reported regarding the low-frequency normal modes of the monomeric protein hen egg white lysozyme, inviting Materials and Solution Preparation. For this study, we employed chicken hen egg white lysozyme (Sigma, St

  8. Effect of secondary structure on the interactions of peptide T4 LYS (11-36) in mixtures of aqueous sodium chloride and 2,2,2,-Trifluoroethanol

    SciTech Connect

    Anderson, Camille O.; Spiegelberg, Susanne; Prausnitz, John M.; Blanch, Harvey W.

    2001-10-01

    The potential of mean force for protein-protein interactions is key to the development of a statistical-mechanical model for salt-induced protein precipitation and crystallization, and for understanding certain disease states, including cataract formation and {beta}-amyloid pathology in Alzheimer's disease. Fluorescence anisotropy provides a method for quantitative characterization of intermolecular interactions due to reversible association. Monomer-dimer equilibria for the peptide T4 LYS(11-36) were studied by fluorescence anisotropy. This peptide, derived from the {beta}-sheet region of the T4 lysozyme molecule, has the potential to form amyloid fibrils. 2,2,2-trifluoroethanol (TFE) induces a change in peptide secondary structure, and was used in aqueous solutions at concentrations from 0 to 50% (v/v) at 25 and 37 C to examine the role of peptide conformation on peptide-peptide interactions. The association constant for dimerization increased with rising TFE concentration and with falling temperature. The peptide-peptide potential of mean force was computed from these association constants. Circular-dichroism measurements showed that the secondary structure of the peptide plays an important role in these strong attractive interactions due to intermolecular hydrogen-bond formation and hydrophobic interactions.

  9. Bacteriophages and Their Role in Food Safety

    PubMed Central

    Sillankorva, Sanna M.; Oliveira, Hugo; Azeredo, Joana

    2012-01-01

    The interest for natural antimicrobial compounds has increased due to alterations in consumer positions towards the use of chemical preservatives in foodstuff and food processing surfaces. Bacteriophages fit in the class of natural antimicrobial and their effectiveness in controlling bacterial pathogens in agro-food industry has led to the development of different phage products already approved by USFDA and USDA. The majority of these products are to be used in farm animals or animal products such as carcasses, meats and also in agricultural and horticultural products. Treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases and ultimately promote safe environments in animal and plant food production, processing, and handling. This is an overview of recent work carried out with phages as tools to promote food safety, starting with a general introduction describing the prevalence of foodborne pathogens and bacteriophages and a more detailed discussion on the use of phage therapy to prevent and treat experimentally induced infections of animals against the most common foodborne pathogens, the use of phages as biocontrol agents in foods, and also their use as biosanitizers of food contact surfaces. PMID:23316235

  10. Bacteriophage Mu sites required for transposition immunity.

    PubMed Central

    Darzins, A; Kent, N E; Buckwalter, M S; Casadaban, M J

    1988-01-01

    Plasmids with bacteriophage Mu sequences receive additional Mu insertions 20-700 times less frequently than plasmids without Mu sequences. The Mu sites required for this transposition immunity were mapped near each end, either of which was sufficient. The left site was between 127 and 203 base pairs from the left end, and the right site was between 22 and 93 base pairs from the right end. These sequences include the innermost but not the outermost of the three binding sites for the Mu A transposition protein at each end of Mu. Transposition immunity was cis-acting and independent of its location on a target plasmid. An additional copy of an immunity site reduced transposition a factor of 10 further. Transposition immunity was seen both during full phage lytic growth, with all the bacteriophage Mu genes, and during normal cellular growth, with a mini-Mu element containing only the Mu c and ner regulatory and A and B transposition genes. PMID:2842794

  11. Making temporal maps using bacterial luciferase: Bacteriophage

    NASA Astrophysics Data System (ADS)

    Kuhn, Jonathan; Broza, Rachel; Verkin, Ekaterina

    2004-06-01

    A method for making temporal maps in bacteria, plasmids and bacteriophages is described. A cassette containing both the genes for bacterial luciferase and kanamycin resistance can be introduced at precise sites. The technique involves clonging followed by genetic recombination. The result is formation of structures that have the luciferase genes in place of the normal DNA and this allows the very precise measurement of transcription/translation of the substituted regions. Very low levels of transcription as well as the kinetics of induction can be easily ascertained. As a specific demonstration of this general method, the technique was used with bacteriophage ?, one of the best known organisms. By measuring light emission, the expression of luciferase was followed after induction for both early and late genes. The exact timing of initial expression of genes was also determined by sampling at very short intervals. The results show that the early genes express almost without delay implying that the function of the N antitermination system is not temporal regulation.

  12. Peering Down the Barrel of a Bacteriophage Portal: The Genome Packaging and Release Valve in P22

    E-print Network

    Baker, Timothy S.

    Structure Article Peering Down the Barrel of a Bacteriophage Portal: The Genome Packaging bacteriophages is packaged to liquid crystalline density through a unique vertex in the procapsid assembly. INTRODUCTION Bacteriophages provide exceptionally tractable model systems to understand the fundamental

  13. Spectroscopic investigations on the interactions of AgTiO2 nanoparticles with lysozyme and its influence on the binding of lysozyme with drug molecule

    NASA Astrophysics Data System (ADS)

    Revathi, R.; Rameshkumar, A.; Sivasudha, T.

    2016-01-01

    Binding of lysozyme with AgTiO2 nanoparticles was analyzed by using absorption, fluorescence, time resolved and synchronous fluorescence measurements. In the presence of AgTiO2 nanoparticles, the fluorescence intensity of lysozyme was decreased. Static type of binding was confirmed through lifetime and ground state absorption measurements. From the fluorescence quenching data, the binding constant and the number of binding sites were found to be 1.5 × 104 M-1 and 1.03, respectively. From the synchronous fluorescence spectroscopic measurements, tryptophan residue in lysozyme was found to have interaction with the nanoparticles. Further, the influence of AgTiO2 nanoparticles on the binding strength of lysozyme with a drug molecule was analyzed through fluorescence quenching methods. The presence of nanoparticles decreases the binding capability of drug with protein. Overall, the observed results will provide basic insights on the utilization of nanoparticles in drug delivery applications.

  14. Spectroscopic investigations on the interactions of AgTiO2 nanoparticles with lysozyme and its influence on the binding of lysozyme with drug molecule.

    PubMed

    Revathi, R; Rameshkumar, A; Sivasudha, T

    2016-01-01

    Binding of lysozyme with AgTiO2 nanoparticles was analyzed by using absorption, fluorescence, time resolved and synchronous fluorescence measurements. In the presence of AgTiO2 nanoparticles, the fluorescence intensity of lysozyme was decreased. Static type of binding was confirmed through lifetime and ground state absorption measurements. From the fluorescence quenching data, the binding constant and the number of binding sites were found to be 1.5×10(4)M(-1) and 1.03, respectively. From the synchronous fluorescence spectroscopic measurements, tryptophan residue in lysozyme was found to have interaction with the nanoparticles. Further, the influence of AgTiO2 nanoparticles on the binding strength of lysozyme with a drug molecule was analyzed through fluorescence quenching methods. The presence of nanoparticles decreases the binding capability of drug with protein. Overall, the observed results will provide basic insights on the utilization of nanoparticles in drug delivery applications. PMID:26210014

  15. Structural Basis of Protein Oxidation Resistance: A Lysozyme Study

    PubMed Central

    Girod, Marion; Enjalbert, Quentin; Brunet, Claire; Antoine, Rodolphe; Lemoine, Jérôme; Lukac, Iva; Radman, Miroslav; Krisko, Anita; Dugourd, Philippe

    2014-01-01

    Accumulation of oxidative damage in proteins correlates with aging since it can cause irreversible and progressive degeneration of almost all cellular functions. Apparently, native protein structures have evolved intrinsic resistance to oxidation since perfectly folded proteins are, by large most robust. Here we explore the structural basis of protein resistance to radiation-induced oxidation using chicken egg white lysozyme in the native and misfolded form. We study the differential resistance to oxidative damage of six different parts of native and misfolded lysozyme by a targeted tandem/mass spectrometry approach of its tryptic fragments. The decay of the amount of each lysozyme fragment with increasing radiation dose is found to be a two steps process, characterized by a double exponential evolution of their amounts: the first one can be largely attributed to oxidation of specific amino acids, while the second one corresponds to further degradation of the protein. By correlating these results to the structural parameters computed from molecular dynamics (MD) simulations, we find the protein parts with increased root-mean-square deviation (RMSD) to be more susceptible to modifications. In addition, involvement of amino acid side-chains in hydrogen bonds has a protective effect against oxidation Increased exposure to solvent of individual amino acid side chains correlates with high susceptibility to oxidative and other modifications like side chain fragmentation. Generally, while none of the structural parameters alone can account for the fate of peptides during radiation, together they provide an insight into the relationship between protein structure and susceptibility to oxidation. PMID:24999730

  16. Lysozyme net charge and ion binding in concentrated aqueous electrolyte solutions

    SciTech Connect

    Kuehner, Daniel E.; Engmann, Jan; Fergg, Florian; Wernick, Meredith; Blanch, Harvey W.; Prausnitz, John M.

    1999-02-01

    Hydrogen-ion titrations were conducted for hen-egg-white lysozyme in solutions of potassium chloride over the range pH 2.5--11.5 and for ionic strengths to 2.0 M. The dependence of lysozyme`s net proton charge, z{sub p}, on pH and ionic strength in potassium chloride solution is measured. From the ionic-strength dependence of z{sub p}, interactions of lysozyme with potassium and chloride ions are calculated using the molecular-thermodynamic theory of Fraaije and Lyklema. Lysozyme interacts preferentially with up to 12 chloride ions at pH 2.5. The observed dependence of ion-protein interactions on pH and ionic strength is explained in terms of electric-double-layer theory. New experimental pK{sub a} data are reported for 11 amino acids in potassium chloride solutions of ionic strength to 3.0 M.

  17. Interaction of fullerenol with lysozyme investigated by experimental and computational approaches.

    PubMed

    Yang, Sheng-Tao; Wang, Haifang; Guo, Lin; Gao, Yang; Liu, Yuanfang; Cao, Aoneng

    2008-10-01

    The potential biomedical applications of fullerenol C(60)(OH)(x) (x?24) have been extensively studied. However, the structural information of the interaction of fullerenol with the bio-system at the molecular level, which is essential for understanding its bioactivity and toxicity, is still missing. In this study, lysozyme was selected as a model protein to investigate the interaction between fullerenol and biomolecules. A strong induced circular dichroism (CD) signal of achiral fullerenol was observed after binding with lysozyme. Activity assay shows that lysozyme activity is inhibited significantly by fullerenol. No heat capacity difference between the folded and unfolded states of lysozyme was measured by differential scanning calorimetry (DSC) in the presence of fullerenol, indicating that fullerenol prefers to bind with the hydrophobic residues. Both experimental and Autodock computational results suggest that the binding site on lysozyme for fullerenol is close to Trp 62, and a ?-? stacking interaction might play an important role in binding. PMID:21832583

  18. The amino acid sequence of monal pheasant lysozyme and its activity.

    PubMed

    Araki, T; Matsumoto, T; Torikata, T

    1998-10-01

    The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly). PMID:9836434

  19. [Gottfried Ewald and the "operation t4" in Göttingen].

    PubMed

    Beyer, C

    2013-09-01

    Gottfried Ewald (1888-1963) had been director of the State Hospital and Nursing Home and the University Clinic for Psychiatry from 1934. In August 1940, he refused his cooperation as a medical expert in the National Socialist's "euthanasia" operation during a discussion of the "Reich Cooperative for State Hospitals and Nursing Homes" (Reichsarbeitsgemeinschaft Heil- und Pflegeanstalten) in Berlin. Shortly afterwards Ewald wrote a comprehensive position paper against the operation which was sent to Werner Heyde, head of the "T4" medical office, and Leonardo Conti, "Reich physician leader" (Reichsärzteführer), among others.While Ewald's protest remained unsuccessful, it did neither result in any disciplinary consequences. By his own account, he decided to remain in his position on order to be able to rescue at least some of the patients of the State Hospital and Nursing Home destined for transport to the "T4" killing centres. In cooperation with colleagues at the hospital and the Provincial Association in Hanover, he partly succeeded to meet this aim through deferrals, leaves of absence, re-assessments and releases. These strategies were, however, not used to prevent the deportation of Jewish and compulsory detention patients. Thus, Ewald's protest was a partial, pragmatic circumvention of the National Socialist's "euthanasia" operation. PMID:23893259

  20. Incorporation of impurity to a tetragonal lysozyme crystal

    NASA Astrophysics Data System (ADS)

    Kurihara, Kazuo; Miyashita, Satoru; Sazaki, Gen; Nakada, Toshitaka; Durbin, Stephen D.; Komatsu, Hiroshi; Ohba, Tetsuhiko; Ohki, Kazuo

    1999-01-01

    Concentration of a phosphor-labeled impurity (ovalbumin) incorporated into protein (hen egg white lysozyme) crystals during growth was measured by fluorescence.This technique enabled us to measure the local impurity concentration in a crystal quantitatively. Impurity concentration increased with growth rate, which could not be explained by two conventional models (equilibrium adsorption model and Burton-Prim-Slichter model); a modified model is proposed. Impurity concentration also increased with the pH of the solution. This result is discussed considering the electrostatic interaction between the impurity and the crystallizing species.

  1. Control of solvent evaporation in hen egg white lysozyme crystallization

    NASA Technical Reports Server (NTRS)

    Wilson, L. J.; Suddath, F. L.

    1992-01-01

    An investigation of the role of solvent evaporation in tetragonal lysozyme crystallization was preformed with a device that employs N2(g) to control the evaporation of solvent from a micro-volume crystallization hanging drop. The number of crystals was found to vary with the rate at which the final supersaturation level was achieved. It was found that the more rapid the approach to supersaturation the larger the number of crystals. Accordingly, the crystals reached a smaller terminal size. Elongation of the (110) face parallel to the four-fold axis was observed with the slower evaporation rates.

  2. Polar solvation dynamics of lysozyme from molecular dynamics studies

    NASA Astrophysics Data System (ADS)

    Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy

    2012-05-01

    The solvation dynamics of a protein are believed to be sensitive to its secondary structures. We have explored such sensitivity in this article by performing room temperature molecular dynamics simulation of an aqueous solution of lysozyme. Nonuniform long-time relaxation patterns of the solvation time correlation function for different segments of the protein have been observed. It is found that relatively slower long-time solvation components of the ?-helices and ?-sheets of the protein are correlated with lower exposure of their polar probe residues to bulk solvent and hence stronger interactions with the dynamically restricted surface water molecules. These findings can be verified by appropriate experimental studies.

  3. Sinorhizobium meliloti Phage ?M9 Defines a New Group of T4 Superfamily Phages with Unusual Genomic Features but a Common T=16 Capsid

    PubMed Central

    Johnson, Matthew C.; Tatum, Kelsey B.; Lynn, Jason S.; Brewer, Tess E.; Lu, Stephen; Washburn, Brian K.

    2015-01-01

    ABSTRACT Relatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of the Sinorhizobium meliloti-infecting T4 superfamily phage ?M9. This phage and its close relative Rhizobium phage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-like S. meliloti phages of the ?M12 group. Structurally, ?M9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the ?M9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. ?M9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, ?M9 and S. meliloti phage ?M12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide. IMPORTANCE Despite recent advances in the phylogenetic and structural characterization of bacteriophages, only a small number of phages of plant-symbiotic nitrogen-fixing soil bacteria have been studied at the molecular level. The effects of phage predation upon beneficial bacteria that promote plant growth remain poorly characterized. First steps in understanding these soil bacterium-phage dynamics are genetic, molecular, and structural characterizations of these groups of phages. The T4 superfamily phages are among the most complex phages; they have large genomes packaged within an icosahedral head and a long, contractile tail through which the DNA is delivered to host cells. This phylogenetic and structural study of S. meliloti-infecting T4 superfamily phage ?M9 provides new insight into the diversity of this family. The comparison of structure-related genes in both ?M9 and S. meliloti-infecting T4 superfamily phage ?M12, which comes from a completely different lineage of these phages, allows the identification of host infection-related factors. PMID:26311868

  4. DYNAMIC INTERACTIONS OF PSEUDOMONAS AERUGINOSA AND BACTERIOPHAGES IN LAKE WATER

    EPA Science Inventory

    The persistence and interaction between newly isolated strains of Pseudomonas aeruginosa and resident bacteriophages indigenous to a freshwater environment was monitored over 45 days in lake water microcosms. he interaction between susceptible and resistant bacteria with pure pha...

  5. Bacteriophage ms2 l protein: genetic and biochemical characterization 

    E-print Network

    McIntosh, Brenley Kathleen

    2009-05-15

    In order to release progeny, bacteriophages must lyse the host cell by compromising the peptidoglycan layer. There are two known strategies of lysis: the holin-endolysin system and single gene lysis (SGL), which are dependent ...

  6. Antimicrobial bacteriophage-derived proteins and therapeutic applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibiotics have the remarkable power to control bacterial infections. Unfortunately, widespread use, whether regarded as prudent or not, has favored the emergence and persistence of antibiotic resistant strains of human pathogenic bacteria, resulting in a global health threat. Bacteriophages (pha...

  7. Genetic engineering of bacteriophage and its applications for biomimetic materials

    E-print Network

    Lee, Soo-Kwan

    2006-01-01

    Filamentous bacteriophage (M13) are excellent biological build block due to their multiple peptide display system including type 8 (complete peptide display at pVIII) and type 83 (complete peptide display at both pVIII and ...

  8. Genome Sequences of Three Novel Bacillus cereus Bacteriophages.

    PubMed

    Grose, Julianne H; Jensen, Jordan D; Merrill, Bryan D; Fisher, Joshua N B; Burnett, Sandra H; Breakwell, Donald P

    2014-01-01

    The Bacillus cereus group is an assemblage of highly related firmicute bacteria that cause a variety of diseases in animals, including insects and humans. We announce three high-quality, complete genome sequences of bacteriophages we isolated from soil samples taken at the bases of fruit trees in Utah County, Utah. While two of the phages (Shanette and JL) are highly related myoviruses, the bacteriophage Basilisk is a siphovirus. PMID:24459255

  9. Bacteriophage-based nanoprobes for rapid bacteria separation

    NASA Astrophysics Data System (ADS)

    Chen, Juhong; Duncan, Bradley; Wang, Ziyuan; Wang, Li-Sheng; Rotello, Vincent M.; Nugen, Sam R.

    2015-10-01

    The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying concentrations were determined. The results indicated a similar bacteria capture efficiency between the two nanoprobes.The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying concentrations were determined. The results indicated a similar bacteria capture efficiency between the two nanoprobes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr03779d

  10. Preliminary investigations into solutal flow about growing tetragonal lysozyme crystals

    NASA Technical Reports Server (NTRS)

    Pusey, Marc; Witherow, William; Naumann, Robert

    1988-01-01

    A series of preliminary experiments were done to investigate solutal flow about growing lysozyme crystals and its effects. Density-gradient-driven flow was observed using a schlieren optical system. Crystals used ranged from 0.3 to 1.72 mm across the (110) face, and protein concentrations were from 3.7 to 23.7 mg/ml. The convective plume velocities were found to be from 10 to 50 microns/s, which correlated with those predicted to occur based upon a diffusive-convective model. When microcrystals of lysozyme, less than 20 microns across the (110) face were subjected to directed solution flows, the growth rate was found to rapidly decrease over the 8-20 h course of the experiment. Solution flow rates used ranged from 18 to 40 microns/s, and protein concentrations were from 7.3 to 11.7 mg/ml, conditions typical of larger (greater than 0.5 mm) crystals in the terminal phases of a typical crystal growth procedure.

  11. Predicting Tensile Stretchability of Trimmed AA6111-T4 Sheets

    SciTech Connect

    Hu, Xiaohua; Sun, Xin; Golovashchenko, Sergey F.

    2014-02-15

    An integrated manufacturing process simulation framework has been developed to predict the trimmed edge tensile stretchability of AA6111-T4 sheets by incorporating the burr geometry, damage, and plastic strain from trimming simulations into subsequent tensile stretchability simulations. The influence of the trimming die clearances on the predicted tensile stretching ductility (stretchability) is studied and quantitatively compared with experimental measurements. Stretchability is found to decrease with increasing cutting clearances, and simulation results have successfully captured experimentally observed edge crack initiation and failure mode variations for different trimming clearances. Subsequent computational sensitivity studies reveal that while deburring of previously trimmed edges has little influence on tensile stretchability, removal of trimmed edge initial plastic strain may significantly enhance the subsequent trimmed edge stretchability.

  12. HyShot-T4 Supersonic Combustion Experiments

    NASA Technical Reports Server (NTRS)

    Paull, A.; Frost, M.; Alesi, H.

    2000-01-01

    A series of experiments were initiated to investigate the operation of a two-dimensional, hypersonic, airbreathing engine (scramjet) inclined at angles of attack to the freestream. The experiments were undertaken to obtain data for use in the Hyshot flight test program. Experiments on the Hyshot scramjet were under taken in the T4 shock tunnel. Experiments were made at a nominal total enthalpy of 3.0MJkg (exp -1) using a nozzle that produced flows with a Mach number of approximately 6.5. The conditions produced correspond to flight at Mach 7.6 at an altitude range of 35.7-21.4km. A summary of the flow conditions is included. The scramjet was tested at 0, plus 2, plus 4, minus 2 and minus 4 degrees angle of attack. Experiments were also undertaken at 2 and 4 degrees angle of skew.

  13. Antimicrobial drug discovery through bacteriophage genomics.

    PubMed

    Liu, Jing; Dehbi, Mohammed; Moeck, Greg; Arhin, Francis; Bauda, Pascale; Bergeron, Dominique; Callejo, Mario; Ferretti, Vincent; Ha, Nhuan; Kwan, Tony; McCarty, John; Srikumar, Ramakrishnan; Williams, Dan; Wu, Jinzi J; Gros, Philippe; Pelletier, Jerry; DuBow, Michael

    2004-02-01

    Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases. PMID:14716317

  14. Escherichia coli Capsule Bacteriophages II. Morphology

    PubMed Central

    Stirm, Stephan; Freund-Mölbert, Elisabeth

    1971-01-01

    The Escherichia coli capsule bacteriophages (K phages) described herein are specific for certain capsular strains of E. coli, all of them test strains for different E. coli K antigens. The phages are not adsorbed to the acapsular mutants of their host organisms nor to similar strains with serologically and chemically different capsular polysaccharides. Thirteen E. coli (and one Klebsiella) K phages were visualized in the electron microscope. Most viruses are similar to P22 and thus belong to Bradley group C; however, one each of group A (long, contractile tail) and group B (long, noncontractile tail) was also found. All K phages were seen to carry spikes but no tail fibers were detected. These results suggest that the structures responsible for the recognition of the thick (about 400 nm or more) capsular polysaccharide gels are located in these spikes. Images PMID:4107543

  15. A Hypothesis for Bacteriophage DNA Packaging Motors

    PubMed Central

    Serwer, Philip

    2010-01-01

    The hypothesis is presented that bacteriophage DNA packaging motors have a cycle comprised of bind/release thermal ratcheting with release-associated DNA pushing via ATP-dependent protein folding. The proposed protein folding occurs in crystallographically observed peptide segments that project into an axial channel of a protein 12-mer (connector) that serves, together with a coaxial ATPase multimer, as the entry portal. The proposed cycle begins when reverse thermal motion causes the connector’s peptide segments to signal the ATPase multimer to bind both ATP and the DNA molecule, thereby producing a dwell phase recently demonstrated by single-molecule procedures. The connector-associated peptide segments activate by transfer of energy from ATP during the dwell. The proposed function of connector/ATPase symmetry mismatches is to reduce thermal noise-induced signaling errors. After a dwell, ATP is cleaved and the DNA molecule released. The activated peptide segments push the released DNA molecule, thereby producing a burst phase recently shown to consist of four mini-bursts. The constraint of four mini-bursts is met by proposing that each mini-burst occurs via pushing by three of the 12 subunits of the connector. If all four mini-bursts occur, the cycle repeats. If the mini-bursts are not completed, a second cycle is superimposed on the first cycle. The existence of the second cycle is based on data recently obtained with bacteriophage T3. When both cycles stall, energy is diverted to expose the DNA molecule to maturation cleavage. PMID:21994710

  16. Montmorillonite-induced Bacteriophage ?6 Disassembly

    NASA Astrophysics Data System (ADS)

    Trusiak, A.; Gottlieb, P.; Katz, A.; Alimova, A.; Steiner, J. C.; Block, K. A.

    2012-12-01

    It is estimated that there are 1031 virus particles on Earth making viruses an order of magnitude more prevalent in number than prokaryotes with the vast majority of viruses being bacteriophages. Clays are a major component of soils and aquatic sediments and can react with RNA, proteins and bacterial biofilms. The clays in soils serve as an important moderator between phage and their host bacteria, helping to preserve the evolutionary balance. Studies on the effects of clays on viral infectivity have given somewhat contradictory results; possibly a consequence of clay-virus interactions being dependent on the unique structure of particular viruses. In this work, the interaction between montmorillonite and the bacteriophage ?6 is investigated. ?6 is a member of the cystovirus family that infects Pseudomonas syringe, a common plant pathogen. As a member of the cystovirus family with an enveloped structure, ?6 serves as a model for reoviruses, a human pathogen. Experiments were conducted with ?6 suspended in dilute, purified homoionic commercial-grade montmorillonite over a range of virus:clay ratios. At a 1:100000 virus:clay ratio, the clay reduced viral infectivity by 99%. The minimum clay to virus ratio which results in a measurable reduction of P. syringae infection is 1:1. Electron microscopy demonstrates that mixed suspensions of smectite and virus co-aggregate to form flocs encompassing virions within the smectite. Both free viral particles as well as those imbedded in the flocs are seen in the micrographs to be missing the envelope- leaving only the nucleocapsid (NC) intact; indicating that smectite inactivates the virus by envelope disassembly. These results have strong implications in the evolution of both the ?6 virus and its P. syringae host cells. TEM of aggregate showing several disassembled NCs.

  17. Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

    PubMed Central

    Jiang, Ming Feng; Hu, Ming Jun; Ren, Hong Hui; Wang, Li

    2015-01-01

    Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZ?A and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity. PMID:26580446

  18. Role of Rabbit Lysozyme in In Vitro Serum and Plasma Serum Bactericidal Reactions Against Bacillus subtilis

    PubMed Central

    Carroll, Stephen F.; Martinez, Rafael J.

    1979-01-01

    The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of “purified ?-lysin,” fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified ?-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components. Images PMID:115789

  19. Lysozyme activity in earthworm (Lumbricus terrestris) coelomic fluid and coelomocytes: Enzyme assay for immunotoxicity of xenobiotics

    SciTech Connect

    Goven, A.J.; Chen, S.C.; Fitzpatrick, L.C. . Dept. of Biological Sciences); Venables, B.J. . Dept. of Biological Sciences TRAC Laboratories Inc., Denton, TX )

    1994-04-01

    Lysozyme activity in earthworm (Lumbricus terrestris) coelomic fluid and coelomocytes appears sufficiently sensitive for use as a nonmammalian biomarker to detect toxic effects of sublethal body burdens of Cu[sup 2+]. Lysozyme, a phylogenetically conserved enzyme, is capable of bactericidal activity via action on peptidoglycan of gram-positive bacterial cell walls and functions as a component of an organism's innate antimicrobial defense mechanism. Coelomic fluid and coelomocyte lysozyme activities, which exhibit temperature-response patterns similar to those of human saliva, plasma, serum and leukocyte extracts, were sensitive to Cu[sup 2+] exposure. Lysozyme activity of coelomic fluid and coelomocyte extracts from earthworms exposed for 5 d to CuSO[sub 4], using filter paper contact exposure, decreased with increasing sublethal Cu[sup 2+] concentrations of 0.05 and 0.1 [mu]g/cm[sup 2]. Compared to controls, coelomic fluid lysozyme activity was suppressed significantly at both exposure concentrations, whereas coelomocyte extract lysozyme activity was suppressed significantly at the 0.1-[mu]g/cm[sup 2] exposure concentration. Low inherent natural variability and sensitivity to sublethal Cu[sup 2+] body burdens indicate that lysozyme activity has potential as a biomarker for assaying immunotoxicity of metals.

  20. [Lysozyme of hydrobionts and its role in self-purification of water reservoirs].

    PubMed

    Solovykh, G N; Nefedova, E M; Ustinova, G M; Raimova, E K; Riabtseva, E A; Ushakova, E I

    2002-01-01

    Model experiments were carried out to study the effects of hydrobiont lysozyme on self-purifying processes of water reservoirs through its participation in the formation of a community of destructive microorganisms. Lysozyme-resistant forms have been found to lie at basis of bacterial cenosis of the Urals, among which there are lysozyme-active and anti-lysozyme-active ones, their population shows an inverse relationship. The exogenous lysozyme that enters the water reservoir in the period when there is a change in the dominant forms of hydrobionts affects the composition of bacterial cenosis by preserving anti-lysozyme-activity with a high activity (6-8 (g/ml), which causes a change in the microbial community of water reservoir, thus affecting the processes of its self-purification. The bacteria having ALA, which do maintain the capacity of a water reservoir for self-clearance from organic substance, persist under the action of endogenous lysozyme of phyto- and zooplankton among saprophytic microorganisms that are destructive agents in the biocenosis. PMID:12476826

  1. Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue.

    PubMed

    Jiang, Ming Feng; Hu, Ming Jun; Ren, Hong Hui; Wang, Li

    2015-12-01

    Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZ?A and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity. PMID:26580446

  2. Control of Electrostatic Interactions Between F-Actin And Genetically Modified Lysozyme in Aqueous Media

    SciTech Connect

    Sanders, L.K.; Xian, W.; Guaqueta, C.; Strohman, M.; Vrasich, C.R.; Luijten, E.; Wong, G.C.L.

    2009-06-04

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  3. Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

    SciTech Connect

    Sanders, Lori K.; Xian, Wujing; Guaqueta, Camilo; Strohman, Michael J.; Vrasich, Chuck R.; Luijten, Erik; Wong, Gerard C.L.

    2008-07-11

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  4. Evaluation of alternative host bacteria as vehicles for oral administration of bacteriophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Survival of bacteriophages through the upper gastrointestinal tract (UGIT) and persistence in the lower gastrointestinal tract (LGIT) is essential for treatment of enteric bacterial infections. We have hypothesized that non-pathogenic Alternative Host Bacteriophage (AHB), originally isolated from p...

  5. The Role of Solution Conditions in the Bacteriophage PP7 Capsid Charge Rikkert J. Nap,1

    E-print Network

    Podgornik, Rudolf

    Article The Role of Solution Conditions in the Bacteriophage PP7 Capsid Charge Regulation Rikkert J concentration on the charge regulation of the bacteriophage PP7 capsid. These effects are found to be extremely

  6. Versatility of M13 bacteriophage in medicine : vaccine storage and cancer diagnostics

    E-print Network

    Shi, Amy (Amy J.)

    2007-01-01

    Two novel ways of engineering the filamentous bacteriophage, M13, for the prevention, diagnosis, and treatment of human disease are proposed. Both ways are founded on the unique structural properties of the M13 bacteriophage ...

  7. Structural similarity of DNA-binding domains of bacteriophage repressors and the globin core

    E-print Network

    Levitt, Michael

    Structural similarity of DNA-binding domains of bacteriophage repressors and the globin core S of the bacteriophage repressor family is almost completely em- bedded in the larger eight-helix fold of the globin

  8. Bacteriophage-based synthetic biology for the study of infectious diseases

    E-print Network

    Citorik, Robert James

    Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic ...

  9. Interaction of T4 UvsW helicase and single-stranded DNA binding protein gp32 through its carboxy terminal acidic tail

    PubMed Central

    Perumal, Senthil K.; Nelson, Scott W.; Benkovic, Stephen J.

    2013-01-01

    Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA binding protein, gp32, is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions. PMID:23732982

  10. [Nature of tryptophan photooxidation products in lysozyme in the presence of methylene blue].

    PubMed

    Churakova, N I; Kravchenko, N A; Serebriakov, E P; Kaverzneva, E D

    1976-05-01

    One out of six trytophan residues in two lysozyme modification, obtained under lysozyme photooxidation in the presence of methylene blue, is found to be oxidized to N'-formylkinurenine (in one modification) and to kinurenine (in the other modification). The transition of one modification into another via detaching of N'-formyl group by soft acid hydrolysis has shown that one and the same tryptophan residue is oxidized in both products, Possible mechanism of tryptophan oxidation to the products mentioned is discu-sed on the basis of the hypothesis on signlet mechanism of lysozyme photooxidation in the presence of methylene blue. PMID:1024586

  11. Bacteriophage cocktail for biocontrol of Salmonella in dried pet food.

    PubMed

    Heyse, Serena; Hanna, Leigh Farris; Woolston, Joelle; Sulakvelidze, Alexander; Charbonneau, Duane

    2015-01-01

    Human salmonellosis has been associated with contaminated pet foods and treats. Therefore, there is interest in identifying novel approaches for reducing the risk of Salmonella contamination within pet food manufacturing environments. The use of lytic bacteriophages shows promise as a safe and effective way to mitigate Salmonella contamination in various food products. Bacteriophages are safe, natural, highly targeted antibacterial agents that specifically kill bacteria and can be targeted to kill food pathogens without affecting other microbiota. In this study, we show that a cocktail containing six bacteriophages had a broadspectrum activity in vitro against a library of 930 Salmonella enterica strains representing 44 known serovars. The cocktail was effective against 95% of the strains in this tested library. In liquid culture dose-ranging experiments, bacteriophage cocktail concentrations of ?10(8) PFU/ml inactivated more than 90% of the Salmonella population (10(1) to 10(3) CFU/ml). Dried pet food inoculated with a mixture containing equal proportions of Salmonella serovars Enteritidis (ATCC 4931), Montevideo (ATCC 8387), Senftenberg (ATCC 8400), and Typhimurium (ATCC 13311) and then surface treated with the six-bacteriophage cocktail (?2.5 ± 1.5 × 10(6) PFU/g) achieved a greater than 1-log (P < 0.001) reduction compared with the phosphate-buffered saline-treated control in measured viable Salmonella within 60 min. Moreover, this bacteriophage cocktail reduced natural contamination in samples taken from an undistributed lot of commercial dried dog food that tested positive for Salmonella. Our results indicate that bacteriophage biocontrol of S. enterica in dried pet food is technically feasible. PMID:25581183

  12. Fast Screening of Whole Blood Samples and Pharmaceutical Compounds for Enantiorecognition of Free L-T3 , L-T4 , and D-T4.

    PubMed

    Mitrofan, Grigorina; Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; van Staden, Jacobus Frederick; Bazylak, Grzegorz; Kapnissi-Christodoulou, Constantina P; Aboul-Enein, Hassan Y

    2015-12-01

    A fast screening method of whole blood was proposed for enantiorecognition of free L-T3 , L-T4 , and D-T4 . Stochastic microsensors based on four inulins (IN, IQ, TEX, and HD) immobilized on diamond paste (DP) were used for recognition of free L-T3 , L-T4 , and D-T4 . For the enantiorecognition of free L-T4 and D-T4 in whole blood and pharmaceutical samples, the best microsensor was the one based on TEX/DP (wide linear concentration ranges, and low limits of quantification). The best limit of detection for the assay of free L-T3 (400 fmol/L) was recorded using the microsensors based on HD/DP, while for the assay of free L-T4, and D-T4 the best limit of determination (1 pmol/L) was recorded using the TX/DP-based microsensor. For the enantiorecognition of free L-T3 in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification - of pmol/L magnitude order). For the enantiorecognition of free L-T3 in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification - of pmol/L magnitude order). Free L-T3 , L-T4 , and D-T4 were recovered with high reliabilities in whole blood samples (recoveries higher than 99.00%, with RSD values lower than 1.00%) and pharmaceutical samples (recoveries higher than 95.00% with RSD values lower than 1.00%). Chirality 27:973-978, 2015. © 2015 Wiley Periodicals, Inc. PMID:26447904

  13. APPLICATION OF DNA PROBES TO ANALYSIS OF BACTERIOPHAGE DISTRIBUTION PATTERNS IN THE ENVIRONMENT

    EPA Science Inventory

    Radiolabeled bacteriophage DNA probes have been used n this study to determine the distribution of Pseudomonas aeruginosa-infecteing bacteriophages in natural samples of lake water, sediment, soil, and sewage. he sensitivity of detection of bacteriophage with the DNA probes was b...

  14. Journal of Theoretical Biology 249 (2007) 411 421 Optimal bacteriophage mutation rates for phage therapy

    E-print Network

    Turner, Paul

    2007-01-01

    Journal of Theoretical Biology 249 (2007) 411 421 Optimal bacteriophage mutation rates for phage of bacteriophages offers a particular advantage in the treatment of bacterial infections not afforded by other; Bacteriophage; Host range 1. Introduction Phage therapy--the therapeutic application of bacterio- phage

  15. Cryoelectron-Microscopy Image Reconstruction of Symmetry Mismatches in Bacteriophage 29

    E-print Network

    Tao, Yizhi Jane

    Cryoelectron-Microscopy Image Reconstruction of Symmetry Mismatches in Bacteriophage 29 Marc C appendages. INTRODUCTION Double-stranded DNA (dsDNA) bacteriophages consist of a capsid, or head infection and through which DNA passes upon injection into the host cell. Bacteriophage tails typically

  16. Rapid MALDI-TOF MS analysis of bacteriophage major capsid proteins with -mercaptoethanol sample pretreatment

    E-print Network

    Rapid MALDI-TOF MS analysis of bacteriophage major capsid proteins with -mercaptoethanol sample Technology Laboratory; Colorado School of Mines, Golden CO 80401 Introduction: ·Bacteriophage (phage bacteriophage -A1122 (which is utilized for plague detection) were analyzed by combining 100µL of phage solution

  17. 40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato....

  18. Linking genetic change to community evolution: insights from studies of bacteria and bacteriophage

    E-print Network

    Bohannan, Brendan

    Linking genetic change to community evolution: insights from studies of bacteria and bacteriophage. In this article, we review recent studies of laboratory communities of bacteria and bacteriophage (viruses that infect bacteria). We focus on the ecology and evolution of bacteriophage-resistance as a case study

  19. 40 CFR 180.1307 - Bacteriophage of Clavibacter michiganensis subspecies michiganensis; exemption from the...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacteriophage of Clavibacter... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1307 Bacteriophage of... exemption from the requirement of a tolerance is established for residues of lytic bacteriophage...

  20. Ion-Dependent Dynamics of DNA Ejections for Bacteriophage l David Van Valen,6

    E-print Network

    Phillips, Rob

    Ion-Dependent Dynamics of DNA Ejections for Bacteriophage l David Wu,6 David Van Valen,6 Qicong Hu studied the control parameters that govern the dynamics of in vitro DNA ejection in bacteriophage l. Previous work demonstrated that bacteriophage DNA is highly pressurized, and this pressure has been

  1. Structural Studies of Bacteriophage a3 Assembly Ricardo A. Bernal1

    E-print Network

    Baker, Timothy S.

    Structural Studies of Bacteriophage a3 Assembly Ricardo A. Bernal1 , Susan Hafenstein2 , Norman H, Tucson AZ 85721, USA Bacteriophage a3 is a member of the Microviridae, a family of small, single Ltd. All rights reserved Keywords: bacteriophage a3; three-dimensional structure; procapsid

  2. Available online at www.sciencedirect.com The bacteriophage l CI protein finds an asymmetric solution

    E-print Network

    Higgins, Darren

    Available online at www.sciencedirect.com The bacteriophage l CI protein finds an asymmetric solution Ann Hochschild1 and Mitchell Lewis2 The CI protein of bacteriophage l (lCI) is both a repressor.12.008 Introduction Bacteriophage l provided one of the earliest paradigms for achieving a mechanistic understanding

  3. Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus

    E-print Network

    Ely, Bert

    Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus propagation. cryo-electron tomography | alpha-proteobacteria Many bacteriophages target host appendages-electron microscopy and image reconstruction (19, 20). In several land- mark studies, it was the first bacteriophage

  4. 40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato....

  5. The NMRRosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope

    E-print Network

    Baker, David

    The NMR­Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope a structural model for the capsid of intact M13 bacteriophage us- ing Rosetta model building guided-molecular-weight molecular assemblies. solid-state NMR | magic-angle spinning | filamentous bacteriophage | structure

  6. POPULATION BIOLOGY/GENETICS No Evidence for Bacteriophage WO orf7 Correlation with Wolbachia-

    E-print Network

    Dobson, Stephen L.

    POPULATION BIOLOGY/GENETICS No Evidence for Bacteriophage WO orf7 Correlation with WolbachiaÞed variation in the orf7 locus of the Wolbachia-associated bacteriophage WO. Here, crosses between eight Culex of bacteriophage WO in determining CI in Culex. Although crossing results show examples of compatibility, partial

  7. DNA Packaging in Bacteriophage: Is Twist Important? Andrew James Spakowitz and Zhen-Gang Wang

    E-print Network

    Spakowitz, Andrew J.

    DNA Packaging in Bacteriophage: Is Twist Important? Andrew James Spakowitz and Zhen-Gang Wang 91125 ABSTRACT We study the packaging of DNA into a bacteriophage capsid using computer simulation ejection of the polymer chain in the absence of external forces. INTRODUCTION The ability of bacteriophage

  8. Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division

    E-print Network

    Erickson, Harold P.

    Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division Ruth for review July 30, 2013) Bacteriophages take over host resources primarily via the activity of proteins that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division in

  9. Complete Bacteriophage Transfer in a Bacterial Endosymbiont (Wolbachia) Determined by Targeted

    E-print Network

    Bordenstein, Seth

    Complete Bacteriophage Transfer in a Bacterial Endosymbiont (Wolbachia) Determined by Targeted Bacteriophage flux can cause the majority of genetic diversity in free-living bacteria. This tenet of bacterial the reductive forces of the intracellular lifestyle. To test whether bacteriophages transfer as single genes

  10. Use of PRD1 bacteriophage in groundwater viral transport, inactivation, and attachment studies

    E-print Network

    Ryan, Joe

    MiniReview Use of PRD1 bacteriophage in groundwater viral transport, inactivation, and attachment (diameter), double-stranded DNA bacteriophage with an internal membrane, has emerged as an important model) sewage [1] and first detailed description by Olsen et al. in 1974 [2], bacteriophage PRD1 (alternative

  11. J. Mol. BioZ.(1983) 171,577~580 Packaging of DNA into Bacteriophage Heads

    E-print Network

    Harrison, Stephen C.

    1983-01-01

    J. Mol. BioZ.(1983) 171,577~580 Packaging of DNA into Bacteriophage Heads: A Model A model is suggestedfor the geometry of DNA entry into a bacteriophage head. It accounts for recent observations indicating absenceof a unique, ordered sequence of windings in the packaged DNA. The heads of bacteriophages

  12. Letter to the Editor Parallel Molecular Evolution of Deletions and Nonsense Mutations in Bacteriophage T7

    E-print Network

    Hillis, David

    in Bacteriophage T7 C. W. Cunningham, * K. Jeng, -/-J. Husti, t M. Badgett,i_ I. J. Molineux, $ D. M. Hillis at the DNA sequence level (e.g. Dykhuizen 1990; Travisano et al. 1995). Phylogenies of bacteriophage T7 grown bifurcating lineages of bacteriophage T7 in the presence of a mutagen accord- ing to an earlier protocol (fig

  13. Three-dimensional architecture of the bacteriophage 29 packaged genome and elucidation of its packaging process

    E-print Network

    Spakowitz, Andrew J.

    Three-dimensional architecture of the bacteriophage 29 packaged genome and elucidation of its packaging in dsDNA bacteriophages. Cryo-EM was used to directly visualize the architecture of the DNA inside curvatures, and the degree of order. We obtained cryo-EM images of bacteriophage that had packaged defined

  14. Evolutionary Genomics of a Temperate Bacteriophage in an Obligate Intracellular Bacteria (Wolbachia)

    E-print Network

    Bordenstein, Seth

    Evolutionary Genomics of a Temperate Bacteriophage in an Obligate Intracellular Bacteria (Wolbachia, the temperate double-stranded DNA bacteriophage WO in Wolbachia persistently transfers between bacterial considered the paradigm of temperate bacteriophage evolution in free-living bacteria, it appears irrelevant

  15. Evaluating Bacteriophage P22 as a Tracer in a Complex Surface Water

    E-print Network

    Evaluating Bacteriophage P22 as a Tracer in a Complex Surface Water System: The Grand River marine and fresh water environments. There is a strong interest in using bacteriophages as tracers the transport of bacteriophages in the subsurface environment, few studies examined phage transport in large

  16. 40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato....

  17. 40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato....

  18. 40 CFR 180.1307 - Bacteriophage of Clavibacter michiganensis subspecies michiganensis; exemption from the...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacteriophage of Clavibacter... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1307 Bacteriophage of... exemption from the requirement of a tolerance is established for residues of lytic bacteriophage...

  19. Ordering of alkali halide salts dissolved in bacteriophage Pf1 solutions: A nuclear magnetic resonance study

    E-print Network

    Augustine, Mathew P.

    Ordering of alkali halide salts dissolved in bacteriophage Pf1 solutions: A nuclear magnetic into filamentous bacteriophage Pf1 solutions display line splittings and shifts consistent with an interaction phospholipid bicelles1 and filamen- tous virus2 and bacteriophage3 particles partially restores an- isotropic

  20. 40 CFR 180.1307 - Bacteriophage of Clavibacter michiganensis subspecies michiganensis; exemption from the...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacteriophage of Clavibacter... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1307 Bacteriophage of... exemption from the requirement of a tolerance is established for residues of lytic bacteriophage...

  1. A library of bacteriophage-displayed antibody fragments directed against proteins of the

    E-print Network

    Hudspeth, A. James

    A library of bacteriophage-displayed antibody fragments directed against proteins of the inner ear, 1999 Bacteriophage display of antibodies provides a method for the generation of immunological reagents against pro- teins from the bullfrog's sacculus. This library was probed for bacteriophage that bound

  2. Visualization of Bacteriophage T3 Capsids with DNA Incompletely Packaged In Vivo

    E-print Network

    Jiang, Wen

    Visualization of Bacteriophage T3 Capsids with DNA Incompletely Packaged In Vivo Ping-An Fang1 in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ip

  3. Single M13 bacteriophage tethering and stretching Matthew J. Lang, and Angela M. Belcher

    E-print Network

    Khalil, Ahmad S.

    Single M13 bacteriophage tethering and stretching Matthew J. Lang, and Angela M. Belcher Ahmad S bacteriophage tethering and stretching Ahmad S. Khalil*, Jorge M. Ferrer , Ricardo R. Brau , Stephen T. Kottmann filamentous bacteriophage is a unique advantage. Where previously this viral template was shown to direct

  4. Oxaliplatin vs. cisplatin: competition experiments on their binding to lysozyme.

    PubMed

    Marasco, Daniela; Messori, Luigi; Marzo, Tiziano; Merlino, Antonello

    2015-06-14

    The model protein hen egg white lysozyme was challenged with oxaliplatin and cisplatin. ESI mass spectrometry, surface plasmon resonance and thermal shift analyses demonstrate the formation of a bis-platinum adduct, though in very small amounts. Crystals of the bis-platinum adduct were obtained using two different preparations and the X-ray structures were solved at 1.85 Å and 1.95 Å resolution. Overall, the obtained data point out that, under the analyzed conditions, the two Pt drugs have similar affinities for the protein, but bind on its surface at two non-overlapping sites. In other words, these two drugs manifest a significantly different reactivity with this model protein and do not compete for the same protein binding sites. PMID:25974859

  5. Dynamics of lysozyme and its hydration water under electric field

    SciTech Connect

    Favi, Pelagie M; Zhang, Qiu; O'Neill, Hugh Michael; Mamontov, Eugene; Omar Diallo, Souleymane; Palmer, Jeremy

    2014-01-01

    The effects of static electric field on the dynamics of lysozyme and its hydration water have been investigated by means of incoherent quasi-elastic neutron scattering (QENS). Measurements were performed on lysozyme samples, hydrated respectively with heavy water (D2O) to capture the protein dynamics, and with light water (H2O), to probe the dynamics of the hydration shell, in the temperature range from 210 < T < 260 K. The hydration fraction in both cases was about 0.38 gram of water per gram of dry protein. The field strengths investigated were respectively 0 kV/mm and 2 kV/mm ( 2 106 V/m) for the protein hydrated with D2O and 0 kV and 1 kV/mm for the H2O-hydrated counterpart. While the overall internal protons dynamics of the protein appears to be unaffected by the application of electric field up to 2 kV/mm, likely due to the stronger intra-molecular interactions, there is also no appreciable quantitative enhancement of the diffusive dynamics of the hydration water, as would be anticipated based on our recent observations in water confined in silica pores under field values of 2.5 kV/mm. This may be due to the difference in surface interactions between water and the two adsorption hosts (silica and protein), or to the existence of a critical threshold field value Ec 2 3 kV/mm for increased molecular diffusion, for which electrical breakdown is a limitation for our sample.

  6. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel; Pusey, Marc

    1998-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk'solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 4(sub 3) axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to greater than 500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 yields PHE or ALA and ASN 113 yields ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 4(sub 3) helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  7. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel J.; Nadarajah, Arunan; Pusey, Marc

    1999-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 43 axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to (3)500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 (Registered) PHE or ALA and ASN 113 (Registered) ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 43 helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  8. Crystallization of Chicken Egg White Lysozyme from Assorted Sulfate Salts

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Snell, Edward H.; Malone, Christine C.; Pusey, Marc L.

    1999-01-01

    Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Crystals have also been grown at 4 C in the absence of any other added salts using isoionic lysozyme which was titrated to pH 4.6 with dilute sulfuric acid. Four different crystal forms have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed. Crystals grown at 15 C were generally tetragonal, with space group P4(sub 3)2(sub 1)2. Crystallization at 20 C typically resulted in the formation of orthorhombic crystals, space group P2(sub 1)2(sub 1)2(sub 1). The tetragonal reversible reaction orthorhombic transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20 C and between 100 and 125 mg/ml protein concentration. Crystallization from 1.2 M magnesium sulfate at pH 7.8 gave a trigonal crystal, space group P3(sub 1)2(sub 1), a = b = 87.4, c = 73.7, gamma = 120 deg, which diffracted to 2.8 A. Crystallization from ammonium sulfate at pH 4.6, generally at lower temperatures, was also found to result in a monoclinic form. space group C2, a = 65.6, b = 95.0, c = 41.2, beta = 119.2 deg. A crystal of approximately 0.2 x 0.2 x 0.5 mm grown from bulk solution diffracted to approximately 3.5 A.

  9. Crystallization of Chicken Egg White Lysozyme from Sulfate Salts

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth; Pusey, Marc

    1998-01-01

    It has been "known" that chicken egg white lysozyme does not crystallize from sulfate, particularly ammonium sulfate, salts, but instead gives amorphous precipitates. This has been the basis of several studies using lysozyme comparing macromolecule crystal nucleation and amorphous precipitation. Recently Ries-Kautt et al (Acta Cryst D50, (1994) 366) have shown that purified isoionic CEWL could be crystallized from low concentrations of sulfate at basic pH, and we subsequently showed that in fact CEWL could be purified in both the tetragonal and orthorhombic forms using ammonium sulfate over the pH range 4.0 to 7.8 (Acta Cryst D53, (1997) 795). We have now extended these observations to include a range of common sulfate salts, specifically sodium, potassium, rubidium, magnesium, and manganese sulfates. In all cases but the manganese sulfates both the familiar tetragonal and orthorhombic forms were obtained, with unit cell dimensions close to those known for the "classic" sodium chloride crystallized forms. Manganese sulfate has only yielded orthorhombic crystals to date. All crystallizations were carried out using low (typically less than or equal to 6 M) salt and high (greater than approximately 90 mg/ml) protein concentrations. As with ammonium sulfate, the tetragonal - orthorhombic phase shift appears to be a function of both the temperature and the protein concentration, with higher temperatures and concentrations favoring the orthorhombic and lower the tetragonal form. The phase change range is somewhat reduced for the sulfate salts, depending upon conditions being typically between approximately 15 - 20 C. Both the magnesium and manganese sulfates gave crystals at salt concentrations over 0.6 M as well, with magnesium sulfate giving a very slowly nucleating and growing hexagonal form. A triclinic crystal form, characterized by aggressively small crystals (typically 0.1 mm in size) has been occasionally obtained from ammonium sulfate. Finally, preliminary spot solubility determinations have suggested that in some cases the solubility increases with increasing salt concentrations.

  10. Bacteriophage Typing of Salmonella. I. Isolation and Host Range Study of Bacteriophages

    PubMed Central

    Ibrahim, Abdel Aziz E.

    1969-01-01

    A series of bacteriophages, lytic for bacteria belonging to the genera Escherichia and Salmonella, were isolated. The phages were isolated from fecal samples, intestinal contents of turkey poults, and carrier cultures of S. typhimurium, S. typhimurium var copenhagen, S. heidelberg, and E. coli. The feasibility of using different habitats as sources of Salmonella phages was evaluated. The carrier cultures were the most promising source for phages active on the serotypes for which the phages were sought. A host range study of the isolated phages was made. Eight phages were selected to develop a phage typing scheme for S. typhimurium, S. typhimurium var copenhagen, and S. heidelberg. PMID:4907005

  11. Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

    PubMed Central

    Burke, Thomas P.; Loukitcheva, Anastasia; Zemansky, Jason; Wheeler, Richard; Boneca, Ivo G.

    2014-01-01

    Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood. PMID:25157076

  12. Small angle neutron scattering study on the structural variation of lysozyme in bioprotectants

    NASA Astrophysics Data System (ADS)

    Koda, Shota; Takayama, Haruki; Shibata, Tomohiko; Mori, Tatsuya; Kojima, Seiji; Park, In-Sung; Shin, Tae-Gyu

    2015-05-01

    The thermal denaturation and subsequent structural variation of lysozyme in various bioprotectant candidate solutions such as trehalose and choline acetate have been investigated by using small angle neutron scattering and differential scanning calorimetry. The gyration radius shows little change with the addition of additives in a native state at room temperature. On heating the lysozyme solution, a remarkable increase in the gyration radius is observed at temperatures above the denaturation temperature without any bioprotectants. Such an increase is suppressed by the additives owing to the intermolecular interactions between the lysozyme molecules and the bioprotectants of trehalose and choline acetate. The fractal dimension of lysozyme varies slightly with the addition of the bioprotectant solutions, and shows a remarkable drop in the vicinity of the denaturation temperature for all the solutions.

  13. Adsorption and desorption studies of lysozyme by Fe3O4-polymer nanocomposite via fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Koc, Kenan; Alveroglu, Esra

    2015-06-01

    The work have been undertaken in this study is to synthesis and characterize Fe3O4-polymer nanocomposites which are having different morphological properties. Also, investigation of the adsorption and desorption behaviour of lysozyme onto Fe3O4-polymer nanocomposites have been studied. Fe3O4 nanoparticles, synthesized by in situ in polyacrylamide hydrogels, show super-paramagnetic behaviour and saturation magnetization of composite material have been tuned by changing the hydrogel conformation. Adsorption and desorption studies of lysozyme were followed by using pure water at room temperature via fluorescence measurements. Fluorescence measurements showed that, the composite materials adsorbed lysozyme molecules less than 20 s and higher monomer concentration of composite materials cause faster adsorption. Besides, structure of lysozyme molecules were not changed during the adsorption and desorption. As a result Fe3O4-polymer nanocomposites could be used for drug delivery, protein separation and PAAm gels could be used for synthesis of magnetic composites with varying magnetic properties.

  14. Potential of mean force for human lysozyme camelid vhh hl6 antibody interaction studies

    NASA Astrophysics Data System (ADS)

    Wang, Yeng-Tseng; Liao, Jun-Min; Chen, Cheng-Lung; Su, Zhi-Yuan; Chen, Chang-Hung; Hu, Jeu-Jiun

    2008-04-01

    Calculating antigen-antibody interaction energies is crucial for understanding antigen-antibody associations in immunology. To shed further light into this equation, we study a separation of human lysozyme-camelid vhh hl6 antibody (cAb-HuL6) complex. The c-terminal end-to-end stretching of the lysozyme-antibody complex structures have been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) and explicit water model. For the lysozyme-antibody complex, there are six important intermediates in the c-terminal extensions process. Inclusion of our simulations may help to understand the binding mechanics of lysozyme-cAb-HuL6 antibody complex.

  15. Unfolding mechanism of lysozyme in various urea solutions: Insights from fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Chen, Bang; Zhang, Hongjia; Xi, Wenying; Zhao, Liqing; Liang, Li; Chen, Yantao

    2014-11-01

    Fluorescence spectroscopic technique is very popular in exploring the folding/unfolding process of proteins. In this paper, unfolding process of hen egg-white lysozyme was investigated in various denaturing solutions. Firstly, polymer solution theory was employed to comprehend the dependence of fluorescence quenching effect on protein concentration, and dynamic contact concentration was suggested as a critical value for related fluorescence experiment. Secondly, it was found that urea alone could not completely unfold lysozyme but did when together with DTT or HCl. Lysozyme was destabilized in concentrated urea solution, but still could maintain its spatial structure. Phase diagram of fluorescence intensities revealed that HCl could enhance the denaturing capacity of urea, resulting in the emergence of intermediate state in the thermodynamic unfolding process of lysozyme.

  16. Lysozyme crystallization by vapor diffusion: characterization and modeling in the absence and presence of exogenous minerals

    NASA Astrophysics Data System (ADS)

    Kimble, W. L.; Rousseau, R. W.; Sambanis, A.

    1995-01-01

    A model accounting for water evaporation and crystal growth was synthesized to simulate protein concentration profiles in the crystallization wells of a vapor-diffusion apparatus. The model calculations were compared with experimental results obtained with chicken egg white lysozyme crystallized in the absence and presence of exogenous mineral particles. The model predicted the increase in protein concentration during water evaporation and the decrease during crystal growth. The effects of magnetite, galena and chalcopyrite on the time profile of dissolved lysozyme concentration appeared minimal, except for the occurrence of earlier nucleation in the presence of magnetite. Few of the lysozyme crystals formed were physically associated with these minerals. More protein crystals were associated with topaz, lepidolite and apophyllite, which exhibit a close match of their crystalline lattice to that of lysozyme.

  17. Lysozyme activity and protein concentration in the haemolymph of the flat oyster Ostrea edulis (L.).

    PubMed

    Cronin, M A; Culloty, S C; Mulcahy, M F

    2001-10-01

    Lysozyme activity and protein concentration in the haemolymph of the flat oyster Ostrea edulis were investigated. These biochemical constituents of the haemolymph could be an indication of the physiological condition and vitality of the defence system of an animal. Haemolymph protein and lysozyme in oysters were examined over an 18 month period to determine their relationship with the strain of oyster, the season, the site, and parasitism by Bonamia ostreae. Haemolymph protein concentration exhibited seasonal fluctuations and varied between strains. Levels of protein in oysters highly infected with B. ostreae were slightly depressed but not significantly so. Haemolymph lysozyme varied greatly between individuals but no correlation was found between lysozyme levels and infection of oysters by B. ostreae. PMID:11592588

  18. [The role of the tryptophan-62 residue in the structure and function of lysozyme].

    PubMed

    Churakova, N I; Cherkasov, I A; Kravchenko, N A

    1977-02-01

    The thermostability and thermodinamics of formation of the enzyme-substrate complex of two oxidation products of chicken egg lysozyme with the tryptophane-62 residue modified to N'-formylkinurenine (with 2.5% activity) and kinurenine (with 27.5% activity) have been studied. In thermostability and pH effect on the substrate binding the lysozyme oxidation products do not differ from native lysozyme. The data obtained and thermodynamical characteristics of the enzyme-substrate complex formation suggest that the chemical nature of the 62 residue does not significantly affect the conformational properties of lysozyme, however, having a strongly pronounced effect on the binding of substrate and hence the total enzyme activity. PMID:15655

  19. Isolation and characterization of a c-type lysozyme from the nurse shark.

    PubMed

    Hinds Vaughan, Nichole; Smith, Sylvia L

    2013-12-01

    Lysozyme is a ubiquitous antibacterial enzyme that occurs in numerous invertebrate and vertebrate species. Three forms have been described c-type, g-type and i-type which differ in primary structure. Shark lysozyme has not been characterized; here we report on the isolation and characterization of lysozyme from unstimulated shark (Ginglymostoma cirratum) leukocytes and provide amino acid sequence data across the highly conserved active site of the molecule identifying it to be a c-type lysozyme. A leukocyte lysate was applied either (a) to the first of two sequential DE-52 cellulose columns or alternatively, (b) to a DEAE-Sepharose column. Lysozyme activity in lysate and active fractions was identified by zones of lysis of Micrococcus lysodeikticus cell walls on lysoplates and zones of growth inhibition in agar diffusion assays using Planococcus citreus as the target organism. SDS-PAGE analysis revealed a 14 kDa protein which was identified as lysozyme by mass spectroscopic analysis of peptides, reactivity against anti-HEWL antibodies on a Western blot, hydrolysis of M. lysodeikticus cell walls, and inhibition of growth of P. citreus on AU-gel blots in which the area of growth inhibition correlated to a 14 kDa protein. PMID:24084042

  20. Stomach lysozymes of the three-toed sloth (Bradypus variegatus), an arboreal folivore from the Neotropics.

    PubMed

    Pacheco, M Andreína; Concepción, Juan Luís; Rangel, José David Rosales; Ruiz, Marie Christine; Michelangeli, Fabián; Domínguez-Bello, María G

    2007-07-01

    Lysozymes are antimicrobial defences that act as digestive enzymes when expressed in the stomach of herbivores with pre-gastric fermentation. We studied this enzyme in the complex stomach of the three-toed sloth (Bradypus variegatus), a folivore with pre-gastric fermentation. Lysozymes were identified by SDS-PAGE and immunoblotting in all portions: diverticulum, pouch, glandular and muscular prepyloric area with 14.3 kDa of molecular mass. Purified lysozymes from all areas but the diverticulum were characterized by MALDI-TOF, optimal pH, optimal ionic strength, and specific activity. The differences observed suggested at least three isoforms. The optimal pHs were similar to the pH of the stomach portion where the enzymes were isolated. The lysozyme from the pouch (fermentation chamber) exhibited higher specific activity and concentration than the others. The specific activity of the enzyme from the acid muscular prepyloric portion was comparable to that reported in the cow abomasums; however, its concentration was lower than that observed in cow. This distinctive pattern of secretion/specific activity and overall low concentration suggests different roles for the lysozymes in this herbivore compared to Artiodactyla. We postulate that sloth stomach lysozymes may still be antimicrobial defences by protecting the microbial flora of the fermentation chamber against foreign bacteria. PMID:16959513

  1. Heat-Denatured Lysozyme Inactivates Murine Norovirus as a Surrogate Human Norovirus

    PubMed Central

    Takahashi, Hajime; Nakazawa, Moemi; Ohshima, Chihiro; Sato, Miki; Tsuchiya, Tomoki; Takeuchi, Akira; Kunou, Masaaki; Kuda, Takashi; Kimura, Bon

    2015-01-01

    Human norovirus infects humans through the consumption of contaminated food, contact with the excrement or vomit of an infected person, and through airborne droplets that scatter the virus through the air. Being highly infectious and highly viable in the environment, inactivation of the norovirus requires a highly effective inactivating agent. In this study, we have discovered the thermal denaturing capacity of a lysozyme with known antimicrobial activity against gram-positive bacteria, as well as its inactivating effect on murine norovirus. This study is the first report on the norovirus-inactivating effects of a thermally denatured lysozyme. We observed that lysozymes heat-treated for 40?min at 100?°C caused a 4.5 log reduction in infectivity of norovirus. Transmission electron microscope analysis showed that virus particles exposed to thermally denatured lysozymes were expanded, compared to the virus before exposure. The amino acid sequence of the lysozyme was divided into three sections and the peptides of each artificially synthesised, in order to determine the region responsible for the inactivating effect. These results suggest that thermal denaturation of the lysozyme changes the protein structure, activating the region responsible for imparting an inactivating effect against the virus. PMID:26134436

  2. Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

    PubMed Central

    Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

    2011-01-01

    Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

  3. Biological and Clinical Implications of Lysozyme Deposition on Soft Contact Lenses.

    PubMed

    Omali, Negar Babaei; Subbaraman, Lakshman N; Coles-Brennan, Chantal; Fadli, Zohra; Jones, Lyndon W

    2015-07-01

    Within a few minutes of wear, contact lenses become rapidly coated with a variety of tear film components, including proteins, lipids, and mucins. Tears have a rich and complex composition, allowing a wide range of interactions and competitive processes, with the first event observed at the interface between a contact lens and tear fluid being protein adsorption. Protein adsorption on hydrogel contact lenses is a complex process involving a variety of factors relating to both the protein in question and the lens material. Among tear proteins, lysozyme is a major protein that has both antibacterial and anti-inflammatory functions. Contact lens materials that have high ionicity and high water content have an increased affinity to accumulate lysozyme during wear, when compared with other soft lens materials, notably silicone hydrogel lenses. This review provides an overview of tear film proteins, with a specific focus on lysozyme, and examines various factors that influence protein deposition on contact lenses. In addition, the impact of lysozyme deposition on various ocular physiological responses and bacterial adhesion to lenses and the interaction of lysozyme with other tear proteins are reviewed. This comprehensive review suggests that deposition of lysozyme on contact lens materials may provide a number of beneficial effects during contact lens wear. PMID:26002002

  4. Transcriptional Onset of Lysozyme Genes during Early Development in Olive Flounder (Paralichthys olivaceus)

    PubMed Central

    Lee, Jang-Wook; Lee, Jeong-Ho; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Kim, Kyung-Kil

    2014-01-01

    The immune system in teleost fish is not completely developed during embryonic and larval stages, therefore effective innate mechanisms is very important for survival in such an environment. However, the knowledge of the development of immune system assumed to be restricted. In many species, lysozymes have been considered as important genes of the first line immune defense. The early detection of lysozyme mRNA in previous reports, led to the investigation of its presence in oocytes. As a result, c-type lysozyme mRNA transcripts were detected in unfertilized oocytes indicating maternal transfer. Therefore, we investigated the expression patterns of lysozymes in flounder, including the matured oocyte. In our results, c-type lysozyme mRNA was first detected in unfertilized oocyte stage, observed the significantly decreased until hatching stage, and was significantly increased after hatching stage. On the other hand, g-type lysozyme mRNA transcripts were first detected at late neurula stage, and the mRNA level was significantly increased after 20 dph. It may be suggest that maternally supplied mRNAs are selectively degraded prior to the activation of embryonic transcription. This study will be help in understanding the maturation and onset of humoral immunity during development of olive flounder immune system. PMID:25949197

  5. Heat-Denatured Lysozyme Inactivates Murine Norovirus as a Surrogate Human Norovirus.

    PubMed

    Takahashi, Hajime; Nakazawa, Moemi; Ohshima, Chihiro; Sato, Miki; Tsuchiya, Tomoki; Takeuchi, Akira; Kunou, Masaaki; Kuda, Takashi; Kimura, Bon

    2015-01-01

    Human norovirus infects humans through the consumption of contaminated food, contact with the excrement or vomit of an infected person, and through airborne droplets that scatter the virus through the air. Being highly infectious and highly viable in the environment, inactivation of the norovirus requires a highly effective inactivating agent. In this study, we have discovered the thermal denaturing capacity of a lysozyme with known antimicrobial activity against gram-positive bacteria, as well as its inactivating effect on murine norovirus. This study is the first report on the norovirus-inactivating effects of a thermally denatured lysozyme. We observed that lysozymes heat-treated for 40 min at 100?°C caused a 4.5 log reduction in infectivity of norovirus. Transmission electron microscope analysis showed that virus particles exposed to thermally denatured lysozymes were expanded, compared to the virus before exposure. The amino acid sequence of the lysozyme was divided into three sections and the peptides of each artificially synthesised, in order to determine the region responsible for the inactivating effect. These results suggest that thermal denaturation of the lysozyme changes the protein structure, activating the region responsible for imparting an inactivating effect against the virus. PMID:26134436

  6. The Lysozyme from Insect (Manduca sexta) is a Cold-Adapted Enzyme

    SciTech Connect

    Sotelo-Mundo,R.; Lopez-Zavala, A.; Garcia-Orozco, K.; Arvizu-Flores, A.; Velazquez-Contreras, E.; Valenzuela-Soto, E.; Rojo-Dominguez, A.; Kanost, M.

    2007-01-01

    Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of {alpha}-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 {sup o}C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.

  7. Complete genome sequence of bacteriophage T5.

    PubMed

    Wang, Jianbin; Jiang, Yan; Vincent, Myriam; Sun, Yongqiao; Yu, Hong; Wang, Jing; Bao, Qiyu; Kong, Huimin; Hu, Songnian

    2005-02-01

    The 121,752-bp genome sequence of bacteriophage T5 was determined; the linear, double-stranded DNA is nicked in one of the strands and has large direct terminal repeats of 10,139 bp (8.3%) at both ends. The genome structure is consistently arranged according to its lytic life cycle. Of the 168 potential open reading frames (ORFs), 61 were annotated; these annotated ORFs are mainly enzymes involved in phage DNA replication, repair, and nucleotide metabolism. At least five endonucleases that believed to help inducing nicks in T5 genomic DNA, and a DNA ligase gene was found to be split into two separate ORFs. Analysis of T5 early promoters suggests a probable motif AAA{3, 4 T}nTTGCTT{17, 18 n}TATAATA{12, 13 W}{10 R} for strong promoters that may strengthen the step modification of host RNA polymerase, and thus control transcription of phage DNA. The distinct protein domain profile and a mosaic genome structure suggest an origin from the common genetic pool. PMID:15661140

  8. Flap endonuclease of bacteriophage T7

    PubMed Central

    Mitsunobu, Hitoshi; Zhu, Bin; Lee, Seung-Joo; Tabor, Stanley; Richardson, Charles C

    2014-01-01

    Gene 6 protein of bacteriophage T7 has 5?-3?-exonuclease activity specific for duplex DNA. We have found that gene 6 protein also has flap endonuclease activity. The flap endonuclease activity is considerably weaker than the exonuclease activity. Unlike the human homolog of gene 6 protein, the flap endonuclease activity of gene 6 protein is dependent on the length of the 5?-flap. This dependency of activity on the length of the 5?-flap may result from the structured helical gateway region of gene 6 protein which differs from that of human flap endonuclease 1. The flap endonuclease activity provides a mechanism by which RNA-terminated Okazaki fragments, displaced by the lagging strand DNA polymerase, are processed. 3?-extensions generated during degradation of duplex DNA by the exonuclease activity of gene 6 protein are inhibitory to further degradation of the 5?-terminus by the exonuclease activity of gene 6 protein. The single-stranded DNA binding protein of T7 overcomes this inhibition. PMID:25105057

  9. Complex kinetics of the ? Bacteriophage genetic switch

    NASA Astrophysics Data System (ADS)

    Finzi, Laura; Manzo, Carlo; Zurla, Chiara; Dunlap, David

    2010-03-01

    The kinetics of the ? bacteriophage repressor-mediated DNA loop formation and breakdown were characterized by Tethered Particle Microscopy (TPM). A generalized likelihood ratio test was first applied to determine the location of change points (cp) in the TPM trace. Expectation-maximization (EM) clustering and the Bayesian information criterion were then used for accurate determination of the number of states accessible to the system. This procedure (cp-EM) allows objective and quantitative determination of TPM change points without the artificial time resolution limitations that arise from filtering and thresholding. Only two states were identified, which corresponded to the looped to the unlooped DNA configurations. The probability distribution function of the looped and unlooped DNA state dwell times revealed a complex kinetics. In particular, it was found that a stretched exponential provided a satisfactory fitting for the probability distribution of the unlooped state dwell times, while the dwell time distribution for the looped DNA state could not be fitted with a standard pdf; we observed, however, that a power law decay fits well the long dwell times. A mechanism is proposed to explain this kinetic behavior, where ? repressor non-specific binding to DNA may play an important physiological role.

  10. Bacteriophage-based biocontrol of biological sludge bulking in wastewater.

    PubMed

    Choi, Jeongdong; Kotay, Shireen Meher; Goel, Ramesh

    2011-01-01

    In a previous paper, the first ever application of lytic bacteriophage (virus)-mediated biocontrol of biomass bulking in the activated sludge process using Haliscomenobacter hydrossis as a model filamentous bacterium was demonstrated. In this work we extended the biocontrol application to another predominant filamentous bacterium, Sphaerotilus natans, notoriously known to cause filamentous bulking in wastewater treatment systems. Very similar to previous study, one lytic bacteriophage was isolated from wastewater that could infect S. natans and cause lysis. Significant reduction in sludge volume index and turbidity of the supernatant was observed in batches containing S. natans biomass following addition of lytic phages. Microscopic examination confirmed that the isolated lytic phage can trigger the bacteriolysis of S. natans. This extended finding further strengthens our hypothesis of bacteriophage-based biocontrol of overgrowth of filamentous bacteria and the possibility of phage application in activated sludge processes, the world's widely used wastewater treatment processes. PMID:21829092

  11. Binding properties and structure-affinity relationships of food antioxidant butylated hydroxyanisole and its metabolites with lysozyme.

    PubMed

    Wu, Di; Yan, Jin; Tang, Peixiao; Li, Shanshan; Xu, Kailin; Li, Hui

    2015-12-01

    Considering the harmful impact of food antioxidants on human bodies, thoroughly exposing their potential effects at the molecular level is important. In this study, the binding interactions of butylated hydroxyanisole (BHA), a phenolic antioxidant, and its different major metabolites tert-butylhydroquinone (TBHQ) and tert-butylbenzoquinone (TBQ) with lysozyme were examined via fluorescence, three-dimensional fluorescence, circular dichroism (CD), and ligand-protein docking studies. The three compounds caused strong quenching of lysozyme fluorescence by a static quenching mechanism but with different quenching efficiencies and different effects on the ?-helix content of the lysozyme. The order of binding affinity of lysozyme for all test compounds is as follows: BHA>TBQ>TBHQ. Thermodynamic parameters indicated that hydrogen bonding and van der Waals forces perform dominant functions in the binding between these compounds and lysozyme. Furthermore, structure-affinity relationships between the model compounds and lysozyme were established on the basis of computational analyses. PMID:26041206

  12. Considerations for using bacteriophages for plant disease control

    PubMed Central

    Jones, Jeffrey B.; Vallad, Gary E.; Iriarte, Fanny B.; Obradovi?, Aleksa; Wernsing, Mine H.; Jackson, Lee E.; Balogh, Botond; Hong, Jason C.; Momol, M.Timur

    2012-01-01

    The use of bacteriophages as an effective phage therapy strategy faces significant challenges for controlling plant diseases in the phyllosphere. A number of factors must be taken into account when considering phage therapy for bacterial plant pathogens. Given that effective mitigation requires high populations of phage be present in close proximity to the pathogen at critical times in the disease cycle, the single biggest impediment that affects the efficacy of bacteriophages is their inability to persist on plant surfaces over time due to environmental factors. Inactivation by UV light is the biggest factor reducing bacteriophage persistence on plant surfaces. Therefore, designing strategies that minimize this effect are critical. For instance, application timing can be altered: instead of morning or afternoon application, phages can be applied late in the day to minimize the adverse effects of UV and extend the time high populations of phage persist on leaf surfaces. Protective formulations have been identified which prolong phage viability on the leaf surface; however, UV inactivation continues to be the major limiting factor in developing more effective bacteriophage treatments for bacterial plant pathogens. Other strategies, which have been developed to potentially increase persistence of phages on leaf surfaces, rely on establishing non-pathogenic or attenuated bacterial strains in the phyllosphere that are sensitive to the phage(s) specific to the target bacterium. We have also learned that selecting the correct phages for disease control is critical. This requires careful monitoring of bacterial strains in the field to minimize development of bacterial strains with resistance to the deployed bacteriophages. We also have data that indicate that selecting the phages based on in vivo assays may also be important when developing use for field application. Although bacteriophages have potential in biological control for plant disease control, there are major obstacles, which must be considered. PMID:23531902

  13. Effects of sunlight on bacteriophage viability and structure.

    PubMed Central

    Wommack, K E; Hill, R T; Muller, T A; Colwell, R R

    1996-01-01

    Current estimates of viral abundance in natural waters rely on direct counts of virus-like particles (VLPs), using either transmission or epifluorescence microscopy. Direct counts of VLPs, while useful in studies of viral ecology, do not indicate whether the observed VLPs are capable of infection and/or replication. Rapid decay in bacteriophage viability under environmental conditions has been observed. However, it has not been firmly established whether there is a corresponding degradation of the virus particles. To address this question, viable and direct counts were carried out employing two Chesapeake Bay bacteriophages in experimental microcosms incubated for 56 h at two depths in the York River estuary. Viruses incubated in situ in microcosms at the surface yielded decay rates in full sunlight of 0.11 and 0.06 h-1 for CB 38 phi and CB 7 phi, respectively. The number of infective particles in microcosms in the dark and at a depth of 1 m was not significantly different from laboratory controls, with decay rates averaging 0.052 h-1 for CB 38 phi and 0.037 h-1 for CB 7 phi. Direct counts of bacteriophages decreased in teh estuarine microcosms, albeit only at a rate of 0.028 h-1, and were independent of treatment. Destruction of virus particles is concluded to be a process separate from loss of infectivity. It is also concluded that strong sunlight affects the viability of bacteriophages in surface waters, with the result that direct counts of VLPs overestimate the number of bacteriophage capable of both infection and replication. However, in deeper waters, where solar radiation is not a significant factor, direct counts should more accurately estimate numbers of viable bacteriophage. PMID:8919794

  14. Fabrication of High-Performance Magnetic Lysozyme-Imprinted Microsphere and Its NIR-Responsive Controlled Release Property.

    PubMed

    Chen, Jinxing; Lei, Shan; Xie, Yunyun; Wang, Mozhen; Yang, Jun; Ge, Xuewu

    2015-12-30

    The preparation of efficient and practical biomacromolecules imprinted polymer materials is still a challenging task because of the spatial hindrance caused by the large size of template and target molecules in the imprinting and recognition process. Herein, we provided a novel pathway to coat a NIR-light responsive lysozyme-imprinted polydopamine (PDA) layer on a fibrous SiO2 (F-SiO2) microsphere grown up from a magnetic Fe3O4 core nanoparticle. The magnetic core-shell structured lysozyme-imprinted Fe3O4@F-SiO2@PDA microspheres (MIP-lysozyme) can be easily separated by a magnet and have a high saturation adsorption capacity of lysozyme of 700 mg/g within 30 min because of the high surface area of 570 m(2)/g and the mesopore size of 12 nm of the Fe3O4@F-SiO2 support. The MIP-lysozyme microspheres also show an excellent selective adsorption of lysozyme (IF > 4). The binding thermodynamic parameters studied by ITC proves that the lysozyme should be restricted by the well-defined 3D structure of MIP-lysozyme microspheres. The MIP-lysozyme can extract lysozyme efficiently from real egg white. Owing to the efficient NIR light photothermal effect of PDA layer, the MIP-lysozyme microspheres show the controlled release property triggered by NIR laser. The released lysozyme molecules still maintain good bioactivity, which can efficiently decompose E. coli. Therefore, this work provides a novel strategy to build practical NIR-light-responsive MIPs for the extraction and application of biomacromolecules. PMID:26642106

  15. Molecular and chemical engineering of bacteriophages for potential medical applications.

    PubMed

    Hodyra, Katarzyna; D?browska, Krystyna

    2015-04-01

    Recent progress in molecular engineering has contributed to the great progress of medicine. However, there are still difficult problems constituting a challenge for molecular biology and biotechnology, e.g. new generation of anticancer agents, alternative biosensors or vaccines. As a biotechnological tool, bacteriophages (phages) offer a promising alternative to traditional approaches. They can be applied as anticancer agents, novel platforms in vaccine design, or as target carriers in drug discovery. Phages also offer solutions for modern cell imaging, biosensor construction or food pathogen detection. Here we present a review of bacteriophage research as a dynamically developing field with promising prospects for further development of medicine and biotechnology. PMID:25048831

  16. Complete genome sequence of Croceibacter bacteriophage P2559S.

    PubMed

    Kang, Ilnam; Kang, Dongmin; Cho, Jang-Cheon

    2012-08-01

    Croceibacter atlanticus HTCC2559(T), a marine bacterium isolated from the Sargasso Sea, is a phylogenetically unique member of the family Flavobacteriaceae. Strain HTCC2559(T) possesses genes related to interaction with primary producers, which makes studies on bacteriophages infecting the strain interesting. Here we report the genome sequence of bacteriophage P2559S, which was isolated off the coast of the Republic of Korea and lytically infects HTCC2559(T). Many genes predicted in the P2559S genome had their homologs in Bacteroides phages. PMID:22843867

  17. Engineered enzymatically active bacteriophages and methods of uses thereof

    DOEpatents

    Collins, James J (Newton, MA); Kobayashi, Hideki (Yokohama, JP); Kearn, Mads (Ottawa, CA); Araki, Michihiro (Minatoku, JP); Friedland, Ari (Boston, MA); Lu, Timothy Kuan-Ta (Palo Alto, CA)

    2012-05-22

    The present invention provides engineered bacteriophages that express at least one biofilm degrading enzyme on their surface and uses thereof for degrading bacterial biofilms. The invention also provides genetically engineered bacteriophages expressing the biofilm degrading enzymes and proteins necessary for the phage to replicate in different naturally occurring biofilm producing bacteria. The phages of the invention allow a method of biofilm degradation by the use of one or only a few administration of the phage because the system using these phages is self perpetuating, and capable of degrading biofilm even when the concentration of bacteria within the biofilm is low.

  18. Bacteriophages of Soft Rot Enterobacteriaceae-a minireview.

    PubMed

    Czajkowski, Robert

    2016-01-01

    Soft rot Enterobacteriaceae (Pectobacterium spp. and Dickeya spp., formerly pectinolytic Erwinia spp.) are ubiquitous necrotrophic bacterial pathogens that infect a large number of different plant species worldwide, including economically important crops. Despite the fact that these bacteria have been studied for more than 50 years, little is known of their corresponding predators: bacteriophages, both lytic and lysogenic. The aim of this minireview is to critically summarize recent ecological, biological and molecular research on bacteriophages infecting Pectobacterium spp. and Dickeya spp. with the main focus on current and future perspectives in that field. PMID:26626879

  19. Genetically engineered acidophilic heterotrophic bacteria by bacteriophage transduction

    SciTech Connect

    Ward, T.E.; Bruhn, D.F.; Bulmer, D.F.

    1989-05-10

    A bacteriophage capable of infecting acidophilic heterotrophic bacteria and processes for genetically engineering acidophilic bacteria for biomining or sulfur removal from coal are disclosed. The bacteriophage is capable of growth in cells existing at pH at or below 3.0. Lytic forms of the phage introduced into areas experiencing acid drainage kill the bacteria causing such drainage. Lysogenic forms of the phage having genes for selective removal of metallic or nonmetallic elements can be introduced into acidophilic bacteria to effect removal of the desired element from ore or coal. 1 fig., 1 tab.

  20. Salmonella and Campylobacter: Antimicrobial resistance and bacteriophage control in poultry.

    PubMed

    Grant, Ar'Quette; Hashem, Fawzy; Parveen, Salina

    2016-02-01

    Salmonella and Campylobacter are major causes of foodborne related illness and are traditionally associated with consuming undercooked poultry and/or consuming products that have been cross contaminated with raw poultry. Many of the isolated Salmonella and Campylobacter that can cause disease have displayed antimicrobial resistance phenotypes. Although poultry producers have reduced on-the-farm overuse of antimicrobials, antimicrobial resistant Salmonella and Campylobacter strains still persist. One method of bio-control, that is producing promising results, is the use of lytic bacteriophages. This review will highlight the current emergence and persistence of antimicrobial resistant Salmonella and Campylobacter recovered from poultry as well as bacteriophage research interventions and limitations. PMID:26678136

  1. Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters.

    PubMed

    Flannery, John; Keaveney, Sinéad; Rajko-Nenow, Paulina; O'Flaherty, Vincent; Doré, William

    2013-09-15

    Norovirus (NoV), the leading cause of adult non-bacterial gastroenteritis can be commonly detected in wastewater but the extent of NoV removal provided by wastewater treatment plants (WWTPs) is unclear. We monitored a newly commissioned WWTP with UV disinfection on a weekly basis over a six month period for NoV using RT-qPCR and for FRNA bacteriophage GA using both RT-qPCR (total concentration) and a plaque assay (infectious concentration). Mean concentrations of NoV GI and GII in influent wastewater were reduced by 0.25 and 0.41 log10 genome copies 100 ml(-1), respectively by the WWTP. The mean concentration of total FRNA bacteriophage GA was reduced by 0.35 log genome copies 100 ml(-1) compared to a reduction of infectious FRNA bacteriophage GA of 2.13 log PFU 100 ml(-1). A significant difference between concentrations of infectious and total FRNA bacteriophage GA was observed in treated, but not in untreated wastewaters. We conclude that RT-qPCR in isolation underestimates the reduction of infectious virus during wastewater treatment. We further compared the concentrations of infectious virus in combined sewer overflow (CSO) and UV treated effluents using FRNA bacteriophage GA. A greater percentage (98%) of infectious virus is released in CSO discharges than UV treated effluent (44%). Following a CSO discharge, concentrations of NoV GII and infectious FRNA bacteriophage GA in oysters from less than the limit of detection to 3150 genome copies 100 g(-1) and 1050 PFU 100 g(-1) respectively. PMID:23850211

  2. Lysozyme stability and amyloid fibrillization dependence on Hofmeister anions in acidic pH.

    PubMed

    Poniková, Slavomíra; Antošová, Andrea; Demjén, Erna; Sedláková, Dagmar; Marek, Jozef; Varha?, Rastislav; Gažová, Zuzana; Sedlák, Erik

    2015-09-01

    We have explored an effect of Hofmeister anions, Na2SO4, NaCl, NaBr, NaNO3, NaSCN and NaClO4, on stability and amyloid fibrillization of hen egg white lysozyme at pH 2.7. The stability of the protein was analyzed by differential scanning calorimetry. The Hofmeister effect of the anions was assessed by the parameter dT trs/d[anion] (T trs, transition temperature). We show that dT trs/d[anion] correlates with anion surface tension effects and anion partition coefficients indicating direct interactions between anions and lysozyme. The kinetic of amyloid fibrillization of lysozyme was followed by Thioflavin T (ThT) fluorescence. Negative correlation between dT trs/d[anion] and the nucleation rate of fibrillization in the presence of monovalent anions indicates specific effect of anions on fibrillization rate of lysozyme. The efficiency of monovalent anions to accelerate fibrillization correlates with inverse Hofmeister series. The far-UV circular dichroism spectroscopy and atomic force microscopy findings show that conformational properties of fibrils depend on fibrillization rate. In the presence of sodium chloride, lysozyme forms typical fibrils with elongated structure and with the secondary structure of the ?-sheet. On the other hand, in the presence of both chaotropic perchlorate and kosmotropic sulfate anions, the fibrils form clusters with secondary structure of ?-turn. Moreover, the acceleration of fibril formation is accompanied by decreased amount of the formed fibrils as indicated by ThT fluorescence. Taken together, our study shows Hofmeister effect of monovalent anions on: (1) lysozyme stability; (2) ability to accelerate nucleation phase of lysozyme fibrillization; (3) amount, and (4) conformational properties of the formed fibrils. PMID:26077813

  3. Effects of bacteriophage traits on plaque formation

    PubMed Central

    2011-01-01

    Background The appearance of plaques on a bacterial lawn is one of the enduring imageries in modern day biology. The seeming simplicity of a plaque has invited many hypotheses and models in trying to describe and explain the details of its formation. However, until now, there has been no systematic experimental exploration on how different bacteriophage (phage) traits may influence the formation of a plaque. In this study, we constructed a series of isogenic ? phages that differ in their adsorption rate, lysis timing, or morphology so that we can determine the effects if these changes on three plaque properties: size, progeny productivity, and phage concentration within plaques. Results We found that the adsorption rate has a diminishing, but negative impact on all three plaque measurements. Interestingly, there exists a concave relationship between the lysis time and plaque size, resulting in an apparent optimal lysis time that maximizes the plaque size. Although suggestive in appearance, we did not detect a significant effect of lysis time on plaque productivity. Nonetheless, the combined effects of plaque size and productivity resulted in an apparent convex relationship between the lysis time and phage concentration within plaques. Lastly, we found that virion morphology also affected plaque size. We compared our results to the available models on plaque size and productivity. For the models in their current forms, a few of them can capture the qualitative aspects of our results, but not consistently in both plaque properties. Conclusions By using a collection of isogenic phage strains, we were able to investigate the effects of individual phage traits on plaque size, plaque productivity, and average phage concentration in a plaque while holding all other traits constant. The controlled nature of our study allowed us to test several model predictions on plaque size and plaque productivity. It seems that a more realistic theoretical approach to plaque formation is needed in order to capture the complex interaction between phage and its bacterium host in a spatially restricted environment. PMID:21827665

  4. Low viscosity highly concentrated injectable nonaqueous suspensions of lysozyme microparticles.

    PubMed

    Miller, Maria A; Engstrom, Joshua D; Ludher, Baltej S; Johnston, Keith P

    2010-01-19

    Subcutaneous injection of concentrated protein and peptide solutions, in the range of 100-400 mg/mL, is often not possible with a 25- to 27-gauge needle, as the viscosity can be well above 50 cP. Apparent viscosities below this limit are reported for suspensions of milled lysozyme microparticles up to nearly 400 mg/mL in benzyl benzoate or benzyl benzoate mixtures with safflower oils through a syringe with a 25- to 27-gauge needle at room temperature. These apparent viscosities were confirmed using a cone-and-plate rheometer. The intrinsic viscosity regressed from the Kreiger-Dougherty model was only slightly above the Einstein value of 2.5, indicating the increase in viscosity relative to that of the solvent was caused primarily by the excluded volume. Thus, the increases in viscosity from electrical double layer interactions (electroviscous effects), solvation of the particles, or deviations of the particle shape from a spherical geometry were minimal, and much smaller than typically observed for proteins dissolved in aqueous solutions. The small electroviscous effects are expected given the negligible zeta potential and thin double layers in the low dielectric constant organic solvent. The suspensions were resuspendable after a year, with essentially constant particle size after two months as measured by static light scattering. The lower apparent viscosities for highly concentrated protein suspensions relative to protein solutions, coupled with these favorable characteristics upon resuspension, may offer novel opportunities for subcutaneous injection of therapeutic proteins. PMID:19803503

  5. ATP-induced noncooperative thermal unfolding of hen lysozyme

    SciTech Connect

    Liu, Honglin; Yin, Peidong; He, Shengnan; Sun, Zhihu; Tao, Ye; Huang, Yan; Zhuang, Hao; Zhang, Guobin; Wei, Shiqiang

    2010-07-02

    To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light-scattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg{sup 2+}-ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the {beta}-domain stability of HEWL, induces a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich {alpha}-helix and less {beta}-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric {beta}-sheet enriched intermediate.

  6. Crystallization of Chicken Egg White Lysozyme from Assorted Sulfate Salts

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Snell, Edward H.; Malone, Christine C.; Pusey, Marc L.

    1998-01-01

    Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Four different crystal morphologies have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed, Crystals grown at 15 C were generally tetragonal, with space group P43212. Crystallization at 20 C typically resulted in the formation of orthorhombic crystals, space group P21212 1. The tetragonal much less than orthorhombic morphology transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20 C and between 100 and 125 mg/ml protein concentration. Crystallization from 0.8 -1.2M magnesium sulfate at pH 7.6 - 8.0 gave a hexagonal (trigonal) crystal form, space group P3121, which diffracted to 2.8 A. Ammonium sulfate was also found to result in a monoclinic form, space group C2. Small twinned monoclinic crystals of approx. 0.2 mm on edge were grown by dialysis followed by seeded sitting drop crystallization.

  7. Crystallization of Chicken Egg-White Lysozyme from Ammonium Sulfate

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Snell, Edward H.; Pusey, Marc L.

    1997-01-01

    Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg/ml. Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(sub 3)2(sub 1)2 space group with crystals grown at ph 4.4 having unit-cell dimensions of a = b = 78.7 1, c=38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(sub 1)2(sub 1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

  8. Hydroanalysis of Animal Lysozymes c and Human Defensins a

    E-print Network

    J. C. Phillips

    2008-08-17

    Proteins appear to be the most dramatic natural example of self-organized criticality (SOC), a concept that explains many otherwise apparently unlikely phenomena. Protein functionality is dominated by long range hydro(phobic/philic) interactions which both drive protein compaction and mediate protein-protein interactions. In contrast to previous reductionist short range hydrophobicity scales, the holistic Moret-Zebende hydrophobicity scale represents a hydroanalytic tool that bioinformatically quantifies SOC in a way fully compatible with evolution. Hydroprofiling identifies chemical trends in the activities and substrate binding abilities of model enzymes and antibiotic animal lysozymes c and antibiotic human defensins, which have been the subject of tens of thousands of experimental studies. The analysis is simple and easily performed, and immediately yields insights not obtainable by traditional methods based on short-range real-space interactions, as described either by classical force fields (CFF) used in molecular dynamics simulations (MDS), or hydrophobicity scales based on transference energies from water to organic solvents.

  9. Bacteriophage and peptidoglycan degrading enzymes with antimicrobial applications.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage are viruses that infect and utilize bacteria as their host. They can reside in the bacterial genome as a prophage, or enter the lytic phase, take over the host gene expression machinery, synthesize new phage particles, lyse the host, and release up to hundreds of phage progeny. Lysis...

  10. BACTERIOPHAGE TRANSPORT IN SANDY SOIL AND FRACTURED TUFF

    EPA Science Inventory

    Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. n the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to 4...

  11. BACTERIOPHAGE TRANSPORT IN SANDY SOIL AND FRACTURED TUFF

    EPA Science Inventory

    Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. In the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to ...

  12. Factors Affecting Survival of Bacteriophage on Tomato Leaf Surfaces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of bacteriophage to persist in the phyllosphere for extended periods is limited by many factors, including sunlight irradiation, especially in the UV zone, temperature, desiccation, and exposure to copper bactericides. The effects of these factors on persistence of phage and formulated p...

  13. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids

    PubMed Central

    Wood, A. Jamie; Dytham, Calvin; Pitchford, Jonathan W.; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A.

    2015-01-01

    ABSTRACT Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. PMID:26037122

  14. Environmental Augmentation with Bacteriophage Prevents Colibacillosis in Broiler Chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages are viruses that kill bacteria. They are plentiful in nature, are safe having no known activity to human or animal cells, and are an attractive alternative to antibiotics. The objectives of this research were to establish an experimental model of colibacillosis induced by indirect e...

  15. Characterization of Salmonella bacteriophages isolated from swine lagoon effluent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four Salmonella bacteriophages originally isolated from swine lagoon effluent were further characterized. Their differences and similarities to known phages and to each other and their potential for biocontrol of Salmonella were assessed. In host inoculation spot tests the lagoon phages produced s...

  16. 21 CFR 866.2050 - Staphylococcal typing bacteriophage.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Staphylococcal typing bacteriophage. 866.2050 Section 866.2050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices §...

  17. 21 CFR 866.2050 - Staphylococcal typing bacteriophage.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Staphylococcal typing bacteriophage. 866.2050 Section 866.2050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices §...

  18. 21 CFR 866.2050 - Staphylococcal typing bacteriophage.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Staphylococcal typing bacteriophage. 866.2050 Section 866.2050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices §...

  19. 21 CFR 866.2050 - Staphylococcal typing bacteriophage.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Staphylococcal typing bacteriophage. 866.2050 Section 866.2050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices §...

  20. 21 CFR 866.2050 - Staphylococcal typing bacteriophage.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Staphylococcal typing bacteriophage. 866.2050 Section 866.2050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices §...

  1. UPTAKE OF BACTERIOPHAGE F2 THROUGH PLANT ROOTS

    EPA Science Inventory

    A model system was designed to measure viral uptake through the roots of plants and translocation to distal plant parts. For this study, uptake of bacteriophage f2 was measured in corn and bean plants growing in hydroponic solutions. Few phage were detected in plants with uncut r...

  2. 21 CFR 172.785 - Listeria-specific bacteriophage preparation.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Listeria-specific bacteriophage preparation. 172.785 Section 172.785 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Other Specific Usage Additives § 172.785...

  3. Bacteriophage for prophylaxis and therapy in cattle, poultry, and pigs.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The successful use of virulent (lytic) bacteriophages (phages) in preventing and treating neonatal enterotoxigenic Escherichia coli infections in calves, lambs and pigs has prompted investigation of other applications phage therapy in food animals. While results have been very variable, some indica...

  4. Natural mummification of the human gut preserves bacteriophage DNA.

    PubMed

    Santiago-Rodriguez, Tasha M; Fornaciari, Gino; Luciani, Stefania; Dowd, Scot E; Toranzos, Gary A; Marota, Isolina; Cano, Raul J

    2016-01-01

    The natural mummification process of the human gut represents a unique opportunity to study the resulting microbial community structure and composition. While results are providing insights into the preservation of bacteria, fungi, pathogenic eukaryotes and eukaryotic viruses, no studies have demonstrated that the process of natural mummification also results in the preservation of bacteriophage DNA. We characterized the gut microbiome of three pre-Columbian Andean mummies, namely FI3, FI9 and FI12, and found sequences homologous to viruses. From the sequences attributable to viruses, 50.4% (mummy FI3), 1.0% (mummy FI9) and 84.4% (mummy FI12) were homologous to bacteriophages. Sequences corresponding to the Siphoviridae, Myoviridae, Podoviridae and Microviridae families were identified. Predicted putative bacterial hosts corresponded mainly to the Firmicutes and Proteobacteria, and included Bacillus, Staphylococcus, Clostridium, Escherichia, Vibrio, Klebsiella, Pseudomonas and Yersinia. Predicted functional categories associated with bacteriophages showed a representation of structural, replication, integration and entry and lysis genes. The present study suggests that the natural mummification of the human gut results in the preservation of bacteriophage DNA, representing an opportunity to elucidate the ancient phageome and to hypothesize possible mechanisms of preservation. PMID:26564967

  5. Restoration of mRNA splicing by a second-site intragenic suppressor in the T4 ribonucleotide reductase (small subunit) self-splicing intron.

    PubMed

    Khan, A U; Ahmad, M; Lal, S K

    2000-02-16

    The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598-base self-splicing intron which is closely related to other group I introns of T4 and eukaryotes. Thirty-one mutants causing splicing defects in the nrdB intron were isolated. Twenty-three EMS-induced revertants for these 31 primary mutants were isolated by the strategic usage of the white halo plaque phenotype. We mapped these revertants by marker rescue using subclones of the nrdB gene. Some of these second-site mutations mapped to regions currently predicted by the secondary structure model of the nrdB intron. One of these suppressor mutants (nrdB753R) was found to be intragenic by marker rescue with the whole nrdB gene. However, this mutation failed to map within the nrdB intron. Splicing assays showed that this pseudorevertant restored splicing proficiency of the nrdB primary mutation to almost wild-type conditions. This is the first example of a mutation within the exons of a gene containing a self-splicing intron that is capable of restoring a self-splicing defect caused by a primary mutation within the intron. In addition, two other suppressor mutations are of interest (nrdB429R and nrdB399R). These suppressors were able to restore their primary 5' defect but in turn create a 3' splicing defect. Both of these revertants mapped in different regions of the intron with respect to their primary mutations. PMID:10679208

  6. Immunoglobulins G and lysozyme concentrations in canine fetal fluids at term of pregnancy.

    PubMed

    Dall'Ara, P; Meloni, T; Rota, A; Servida, F; Filipe, J; Veronesi, M C

    2015-03-01

    In the dog, the endotheliochorial placenta allows only the 5% to 10% transfer of maternal antibodies to the fetus, but the timing and the factors influencing the immunoglobulin G (IgG) transplacental transport were not fully investigated. The aims of the present study were the following: (1) to assess the presence of both IgG and lysozyme in amniotic and allantoic fluids collected from fully developed and viable newborn puppies born by elective cesarean section at term and possible correlations between amniotic and allantoic IgG and lysozyme levels; (2) to verify possible differences in IgG and lysozyme concentrations between the two fluids; and (3) to detect possible differences in IgG and lysozyme fetal fluid levels in relation to the maternal breed body size and parity, as well as to the neonatal gender. The study, performed on 41 purebred bitches submitted to elective cesarean section at term, enrolled 142 puppies, 74 males and 68 females, born mature, viable, without gross malformations, and with a normal weight. At surgery, a total of 129 amniotic and 84 allantoic samples were collected for IgG and lysozyme analysis. Class G immunoglobulins and lysozyme were detected in both fluids, but IgG concentrations were higher (P < 0.01) in amniotic fluid. Moreover, a significant positive correlation (P < 0.01) between IgG amniotic and allantoic levels, but not for lysozyme, was observed. A significant effect of the maternal parity (P < 0.05), but not of the breed body size, on the amniotic IgG concentrations was found, whereas the newborn gender was not associated to different IgG or lysozyme amniotic or allantoic levels. Given the significant contributions of fetal fluids to fetal and neonatal health, the results reported that the amniotic and allantoic fluids play a role in the immune protection of the fetus/newborn also in canine species. However, additional research is needed to better elucidate both the origin of IgG and lysozyme and the factors influencing the wide interindividual variations. PMID:25488792

  7. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances § 721.1880 Borate(1-), tris(acetato-.kappa.O)hydro-, sodium... substance identified as borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

  8. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances § 721.1880 Borate(1-), tris(acetato-.kappa.O)hydro-, sodium... substance identified as borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

  9. Characterization of expression, activity and role in antibacterial immunity of Anopheles gambiae lysozyme c-1

    PubMed Central

    Kajla, Mayur K.; Andreeva, Olga; Gilbreath, Thomas M.; Paskewitz, Susan M.

    2009-01-01

    There are eight lysozyme genes in the Anopheles gambiae genome. Transcripts of one of these genes, LYSC-1, increased in Anopheles gambiae cell line 4a3B by 24 h after exposure to heat-killed Micrococcus luteus. Lysozyme activity was also identified in conditioned media from the cell line from which the protein was purified to homogeneity using ion exchange and gel filtration. Mass spectrometric analysis of the purified protein showed 100% identity to lysozyme c-1. Purified lysozyme c-1 was tested against non-mosquito derived as well as culturable bacteria isolated from mosquito midguts. Lysozyme c-1 had negligible effects on the growth of most mosquito-derived bacteria in vitro but did inhibit the growth of M. luteus. Although Lys c-1 did not directly kill most bacteria, knockdown of LYSC-1 resulted in significant mortality in mosquitoes subjected to hemocoelic infections with Escherichia coli but not M. luteus thus suggesting that this protein plays an important role in antibacterial defense against selected bacteria. PMID:19932188

  10. Lysozyme Activity in the Plasma of Rodents Infected With Their Homologous Trypanosomes

    PubMed Central

    Maraghi, S; Molyneux, DH; Wallbanks, KR

    2012-01-01

    Background In this study the concentration of lysozyme in blood plasma of Microtus agrestis, Clethrinomys glareolus, Apodemus sylvaticus, BK rats and outbred white mice before and after infection with culture forms of Trypanosoma microti, T, evotomys, T. grosi, T. lewisi and T. musculi respectively was measured. Methods Blood samples of rodents, Microtus agrestis, Clethrionomys glareolus, Apodemus sylvaticus, BK rats and outbred mice infected with T. microti, T. evotomys, T. grosi, T. lewisi and T. musculi respectively were collected in heparinized micro- tubes immediately before inoculation and 3, 6, 12, 24, 48, 96 and more than 400 days after intra- perituneal inoculation with 5×105of their homologous trypanosome parasites of which more than half were metacyclic trypomastigote in 0.2 ml of culture medium. Micro- tubes were centrifuged and plasma samples were separated and the lysozyme activity was measured by the agar method. Results Levels of lysozyme rose rapidly three to six days after the inoculation to ten to twenty than their pre- infection levels. They then gradually decreased, although after more than one year they were still two to ten folds higher than controls. The highest level measured occurred in rats infected with T. lewisi and the lowest in A. sylvaticus infected with T. grosi. After one year the highest concentration of lysozyme was in mice infected with T. musculi and lowest in A. sylvaticus. Conclusion Persistent enhanced lysozyme levels may prevent re- infection with trypanosomes. PMID:23323096

  11. Synthesis of Ag{sub 2}S nanorods by biomimetic method in the lysozyme matrix

    SciTech Connect

    Qin, Dezhi Zhang, Li; He, Guoxu; Zhang, Qiuxia

    2013-09-01

    Graphical abstract: - Highlights: • Firstly, Ag{sub 2}S nanorods were synthesized by biomimetic method in the lysozyme solutions. • The study of the interaction between Ag{sup +} and the lysozyme. • Discussion of possible formation mechanism of Ag{sub 2}S nanorods. • The synthesis process of lyso-conjugated Ag{sub 2}S nanocrystals is facile, effective and environment friendly. - Abstract: Ag{sub 2}S nanorods were successfully synthesized by biomimetic route in the lysozyme solution at physiological temperature and atmospheric pressure. The transmission electron microscopy (TEM) images revealed that the prepared nanorods are uniform and monodisperse with homogeneous size about 50 nm in diameter and 150 nm in length. The optical property of Ag{sub 2}S nanocrystals was studied by the ultraviolet–visible (UV–vis) and photoluminescence (PL) spectroscopy, the results show that the products exhibit well-defined emission at 471 nm and 496 nm excited by 292 nm. The interaction of Ag{sup +}/Ag{sub 2}S with the lysozyme was investigated through Fourier transform infrared (FT-IR) spectroscopy, which shows that the cooperation effect of the lysozyme and Ag{sup +} could be responsible for the formation of as obtained Ag{sub 2}S nanorods.

  12. Lysozyme as an alternative to growth promoting antibiotics in swine production.

    PubMed

    Oliver, W T; Wells, J E

    2015-01-01

    Lysozyme is a naturally occurring enzyme found in bodily secretions such as tears, saliva, and milk. It functions as an antimicrobial agent by cleaving the peptidoglycan component of bacterial cell walls, which leads to cell death. Antibiotics are also antimicrobials and have been fed at subtherapeutic levels to swine as growth promoters. These compounds benefit swine producers by minimizing production losses by increasing feed efficiency and decreasing susceptibility to bacterial infection and disease. This manuscript reviews the knowledge of the effects of lysozyme, as compared to traditional subtherapeutic antibiotics in swine feed, on pig performance and health. It is clear from decades of studies that antibiotic use in feeds increases pig performance, particularly in the nursery. Similarly, lysozyme, as a feed additive, increases growth and feed efficiency. While the mechanism by which antibiotics and lysozyme improve performance is not clearly understood, both of these feed additives improve gastrointestinal health, improve the metabolic profile, and alter the gastrointestinal bacteria ecology of swine. Therefore, lysozyme is a suitable alternative to growth-promoting subtherapeutic antibiotic use in swine feed. PMID:26273432

  13. The extracellular chaperone clusterin potently inhibits human lysozyme amyloid formation by interacting with prefibrillar species.

    PubMed

    Kumita, Janet R; Poon, Stephen; Caddy, Gemma L; Hagan, Christine L; Dumoulin, Mireille; Yerbury, Justin J; Stewart, Elise M; Robinson, Carol V; Wilson, Mark R; Dobson, Christopher M

    2007-05-25

    We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid disease. The aggregation of the amyloidogenic variant I56T is inhibited significantly at clusterin to lysozyme ratios as low as 1:80 (i.e. one clusterin molecule per 80 lysozyme molecules). Experiments indicate that under the conditions where inhibition of aggregation occurs, clusterin does not bind detectably to the native or fibrillar states of lysozyme, or to the monomeric transient intermediate known to be a key species in the aggregation reaction. Rather, it seems to interact with oligomeric species that are present at low concentrations during the lag (nucleation) phase of the aggregation reaction. This behavior suggests that clusterin, and perhaps other extracellular chaperones, could have a key role in curtailing the potentially pathogenic effects of the misfolding and aggregation of proteins that, like lysozyme, are secreted into the extracellular environment. PMID:17407782

  14. A zinc complex of heparan sulfate destabilises lysozyme and alters its conformation

    SciTech Connect

    Hughes, Ashley J.; Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire OX11 0DE ; Hussain, Rohanah; Cosentino, Cesare; Guerrini, Marco; Siligardi, Giuliano; Yates, Edwin A.; Rudd, Timothy R.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Zinc-heparan sulfate complex destabilises lysozyme, a model amyloid protein. Black-Right-Pointing-Pointer Addition of zinc, without heparan sulfate, stabilises lysozyme. Black-Right-Pointing-Pointer Heparan sulfate cation complexes provide alternative protein folding routes. -- Abstract: The naturally occurring anionic cell surface polysaccharide heparan sulfate is involved in key biological activities and is implicated in amyloid formation. Following addition of Zn-heparan sulfate, hen lysozyme, a model amyloid forming protein, resembled {beta}-rich amyloid by far UV circular dichroism (increased {beta}-sheet: +25%), with a significantly reduced melting temperature (from 68 to 58 Degree-Sign C) by fluorescence shift assay. Secondary structure stability of the Zn-heparan sulfate complex with lysozyme was also distinct from that with heparan sulfate, under stronger denaturation conditions using synchrotron radiation circular dichroism. Changing the cation associated with heparan sulfate is sufficient to alter the conformation and stability of complexes formed between heparan sulfate and lysozyme, substantially reducing the stability of the protein. Complexes of heparan sulfate and cations, such as Zn, which are abundant in the brain, may provide alternative folding routes for proteins.

  15. The effect of protein contaminants on the crystallization of turkey egg white lysozyme

    NASA Astrophysics Data System (ADS)

    Abergel, Chantal; Nesa, Marie P.; Fontecilla-Camps, Juan C.

    1991-03-01

    We report here a series of studies on the controlled contamination of crystallizing solutions of the hexagonal form of turkey egg white lysozyme (TEWL) carried out to understand the effects of impurities on the nucleation and growth of protein crystals. The contamination of TEWL solutions with any of three other avian lysozymes affects both the nucleation and the growth processes. For hen and quail egg white lysozymes, low and medium levels of contamination result in partial inhibition of nucleation and shortening of the c-axis. Further increase of the contaminant concentration leads to detectable co-crystallization. A different effect is obtained when using the pheasant egg white lysozyme. Contamination by an unrelated protein, ribonuclease A, has an effect on the nucleation levels that is similar to those observed with the avian lysozymes. However, no effect on TEWL crystal morphology is observed. Thus, in the case of TEWL crystals, one can distinguish between a specific effect on crystal morphology induced by related proteins and a more general inhibitory effect on the nucleation levels observed in all cases studied here.

  16. Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica

    SciTech Connect

    Marana, S. R.; Cançado, F. C.; Valério, A. A.; Ferreira, C.; Terra, W. R.; Barbosa, J. A. R. G.

    2006-08-01

    The digestive lysozymes 1 and 2 from M. domestica were crystallized by vapour diffusion. The crystallographic data were processed to a maximum resolution of 1.9 Å in both cases. Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 Å using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P2{sub 1} (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 Å, ? = 102.97°) and the orthorhombic space group P2{sub 1}2{sub 1}2 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 Å), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.

  17. One-step assay for the quantification of T4 DNA ligase.

    PubMed

    Franke, Steffi; Kreisig, Thomas; Buettner, Karin; Zuchner, Thole

    2015-02-01

    As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays. PMID:25503935

  18. Genomic characteristics and environmental distributions of the uncultivated Far-T4 phages

    PubMed Central

    Roux, Simon; Enault, François; Ravet, Viviane; Pereira, Olivier; Sullivan, Matthew B.

    2015-01-01

    Viral metagenomics (viromics) is a tremendous tool to reveal viral taxonomic and functional diversity across ecosystems ranging from the human gut to the world's oceans. As with microbes however, there appear vast swaths of “dark matter” yet to be documented for viruses, even among relatively well-studied viral types. Here, we use viromics to explore the “Far-T4 phages” sequence space, a neighbor clade from the well-studied T4-like phages that was first detected through PCR study in seawater and subsequently identified in freshwater lakes through 454-sequenced viromes. To advance the description of these viruses beyond this single marker gene, we explore Far-T4 genome fragments assembled from two deeply-sequenced freshwater viromes. Single gene phylogenetic trees confirm that the Far-T4 phages are divergent from the T4-like phages, genome fragments reveal largely collinear genome organizations, and both data led to the delineation of five Far-T4 clades. Three-dimensional models of major capsid proteins are consistent with a T4-like structure, and highlight a highly conserved core flanked by variable insertions. Finally, we contextualize these now better characterized Far-T4 phages by re-analyzing 196 previously published viromes. These suggest that Far-T4 are common in freshwater and seawater as only four of 82 aquatic viromes lacked Far-T4-like sequences. Variability in representation across the five newly identified clades suggests clade-specific niche differentiation may be occurring across the different biomes, though the underlying mechanism remains unidentified. While complete genome assembly from complex communities and the lack of host linkage information still bottleneck virus discovery through viromes, these findings exemplify the power of metagenomics approaches to assess the diversity, evolutionary history, and genomic characteristics of novel uncultivated phages. PMID:25852662

  19. Genomic characteristics and environmental distributions of the uncultivated Far-T4 phages.

    PubMed

    Roux, Simon; Enault, François; Ravet, Viviane; Pereira, Olivier; Sullivan, Matthew B

    2015-01-01

    Viral metagenomics (viromics) is a tremendous tool to reveal viral taxonomic and functional diversity across ecosystems ranging from the human gut to the world's oceans. As with microbes however, there appear vast swaths of "dark matter" yet to be documented for viruses, even among relatively well-studied viral types. Here, we use viromics to explore the "Far-T4 phages" sequence space, a neighbor clade from the well-studied T4-like phages that was first detected through PCR study in seawater and subsequently identified in freshwater lakes through 454-sequenced viromes. To advance the description of these viruses beyond this single marker gene, we explore Far-T4 genome fragments assembled from two deeply-sequenced freshwater viromes. Single gene phylogenetic trees confirm that the Far-T4 phages are divergent from the T4-like phages, genome fragments reveal largely collinear genome organizations, and both data led to the delineation of five Far-T4 clades. Three-dimensional models of major capsid proteins are consistent with a T4-like structure, and highlight a highly conserved core flanked by variable insertions. Finally, we contextualize these now better characterized Far-T4 phages by re-analyzing 196 previously published viromes. These suggest that Far-T4 are common in freshwater and seawater as only four of 82 aquatic viromes lacked Far-T4-like sequences. Variability in representation across the five newly identified clades suggests clade-specific niche differentiation may be occurring across the different biomes, though the underlying mechanism remains unidentified. While complete genome assembly from complex communities and the lack of host linkage information still bottleneck virus discovery through viromes, these findings exemplify the power of metagenomics approaches to assess the diversity, evolutionary history, and genomic characteristics of novel uncultivated phages. PMID:25852662

  20. Engineering bacteriophage P22 as a nanomaterial

    NASA Astrophysics Data System (ADS)

    O'Neil, Alison Linsley

    The precise architectures of viruses and virus-like particles are highly advantageous in synthetic materials applications. These nano-size compartments are perfectly suited to act as containers of designed cargo. Not only can these nano-containers be harnessed as active materials, but they can be exploited for examining the effects of in vivo "cell-like" crowding and confinement on the properties of the encapsulated cargo. The high concentration of many different types of mutually volume excluding macromolecules in the cell causes it to be a crowded and confining environment in which to carry out reactions. Herein, the molecular design of the bacteriophage P22 encapsulation system is described and utilized for the synthesis of active nanomaterials and to explore the effect of encapsulation on the entrapped proteins' properties. In the designed system, any gene can be inserted and results in the fusion of the insert to a truncated form of the P22 scaffold protein. This scaffold protein fusion templates the spontaneous in vivo assembly of P22 capsids and also acts as an encapsulation signal. Once encapsulated, we can examine how crowding and confinement affect inter-molecular communication and activity of the cargo molecules. The P22 system is unique in that the capsid morphology can be altered, without losing the encapsulated cargo, resulting in a doubling of the capsid volume. Thus, the encapsulated fusions can be examined at two different internal concentrations. The packaged capsids contain up to 300 copies of fusion and fill more than 24% of the internal volume with the internal concentration of the fusions in the millimolar range. Not only are these fusions densely and efficiently packaged, but they retain their activity. Described herein is the packaging of fluorescent proteins, enzymes, and active peptides. In all cases, it is shown that the enforced proximity via encapsulation greatly affects the fusions activity compared to the fusion free in solution. To expand the utility of the P22 capsid as a nanomaterial, the inherent asymmetry implored by the portal complex has also been exploited. The P22 encapsulation system has proved to be an effective and versatile vehicle for nanomaterials design.

  1. The presence of peptidoglycan O-acetyltransferase in various staphylococcal species correlates with lysozyme resistance and pathogenicity.

    PubMed

    Bera, Agnieszka; Biswas, Raja; Herbert, Silvia; Götz, Friedrich

    2006-08-01

    Human-pathogenic bacteria that are able to cause persistent infections must have developed mechanisms to resist the immune defense system. Lysozyme, a cell wall-lytic enzyme, is one of the first defense compounds induced in serum and tissues after the onset of infection. Recently, we showed that Staphylococcus aureus is resistant to lysozyme by O acetylating its peptidoglycan (PG) by O-acetyltransferase (OatA). We asked the question of which staphylococcal species PG is O acetylated. We applied various methods, such as genome analysis, PCR, Southern blotting, lysozyme sensitivity assay, and verification of O acetylation of PG by high-performance liquid chromatography (HPLC) analysis. PCR analysis using S. aureus-derived oatA primers and Southern blotting did not yield reliable results with other staphylococcal species. Therefore, we used the HPLC-based assay to directly detect PG O acetylation. Our studies revealed that the muramic acid was O acetylated only in pathogenic, lysozyme-resistant staphylococci (e.g., S. aureus, S. epidermidis, S. lugdunensis, and others). All nonpathogenic species were lysozyme sensitive. They can be divided into sensitive species (e.g., S. carnosus, S. gallinarum, and S. xylosus) and hypersensitive species (e.g., S. equorum, S. lentus, and S. arlettae). In all lysozyme-sensitive species, the analyzed PG was de-O-acetylated. When we transformed the oatA gene from lysozyme-resistant S. aureus into S. carnosus, the corresponding transformants also became lysozyme resistant. PMID:16861647

  2. The Presence of Peptidoglycan O-Acetyltransferase in Various Staphylococcal Species Correlates with Lysozyme Resistance and Pathogenicity

    PubMed Central

    Bera, Agnieszka; Biswas, Raja; Herbert, Silvia; Götz, Friedrich

    2006-01-01

    Human-pathogenic bacteria that are able to cause persistent infections must have developed mechanisms to resist the immune defense system. Lysozyme, a cell wall-lytic enzyme, is one of the first defense compounds induced in serum and tissues after the onset of infection. Recently, we showed that Staphylococcus aureus is resistant to lysozyme by O acetylating its peptidoglycan (PG) by O-acetyltransferase (OatA). We asked the question of which staphylococcal species PG is O acetylated. We applied various methods, such as genome analysis, PCR, Southern blotting, lysozyme sensitivity assay, and verification of O acetylation of PG by high-performance liquid chromatography (HPLC) analysis. PCR analysis using S. aureus-derived oatA primers and Southern blotting did not yield reliable results with other staphylococcal species. Therefore, we used the HPLC-based assay to directly detect PG O acetylation. Our studies revealed that the muramic acid was O acetylated only in pathogenic, lysozyme-resistant staphylococci (e.g., S. aureus, S. epidermidis, S. lugdunensis, and others). All nonpathogenic species were lysozyme sensitive. They can be divided into sensitive species (e.g., S. carnosus, S. gallinarum, and S. xylosus) and hypersensitive species (e.g., S. equorum, S. lentus, and S. arlettae). In all lysozyme-sensitive species, the analyzed PG was de-O-acetylated. When we transformed the oatA gene from lysozyme-resistant S. aureus into S. carnosus, the corresponding transformants also became lysozyme resistant. PMID:16861647

  3. "Click on the bidirectional switch": the aptasensor for simultaneous detection of lysozyme and ATP with high sensitivity and high selectivity.

    PubMed

    Chen, Feng; Cai, Changqun; Chen, Xiaoming; Chen, Chunyan

    2016-01-01

    A bifunctional and simple aptasensor was designed to one-spot simultaneously detect two analytes, lysozyme and ATP. The aptasensor was obtained by the electronic interaction between methyl violet (MV) and dsDNA. The dsDNA was obtained by hybridization of ATP aptamer and lysozyme aptamer. And we used the resonance light scattering (RLS) technique to detect the concentration of lysozyme and ATP. During the procedure of detection, the aptasensor works like a bidirectional switch, the corresponding side of the dsDNA will open when the target (lysozyme or ATP) "click" the aptamer, which results in corresponding RLS signal change. By the combination of the RLS technique, it is found that the changed RLS intensity was proportional to the concentration of lysozyme and ATP. The mixtures of ATP and lysozyme also met two binary function relations. The results indicated that the aptasensor could achieve simultaneous detection of ATP and lysozyme, the detection limits of ATP and lysozyme could reach 10(-11)?M and 10(-12)?M, respectively. The aptasensor shows potential application for small molecule and protein detection by RLS, it could extend the application of RLS technique. PMID:26742854

  4. CRISPR/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4

    E-print Network

    Yaung, Stephanie J.

    Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain ...

  5. [Lysozyme-antilysozyme interactions in protozoa-bacteria communities (a model Tetrahymena-Escherichia community)].

    PubMed

    Bukharin, O V; Nemtseva, N V

    2001-01-01

    Lysozyme and antilysozyme activities present in a wide range of microorganisms determine the so-called lysozyme-antilysozyme system of hydrobionts, which greatly contribute to the formation of aquatic biocenoses. However, the mechanism of the functioning of this system in natural freshwater communities remains obscure. The experimental investigation of lysozyme-antilysozyme interactions in a model Tetrahymena--Escherichia community showed that the antilysozyme activity of Escherichia coli leads to incomplete phagocytosis, thus enhancing bacterial survival in a mixed culture with infusoria. The selection and reproduction of bacterial cells resistant to grazing by infusoria determine the character of host-parasite interactions and allow bacteria to survive. It was demonstrated that the antilysozyme activity of microorganisms, which is responsible for bacterial persistency in natural biocenoses, is involved in the maintenance of protozoa-bacteria communities in bodies of water. PMID:11763786

  6. A zinc complex of heparan sulfate destabilises lysozyme and alters its conformation.

    PubMed

    Hughes, Ashley J; Hussain, Rohanah; Cosentino, Cesare; Guerrini, Marco; Siligardi, Giuliano; Yates, Edwin A; Rudd, Timothy R

    2012-09-01

    The naturally occurring anionic cell surface polysaccharide heparan sulfate is involved in key biological activities and is implicated in amyloid formation. Following addition of Zn-heparan sulfate, hen lysozyme, a model amyloid forming protein, resembled ?-rich amyloid by far UV circular dichroism (increased ?-sheet: +25%), with a significantly reduced melting temperature (from 68 to 58 °C) by fluorescence shift assay. Secondary structure stability of the Zn-heparan sulfate complex with lysozyme was also distinct from that with heparan sulfate, under stronger denaturation conditions using synchrotron radiation circular dichroism. Changing the cation associated with heparan sulfate is sufficient to alter the conformation and stability of complexes formed between heparan sulfate and lysozyme, substantially reducing the stability of the protein. Complexes of heparan sulfate and cations, such as Zn, which are abundant in the brain, may provide alternative folding routes for proteins. PMID:22884801

  7. Lysozyme Net Charge and Ion Binding in Concentrated Aqueous Electrolyte Solutions

    SciTech Connect

    Kuehner, Daniel E.; Engmann, Jan; Fergg, Florian; Wernick, Meredith; Blanch, Harvey W.; Prausnitz, John M.

    1999-02-01

    Hydrogen-ion titrations were conducted for hen-egg-white lysozyme in solutions of potassium chloride, over the range of pH 2.5 - 11.5 and for ionic strengths to 2. 0 M. The dependence of lysozyme's net proton charge, zP' on pH and ionic-strength in potassium-chloride solution is measured. From the ionic-strength dependence of zP' interactions of lysozynie with potassium and chloride ions are calculated using the molecular-thennodynamic theory of Fraaije and Lyklema 1. Lysozyme interacts preferentially with up to 12 chloride ions at pH 2.5. The observed dependence of ion-protein interactions on pH and ionic strength is explained in terms of electricdouble-layer theory. New experimental pKa data are reported for eleven ammo acids in potassium-chloride solutions of ionic strength to 3.0 M.

  8. Study on the conformation changes of Lysozyme induced by Hypocrellin A: The mechanism investigation

    NASA Astrophysics Data System (ADS)

    Ma, Fei; Huang, He-Yong; Zhou, Lin; Yang, Chao; Zhou, Jia-Hong; Liu, Zheng-Ming

    2012-11-01

    The interactions between Lysozyme and Hypocrellin A are investigated in details using time-resolved fluorescence, fourier transform infrared spectroscopy (FTIR), circular dichroism spectroscopy (CD), three-dimensional fluorescence spectra, and thermal gravimetric analysis (TGA) techniques. The results of time-resolved fluorescence suggest that the quenching mechanism is static quenching. FTIR and CD spectroscopy provide evidences of the reducing of ?-helix after interaction. Hypocrellin A could change the micro-environmental of Lysozyme according to hydrophobic interaction between the aromatic ring and the hydrophobic amino acid residues, and the altered polypeptide backbone structures induce the reduction of ?-helical structures. Moreover, TGA study further demonstrates the structure changes of Lysozyme on the effect of Hypocrellin A. This study could provide some important information for the derivatives of HA in pharmacy, pharmacology and biochemistry.

  9. Role of ionic liquids in protein refolding: native/fibrillar versus treated lysozyme

    E-print Network

    Sara Mangialardo; Lorenzo Gontrani; Ruggero Caminiti; Paolo Postorino

    2012-04-21

    Several ionic liquids (ILs) are known to revert aggregation processes and improve the in vitro refolding of denatured proteins. In this paper the capacity of a particular class of ammonium based ILs to act as refolding enhancers was tested using lysozyme as a model protein. Raman spectra of ILs treated fibrillar lysozyme as well as lysozyme in its native and fibrillar conformations were collected and carefully analyzed to characterize the refolding extent under the effect of the IL interaction. Results obtained confirm and largely extend the earlier knowledge on this class of protic ILs and indicate Ethyl Ammonium Nitrate (EAN) as the most promising additive for protein refolding. The experiment provides also the demonstration of the high potentiality of Raman spectroscopy as a comprehensive diagnostic tool in this field.

  10. Role of ionic liquids in protein refolding: native/fibrillar versus treated lysozyme

    E-print Network

    Mangialardo, Sara; Caminiti, Ruggero; Postorino, Paolo

    2012-01-01

    Several ionic liquids (ILs) are known to revert aggregation processes and improve the in vitro refolding of denatured proteins. In this paper the capacity of a particular class of ammonium based ILs to act as refolding enhancers was tested using lysozyme as a model protein. Raman spectra of ILs treated fibrillar lysozyme as well as lysozyme in its native and fibrillar conformations were collected and carefully analyzed to characterize the refolding extent under the effect of the IL interaction. Results obtained confirm and largely extend the earlier knowledge on this class of protic ILs and indicate Ethyl Ammonium Nitrate (EAN) as the most promising additive for protein refolding. The experiment provides also the demonstration of the high potentiality of Raman spectroscopy as a comprehensive diagnostic tool in this field.

  11. Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease.

    PubMed Central

    Hughey, V L; Johnson, E A

    1987-01-01

    Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant. The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C. botulinum and L. monocytogenes. PMID:3118808

  12. Preparation and Characterization of Fluorescent Derivatives of Lysozyme

    NASA Technical Reports Server (NTRS)

    Smith, Lori; Pusey, Marc

    1998-01-01

    Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. However, its use in macromolecular crystal growth studies is hampered by the necessity of preparing fluorescent derivatives where the probe does not markedly affect the crystal packing. Alternatively, one can prepare derivatives of limited utility if it is known that they will not affect the specific goals of a given study. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme, covalently attaching fluorescent probes to two different sites on the protein molecule. The first site is the side chain carboxyl group of ASP 101. Amine containing probes such as lucifer yellow, cascade blue, and 5- (2-aminoethyl) aminonapthalene-l-sulfonic acid (EDANS) have been attached using a carbodiimide coupling procedure. ASP 101 lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. This is supported by the fact that all such derivatives have been found to crystallize, with the crystals being fluorescent. Tetragonal crystals of the lucifer yellow derivative have been found to diffract to at least 1.9 A resolution. X-ray diffraction data has been acquired and we are now working on the structure of this derivative. The second group of derivatives is to the N-terminal amine group. The derivatization reaction is performed by using a succinimidyl ester of the probe to be attached. Fluorescent probes such as pyrene acetic acid, 5-carboxyfluorescein, and Oregon green have been attached to this site. We have had little success in crystallizing these derivatives, probably because this site is part of the contact region between the 43 helix chains. However, these sites do not interfere with formation of the 43 helices and the derivatives are suitable for study of their formation in solution. The derivatives are being characterized by steady state and lifetime fluorescence methods, and the presentation will discuss these results.

  13. The protective effect of salicylic acid on lysozyme against riboflavin-mediated photooxidation

    NASA Astrophysics Data System (ADS)

    Li, Kun; Wang, Hongbao; Cheng, Lingli; Zhu, Hui; Wang, Mei; Wang, Shi-Long

    2011-06-01

    As a metabolite of aspirin in vivo, salicylic acid was proved to protect lysozyme from riboflavin-mediated photooxidation in this study. The antioxidative properties of salicylic acid were further studied by using time-resolved laser flash photolysis of 355 nm. It can quench the triplet state of riboflavin via electron transfer from salicylic acid to the triplet state of riboflavin with a reaction constant of 2.25 × 10 9 M -1 s -1. Mechanism of antioxidant activities of salicylic acid on lysozyme oxidation was discussed. Salicylic acid can serve as a potential antioxidant to quench the triplet state of riboflavin and reduce oxidative pressure.

  14. Dynamic light scattering analysis of solutions from which lysozyme crystals grow

    NASA Technical Reports Server (NTRS)

    Bishop, J. B.; Fredericks, W. J.; Howard, S. B.; Sawada, T.

    1992-01-01

    The variation of the friction factor was measured by photon correlation spectroscopy during isothermal growth of chicken egg white lysozyme crystals. It was observed to initially increase to a maximum as crystals formed, and then to decrease to a constant value. The change in the friction factors was measured as a function of temperature at concentrations of lysozyme varying from one sufficiently low that molecular interactions were undetectable to concentrations near those at which crystals grow in solutions, in all cases the ionic strength was below that required for crystallization.

  15. Measurement of serum T4 concentration by high performance liquid chromatography.

    PubMed

    Burman, K D; Bongiovanni, R; Garis, R K; Wartofsky, L; Boehm, T M

    1981-11-01

    Although RIA techniques for the measurement of serum T4 have been extremely useful, this methodology has several disadvantages, including the requirement for the use of radioisotopes, various levels of thyronine cross-reactivity, and the ability to measure only a single iodothyronine in one assay. We have developed a high performance liquid chromatography (HPLC) method for quantitating serum T4 that utilizes the detection of dansyl-T4 compounds and obviates the problems described for RIA techniques. Serum samples were extracted with ethanol and then chloroform, reacted with dansyl chloride, and, after n-heptane extraction, placed directly on column. Utilizing this technique, dansyl-T4 was easily separated and identified. The sensitivity of detection of the dansylated-T4 in serum was 1 microgram/dl, and linearity was observed when increasing standard T4 concentrations were employed. Sensitivity to 10 ng/dl (100 fmol on column) was achieved when T4 was added to buffer. The coefficients of variation were 4.8% and 2.1% for normal and high serum samples, respectively. When 39 random serum samples were anayzed both by HPLC and RIA, there was concordance of these techniques, since the derived correlation coefficient was 0.94. In summary, the present study demonstrates that serum T4 concentrations can be measured by HPLC and that these measurements agree remarkably well with those obtained by RIA. Because of the inherent advantages of HPLC methodology over that of RIA, this technique of measurement of T4 may have wide applicability to the measurement of iodothyronines. PMID:7287878

  16. A new bacteriophage, which infects acidophilic, heterotrophic bacteria from acidic mining environments

    SciTech Connect

    Ward, T.E.; Bruhn, D.F.; Bulmer, D.K.; Watkins, C.S.; Rowland, M.L.; Winston, V.

    1989-01-01

    The genetic characteristics of members of the genus Acidiphilium are poorly understood. As part of our study of the genetics of these bacteria, a search was made for an endogenous bacteriophage. Such a bacteriophage has been discovered. Several properties of the phage have been investigated. The phage has a lambdoid morphology and is somewhat larger than lambda. A variety of factors which affect phage stability have been investigated. The bacteriophage infects several of the strains that have been tested. Study of this bacteriophage should greatly increase our understanding of genetic mechanisms in Acidiphilium. 20 refs., 3 figs., 1 tab.

  17. Bacteriophage-based synthetic biology for the study of infectious diseases

    PubMed Central

    Lu, Timothy K.

    2014-01-01

    Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic engineering and synthetic biology. Here, we discuss bacteriophage-based technologies and their application to the study of infectious diseases. New strategies for engineering genomes have the potential to accelerate the design of novel phages as therapies, diagnostics, and tools. Though almost a century has elapsed since their discovery, bacteriophages continue to have a major impact on modern biological sciences, especially with the growth of multidrug-resistant bacteria and interest in the microbiome. PMID:24997401

  18. Methods for generation of reporter phages and immobilization of active bacteriophages on a polymer surface

    NASA Technical Reports Server (NTRS)

    Applegate, Bruce Michael (Inventor); Perry, Lynda Louise (Inventor); Morgan, Mark Thomas (Inventor); Kothapalli, Aparna (Inventor)

    2012-01-01

    Novel reporter bacteriophages are provided. Provided are compositions and methods that allow bacteriophages that are used for specific detection or killing of E. coli 0157:H7 to be propagated in nonpathogenic E. coli, thereby eliminating the safety and security risks of propagation in E. coli 0157:H7. Provided are compositions and methods for attaching active bacteriophages to the surface of a polymer in order to kill target bacteria with which the phage comes into contact. Provided are modified bacteriophages immobilized to a surface, which capture E. coli 0157:H7 and cause the captured cells to emit light or fluorescence, allowing detection of the bacteria in a sample.

  19. Removal of particle-associated bacteriophages by dual-media filtration at different filter cycle stages and impacts on subsequent UV disinfection.

    PubMed

    Templeton, Michael R; Andrews, Robert C; Hofmann, Ron

    2007-06-01

    This bench-scale study investigated the passage of particle-associated bacteriophage through a dual-media (anthracite-sand) filter over a complete filter cycle and the effect on subsequent ultraviolet (UV) disinfection. Two model viruses, bacteriophages MS2 and T4, were considered. The water matrix was de-chlorinated tap water with either kaolin or Aldrich humic acid (AHA) added and coagulated with alum to form floc before filtration. The turbidity of the influent flocculated water was 6.4+/-1.5 NTU. Influent and filter effluent turbidity and particle counts were measured as well as headloss across the filter media. Filter effluent samples were collected for phage enumeration during three filter cycle stages: (i) filter ripening; (ii) stable operation; and (iii) end of filter cycle. Stable filter operation was defined according to a filter effluent turbidity goal of <0.3 NTU. Influent and filter effluent samples were subsequently exposed to UV light (254 nm) at 40 mJ/cm(2) using a low pressure UV collimated beam. The study found statistically significant differences (alpha=0.05) in the quantity of particle-associated phage present in the filter effluent during the three stages of filtration. There was reduced UV disinfection efficiency due to the presence of particle-associated phage in the filter effluent in trials with bacteriophage MS2 and humic acid floc. Unfiltered influent water samples also resulted in reduced UV inactivation of phage relative to particle-free control conditions for both phages. Trends in filter effluent turbidity corresponded with breakthrough of particle-associated phage in the filter effluent. The results therefore suggest that maintenance of optimum filtration conditions upstream of UV disinfection is a critical barrier to particle-associated viruses. PMID:17433406

  20. Bacteriophages and their implications on future biotechnology: a review

    PubMed Central

    2012-01-01

    Recently it has been recognized that bacteriophages, the natural predators of bacteria can be used efficiently in modern biotechnology. They have been proposed as alternatives to antibiotics for many antibiotic resistant bacterial strains. Phages can be used as biocontrol agents in agriculture and petroleum industry. Moreover phages are used as vehicles for vaccines both DNA and protein, for the detection of pathogenic bacterial strain, as display system for many proteins and antibodies. Bacteriophages are diverse group of viruses which are easily manipulated and therefore they have potential uses in biotechnology, research, and therapeutics. The aim of this review article is to enable the wide range of researchers, scientists, and biotechnologist who are putting phages into practice, to accelerate the progress and development in the field of biotechnology. PMID:22234269

  1. The Molecular Genetics of Bacteriophage: The Work of Norton Zinder

    PubMed Central

    Kresge, Nicole; Simoni, Robert D.; Hill, Robert L.

    2011-01-01

    In 1966, Norton Zinder and Joshua Lederberg discovered that Salmonella could exchange genes via bacteriophages. They named this phenomenon “genetic transduction.” This discovery set Zinder on a lifelong journey researching bacteriophage. In the two Journal of Biological Chemistry (JBC) Classic papers reprinted here, Zinder and Nina Fedoroff present their findings on the phage f2 replicase. Properties of the Phage f2 Replicase. I. Optimal Conditions for Replicase Activity and Analysis of the Polynucleotide Product Synthesized in Vitro (Fedoroff, N. V., and Zinder, N. D. (1972) J. Biol. Chem. 247, 4577–4585) Properties of the Phage f2 Replicase. II. Comparative Studies on the Ribonucleic Acid-dependent and Poly(C)-dependent Activities of the Replicase (Fedoroff, N. V., and Zinder, N. D. (1972) J. Biol. Chem. 247, 4586–4592) PMID:21830328

  2. Sequence and comparative analysis of Leuconostoc dairy bacteriophages.

    PubMed

    Kot, Witold; Hansen, Lars H; Neve, Horst; Hammer, Karin; Jacobsen, Susanne; Pedersen, Per D; Sørensen, Søren J; Heller, Knut J; Vogensen, Finn K

    2014-04-17

    Bacteriophages attacking Leuconostoc species may significantly influence the quality of the final product. There is however limited knowledge of this group of phages in the literature. We have determined the complete genome sequences of nine Leuconostoc bacteriophages virulent to either Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides strains. The phages have dsDNA genomes with sizes ranging from 25.7 to 28.4 kb. Comparative genomics analysis helped classify the 9 phages into two classes, which correlates with the host species. High percentage of similarity within the classes on both nucleotide and protein levels was observed. Genome comparison also revealed very high conservation of the overall genomic organization between the classes. The genes were organized in functional modules responsible for replication, packaging, head and tail morphogenesis, cell lysis and regulation and modification, respectively. No lysogeny modules were detected. To our knowledge this report provides the first comparative genomic work done on Leuconostoc dairy phages. PMID:24561391

  3. Combined Use of Bacteriophage K and a Novel Bacteriophage To Reduce Staphylococcus aureus Biofilm Formation

    PubMed Central

    Alves, D. R.; Gaudion, A.; Bean, J. E.; Perez Esteban, P.; Arnot, T. C.; Harper, D. R.; Kot, W.; Hansen, L. H.; Enright, M. C.

    2014-01-01

    Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms. PMID:25149517

  4. Steel tanks T5 and T4 with overhead pipeline between. Redwood ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Steel tanks T5 and T4 with overhead pipeline between. Redwood tanks seen in background - Hawaii Volcanoes National Park Water Collection System, Hawaii Volcanoes National Park, Volcano, Hawaii County, HI

  5. Effects of Microheterogeneity in Hen Egg-White Lysozyme Crystallization

    NASA Technical Reports Server (NTRS)

    Thomas, B. R.; Vekilov, P. G.; Rosenberger, F.

    1998-01-01

    In earlier sodium dodecylsulfate polyacylamide gel electrophoresis (SDS-PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026-0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS-PAGE analysis of OHEWL yielded two closely spaced dimer bands with M(sub r) = 28 000 and 27 500. In addition, larger HEWL oligomers with M, = 1.7 million and 320 000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 microgram(exp -1) mg ml(exp -1) OHEWL to 32 mg ml(exp -1) HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03-9,3 M Hydrogen peroxide. Repartioning of the dimer during crystallzation aat various temperatures between 277 and 293 K was analyzed by SDS-PAGE. The crystals contained <= 25 % weight by volume of the oligomers in the solution, with no apparent temperature dependence of the repartioning.

  6. A novel convenient method for high bacteriophage titer assay.

    PubMed

    Yoshida, Kazuo; Nasu, Yuki; Shitami, Nozomi; Toyoda, Hitoko; Takemura, Hitomi; Oomori, Kiyomi

    2009-01-01

    The recent various applications of phages (bacteriophages) including phage therapy have brought about a revival of phage investigation. The phage titer assay is indispensable for phage experiments. However, the conventional standard method is a plaque counting method which requires a little skill with tedious repeating operation. Furthermore, it is not directly applicable to high phage titers. In this paper, we describe a novel convenient "cross streak and paper disk assay method" for high titer concentration without plaque counting. PMID:19749387

  7. Alternatives to antibiotics: utilization of bacteriophage to treat colibacillosis and prevent foodborne pathogens.

    PubMed

    Huff, W E; Huff, G R; Rath, N C; Balog, J M; Donoghue, A M

    2005-04-01

    Bacteriophages are viruses that infect and kill bacteria. Bacteriophage do not infect animal and plant cells, which makes them a potentially safe alternative to antibiotics. We have been conducting research on the efficacy of bacteriophage to prevent and treat colibacillosis in poultry. Bacteriophages that were lytic to a non-motile, serotype 02 isolate of Escherichia coli were isolated from municipal wastewater treatment plants and poultry processing plants. This E. coli isolate is pathogenic to poultry, causing severe respiratory and systemic infections. Two bacteriophage isolates were selected for use in studies designed to determine the efficacy of these bacteriophage to prevent and treat severe colibacillosis in poultry. Colibacillosis was induced by injecting 6 x 10(4) cfu of E. coli into the thoracic air sac when birds were 1 wk of age. Initial studies demonstrated that mortality was significantly reduced from 85 to 35% when the challenge culture was mixed with equal titers of bacteriophage, and the birds were completely protected when the challenge culture was mixed with 10 pfu of bacteriophage. In subsequent studies, we have shown that an aerosol spray of bacteriophage given to birds prior to this E. coli challenge could significantly reduce mortality even when given 3 d prior to the E. coli challenge. Our research on treating colibacillosis in poultry has demonstrated that an intramuscular injection of bacteriophage given 24 or 48 h after the birds were challenged rescued the birds from this severe E. coli infection. We have demonstrated that bacteriophage can be used to prevent and treat colibacillosis in poultry and may provide an effective alternative to antibiotic use in animal production. PMID:15844825

  8. Host adaption to the bacteriophage carrier state of Campylobacter jejuni

    PubMed Central

    Brathwaite, Kelly J.; Siringan, Patcharin; Connerton, Phillippa L.; Connerton, Ian F.

    2015-01-01

    The carrier state of the foodborne pathogen Campylobacter jejuni represents an alternative life cycle whereby virulent bacteriophages can persist in association with host bacteria without commitment to lysogeny. Host bacteria exhibit significant phenotypic changes that improve their ability to survive extra-intestinal environments, but exhibit growth-phase-dependent impairment in motility. We demonstrate that early exponential phase cultures become synchronised with respect to the non-motile phenotype, which corresponds with a reduction in their ability to adhere to and invade intestinal epithelial cells. Comparative transcriptome analyses (RNA-seq) identify changes in gene expression that account for the observed phenotypes: downregulation of stress response genes hrcA, hspR and per and downregulation of the major flagellin flaA with the chemotactic response signalling genes cheV, cheA and cheW. These changes present mechanisms by which the host and bacteriophage can remain associated without lysis, and the cultures survive extra-intestinal transit. These data provide a basis for understanding a critical link in the ecology of the Campylobacter bacteriophage. PMID:26004283

  9. A Bacteriophage-Related Chimeric Marine Virus Infecting Abalone

    PubMed Central

    Zhuang, Jun; Cai, Guiqin; Lin, Qiying; Wu, Zujian; Xie, Lianhui

    2010-01-01

    Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV) can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin). The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs), eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB) protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria. PMID:21079776

  10. Bacteriophage-insensitive mutants for high quality Crescenza manufacture.

    PubMed

    Chirico, Donatella; Gorla, Arianna; Verga, Viola; Pedersen, Per D; Polgatti, Eliseo; Cava, Antonio; Dal Bello, Fabio

    2014-01-01

    Streptococcus thermophilus is a thermophilic lactic acid bacterium used as starter culture for the manufacture of fermented dairy products. For the production of Crescenza and other soft cheeses, Sacco has developed and provides dairies with three different defined blends of S. thermophilus strains. Each blend contains two different S. thermophilus strains. The strains were selected based on their unique technological properties as well as different phage profiles. Analysis of 133 whey samples collected in 2009-2010 from Italian dairies showed a high prevalence (about 50%) of bacteriophage attacks on the blend ST020. More specifically, the strain S. thermophilus ST1A was found to be the preferred target of the bacteriophages. A bacteriophage insensitive mutant (BIM5) of the phage-sensitive strain ST1A was successfully developed and used to substitute strain ST1A in the Crescenza starter culture ST020. The strain BIM5 showed identical technological and industrial traits as those of the phage-sensitive strain ST1A. The improved resistance of the modified Crescenza starter culture ST020R was confirmed at Italian dairies, and its effectiveness monitored on 122 whey samples collected in 2011-2012. Compared to the previous values (2009-2010), the use of the phage-hardened blend ST020R allowed reducing of frequency of phage attacks from about 50 to less than 5% of the whey samples investigated. PMID:24834065

  11. Bacteriophage-insensitive mutants for high quality Crescenza manufacture

    PubMed Central

    Chirico, Donatella; Gorla, Arianna; Verga, Viola; Pedersen, Per D.; Polgatti, Eliseo; Cava, Antonio; Dal Bello, Fabio

    2014-01-01

    Streptococcus thermophilus is a thermophilic lactic acid bacterium used as starter culture for the manufacture of fermented dairy products. For the production of Crescenza and other soft cheeses, Sacco has developed and provides dairies with three different defined blends of S. thermophilus strains. Each blend contains two different S. thermophilus strains. The strains were selected based on their unique technological properties as well as different phage profiles. Analysis of 133 whey samples collected in 2009–2010 from Italian dairies showed a high prevalence (about 50%) of bacteriophage attacks on the blend ST020. More specifically, the strain S. thermophilus ST1A was found to be the preferred target of the bacteriophages. A bacteriophage insensitive mutant (BIM5) of the phage-sensitive strain ST1A was successfully developed and used to substitute strain ST1A in the Crescenza starter culture ST020. The strain BIM5 showed identical technological and industrial traits as those of the phage-sensitive strain ST1A. The improved resistance of the modified Crescenza starter culture ST020R was confirmed at Italian dairies, and its effectiveness monitored on 122 whey samples collected in 2011–2012. Compared to the previous values (2009–2010), the use of the phage-hardened blend ST020R allowed reducing of frequency of phage attacks from about 50 to less than 5% of the whey samples investigated. PMID:24834065

  12. Temperate bacteriophages collected by outer membrane vesicles in Komagataeibacter intermedius.

    PubMed

    Kharina, Alla; Podolich, Olga; Faidiuk, Iuliia; Zaika, Sergiy; Haidak, Andriy; Kukharenko, Olga; Zaets, Iryna; Tovkach, Fedor; Reva, Oleg; Kremenskoy, Maxim; Kozyrovska, Natalia

    2015-04-01

    The acetic acid bacteria have mainly relevance for bacterial cellulose production and fermented bio-products manufacture. The purpose of this study was to identify temperate bacteriophages in a cellulose-producing bacterial strain Komagataeibacter intermedius IMBG180. Prophages from K. intermedius IMBG180 were induced with mitomycin C and nalidixic acid. Transmission electron microscopy analysis exhibited tailed bacteriophages belonging to Myoviridae. A PCR assay targeting the capsid gene of the myoviruses proved phylogenetic position of induced phages. Nalidixic acid was poor inducer of prophages, however, it induced the OMV-like particles release. Size of OMVs depended on an antibiotic applied for phage induction and varied in the range of 30-80 and 120-200?nm. Inside some of them, tails of phages have been visible. Under conditions, inducing prophages, OMVs acted as the collectors of formed phage particles, using outer membrane receptors for phage detection (in this case, outer membrane siderophore receptor), and fulfilled therefore "a cleaning," as well as defensive functions, preventing bacteriophage spread outside population. This is the first description of myoviruses affiliated to K. intermedius, as well as outer membrane vesicles interaction with phages within this host. PMID:25589010

  13. Structure of T4moF, the Toluene 4-Monooxygenase Ferredoxin Oxidoreductase.

    PubMed

    Acheson, Justin F; Moseson, Hannah; Fox, Brian G

    2015-09-29

    The 1.6 Å crystal structure of toluene 4-monooxygenase reductase T4moF is reported. The structure includes ferredoxin, flavin, and NADH binding domains. The position of the ferredoxin domain relative to the other two domains represents a new configuration for the iron-sulfur flavoprotein family. Close contacts between the C8 methyl group of FAD and [2Fe-2S] ligand Cys36-O represent a plausible pathway for electron transfer between the redox cofactors. Energy-minimized docking of NADH and calculation of hingelike motions between domains suggest how simple coordinated shifts of residues at the C-terminus of the enzyme could expose the N5 position of FAD for productive interaction with the nicotinamide ring. The domain configuration revealed by the T4moF structure provides an excellent steric and electrostatic match to the obligate electron acceptor, Rieske-type [2Fe-2S] ferredoxin T4moC. Protein-protein docking and energy minimization of the T4moFC complex indicate that T4moF [2Fe-2S] ligand Cys41 and T4moC [2Fe-2S] ligand His67, along with other electrostatic interactions between the protein partners, form the functional electron transfer interface. PMID:26309236

  14. Phage T4-induced DNA breaks activate a tRNA repair-defying anticodon nuclease.

    PubMed

    Bitton, Lital; Klaiman, Daniel; Kaufmann, Gabriel

    2015-09-01

    The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4-excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction-modification (RM) protein and turned on by a phage-encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double-stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi?RloC expressed in Escherichia coli is activated by wild-type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction. PMID:26031711

  15. An N-acetyllactosamine-specific lectin, PFA, isolated from a moth (Phalera flavescens), structurally resembles an invertebrate-type lysozyme.

    PubMed

    Yokoyama, Kazutaka; Sato, Michihiko; Haneda, Toshihiro; Yamazaki, Kentaro; Kitano, Takashi; Umetsu, Kazuo

    2014-11-01

    PFA (Phalera flavescens agglutinin) lectin purified from larvae of the lobster moth (P. flavescens) shows a strong binding ability specific to the N-acetyllactosamine (Gal?1-4GlcNAc) site. We determined the genomic and cDNA sequences of the PFA gene, which consists of five exons and spans approximately 5 kb of a genomic region. Surprisingly, the amino acid sequence (149 amino acids) was similar to invertebrate-type lysozymes and related proteins. The predicted tertiary structure of the PFA protein was similar to the lysozymes of clams such as the common orient clam (Meretrix lusoria) and Japanese littleneck (Venerupis philippinarum (Tapes japonica)). The PFA, however, lacks a catalytically essential amino acid, an Asp (D), which is one of the two important amino acids (Glu (E) and D) express the function of lysozyme. As a result, lysozyme activity assays indicated that PFA does not have lysozyme activity. Results suggest that the PFA gene evolved from a lysozyme gene through the loss of lysozyme activity sites and the acquisition of lectin activity during evolution of the genus Phalera. PMID:25257940

  16. Efficient purification of lysozyme from egg white by 2-mercapto-5-benzimidazolesulfonic acid modified Fe3O4/Au nanoparticles.

    PubMed

    Zhu, Xinjun; Zhang, Lianying; Fu, Aiyun; Yuan, Hao

    2016-02-01

    2-Mercapto-5-benzimidazolesulfonic acid (MBISA) modified Fe3O4/Au nanoparticles were synthesized in aqueous solution and characterized by photo correlation spectroscopy (PCS) and vibrating sample magnetometer (VSM). The so-obtained Fe3O4/Au-MBISA nanoparticles were capable of specific adsorbing lysozyme. The maximum amount of lysozyme adsorbed on 1.0mg Fe3O4/Au-MBISA nanoparticles was 346?g. The lysozyme desorption behavior was studied and the lysozyme recovery from Fe3O4/Au-MBISA nanoparticles approached 100% under optimal conditions, and the reusability studies showed that the nanoparticles could maintain about 91% of the initial lysozyme adsorption capacity after 7 repeated adsorption-elution cycles. The Fe3O4/Au-MBISA nanoparticles were used in the purification of lysozyme from chicken egg white, which was verified by a single SDS-PAGE band. Therefore, the obtained Fe3O4/Au-MBISA nanoparticles exhibited excellent performance in the direct purification of lysozyme from egg white. PMID:26652366

  17. Logarithmic Decay in Single-Particle Relaxation of Hydrated Lysozyme Powder Marco Lagi,1,2

    E-print Network

    Chen, Sow-Hsin

    -dynamics of protein amino acids of hydrated lysozyme powder around the physiological temperature by means of molecular dynamics simulations. The self-intermediate scattering functions of the amino acid residue center of mass, 61.20.Ja, 64.70.kj It is well known that the dynamics of native globular proteins has much in common

  18. Synthesis of pH- and ionic strength-responsive microgels and their interactions with lysozyme.

    PubMed

    Zhang, Bao; Sun, Binghua; Li, Xiaoxiao; Yu, Yun; Tian, Yaoqi; Xu, Xueming; Jin, Zhengyu

    2015-08-01

    Microgels composed of carboxymethyl cellulose (CMC) polymers via chemical crosslinking with sodium trimetaphosphate were synthesized and characterized using thermogravimetric analysis (TGA), swelling, and rheological analysis. The effects of pH, ionic strength, and crosslinking density on lysozyme loading in microgels were also studied. The microgel particle size ranged primarily from 10 to 20 ?m. TGA revealed that the crosslinking increased the thermal stability of CMC. The swelling degree increased as pH increased from 3 to 5, and remained almost constant from pH 5 to 8. However, the swelling degree decreased with increasing ionic strength. The rheological analysis was in good agreement with the results of swelling degree. The protein uptake decreased with increasing ionic strength and crosslinking density. The pH 6 was the optimal pH for lysozyme absorption at ionic strength 0.05 M. The lysozyme-microgel complex was identified by confocal laser scanning microscopy, and the lysozyme distribution in the microgel was observed to be rather homogeneous. PMID:26001494

  19. Mechanic Insight into Aggregation of Lysozyme by Ultrasensitive Differential Scanning Calorimetry and Sedimentation Velocity.

    PubMed

    Wu, Sha; Ding, Yanwei; Zhang, Guangzhao

    2015-12-31

    Folding and aggregation of proteins profoundly influence their functions. We have investigated the effects of thermal history, concentration and pH on the denaturation and refolding of lysozyme by using ultrasensitive differential scanning calorimetry (US-DSC) and sedimentation velocity (SV) via analytical ultracentrifugation (AUC). The former is sensitive to small energy change whereas the latter can differentiate the oligomers such as dimer and trimer from individual protein molecules. Our studies reveal that the degree of denaturation irreversibility increases as heating times increases. The denaturation temperature (Td) and enthalpy change (?H) are influenced by heating rate since the denaturation is not in equilibrium during the heating. We can obtain Td and ?H in equilibrium by extrapolation of heating rate to zero. In a dilute solution, no aggregation but unfolding happens in the denaturation. However, when the concentration is above a critical value (?15.0 mg/mL), lysozyme molecules readily form trimers or other oligomers. Lysozyme molecules unfold into stretched chains at pH > 6.0, which would further forms large aggregates. The formation of aggregates makes the refolding of lysozyme impossible. PMID:26633732

  20. Lack of Evidence for Prenucleation Aggregate Formation in Lysozyme Crystal Growth Solutions

    NASA Technical Reports Server (NTRS)

    Muschol, Martin; Rosenberger, Franz

    1996-01-01

    There have been numerous claims of large concentrations of prenucleation aggregates in supersaturated as well as undersaturated lysozyme solutions at high salt concentrations. The presence of these aggregates was derived from measurements of the light or neutron scattering intensity, ultracentrifugation and dialysis behavior, as well as over-simplified crystal growth kinetics considerations. In all these interpretations it has been assumed that lysozyme solutions are either ideal or that protein interactions are independent of salt concentration. Contrary to these presumptions, our static and dynamic light scattering experiments provide evidence that lysozyme forms highly non-ideal, strongly interacting solutions. At low salt concentrations, the scattering intensities fall well below the values expected for an ideal, monomeric solution at the same protein concentration, while diffusivities increase with increasing protein concentration. Upon increase in salt concentration, these trends are eventually reversed. This enhancement in scattering intensity and decrease in diffusivity was widely interpreted as sign of aggregate formation. Yet, a quantitative interpretation of the scattering behavior over the whole salt concentration range can only be given in terms of a transition from net repulsion to net attraction between lysozyme monomers. Increased salt screening of the electrostatic repulsion among the protein macro-ions, together with attractive protein interactions, such as van der Waals, hydrophobic and hydration forces, provide an unambiguous mechanism for the observed transition and a more physical interpretation of the various observations.

  1. Characterization of a Novel Lysozyme-Like 4 Gene in the Rat

    PubMed Central

    Narmadha, Ganapathy; Muneswararao, Katakam; Rajesh, Angireddy; Yenugu, Suresh

    2011-01-01

    Lysozyme-like proteins (LYZLs) belong to the class of c-type lysozymes and are not well characterized in many species including the rat. In this study, using in silico and molecular biology techniques, we report the identification, cloning and characterization of rat Lyzl4 gene and also determine the expression pattern of Lyzl1, Lyzl3 and Lyzl6. The rat Lyzl genes were found to be distributed on three chromosomes and all of them retained the characteristic eight cysteine signature of c-type lysozyme. Homology modeling of rat LYZL4 indicated that its structure is similar to that of the mouse SLLP1. In the male reproductive tract of rat, Lyzl gene expression was confined to the testis. Lyzl1 and Lyzl4 were found to be expressed in tissues beyond the male reproductive tract, whereas Lyzl3 and Lyzl6 were not. Lyzl expression in the developing (10–60 day old) rats was androgen dependent in the testis. Immunodetection using antibodies against rat LYZL4 revealed the presence of LYZL4 protein in the germinal layer of the testes and on the sperm tail. Recombinant LYZL4 did not exhibit antibacterial, muramidase and isopeptidase activities characteristic to c-type lysozyme. To the best of our knowledge, for the first time we report the characterization of Lyzl genes in the rat. Results of our study indicate that rat LYZL proteins may have an important role in male reproductive tract function. PMID:22110709

  2. Lysozyme as an alternative to growth promoting antibiotics in swine production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lysozyme is a naturally occurring enzyme found in bodily secretions such as tears, saliva, and milk. It functions as an antimicrobial agent by cleaving the peptidoglycan component of bacterial cell walls, which leads to cell death. Antibiotics are also antimicrobials and have been fed at subtherape...

  3. Characterization of fish-skin gelatin gels and films containing the antomicrobial enzyme lysozyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish skins are rich in collagen and can be used to produce food-grade gelatin. Films cast from fish-skin gelatins are stable at room temperature and can act as a barrier when applied to foods. Lysozyme is a food-safe, antimicrobial enzyme that can also produce gels and films. When cold-water, fish-s...

  4. Expression of lysozyme in the life history of the house fly (Musca domestica L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    From egg to adult, all life history stages of house flies associate with septic environments teeming with bacteria. House fly lysozyme was first identified in the larval midgut, where it is used for digestion of microbe-rich meals due to its broad-spectrum activity against Gram positive and Gram neg...

  5. Experimental antibacterial therapy with puroindolines, lactoferrin and lysozyme in Listeria monocytogenes-infected mice.

    PubMed

    Palumbo, Daniela; Iannaccone, Marco; Porta, Amalia; Capparelli, Rosanna

    2010-07-01

    Puroindoline A and puroindoline B from plant seeds, bovine lactoferrin and chicken eggs lysozyme are antimicrobial proteins of innate immune system that lyse invading organisms. We investigate their potential antibacterial activity against Listeria monocytogenes in a mouse model. Bacteria were isolated from various organs for 7 days after challenge. Livers displayed consistently higher bacterial count (up to 10(7)cfu/g) than spleens, kidneys and brains. The efficacy of the AMPs was therefore established by measuring the infection level (cfu number) of these organs. Puroindoline A and puroindoline B (5mg/mouse), lactoferrin and lysozyme (1.25mg/mouse), intravenously injected individually, inhibited bacterial growth completely. Puroindoline A, puroindoline B and lactoferrin were effective when administered 24h before infection; lysozyme was effective at the time of infection or 5 days after. Their combined use resulted in the enhancement of individual antibacterial activities. Complete inhibition of bacterial growth was observed using concurrently 0.059mg/mouse of puroindoline A and 0.019mg/mouse of puroindoline B, lactoferrin and lysozyme. Individual antimicrobial proteins reduced significantly the expression level of pro-inflammatory cytokines (IL-6, IL-8, INF-gamma and TNF-alpha), acute phase proteins (C-reactive protein and fibrinogen) and the T lymphocyte antigens CD4, CD8a, CD8b and CD25. These results suggest their potential use for the control of L. monocytogenes infections. PMID:20348006

  6. Effect of lysozyme or antibiotics on fecal zoonotic pathogens in nursery pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to determine the effect of lysozyme and antibiotics on zoonotic pathogen shedding in feces from nursery pigs housed without and with an indirect disease challenge. Two replicates of 600 pigs each were weaned and randomly assigned to one of 24 pens in either a nursery...

  7. Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory

    ERIC Educational Resources Information Center

    Schwinefus, Jeffrey J.; Schaefle, Nathaniel J.; Muth, Gregory W.; Miessler, Gary L.; Clark, Christopher A.

    2008-01-01

    As part of an effort to infuse our physical chemistry laboratory with biologically relevant, investigative experiments, we detail four integrated thermodynamic experiments that characterize the denaturation (or unfolding) and self-interaction of hen egg white lysozyme as a function of pH and ionic strength. Students first use Protein Explorer to…

  8. The Effect of Ethylene Glycol, Glycine Betaine, and Urea on Lysozyme Thermal Stability

    ERIC Educational Resources Information Center

    Schwinefus, Jeffrey J.; Leslie, Elizabeth J.; Nordstrom, Anna R.

    2010-01-01

    The four-week student project described in this article is an extension of protein thermal denaturation experiments to include effects of added cosolutes ethylene glycol, glycine betaine, and urea on the unfolding of lysozyme. The transition temperatures and van't Hoff enthalpies for unfolding are evaluated for six concentrations of each cosolute,…

  9. Spectroscopic studies on the interaction between novel polyvinylthiol-functionalized silver nanoparticles with lysozyme

    NASA Astrophysics Data System (ADS)

    Ali, Mohd. Sajid; Al-Lohedan, Hamad A.; Rafiquee, M. Z. A.; Atta, Ayman M.; Ezzat, Abdurrahman O.

    2015-01-01

    Silver nanoparticles were functionalized with polyvinylthiol (Ag-PVT) and their effect on the conformation of hen-egg white lysozyme was seen by means of spectroscopic techniques, viz., UV visible, fluorescence (intrinsic and synchronous), resonance Rayleigh scattering and circular dichroism. UV absorption spectra of lysozyme show a hyperchromic shift on the addition of Ag-PVT nanoparticles indicating the complex formation between the two. The interaction between lysozyme and Ag-PVT nanoparticles was takes place via static quenching with 1:1 binding ratio as revealed by the analysis of fluorescence measurements. Circular dichroism spectroscopic data show a decrease in ?-helical content of lysozyme on interaction with Ag-PVT nanoparticles which was due to the partial unfolding of the protein. Synchronous fluorescence spectroscopy disclosed that the microenvironments of both tryptophan and tyrosine residues were perturbed in the presence of Ag-PVT nanoparticles and perturbation in the tryptophan environment was more prominent. Rayleigh scattering (RRS) intensity increases on increasing the Ag-PVT nanoparticles concentration till it reaches to the saturation. The RRS intensity increases four times as compared to the native protein indicating the possibility of protein aggregation at higher concentrations of nanoparticles.

  10. Lysozyme gene expression by hemocytes of Pacific white shrimp, Litopenaeus vannamei, after injection with Vibrio

    E-print Network

    Burnett, Louis E.

    injection with Vibrio Erin J. Burge*, Daniel J. Madigan, Louis E. Burnett, Karen G. Burnett Grice Marine and in circulating hemocytes for 48 h following challenge with the shrimp pathogen Vibrio campbellii Baumann rights reserved. Keywords: Litopenaeus vannamei; Shrimp; Hemocyte; Vibrio; Lysozyme; Gene expression

  11. Gene, 65 (1988) 259-268 Purification and characterization of the DNA-binding protein Ner of bacteriophage Mu

    E-print Network

    Clore, G. Marius

    1988-01-01

    -binding protein Ner of bacteriophage Mu (Recombinant DNA; phage I pL promoter; c repressor; clear-plaque phenotype directs the high-level production of the bacteriophage Mu Ner protein in Escherichia cob. The protein to synthesise a functional Mu repressor. INTRODUCTION The Ner protein of bacteriophage Mu consists of the 74 aa

  12. From the Cover: Single M13 bacteriophage tethering and stretching Matthew J. Lang, and Angela M. Belcher

    E-print Network

    Lang, Matthew

    From the Cover: Single M13 bacteriophage tethering and stretching Matthew J. Lang, and Angela M.pnas.org/misc/reprints.shtml To order reprints, see: Notes: #12;Single M13 bacteriophage tethering and stretching Ahmad S. Khalil biomolecules on the highly organized struc- ture of M13 filamentous bacteriophage is a unique advantage. Where

  13. The effect of genome length on ejection forces in bacteriophage lambda Paul Grayson a,, Alex Evilevitch b,1

    E-print Network

    Purohit, Prashant

    The effect of genome length on ejection forces in bacteriophage lambda Paul Grayson a,, Alex into protein capsids that are barely large enough to enclose the genome. In particular, in bacteriophages of the ejection forces for two different mutants of bacteriophage , b221cI26 and cI60, which differ in genome

  14. Forces during Bacteriophage DNA Packaging and Ejection Prashant K. Purohit,* Mandar M. Inamdar,* Paul D. Grayson,y

    E-print Network

    Phillips, Rob

    Forces during Bacteriophage DNA Packaging and Ejection Prashant K. Purohit,* Mandar M. InamdarDNA bacteriophages and show that the bending elasticity of DNA and its electrostatics in solution can be combined packaging. Finally, we present a model for DNA ejection from bacteriophages based on the hypothesis

  15. Determinants of bacteriophage P22 polyhead formation: the role of coat protein flexibility in conformational switchingmmi_7311 1568..1582

    E-print Network

    Baker, Timothy S.

    Determinants of bacteriophage P22 polyhead formation: the role of coat protein flexibility determinants of polyhead for- mation in bacteriophage P22 in order to understand the molecular mechanism unique, virally encoded proteins. Many double-stranded DNA (dsDNA) viruses, such as bacteriophage P22

  16. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 2011-07-01 false Escherichia coli O157:H7 specific bacteriophages; temporary...Tolerances § 180.1301 Escherichia coli O157:H7 specific bacteriophages...bacteriophages that are specific to Escherichia coli O157:H7, sequence negative...

  17. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 2014-07-01 false Escherichia coli O157:H7 specific bacteriophages; temporary...Tolerances § 180.1301 Escherichia coli O157:H7 specific bacteriophages...bacteriophages that are specific to Escherichia coli O157:H7, sequence negative...

  18. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 2012-07-01 false Escherichia coli O157:H7 specific bacteriophages; temporary...Tolerances § 180.1301 Escherichia coli O157:H7 specific bacteriophages...bacteriophages that are specific to Escherichia coli O157:H7, sequence negative...

  19. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 2013-07-01 false Escherichia coli O157:H7 specific bacteriophages; temporary...Tolerances § 180.1301 Escherichia coli O157:H7 specific bacteriophages...bacteriophages that are specific to Escherichia coli O157:H7, sequence negative...

  20. Expression of Recombinant Human Lysozyme in Egg Whites of Transgenic Hens

    PubMed Central

    Cao, Dainan; Wu, Hanyu; Li, Qingyuan; Sun, Yingmin; Liu, Tongxin; Fei, Jing; Zhao, Yaofeng; Wu, Sen; Hu, Xiaoxiang; Li, Ning

    2015-01-01

    Chicken egg lysozyme (cLY) is an enzyme with 129 amino acid (AA) residue enzyme. This enzyme is present not only in chicken egg white but also in mucosal secretions such as saliva and tears. The antibacterial properties of egg white can be attributed to the presence of lysozyme, which is used as an anti-cancer drug and for the treatment of human immunodeficiency virus (HIV) infection. In this study, we constructed a lentiviral vector containing a synthetic cLY signal peptide and a 447 bp synthetic human lysozyme (hLY) cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. The transgene inserted in the chicken chromosomes directs the synthesis and secretion of hLY which has three times higher specific activity than cLY. Three G1 transgenic chickens were identified, the only female of which expressed recombinant human lysozyme (rhLY) at 57.66 ± 4.10 ?g/ml in the egg white and the G2 transgenic hens of the G1 transgenic cock A011 expressed rhLY at 48.72 ± 1.54 ?g/ml. This experiment demonstrated that transgenic hens with stable oviduct-specific expression of recombinant human lysozyme proteins can be created by microinjection of lentiviral vectors. The results of this research could be contribute to the technological development using transgenic hens as a cost-effective alternative to other mammalian systems, such as cow, sheep and goats, for the production of therapeutic proteins and other applications. PMID:25706123

  1. Sequential separation of lysozyme, ovomucin, ovotransferrin, and ovalbumin from egg white.

    PubMed

    Abeyrathne, E D N S; Lee, H Y; Ahn, D U

    2014-04-01

    Ovalbumin, ovotransferrin, ovomucin, and lysozyme are a few of the egg white proteins that can be used as functional components. The objective of this study was to develop a simple, sequential separation method for multiple proteins from egg white. Separated proteins are targeted for human use, and thus any toxic compounds were excluded. The methods for individual components and the sequential separation were practiced in laboratory scale first, and then tested for scale-up. Lysozyme was separated first using FPC3500 cation exchange resin and then ovomucin using isoelectric precipitation. Ovalbumin and ovotransferrin were separated from the lysozyme- and ovomucin-free egg white by precipitating ovotransferrin first using 5.0% (wt/vol) (NH4)2SO4 and 2.5% (wt/vol) citric acid combination. After centrifugation, the supernatant (S1) was used for ovalbumin separation and the precipitant was dissolved in water, and reprecipitated using 2.0% ammonium sulfate (wt/vol) and 1.5% citric acid (wt/vol) combination. The precipitant was used as ovotransferrin fraction, and the supernatant (S2) was pooled with the first supernatant (S1), desalted using ultrafiltration, and then heat-treated to remove impurities. The yield of ovomucin and ovalbumen was >98% and that of ovotransferrin and lysozyme was >82% for both laboratory and scale-up preparations. The SDS-PAGE and western blotting of the separated proteins, except for ovomucin, showed >90% purity. The ELISA results indicated that the activities of separated ovalbumin, ovotransferrin, and lysozyme were >96%. The protocol separated 4 major proteins in sequence, and the method was simple and easily scaled up. PMID:24706978

  2. 40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of a tolerance is established for residues of Xanthomonas campestris pv. vesicatoria and...

  3. The Genome Sequence of Bacteriophage CPV1 Virulent for Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents. P...

  4. ISOLATION AND USE OF BACTERIOPHAGE TO REDUCE E. COLI O157:H7 POPULATIONS IN RUMINANTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage are viruses that prey on bacteria and are specific to the species or even strain level. Bacteriophage that target the food-borne pathogen E. coli O157:H7 have been previously isolated from several environments. However, no systematic survey has been performed to determine the inciden...

  5. Inactivation of Recombinant Bacteriophage Lambda by Use of Chemical Agents and UV Radiation

    PubMed Central

    Clark, Ewan M.; Wright, Harry; Lennon, Kelly-Anne; Craik, Vicki A.; Clark, Jason R.

    2012-01-01

    Several approaches for the inactivation of bacteriophage lambda, including UV germicidal irradiation (UVGI) and the chemical agents Virkon-S, Chloros, Decon-90, and sodium hydroxide (NaOH), were compared. Virkon, NaOH, and UVGI caused a ?7-log10 reduction in phage titers. This study successfully describes several methods with potential for bacteriophage inactivation in industrial settings. PMID:22327583

  6. Inactivation of recombinant bacteriophage lambda by use of chemical agents and UV radiation.

    PubMed

    Clark, Ewan M; Wright, Harry; Lennon, Kelly-Anne; Craik, Vicki A; Clark, Jason R; March, John B

    2012-04-01

    Several approaches for the inactivation of bacteriophage lambda, including UV germicidal irradiation (UVGI) and the chemical agents Virkon-S, Chloros, Decon-90, and sodium hydroxide (NaOH), were compared. Virkon, NaOH, and UVGI caused a ?7-log(10) reduction in phage titers. This study successfully describes several methods with potential for bacteriophage inactivation in industrial settings. PMID:22327583

  7. Generation of random numbers on graphics processors: Forced indentation in silico of the bacteriophage HK97

    E-print Network

    Barsegov, Valeri

    of the bacteriophage HK97 A. Zhmurov1,3 , K. Rybnikov2 , Y. Kholodov3 and V. Barsegov1,3 1Department of Chemistry measurements in silico to explore the mechanical properties of the bacteriophage HK97 in the experimental

  8. Polymer-based delivery systems for support and delivery of bacteriophages

    NASA Astrophysics Data System (ADS)

    Brown, Alyssa Marie

    One of the most urgent problems in the fields of medicine and agriculture is the decreasing effectiveness of antibiotics. Once a miracle drug, antibiotics have recently become associated with the creation of antibiotic-resistant bacteria. The main limitations of these treatments include lack of both adaptability and specificity. To overcome these shortcomings of current antibiotic treatments, there has been a renewed interest in bacteriophage research. Bacteriophages are naturally-occurring viruses that lyse bacteria. They are highly specific, with each bacteriophage type lysing a narrow range of bacteria strains. Bacteriophages are also ubiquitous biological entities, populating environments where bacterial growth is supported. Just as humans are exposed to bacteria in their daily lives, we are exposed to bacteriophages as well. To use bacteriophages in practical applications, they must be delivered to the site of an infection in a controlled-release system. Two systems were studied to observe their support of bacteriophage lytic activity, as well as investigate the possibility of controlling bacteriophage release rates. First, hydrogels were studied, using crosslinking and blending techniques to achieve a range of release profiles. Second, polyanhydride microparticles were studied, evaluating release rates as a function of monomer chemistries.

  9. Complete Genome Sequence of Salmonella enterica Serovar Enteritidis Bacteriophage f18SE, Isolated in Chile

    PubMed Central

    Segovia, Cristopher; Vasquez, Ignacio; Maracaja-Coutinho, Vinicius; Robeson, James

    2015-01-01

    Bacteriophage f18SE was isolated from poultry sewage in Olmue, Chile, and lytic activity was demonstrated against Salmonella enterica serovar Enteritidis and serovar Pullorum strains. This bacteriophage has a 41,868-bp double-stranded DNA (ds-DNA) genome encoding 53 coding sequences (CDSs) and belongs to the family Siphoviridae, subfamily Jerseyvirinae. PMID:26450716

  10. Immune interference with bacteriophage efficacy to treat colibacillosis in broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage are viruses that kill bacteria, and may provide a natural and safe alternative to antibiotics. Colibacillosis is an important poultry disease caused by Escherichia coli. Previous work has indicated that bacteriophage could be used to both prevent and treat colibacillosis. However, b...

  11. Genotyping Staphylococcus aureus allows one to identify bacteriophages harboring unknow endolysins.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background and Objectives. The search of new bacteriophage endolysins is important in view of the ability of staphylococci to acquire resistance to commonly used antibiotics. Most known genomes of Staphylococcus aureus strains contain two or more temperate bacteriophages. For example, the chromosome...

  12. Complete Genome Sequence of Salmonella enterica Serovar Enteritidis Bacteriophage f18SE, Isolated in Chile.

    PubMed

    Segovia, Cristopher; Vasquez, Ignacio; Maracaja-Coutinho, Vinicius; Robeson, James; Santander, Javier

    2015-01-01

    Bacteriophage f18SE was isolated from poultry sewage in Olmue, Chile, and lytic activity was demonstrated against Salmonella enterica serovar Enteritidis and serovar Pullorum strains. This bacteriophage has a 41,868-bp double-stranded DNA (ds-DNA) genome encoding 53 coding sequences (CDSs) and belongs to the family Siphoviridae, subfamily Jerseyvirinae. PMID:26450716

  13. THE GENOME SEQUENCE OF BACTERIOPHAGE CpV1 LYTIC FOR CLOSTRIDIUM PERFRINGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Application of bacteriophages and their lytic enzymes to control Clostri-dium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. We have established a collection of 30 bacteriophages lytic for C. perfringens. These were isolated from s...

  14. Biocontrol of Escherichia coli O157:H7 using a bacteriophage cocktail in laboratory media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages are natural enemies of bacteria, and therefore, logical candidates to evaluate as antibacterial agents for the control of foodborne pathogens. The effect of a bacteriophage treatment on the prevention of E. coli O157:H7 growth was investigated in Tryptic Soy Broth (TSB) laboratory med...

  15. Escherichia coli capsule bacteriophages. VII. Bacteriophage 29-host capsular polysaccharide interactions.

    PubMed Central

    Fehmel, F; Feige, U; Niemann, H; Stirm, S

    1975-01-01

    Different interactions between particles of Escherichia coli capsule bacteriophage 29 and its receptor, the E. coli serotype 29 capsular polysaccharide have been studied. The inactivation of phage 29 (8 x 10(3) PFU/ml) by isolated host capsular glycan was found to be physiologically insignificant (50% inactivation dose equals 100 mug after 1 h at 37 C). No adsorption (less than 2 x 10(4) PFU/mug) of the viruses to K29 polysaccharide-coated erythroyctes (at 0 or 37 C) was observed either. The phage particles were, however, found to catalyze the hydrolysis of beta-D-glucosido-(1leads to 3)-D-glucuronic acid bonds (arrow) in the receptor polymer, leading, ultimately, to the formation of a mixture of K29 hexasaccharide (one repeating unit), dodecasaccharide, and octadecasaccharide: (see article). Testing derivatives of K29 polysaccharide, as well as 82 heterologous bacterial (mainly Enteriobactericeae) capsular glycans, the viral glycanase was found to be highly specific; in accordance with the host range of phage 29, only one enzymatic cross-reaction (with the Klebsiella K31 polysaccharide) was observed. These and previous results, as well as the electron optical findings of M. E. Bayer and H. Thurow (submitted for publication), are discussed in terms of a unifying mechanism of phage 29-host capsule interaction. We propose that the viruses penetrate the capsules by means of their spike-associated glycanase activity, which leads them along capsular polysaccharide strands to membrane-cell wall adhesions where ejection of the viral genomes occurs. PMID:1099233

  16. Removal of MS2, Q? and GA bacteriophages during drinking water treatment at pilot scale.

    PubMed

    Boudaud, Nicolas; Machinal, Claire; David, Fabienne; Fréval-Le Bourdonnec, Armelle; Jossent, Jérôme; Bakanga, Fanny; Arnal, Charlotte; Jaffrezic, Marie Pierre; Oberti, Sandrine; Gantzer, Christophe

    2012-05-15

    The removal of MS2, Q? and GA, F-specific RNA bacteriophages, potential surrogates for pathogenic waterborne viruses, was investigated during a conventional drinking water treatment at pilot scale by using river water, artificially and independently spiked with these bacteriophages. The objective of this work is to develop a standard system for assessing the effectiveness of drinking water plants with respect to the removal of MS2, Q? and GA bacteriophages by a conventional pre-treatment process (coagulation-flocculation-settling-sand filtration) followed or not by an ultrafiltration (UF) membrane (complete treatment process). The specific performances of three UF membranes alone were assessed by using (i) pre-treated water and (ii) 0.1 mM sterile phosphate buffer solution (PBS), spiked with bacteriophages. These UF membranes tested in this work were designed for drinking water treatment market and were also selected for research purpose. The hypothesis serving as base for this study was that the interfacial properties for these three bacteriophages, in terms of electrostatic charge and the degree of hydrophobicity, could induce variations in the removal performances achieved by drinking water treatments. The comparison of the results showed a similar behaviour for both MS2 and Q? surrogates whereas it was particularly atypical for the GA surrogate. The infectious character of MS2 and Q? bacteriophages was mostly removed after clarification followed by sand filtration processes (more than a 4.8-log reduction) while genomic copies were removed at more than a 4.0-log after the complete treatment process. On the contrary, GA bacteriophage was only slightly removed by clarification followed by sand filtration, with less than 1.7-log and 1.2-log reduction, respectively. After the complete treatment process achieved, GA bacteriophage was removed with less than 2.2-log and 1.6-log reduction, respectively. The effectiveness of the three UF membranes tested in terms of bacteriophages removal showed significant differences, especially for GA bacteriophage. These results could provide recommendations for drinking water suppliers in terms of selection criteria for membranes. MS2 bacteriophage is widely used as a surrogate for pathogenic waterborne viruses in Europe and the United States. In this study, the choice of MS2 bacteriophage as the best surrogate to be used for assessment of the effectiveness of drinking water treatment in removal of pathogenic waterborne viruses in worst conditions is clearly challenged. It was shown that GA bacteriophage is potentially a better surrogate as a worst case than MS2. Considering GA bacteriophage as the best surrogate in this study, a chlorine disinfection step could guaranteed a complete removal of this model and ensure the safety character of drinking water plants. PMID:22421032

  17. Correlation of the Genetic Map and the Endonuclease Site Map of Bacillus subtilis Bacteriophage SP02

    PubMed Central

    Graham, S.; Sutton, S.; Yoneda, Y.; Young, F. E.

    1982-01-01

    By marker rescue of bacteriophage SP02 sus mutants with purified bacteriophage SP02 DNA fragments, 11 of the 17 known bacteriophage SP02 sus loci were assigned to discrete DNA fragments. The left-most genetic locus, susA, was found to reside near one bacteriophage SP02 terminus (EcoRI-C1 fragment), whereas the right-most genetic locus, susP, was found to reside near the other bacteriophage SP02 terminus (EcoRI-C2 fragment). The physical locations of the intervening genetic loci were found to be consistent with the previously determined genetic order. Evidence was also obtained which suggested that at least one end of a transforming DNA fragment is degraded during DNA uptake by the competent bacterium. PMID:16789220

  18. Inactivation of Escherichia coli O157 Bacteriophages by Using a Mixture of Ferrous Sulfate and Tea Extract.

    PubMed

    Liu, Hui; Meng, Ruiqiang; Wang, Jiaying; Niu, Yan D; Li, Jinquan; Stanford, Kim; McAllister, Tim A

    2015-12-01

    Bacteriophages (phages) have been used for biocontrol of Escherichia coli O157 and other pathogenic bacteria in many different matrices and foods, but few studies have included inactivation of residual phages in culture medium before plating and enumeration of surviving host bacteria for the assessment of phage efficacy. This oversight may lead to overestimation of phage efficacy. The ability of virucidal solution containing a mixture of ferrous sulfate [iron(II) sulfate, FeSO4] and tea extract [Fe(II)T] to inactivate residual T5-like, T1-like, T4-like, and rV5-like phages was assessed using E. coli O157 as the host. At concentrations of ?10 mM FeSO4, all phages were not detected after 20 min in a broth culture model. Compared with the virucidal solution-free samples (1 to 96% recovery), Fe(II)T (10 mM FeSO4 plus 15% tea extract) recovered a greater (P < 0.01) number of E. coli O157 from phage-treated broth culture (97 to 100% recovery) and beef samples (52 to 100% recovery). Moreover, with the addition of Fe(II)T, the number of bacteria surviving after exposure to T5-like or T4-like phages was greater (P < 0.01) than that after exposure to T1-like or rV5-like phages. Consequently, use of a virucide for phage inactivation is recommended to improve the accuracy of evaluations of phage efficacy for biocontrol of E. coli O157. PMID:26613917

  19. Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multi-protein system.

    PubMed

    Wu, Li; Zhao, Wei; Yang, Ruijin; Yan, Wenxu

    2015-05-15

    The aggregation of multi-proteins is of great interest in food processing and a good understanding of the formation of aggregates during PEF processing is needed for the application of the process to pasteurize protein-based foods. The aggregates formation of a multi-protein system (containing ovalbumin, ovotransferrin and lysozyme) was studied through turbidity, size exclusion chromatography and SDS-PAGE patterns for interaction studies and binding forces. Results from size exclusion chromatography indicated that there was no soluble aggregates formed during PEF processing. The existence of lysozyme was important to form insoluble aggregates in the chosen ovalbumin solution. The results of SDS-PAGE patterns indicated that lysozyme was prone to precipitate, and was relatively the higher component of aggregates. Citric acid could be effective in inhibiting lysozyme from interacting with other proteins during PEF processing. Blocking the free sulphydryl by N-ethylmaleimide (NEM) did not affect aggregation inhibition. PMID:25577059

  20. Cloning and characterization of an invertebrate type lysozyme from Venerupis philippinarum.

    PubMed

    Zhao, Jianmin; Qiu, Lihua; Ning, Xuanxuan; Chen, Aiqin; Wu, Huifeng; Li, Chenghua

    2010-05-01

    Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections and providing nutrition as digestion enzymes. In the present study, an invertebrate type lysozyme (denoted as VpLYZ) was identified from Venerupis philippinarum haemocytes by cDNA library and RACE approaches. The full-length cDNA of VpLYZ consisted of 805 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open-reading frame of 558bp encoding a polypeptide of 185 amino acids with a calculated molecular mass of 20.87kD and theoretical pI of 8.44. The high similarity of VpLYZ with other i-type lysozymes from mollusk indicated that VpLYZ should be a new member of i-type lysozyme family. Similar to most i-type lysozymes, VpLYZ possessed all conserved features critical for the fundamental structure and function of i-type lysozymes, such as three catalytic residues (Glu19, Asn72 and Ser75) and i-type specific motif CL(E/L/R/H)C(I/M)C. By semi-quantitative RT-PCR analysis, mRNA transcript of VpLYZ was found to be most abundantly expressed in the tissues of gills, hepatopancreas and haemocytes, weakly expressed in the tissues of muscle, foot and mantle. After clams were challenged by Vibrio anguillarum, the mRNA level of VpLYZ in overall haemocyte population was recorded by quantitative real-time RT-PCR. VpLYZ mRNA was down-regulated sharply from 6h to 12h post-infection. Then, the expression level increased to the peak at 72h and recovered to the original level at 96h. All these results indicated that VpLYZ was involved in the immune response against microbe infection and contributed to the clearance of bacterial pathogens. PMID:20144728