Sample records for bacteriophage t4 lysozyme

  1. Dynamics of xenon binding inside the hydrophobic cavity of pseudo-wild-type bacteriophage T4 lysozyme explored through xenon-based NMR spectroscopy.

    PubMed

    Desvaux, Hervé; Dubois, Lionel; Huber, Gaspard; Quillin, Michael L; Berthault, Patrick; Matthews, Brian W

    2005-08-24

    Wild-type bacteriophage T4 lysozyme contains a hydrophobic cavity with binding properties that have been extensively studied by X-ray crystallography and NMR. In the present study, the monitoring of 1H chemical shift variations under xenon pressure enables the determination of the noble gas binding constant (K = 60.2 M(-1)). Although the interaction site is highly localized, dipolar cross-relaxation effects between laser-polarized xenon and nearby protons (SPINOE) are rather poor. This is explained by the high value of the xenon-proton dipolar correlation time (0.8 ns), much longer than the previously reported values for xenon in medium-size proteins. This indicates that xenon is highly localized within the protein cavity, as confirmed by the large chemical shift difference between free and bound xenon. The exploitation of the xenon line width variation vs xenon pressure and protein concentration allows the extraction of the exchange correlation time between free and bound xenon. Comparison to the exchange experienced by protein protons indicates that the exchange between the open and closed conformations of T4 lysozyme is not required for xenon binding. PMID:16104744

  2. Bacteriophage T4 Genome†

    PubMed Central

    Miller, Eric S.; Kutter, Elizabeth; Mosig, Gisela; Arisaka, Fumio; Kunisawa, Takashi; Rüger, Wolfgang

    2003-01-01

    Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth. PMID:12626685

  3. 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.

  4. Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme

    PubMed Central

    Yang, Guoliang; Cecconi, Ciro; Baase, Walter A.; Vetter, Ingrid R.; Breyer, Wendy A.; Haack, Julie A.; Matthews, Brian W.; Dahlquist, Frederick W.; Bustamante, Carlos

    2000-01-01

    Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 ± 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol. PMID:10618384

  5. T4 bacteriophage as a phage display platform.

    PubMed

    Gamkrelidze, Mariam; D?browska, Krystyna

    2014-07-01

    Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems. PMID:24828789

  6. Structure and assembly of bacteriophage T4 head

    Microsoft Academic Search

    Venigalla B Rao; Lindsay W Black

    2010-01-01

    The bacteriophage T4 capsid is an elongated icosahedron, 120 nm long and 86 nm wide, and is built with three essential proteins; gp23*, which forms the hexagonal capsid lattice, gp24*, which forms pentamers at eleven of the twelve vertices, and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. The past

  7. Direct Observation of T4 Lysozyme Hinge-Bending Motion by Fluorescence Correlation Spectroscopy

    PubMed Central

    Yirdaw, Robel B.; Mchaourab, Hassane S.

    2012-01-01

    Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ?15 ?s relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants. PMID:23062345

  8. Coordinated DNA Replication by the Bacteriophage T4 Replisome

    PubMed Central

    Noble, Erin; Spiering, Michelle M.; Benkovic, Stephen J.

    2015-01-01

    The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been used to reconstitute DNA synthesis in vitro and are a well-characterized model system. Recent work on the T4 replisome has yielded more detailed insight into the dynamics and coordination of proteins at the replication fork. Since the leading and lagging strands are synthesized in opposite directions, coordination of DNA synthesis as well as priming and unwinding is accomplished by several protein complexes. These protein complexes serve to link catalytic activities and physically tether proteins to the replication fork. Essential to both leading and lagging strand synthesis is the formation of a holoenzyme complex composed of the polymerase and a processivity clamp. The two holoenzymes form a dimer allowing the lagging strand polymerase to be retained within the replisome after completion of each Okazaki fragment. The helicase and primase also form a complex known as the primosome, which unwinds the duplex DNA while also synthesizing primers on the lagging strand. Future studies will likely focus on defining the orientations and architecture of protein complexes at the replication fork. PMID:26102578

  9. Coordinated DNA Replication by the Bacteriophage T4 Replisome.

    PubMed

    Noble, Erin; Spiering, Michelle M; Benkovic, Stephen J

    2015-01-01

    The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been used to reconstitute DNA synthesis in vitro and are a well-characterized model system. Recent work on the T4 replisome has yielded more detailed insight into the dynamics and coordination of proteins at the replication fork. Since the leading and lagging strands are synthesized in opposite directions, coordination of DNA synthesis as well as priming and unwinding is accomplished by several protein complexes. These protein complexes serve to link catalytic activities and physically tether proteins to the replication fork. Essential to both leading and lagging strand synthesis is the formation of a holoenzyme complex composed of the polymerase and a processivity clamp. The two holoenzymes form a dimer allowing the lagging strand polymerase to be retained within the replisome after completion of each Okazaki fragment. The helicase and primase also form a complex known as the primosome, which unwinds the duplex DNA while also synthesizing primers on the lagging strand. Future studies will likely focus on defining the orientations and architecture of protein complexes at the replication fork. PMID:26102578

  10. A Promiscuous DNA Packaging Machine from Bacteriophage T4

    PubMed Central

    Zhang, Zhihong; Kottadiel, Vishal I.; Vafabakhsh, Reza; Dai, Li; Chemla, Yann R.; Ha, Taekjip; Rao, Venigalla B.

    2011-01-01

    Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%–25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles. PMID:21358801

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

    Microsoft Academic Search

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

    2007-01-01

    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

  12. Phylogenetic diversity of T4-like bacteriophages in Lake Baikal, East Siberia.

    PubMed

    Butina, Tatyana Vladimirovna; Belykh, Olga I; Maksimenko, Svetlana Yu; Belikov, Sergey I

    2010-08-01

    Among the tailed phages, the myoviruses, those with contractile tails, are widespread and diverse. An important component of the Myoviridae family is the genus 'T4-like viruses'. The present study was aimed at elucidating the molecular diversity of T4-type bacteriophages in Lake Baikal by partial sequencing of g23 genes of T4-type bacteriophages. Our study revealed that the g23 gene sequences investigated were highly diverse and different from those of T4-like bacteriophages and from g23 clones obtained from different environments. Phylogenetic analysis showed that all g23 fragments from Lake Baikal, except for the one sequence, were more closely related to marine T4 cyanophages and to previously described subgroups of uncultured T4 phages from marine and rice field environments. PMID:20579103

  13. 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.

  14. On the origin of the polar order of T4 lysozyme on planar model surfaces.

    PubMed

    Jacobsen, Kerstin; Risse, Thomas

    2008-01-24

    Site directed spin labeling is used to investigate the origin of the macroscopic alignment of T4 lysozyme vectorially tethered to planar biomimetic surfaces. T4 lysozyme was adsorbed to a quartz-supported dioleoylphosphatidylcholine (DOPC) bilayer by selective binding of the histidine-tagged protein to functionalized headgroups (1,2-dioleoyl-sn-glycero-3-[[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl], DOGS NTA) of the bilayer. This results in a polar oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance (EPR) spectra. In order to reveal the mechanism of the protein alignment, the influence of protein coverage on the order of the molecules was addressed. Along the lines described previously for a full monolayer (Jacobsen, et al. Biophys. J. 2005, 88, 4351), the polar orientation of the molecules was inferred from an analysis of the EPR line shape using the stochastic Liouville equation (SLE) approach developed by Freed and co-workers. The simulations reveal that the orientation of the protein is strongly determined by lateral protein-protein interactions. In comparison to the lipid bilayer, a fusion protein of T4 lysozyme (T4L) with Annexin XII was investigated, where the two-dimensional crystallization of Annexin XII on a dioleoylphosphatidylserine (DOPS) bilayer provides a surface layer of regularly anchored T4L molecules. For this system, it is found that the interaction between T4L and Annexin plays a more important role for understanding the structure in the adsorbed state. PMID:18171040

  15. 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

  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. Structure and Orientation of T4 Lysozyme Bound to the Small Heat Shock Protein ?-Crystallin

    PubMed Central

    Claxton, Derek P.; Zou, Ping; Mchaourab, Hassane S.

    2008-01-01

    Summary We have determined the structural changes that accompany the formation of a stable complex between a destabilized mutant of T4 lysozyme (T4L) and the small heat-shock protein ?-crystallin. Using pairs of fluorescence or spin label probes to fingerprint the T4L tertiary fold, we demonstrate that binding disrupts tertiary packing in the two domains as well as across the active site cleft. Furthermore, increased distances between i and i+4 residues of helices support a model in which the bound structure is not native-like but significantly unfolded. In the confines of the oligomer, T4L has a preferential orientation with residues in the more hydrophobic C-terminal domain sequestered in a buried environment while residues in the N-terminal domain are exposed to the aqueous solvent. Furthermore, EPR spectral lineshapes of sites in the N-terminal domain are narrower than in the folded, unbound T4L reflecting an unstructured backbone and an asymmetric pattern of contacts between T4L and ?-crystallin. The net orientation is not affected by the location of the destabilizing mutation consistent with the notion that binding is not triggered by recognition of localized unfolding. Together, the structural and thermodynamic data indicate that the stably bound conformation of T4L is unfolded and support a model in which the two-modes of substrate binding originate from two discrete binding sites on the chaperone. PMID:18062989

  18. Carbon loss during irradiation of T4 bacteriophages and E. coli bacteria in electron microscopes

    Microsoft Academic Search

    J DUBOCHET

    1975-01-01

    The loss of ¹⁴C due to electron irradiation has been measured on labeled T4 bacteriophages and E. coli bacteria under conditions relevant for practical electron microscopy for fixed and scanning beam exposure. During irradiation, the remaining material became less and less sensitive to further carbon loss. Surface migration of molecular fragments and adsorbed molecules is involved in the process of

  19. 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 form a large "hole...

  20. Inactivation of Escherichia coli and bacteriophage T4 by high levels of dissolved CO 2

    Microsoft Academic Search

    Xuehang Cheng; Tsuyoshi Imai; Jantima Teeka; Junki Yamaguchi; Mami Hirose; Takaya Higuchi; Masahiko Sekine

    2011-01-01

    Little information is available regarding the effectiveness of water disinfection by CO2 at low pressure. The aim of this study was to evaluate the use of high levels of dissolved CO2 at 0.3–0.6 MPa for the inactivation of microorganisms. Bacteriophage T4 was chosen as the model virus and Escherichia coli was selected as the representative bacterium. The results of the study

  1. Inactivation of bacteriophage T4 by organic and inorganic tin compounds

    Microsoft Academic Search

    Mark M. Doolittle; Joseph J. Cooney

    1992-01-01

    Summary Butyltins and inorganic tins inactivated bacteriophage T4. The effect was on the phage and not on its host,Escherichia coli. The order of effectiveness was SnCl4=monobutyltin>dibutyltin>tributyltin=SnCl2. For the butyltins and SnCl4 this was the reverse of the order of effectiveness usually observed for plants, animals, and microorganisms. This pattern suggests that degradation of tributyltin does not always detoxify it. Monobutyltin

  2. EPR Relaxation-Enhancement-Based Distance Measurements on Orthogonally Spin-Labeled T4-Lysozyme

    PubMed Central

    Razzaghi, Sahand; Brooks, Evan K.; Bordignon, Enrica; Hubbell, Wayne L.; Yulikov, Maxim; Jeschke, Gunnar

    2013-01-01

    Lanthanide-induced enhancement of the longitudinal relaxation of nitroxide radicals in combination with orthogonal site-directed spin labeling is presented as a systematic distance measurement method intended for studies of biomacromolecules and biomacromolecular complexes. The approach is tested on a water soluble protein (T4-lysozyme) for two different commercially available lanthanide labels, and complemented by previously reported data on a membrane inserted polypeptide. Single temperature measurements are shown to be sufficient for reliable distance determination, with an upper measurable distance limit of about 5-6 nm. The extracted averaged distances represent the closest approach in LnIII-nitroxide distance distributions. Studies of conformational changes and of biomacromolecule association-dissociation are proposed as possible application area of the RE-based distance measurements. PMID:23775845

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

    SciTech Connect

    Fokine, Andrei; Bowman, Valorie D.; Battisti, Anthony J. [Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 (United States); Li Qin [Department of Biology, Catholic University of America, 620 Michigan Avenue NE, Washington, DC 20064 (United States); Chipman, Paul R. [Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 (United States); Rao, Venigalla B. [Department of Biology, Catholic University of America, 620 Michigan Avenue NE, Washington, DC 20064 (United States); Rossmann, Michael G. [Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 (United States)], E-mail: mr@purdue.edu

    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.

  4. New epistasis group for the repair of DNA damage in bacteriophage T4: replication repair

    SciTech Connect

    Wachsman, J.T.; Drake, J.W.

    1987-03-01

    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 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 encode the major T4 ssDNA-binding protein (the scaffolding of the DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication. The authors conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before of after DNA replication.

  5. Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    PubMed Central

    Bryson, Alexandra L.; Hwang, Young; Sherrill-Mix, Scott; Wu, Gary D.; Lewis, James D.; Black, Lindsay; Clark, Tyson A.

    2015-01-01

    ABSTRACT The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack. PMID:26081634

  6. Suppression of Amber Mutations of Bacteriophage T4 Gene 43 (DNA Polymerase) by Translational Ambiguity

    PubMed Central

    Karam, J. D.; O'Donnell, P. V.

    1973-01-01

    The growth properties of twelve different amber (am) mutants of bacteriophage T4 gene 43 (DNA polymerase) were examined by using nonpermissive (su?) as well as permissive (su+) Escherichia coli hosts. It was found that most of these mutants were measurably suppressed in su? hosts by translational ambiguity (misreading of codons during protein synthesis). The ability of these mutants to grow in response to this form of weak suppression probably means that the T4 gene 43 DNA polymerase can be effective in supporting productive DNA replication when it is supplied in small amounts. By similar criteria, studies with other phage mutants suggested that the products of T4 genes 62 (uncharacterized), 44 (uncharacterized), 42 (dCMP-hydroxymethylase), and 56 (dCTPase) are also effective in small amounts. Some T4 gene products, such as the product of gene 41 (uncharacterized), seem to be partially dispensable for phage growth since am mutants of such genes do propagate, although weakly, in streptomycin-resistant su? hosts which appear to have lost the capacity to suppress am mutations by ambiguity. PMID:4351461

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

    PubMed Central

    Ando, Nozomi; Barstow, Buz; Baase, Walter A.; Fields, Andrew; Matthews, Brian W.

    2009-01-01

    Using small angle X-ray scattering (SAXS) and tryptophan fluorescence spectroscopy, we have identified multiple compact denatured states of a series of T4 lysozyme mutants that are stabilized by high pressures. Recent studies imply that the mechanism of pressure denaturation is the penetration of water into the protein rather than the transfer of hydrophobic residues into water. To investigate water penetration and the volume change associated with pressure denaturation, we studied the solution behavior of four T4 lysozyme mutants having different cavity volumes at low and neutral pH up to a pressure of 400 MPa (0.1 MPa = 0.9869 atm). At low pH, L99A T4 lysozyme expanded from a compact folded state to a partially unfolded state with a corresponding change in radius of gyration from 17 to 32 Å. The volume change upon denaturation correlated well with the total cavity volume, indicating that all of the molecule's major cavities are hydrated with pressure. As a direct comparison to high-pressure crystal structures of L99A T4 lysozyme solved at neutral pH [Collins, M. D., Hummer, G., Quillin, M. L., Matthews, B. W., and Gruner, S. M. (2005), PNAS 102, 16668-16671], pressure denaturation of L99A and the structurally similar L99G/E108V mutant was studied at neutral pH. The pressure-denatured state at neutral pH is even more compact than at low pH, and the small volume changes associated with denaturation suggest that the preferential filling of large cavities is responsible for the compactness of the pressure-denatured state. These results confirm that pressure denaturation is characteristically distinct from thermal or chemical denaturation. PMID:18816066

  8. 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

  9. Marine T4-type bacteriophages, a ubiquitous component of the dark matter of the biosphere

    NASA Astrophysics Data System (ADS)

    Filée, Jonathan; Tétart, Françoise; Suttle, Curtis A.; Krisch, H. M.

    2005-08-01

    Tailed bacteriophages are the most abundant biological entities in marine environments. However, most of these marine phages are uncharacterized because few of their hosts have been cultivated. To learn more about such phages, we designed a set of degenerate PCR primers for phage T4 g23, which encodes the major capsid protein in all of the T4-type phages, an important family of the tailed phage. These primers were used to amplify g23-related sequences from diverse marine environments (fjords and bays of British Columbia, the eastern Gulf of Mexico, and the western Arctic Ocean) revealing a remarkable level of molecular diversity, which in some cases was correlated with morphological variation of the virions. Phylogenetic analysis showed that although some of these sequences were closely related to well studied subgroups of the T4-type phage, such as the T-evens, the majority of them belong to five previously uncharacterized subgroups. These data indicate that the host range of T4-type phages is much broader than previously imagined and that the laboratory isolate T4 belongs to a phage family that is extraordinarily widespread and diverse in the biosphere. Author contributions: J.F., F.T., and H.M.K. designed research; J.F. and F.T. performed research; C.A.S. contributed new reagents/analytic tools; J.F., C.A.S., and H.M.K. analyzed data; and J.F., C.A.S., and H.M.K. wrote the paper.This paper was submitted directly (Track II) to the PNAS office.Data deposition: The nucleotide sequences of g23 reported in this paper have been deposited in the GenBank database (accession nos. DQ105858-DQ105942).

  10. Expression of a cloned denV gene of bacteriophage T4 in Escherichia coli

    SciTech Connect

    Valerie, K.; Henderson, E.E.; de Riel, J.K.

    1985-07-01

    A 713-base-pair Hae III fragment from bacteriophage T4 encompassing the denV gene with its preceding promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains. The denV gene was found to be expressed, probably from its own promoter, causing pyrimidine dimer incision-deficient uvrA, uvrB, uvrC strains to be rescued by the denV gene. A uvrD (DNA helicase II) strain was also complemented, but to a lesser extent. A wild-type strain did not seem to be affected at the UV doses tested. Surprisingly, all recA, recB, and recC strains tested also showed an increased UV resistance, perhaps by reinforcement of the intact uvr system in these strains. Complementation of denV- T4 strains and host-cell reactivation of lambda phage was also observed in denV+ E. coli strains. Equilibrium sedimentation showed that DNA repair synthesis occurred in a UV-irradiated uvrA E. coli strain carrying the cloned denV gene. Southern blotting confirmed earlier results that the denV gene is located at 64 kilobases on the T4 map. Phage T2 (denV-) did not hybridize to a denV-specific probe.

  11. Evidence that bacteriophage T4 ephl is a missense hoc mutation

    SciTech Connect

    Childs, J.D.; Pilon, R.

    1983-05-01

    An electrophoretic mutation of bacteriophage T4, ephl, appears to code for a missense hoc (highly antigenic outer capsid) protein. This is based on the observation that particles lacking (hoc protein hoc/sup -/ particles), after incubation in a crude extract of Escherichia coli infected with phage carrying the ephl mutation acquired the electrophoretic mobility of the ephl strain (the electrophoretic mobility of the ephl strain itself is slower than that of hoc/sup -/ particles). Thus, it is likely that during infection of E. coli with the ephl strain, a hoc protein is made that has a lower negative charge than normal hoc protein but can nevertheless bind to particles lacking hoc protein. These results confirm that ephl is a hoc mutation.

  12. 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.

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

    PubMed

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

    2014-01-01

    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. PMID:24937091

  14. Bacteriophage T4 and human type I DNA ligases relax DNA under joining conditions.

    PubMed Central

    Ciarrocchi, G; Lestingi, M; Wright, G; Montecucco, A

    1993-01-01

    Both bacteriophage T4 and human type I DNA ligases in the presence of a mixture of ATP, AMP and PPi altered the topological properties of a supercoiled substrate by a step-wise reaction eventually leading to a population of fully relaxed, covalently closed products. In the presence of only AMP and PPi DNA products containing nicks with 3'OH/5'P termini accumulated in the presence of bacteriophage T4 DNA ligase, suggesting reversal of the entire joining reaction, but not in the presence of human DNA ligase I. Both DNA ligases became deoxyadenylylated in the presence of dATP, but the joining reaction did not proceed to completion. However, with both enzymes the full relaxing reaction took place in the presence of dAMP alone and in the presence of a mixture of dATP, dAMP and PPi. In no case could the joining reaction be reversed by dAMP and PPi. Related experiments with modified derivatives of deoxyribonucleoside 5'-triphosphates and PPi gave results in accord with these observations. The AMP dependent DNA relaxation catalysed by DNA ligases was insensitive to the presence of exonuclease III. These results indicate that controlled relaxation of the substrate by both DNA ligases occurs as a separate reaction rather than by simple reversal of the joining reaction. These findings support the hypothesis that in vivo the DNA topoisomerising ligases relax their substrate at the replication fork both during and separately from ligation of a pre-existing nick. Images PMID:8290355

  15. 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.

  16. In vitro synthesis of large peptide molecules using glucosylated single-stranded bacteriophage T4D DNA template.

    PubMed Central

    Hulen, C; Legault-Demare, J

    1975-01-01

    Denatured Bacteriophage T4D DNA is able to stimulate aminoacid incorporation into TCA-precipitable material in an in vitro protein synthesis system according to base DNA sequences. Newly synthesized polypeptides remain associated with ribosomes and have a molecular weight in range of 15,000 to 45,000 Daltons. PMID:1052527

  17. Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation*

    PubMed Central

    Hsieh, Meng-Lun; James, Tamara D.; Knipling, Leslie; Waddell, M. Brett; White, Stephen; Hinton, Deborah M.

    2013-01-01

    Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called ? appropriation, the T4 co-activator AsiA structurally remodels the ?70 subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of ?70 and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the ?-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotANTD, MotACTD, and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual “double wing” motif present within MotACTD resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses. PMID:23902794

  18. 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

  19. Protein hinge bending as seen in molecular dynamics simulations of native and M61 mutant T4 lysozymes.

    PubMed

    Arnold, G E; Ornstein, R L

    1997-04-15

    A dynamical model of interdomain "hinge bending" of T4 lysozyme in aqueous solution has been developed on the basis of molecular dynamics (MD) simulation. The MD model study provides a description of the conformational reorganization expected to occur for the protein in aqueous solution as compared to the crystalline environment. Three different 500 ps molecular dynamics simulations were calculated, each using a distinctly different crystal conformation of T4 lysozyme as the starting points of the MD simulations. Crystal structures of wild-type lysozyme and "open" and "closed" forms of M61 variant structures were analyzed in this study. Large-scale, molecular-conformational rearrangements were observed in all three simulations, and the largest structural change was found for the open form of the M61 allomorph. All three simulated proteins had closed relative to the wild-type crystal structure, and the closure of the "jaws" of the active site cleft occurred gradually over the time course of the trajectories. The time average MD structures, calculated over the final 50 ps of each trajectory, had all adapted to conformations more similar to each other than to their incipient crystal forms. Using a similar MD protocol on cytochrome P450BM-3 [M. D. Paulsen and R. L. Ornstein (1995) Proteins: Structure Function and Genetics, Vol. 27, pp. 237-243] we have found that the opposite type of motion relative to the starting crystal structure, that is, the open form of the crystal structure, had opened to a greater degree relative to the incipient crystal structure form. Therefore we do not believe that either result is merely a simulation artifact, but rather the protein dynamics are due to protein relaxation in the absence of crystal packing forces in the simulated solution environments. PMID:9095676

  20. 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

  1. 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

  2. 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

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

    PubMed

    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

  4. Structural characteristics and plant-beneficial effects of bacteria colonizing the shoots of field grown conventional and genetically modified T4-lysozyme producing potatoes

    Microsoft Academic Search

    Frank Rasche; Ester Marco-Noales; Henk Velvis; Leo S. van Overbeek; María M. López; Jan D. van Elsas; Angela Sessitsch

    2006-01-01

    Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse effects on potato-associated bacteria which frequently exhibit plant beneficial functions such as plant growth promotion and antagonism towards pathogens invading the plant.

  5. Necessity of Quantum Coherence to Account for the Spectrum of Time-Dependent Mutations Exhibited by Bacteriophage T4

    Microsoft Academic Search

    W. Grant Cooper

    2009-01-01

    Transcriptase measurements of quantum expectations due to time-dependent coherent states populating informational DNA base-pair\\u000a sites, designated by G–C ? *G–*C, G–C ? G?–C?, and A–T ? *A–*T, provide a model for transcription and replication of time-dependent\\u000a DNA lesions exhibited by bacteriophage T4. Coherent states are introduced as consequences of hydrogen bond arrangement, keto-amino ? enol-imine, where product protons are shared between two sets of indistinguishable electron lone-pairs and

  6. Structural Refinement from Restrained-Ensemble Simulations Based on EPR/DEER Data: Application to T4 Lysozyme

    PubMed Central

    Islam, Shahidul M.; Stein, Richard A.; Mchaourab, Hassane; Roux, Benoît

    2013-01-01

    DEER (Double Electron Electron Resonance) is a powerful pulsed ESR (electron spin resonance) technique allowing the determination of distance histograms between pairs of nitroxide spin-labels linked to a protein in a native-like solution environment. However, exploiting the huge amount of information provided by ESR/DEER histograms to refine structural models is extremely challenging. In this study, a restrained ensemble (RE) molecular dynamics (MD) simulation methodology is developed to address this issue. In RE simulation, the spin-spin distance distribution histograms calculated from a multiple-copy MD simulation are enforced, via a global ensemble-based energy restraint, to match those obtained from ESR/DEER experiments. The RE simulation is applied to 51 ESR/DEER distance histogram data from spin-labels inserted at 37 different positions in T4 lysozyme (T4L). The rotamer population distribution along the five dihedral angles connecting the nitroxide ring to the protein backbone is determined and shown to be consistent with available information from X-ray crystallography. For the purpose of structural refinement, the concept of a simplified nitroxide dummy spin-label is designed and parameterized on the basis of these all-atom RE simulations with explicit solvent. It is demonstrated that RE simulations with the dummy nitroxide spin-labels imposing the ESR/DEER experimental distance distribution data are able to systematically correct and refine a series of distorted T4L structures, while simple harmonic distance restraints are unsuccessful. This computationally efficient approach allows experimental restraints from DEER experiments to be incorporated into RE simulations for efficient structural refinement. PMID:23510103

  7. Phylogeny of the Major Head and Tail Genes of the Wide-Ranging T4-Type Bacteriophages†

    PubMed Central

    Tétart, Françoise; Desplats, Carine; Kutateladze, Mzia; Monod, Caroline; Ackermann, Hans-Wolfgang; Krisch, H. M.

    2001-01-01

    We examined a number of bacteriophages with T4-type morphology that propagate in different genera of enterobacteria, Aeromonas, Burkholderia, and Vibrio. Most of these phages had a prolate icosahedral head, a contractile tail, and a genome size that was similar to that of T4. A few of them had more elongated heads and larger genomes. All these phages are phylogenetically related, since they each had sequences homologous to the capsid gene (gene 23), tail sheath gene (gene 18), and tail tube gene (gene 19) of T4. On the basis of the sequence comparison of their virion genes, the T4-type phages can be classified into three subgroups with increasing divergence from T4: the T-evens, pseudoT-evens, and schizoT-evens. In general, the phages that infect closely related host species have virion genes that are phylogenetically closer to each other than those of phages that infect distantly related hosts. However, some of the phages appear to be chimeras, indicating that, at least occasionally, some genetic shuffling has occurred between the different T4-type subgroups. The compilation of a number of gene 23 sequences reveals a pattern of conserved motifs separated by sequences that differ in the T4-type subgroups. Such variable patches in the gene 23 sequences may determine the size of the virion head and consequently the viral genome length. This sequence analysis provides molecular evidence that phages related to T4 are widespread in the biosphere and diverged from a common ancestor in acquiring the ability to infect different host bacteria and to occupy new ecological niches. PMID:11114936

  8. Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

    PubMed Central

    2010-01-01

    Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context. PMID:21129205

  9. 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. (CUA); (Purdue)

    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.

  10. Inhibition of transcription of cytosine-containing DNA in vitro by the alc gene product of bacteriophage T4

    SciTech Connect

    Drivdahl, R.H.; Kutter, E.M. (Evergreen State College, Olympia, WA (USA))

    1990-05-01

    The alc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. The authors examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D{sup +} or alc mutants. A 50 to 60% decline in RNA polymerase activity, measured on phage T7 DNA, was observed by 1 min after infection with either T4D{sup +} or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D{sup +} strain but not alc mutants, this was followed by a further decrease. By 5 min after infection the activity of alc mutants was 1.5 to 2.5 times greater than that of the wild type on various cytosine-containing DNA templates, whereas there was little or no difference in activity on T4 HMdC-DNA, in agreement with the in vivo specificity. Effects on transcript initiation and elongation were distinguished by using a T7 phage DNA template. Rifampin challenge, end-labeling with ({gamma}-{sup 32}P)ATP, and selective initiation with a dinucleotide all indicate that the decreased in vitro activity of the wild-type polymerase relative to that of the alc mutants was due to inhibition of elongation, not to any difference in initiation rates. Wild-type (but not mutated) gpalc copurified with RNA polymerase on heparin agarose but not in subsequent steps. Immunoprecipitation of modified RNA polymerase also indicated that gpalc was not tightly bound to RNA polymerase intracellularly.

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

    E-print Network

    Tran, Tram Anh Thi

    2009-05-15

    , and the anti-microbe could not attack heat-killed bacilli whereas they grew perfectly well on saline washed bacilli. He concluded that the anti-dysentery microbe was an obligatory ?bacteriophage?, literally meaning bacteria-eating and also known as phage... at that time. The problems with phage therapy were mostly due to ignorance, such as a lack of a systematic testing of virulence of phages against infectious bacteria and of the knowledge of proper dosage used in patients (53). D?Herelle was interested...

  12. 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 ...

  13. Studies of viral DNA packaging motors with optical tweezers: a comparison of motor function in bacteriophages ?29, ?, and T4

    NASA Astrophysics Data System (ADS)

    Smith, Douglas E.; Fuller, Derek N.; Raymer, Dorian M.; Rickgauer, Peter; Grimes, Shelley; Jardine, Paul J.; Anderson, Dwight L.; Catalano, Carlos E.; Kottadiel, Vishal; Rao, Venigalla B.

    2007-09-01

    A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the capsid against resisting forces. In addition to studying bacteriophage ?29, we have recently extended these methods to study the E. coli bacteriophages ? and T4, two important model systems in molecular biology. The three systems have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and dynamic disorder, responses to load, and pausing and slipping dynamics.

  14. Contributions of all 20 amino acids at site 96 to the stability and structure of T4 lysozyme

    PubMed Central

    Mooers, Blaine H M; Baase, Walter A; Wray, Jonathan W; Matthews, Brian W

    2009-01-01

    To try to resolve the loss of stability in the temperature-sensitive mutant of T4 lysozyme, Arg 96 ? His, all of the remaining 18 naturally occurring amino acids were substituted at site 96. Also, in response to suggestions that the charged residues Lys85 and Asp89, which are 5–8 Å away, may have important effects, each of these amino acids was replaced with alanine. Crystal structures were determined for many of the variants. With the exception of the tryptophan and valine mutants R96W and R96V, the crystallographic analysis shows that the substituted side chain following the path of Arg96 in wildtype (WT). The melting temperatures of the variants decrease by up to ?16°C with WT being most stable. There are two site 96 replacements, with lysine or glutamine, that leave the stability close to that of WT. The only element that the side chains of these residues have in common with the WT arginine is the set of three carbon atoms at the C?, C?, and C? positions. Although each side chain is long and flexible with a polar group at the distal position, the details of the hydrogen bonding to the rest of the protein differ in each case. Also, the glutamine replacement lacks a positive charge. This shows that there is some adaptability in achieving full stabilization at this site. At the other extreme, to be maximally destabilizing a mutation at site 96 must not only eliminate favorable interactions but also introduce an unfavorable element such as steric strain or a hydrogen-bonding group that remains unsatisfied. Overall, the study highlights the essential need for atomic resolution site-specific structural information to understand and to predict the stability of mutant proteins. It can be very misleading to simply assume that conservative amino acid substitutions cause small changes in stability, whereas large stability changes are associated with nonconservative replacements. PMID:19384988

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

    PubMed

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

    2014-07-01

    The phage-proximal part of the long tail fibres of bacteriophage T4 consists of a trimer of the 1289 amino-acid gene product 34 (gp34). Different carboxy-terminal parts of gp34 have been produced and crystallized. Crystals of gp34(726-1289) diffracting X-rays to 2.9?Å resolution, crystals of gp34(781-1289) diffracting to 1.9?Å resolution and crystals of gp34(894-1289) diffracting to 3.0 and 2.0?Å resolution and belonging to different crystal forms were obtained. Native data were collected for gp34(726-1289) and gp34(894-1289), while single-wavelength anomalous diffraction data were collected for selenomethionine-containing gp34(781-1289) and gp34(894-1289). For the latter, high-quality anomalous signal was obtained. PMID:25005101

  16. The Bacteriophage T4 Rapid-Lysis Genes and Their Mutational Proclivities ?

    PubMed Central

    Burch, Lauranell H.; Zhang, Leilei; Chao, Frank G.; Xu, Hong; Drake, John W.

    2011-01-01

    Like most phages with double-stranded DNA, phage T4 exits the infected host cell by a lytic process requiring, at a minimum, an endolysin and a holin. Unlike most phages, T4 can sense superinfection (which signals the depletion of uninfected host cells) and responds by delaying lysis and achieving an order-of-magnitude increase in burst size using a mechanism called lysis inhibition (LIN). T4 r mutants, which are unable to conduct LIN, produce distinctly large, sharp-edged plaques. The discovery of r mutants was key to the foundations of molecular biology, in particular to discovering and characterizing genetic recombination in T4, to redefining the nature of the gene, and to exploring the mutation process at the nucleotide level of resolution. A number of r genes have been described in the past 7 decades with various degrees of clarity. Here we describe an extensive and perhaps saturating search for T4 r genes and relate the corresponding mutational spectra to the often imperfectly known physiologies of the proteins encoded by these genes. Focusing on r genes whose mutant phenotypes are largely independent of the host cell, the genes are rI (which seems to sense superinfection and signal the holin to delay lysis), rIII (of poorly defined function), rIV (same as sp and also of poorly defined function), and rV (same as t, the holin gene). We did not identify any mutations that might correspond to a putative rVI gene, and we did not focus on the famous rII genes because they appear to affect lysis only indirectly. PMID:21571993

  17. Identification of a New Motif in Family B DNA Polymerases by Mutational Analyses of the Bacteriophage T4 DNA Polymerase

    PubMed Central

    Li, Vincent; Hogg, Matthew; Reha-Krantz, Linda J.

    2011-01-01

    Structure-based protein sequence alignments of family B DNA polymerases revealed a conserved motif that is formed from interacting residues between loops from the N-terminal and palm domains and between the N-terminal loop and a conserved proline residue. The importance of the motif for function of the bacteriophage T4 DNA polymerase was revealed by suppressor analysis. T4 DNA polymerases that form weak replicating complexes cannot replicate DNA when the dGTP pool is reduced. The conditional lethality provides the means to identify amino acid substitutions that restore replication activity under low dGTP conditions by either correcting the defect produced by the first amino acid substitution or by generally increasing the stability of polymerase complexes; the second type are global suppressors that can effectively counter the reduced stability caused by a variety of amino acid substitutions. Some amino acid substitutions that increase the stability of polymerase complexes produce a new phenotype - sensitivity to the antiviral drug phosphonoacetic acid. Amino acid substitutions that confer decreased ability to replicate DNA under low dGTP conditions or drug sensitivity were identified in the new motif, which suggests that the motif functions in regulating the stability of polymerase complexes. Additional suppressor analyses revealed an apparent network of interactions that link the new motif to the fingers domain and to two patches of conserved residues that bind DNA. The collection of mutant T4 DNA polymerases provides a foundation for future biochemical studies to determine how DNA polymerases remain stably associated with DNA while waiting for the next available dNTP, how DNA polymerases translocate, and the biochemical basis for sensitivity to antiviral drugs. PMID:20493878

  18. Bacteriophage T4 development depends on the physiology of its host Escherichia coli.

    PubMed

    Hadas, H; Einav, M; Fishov, I; Zaritsky, A

    1997-01-01

    Several parameters of phage T4 adsorption to and growth in Escherichia coli B/r were determined. All changed monotonously with the bacterial growth rate (mu), which was modified by nutritional conditions. Adsorption rate was faster at higher mu values, positively correlated to cell size, and increased by pretreatment with low penicillin (Pn) concentrations; it was directly proportional to total cellular surface area, indicating a constant density of T4 receptors on cell envelopes irrespective of growth conditions. Parameters of phage development and cell lysis were mu-dependent. The rate of phage release and burst size increased, while the eclipse and latent periods decreased with increasing mu. Differentiation between the contribution of several physiological parameters to the development of T4 was performed by manipulating the host cells. A competitive inhibitor of glucose uptake, methyl alpha-D-glucoside, was exploited to reduce the growth rate in the same effective carbon source. Synchronous cells were obtained by the "baby-machine' and large cells were obtained by pretreatment with low Pn concentrations. Lysis was delayed by superinfection, and DNA content and concentration were modified by growing a thy mutant in limiting thymine concentrations. The results indicate that burst size is not limited by cell size or DNA composition, nor directly by the rate of metabolism, but rather by the rates of synthesis and assembly of phage components and by lysis time. The rates of synthesis and assembly of phage components seem to depend on the content of the protein-synthesizing system and lysis time seems to depend on cellular dimensions. PMID:9025292

  19. Partial replicas of uv-irradiated bacteriophage T4 genomes and their role in multiplicity reactivation

    SciTech Connect

    Rayssiguier, C. (Univ. of Pennsylvania, Philadelphia); Kozinski, A.W.; Doermann, A.H.

    1980-08-01

    A physicochemical study was made of the replication and transmission of uv-irradiated T4 genomes. The data presented in this paper justify the following conclusions. (i) For both low and high multiplicity of infection there was abundant replication from uv-irradiated parental templates. It exceeded by far the efficiency predicted by the hypothesis that a single lethal hit completely prevents replication of the killed phage DNA: i.e., some dead phage particles must replicate parts of their DNA. (ii) Replication of the uv-irradiated DNA was repetitive as shown by density reversal experiments. (iii) Newly synthesized progeny DNA originating from uv-irradiated templates appeared as significantly shorter segments of the genomes than progeny DNA produced from non-uv-irradiated templates. A good correlation existed between the number of uv hits and the number of random cuts that would be needed to reduce replication fragments to the length observed. (iv) The contribution of uv-irradiated parental DNA among progeny phage in multiplicity reactivation was disposed in shorter subunits than was the DNA from unirradiated parental phage. It is important to emphasize that it was mainly in the form of replicative hybrid. These conclusions appear to justify excluding interparental recombination as a prerequisite for multiplicity reactivation. They lead directly to some form of partial replica hypothesis for multiplicity reactivation.

  20. Genetic and amber fragment maps of genes 46 and 47 of bacteriophage T4D.

    PubMed Central

    Wiberg, J S; Cardillo, T S; Mickelson, C

    1981-01-01

    We constructed genetic recombinational maps of genes 46 and 47 by using five amber mutants in gene 46, nine amber mutants in gene 47, and two-factor crosses. Two different amber fragments in gene 46 and three different amber fragments in gene 47 were detected on polyacrylamide slab gels in the presence of sodium dodecyl sulfate. The genetic maps agreed with the amber fragment maps; taken together, the data oriented all of the sites in both genes with respect to each other. Given the relative map positions of genes 46 and 47 determined genetically by Epstein et al. (Cold Spring Harbor Symp. Quant. Biol. 28:375-394, 1963), our results extend and reinforce the work of Hercules and Sauerbier (J. Virol. 12: 872-881, 1973) and that of Minner and Bernstein (J. Gen. Virol. 31:277-280, 1976), which indicated that the direction of transcription and translation of these genes if counterclockwise on the T4 genetic map (i.e., from gene 47 toward gene 46). Images PMID:7288925

  1. The Mechanism of Inactivation of T4 Bacteriophage by Tritium Decay

    PubMed Central

    Funk, Fred; Person, Stanley; Bockrath, Richard C.

    1968-01-01

    Coliphage T4 was used as a model system to study the mechanism of biological inactivation produced by tritium decay. Experimentally, tritiated precursors were incorporated into phage DNA (thymidine-3H) or into phage protein (3H-amino acids). The ratio of killing efficiencies for decays originating in phage DNA to those originating in phage protein was 2.6. Inactivation by decays from labeled amino acids was assumed to occur exclusively from ?-particle irradiation of phage DNA. If decays originating in DNA are due solely to irradiation of DNA, then the killing efficiencies reflect the energy transfer paths in phage DNA for decays originating in phage DNA and in the protein coat. The energy transfer paths were determined for the two cases with the help of a computer and found to be very nearly equal to the experimentally determined ratio (2.6). The killing efficiencies for decays originating in phage DNA were 0.12 and for decays originating in protein 0.046. PMID:5678320

  2. Cryoelectron microscopy analysis of small heat shock protein 16.5 (Hsp16.5) complexes with T4 lysozyme reveals the structural basis of multimode binding.

    PubMed

    Shi, Jian; Koteiche, Hanane A; McDonald, Ezelle T; Fox, Tara L; Stewart, Phoebe L; McHaourab, Hassane S

    2013-02-15

    Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used cryoelectron microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the ?-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human ?B-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition. PMID:23277356

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

    PubMed Central

    Esvelt, Kevin M.; Church, George M.

    2015-01-01

    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 T4, RB55, and RB59; RB32 and RB33; and RB3, RB5, RB6, RB7, RB9, and RB10 phages. PMID:25555735

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

    PubMed

    Yaung, Stephanie J; Esvelt, Kevin M; Church, George M

    2015-01-01

    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 T4, RB55, and RB59; RB32 and RB33; and RB3, RB5, RB6, RB7, RB9, and RB10 phages. PMID:25555735

  5. Control of Escherichia coli O157 on beef at 37, 22 and 4 °C by T5-, T1-, T4-and O1-like bacteriophages.

    PubMed

    Liu, H; Niu, Y D; Meng, R; Wang, J; Li, J; Johnson, R P; McAllister, T A; Stanford, K

    2015-10-01

    Efficacy of four bacteriophages (phages) and a cocktail for biocontrol of Escherichia coli O157 was assessed on beef samples stored at 4, 22 and 37 °C. Samples (3 × 3 × 1 cm) were contaminated with E. coli O157 (10(4) CFU/cm(2)) and treated with single phages: T5-like (T5), T1-like (T1), T4-like (T4) and O1-like (O1), or a cocktail at two titers: multiplicity of infection (MOI) = 1000 and MOI = 10. In contrast to previous studies, use of virucidal solution prevented over-estimation of phage efficacy. Irrespective of temperature and MOIs, T5 was most (P < 0.001) and O1 least (P < 0.05) effective for biocontrol of E. coli O157, with relative efficacy of other phages temperature dependent. At 4 °C, T1 (P < 0.05) and cocktail (P < 0.001) were more effective than T4. In contrast, T4 was equally (P = 0.08, at 37 °C) or less effective (P = 0.003, at 22 °C) than T5. Phages were more effective (P < 0.001) against E. coli O157 at warmer temperatures and high MOI. As the beef supply chain includes hours of storage or transport at temperatures near 4 °C, this study demonstrates phages could significantly reduce E. coli O157 during this period. PMID:26187829

  6. Computational stability ranking of mutated hydrophobic cores in staphylococcal nuclease and T4 lysozyme using hard-sphere and stereochemical constraints

    NASA Astrophysics Data System (ADS)

    Virrueta, Alejandro; Zhou, Alice; O'Hern, Corey; Regan, Lynne

    2014-03-01

    Molecular dynamics methods have significantly advanced the understanding of protein folding and stability. However, current force-fields cannot accurately calculate and rank the stability of modified or de novo proteins. One possible reason is that current force-fields use knowledge-based corrections that improve dihedral angle sampling, but do not satisfy the stereochemical constraints for amino acids. I propose the use of simple hard-sphere models for amino acids with stereochemical constraints taken from high-resolution protein crystal structures. This model can enable a correct consideration of the entropy of side-chain rotations, and may be sufficient to predict the effects of single residue mutations in the hydrophobic cores of staphylococcal nuclease and T4 lysozyme on stability changes. I will computationally count the total number of allowed side-chain conformations ? and calculate the associated entropy, S = kBln(?) , before and after each mutation. I will then rank the stability of the mutated cores based on my computed entropy changes, and compare my results with structural and thermodynamic data published by the Stites and Matthews groups. If successful, this project will provide a novel framework for the evaluation of entropic protein stabilities, and serve as a possible tool for computational protein design.

  7. Structural determinants of nitroxide motion in spin-labeled proteins: tertiary contact and solvent-inaccessible sites in helix G of T4 lysozyme.

    PubMed

    Guo, Zhefeng; Cascio, Duilio; Hideg, Kálmán; Kálái, Támás; Hubbell, Wayne L

    2007-06-01

    A nitroxide side chain (R1) has been substituted at single sites along a helix-turn-helix motif in T4 lysozyme (residues 114-135). Together with previously published data, the new sites reported complete a continuous scan through the motif. Mutants with R1 at sites 115 and 118 were selected for crystallographic analysis to identify the structural origins of the corresponding two-component EPR spectra. At 115, R1 is shown to occupy two rotamers in the room temperature crystal structure, one of which has not been previously reported. The two components in the EPR spectrum apparently arise from differential interactions of the two rotamers with the surrounding structure, the most important of which is a hydrophobic interaction of the nitroxide ring. Interestingly, the crystal structure at 100 K reveals a single rotamer, emphasizing the possibility of rotamer selection in low-temperature crystal structures. Residue 118 is at a solvent-inaccessible site in the protein core, and the structure of 118R1, the first reported for the R1 side chain at a buried site, reveals how the side chain is accommodated in an overpacked core. PMID:17473014

  8. Cavity as a Source of Conformational Fluctuation and High-Energy State: High-Pressure NMR Study of a Cavity-Enlarged Mutant of T4Lysozyme

    NASA Astrophysics Data System (ADS)

    Maeno, Akihiro; Sindhikara, Daniel; Hirata, Fumio; Otten, Renee; Dahlquist, Frederick W.; Yokoyama, Shigeyuki; Akasaka, Kazuyuki; Mulder, Frans A. A.; Kitahara, Ryo

    2015-01-01

    Although the structure, function, conformational dynamics, and controlled thermodynamics of proteins are manifested by their corresponding amino acid sequences, the natural rules for molecular design and their corresponding interplay remain obscure. In this study, we focused on the role of internal cavities of proteins in conformational dynamics. We investigated the pressure-induced responses from the cavity-enlarged L99A mutant of T4 lysozyme, using high-pressure NMR spectroscopy. The signal intensities of the methyl groups in the 1H/13C HSQC spectra, particularly those around the enlarged cavity, decreased with the increasing pressure, and disappeared at 200 MPa, without the appearance of new resonances, thus indicating the presence of heterogeneous conformations around the cavity within the ground state ensemble. Above 200 MPa, the signal intensities of more than 20 methyl groups gradually decreased with the increasing pressure, without the appearance of new resonances. Interestingly, these residues closely matched those sensing a large conformational change between the ground- and high-energy states, at atmospheric pressure. 13C and 1H NMR line-shape simulations showed that the pressure-induced loss in the peak intensity could be explained by the increase in the high-energy state population. In this high-energy state, the aromatic side chain of F114 gets flipped into the enlarged cavity. The accommodation of the phenylalanine ring into the efficiently packed cavity may decrease the partial molar volume of the high-energy state, relative to the ground state. We suggest that the enlarged cavity is involved in the conformational transition to high-energy states and in the volume fluctuation of the ground state.

  9. 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.

  10. Effect of Freezing Conditions on Distances and Their Distributions Derived from Double Electron Electron Resonance (DEER): A Study of Doubly-Spin-Labeled T4 Lysozyme

    PubMed Central

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

    2012-01-01

    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, 1s 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. PMID:22341208

  11. Coordination and Processing of DNA Ends During Double-Strand Break Repair: The Role of the Bacteriophage T4 Mre11/Rad50 (MR) Complex

    PubMed Central

    Almond, Joshua R.; Stohr, Bradley A.; Panigrahi, Anil K.; Albrecht, Dustin W.; Nelson, Scott W.; Kreuzer, Kenneth N.

    2013-01-01

    The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of double-strand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections. PMID:23979587

  12. The C-terminal domain of the bacteriophage T4 terminase docks on the prohead portal clip region during DNA packaging

    PubMed Central

    Dixit, Aparna Banerjee; Ray, Krishanu; Thomas, Julie A.; Black, Lindsay W.

    2013-01-01

    Bacteriophage ATP-based packaging motors translocate DNA into a pre-formed prohead through a dodecameric portal ring channel to high density. We investigated portal–terminase docking interactions at specifically localized residues within a terminase-interaction region (aa279–316) in the phage T4 portal protein gp20 equated to the clip domain of the SPP1 portal crystal structure by 3D modeling. Within this region, three residues allowed A to C mutations whereas three others did not, consistent with informatics analyses showing the tolerated residues are not strongly conserved evolutionarily. About 7.5 nm was calculated by FCS-FRET studies employing maleimide Alexa488 dye labeled A316C proheads and gp17 CT-ReAsH supporting previous work docking the C-terminal end of the T4 terminase (gp17) closer to the N-terminal GFP-labeled portal (gp20) than the N-terminal end of the terminase. Such a terminase–portal orientation fits better to a proposed “DNA crunching” compression packaging motor and to portal determined DNA headful cutting. PMID:24074593

  13. Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia pestis as Next Generation Plague Vaccines

    PubMed Central

    Tao, Pan; Mahalingam, Marthandan; Kirtley, Michelle L.; van Lier, Christina J.; Sha, Jian; Yeager, Linsey A.; Chopra, Ashok K.; Rao, Venigalla B.

    2013-01-01

    Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal ?-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the ?-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines. PMID:23853602

  14. Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

    PubMed Central

    Mosig, Gisela; Gewin, John; Luder, Andreas; Colowick, Nancy; Vo, Daniel

    2001-01-01

    Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses. PMID:11459968

  15. Analysis of near-neighbor contacts in bacteriophage T4 wedges and hubless baseplates by using a cleavable chemical cross-linker.

    PubMed Central

    Watts, N R; Coombs, D H

    1989-01-01

    Although bacteriophage T4 baseplate morphogenesis has been analyzed in some detail, there is little information available on the spatial arrangement and associations of its 150 subunits. We have therefore carried out the first analysis of its near-neighbor interactions by using the cleavable chemical cross-linker ethylene glycolbis(succinimidylsuccinate). In this report, we describe the cross-linked complexes that have been identified in the one-sixth arms or wedges and also in baseplatelike structures called rings consisting of six wedges but lacking the central hub, both of which are purified from T4 gene 5- -infected cells. Thirty different complexes were identified, of which about half contain multimers of a single species and half contain two different species. In general, the complexes reflect and support the assembly pathway derived by Kikuchi and King (Y. Kikuchi and J. King, J. Mol. Biol. 99:695-716, 1975) but broaden its scope to include such complexes as gp25-gp53, gp25-gp48, and gp48-gp53, which locate the gp48 binding site over the inner edge of the ring but outside the central hub. The data also supports the view that wedges are assembled from the outer edge inward toward the central hub. Wedge-wedge contact in rings was mediated primarily by gp12 and gp9, the absence of which dramatically destabilized the ring----wedge equilibrium in favor of wedges. Although no heterologous complexes containing gp9 were identified, gp12 contacts unique to rings were observed with both gp10 and gp11. Images PMID:2724408

  16. 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

  17. An interaction between the Walker A and D-loop motifs is critical to ATP hydrolysis and cooperativity in bacteriophage T4 Rad50.

    PubMed

    De la Rosa, Metzere Bierlein; Nelson, Scott W

    2011-07-22

    The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the ?-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a ?-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site. PMID:21610075

  18. Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases

    Microsoft Academic Search

    Shujie Dong; H. David Shew; Jianli Lu; Elumalai Sivamani; Eric S. Miller; Rongda Qu

    2008-01-01

    Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance\\u000a of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by\\u000a Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of

  19. 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. [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States); Rao, Mangala [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Janosi, Laszlo [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Sathaliyawala, Taheri [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States); Matyas, Gary R. [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Alving, Carl R. [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Leppla, Stephen H. [Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases, NIH, 30 Convent Dr., Bethesda, MD 20892 (United States); Rao, Venigalla B. [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States)]. 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.

  20. Deletion of the Hoc and Soc capsid proteins affects the surface and cellular uptake properties of bacteriophage T4 derived nanoparticles

    PubMed Central

    Robertson, Kelly; Furukawa, Yoko; Underwood, Alison; Black, Lindsay; Liu, Jinny L.

    2014-01-01

    Recently the use of engineered viral scaffolds in biotechnology and medical applications has been increasing dramatically. T4 phage capsid derived nanoparticles (NPs) have potential advantages as sensors and in biotechnology. These applications require that the physical properties and cellular uptake of these NPs be understood. In this study we used a T4 deletion mutant to investigate the effects of removing both the Hoc and Soc proteins from the capsid surface on T4 tailless NPs. The surface charge, zeta potential, size, and cellular uptake efficiencies for both the T4 NP and T4?Hoc?Soc NP mutant were measured and compared using dynamic light scattering and flow cytometry and significant differences were detected. PMID:22285187

  1. Head maturation pathway of bacteriophages T4 and T2. III. Isolation and characterization of particles produced by mutants in gene 17.

    PubMed Central

    Carrascosa, J L; Kellenberger, E

    1978-01-01

    We have isolated and characterized two types of particles produced in comparable amounts by mutants in gene 17: the empty large particle and the empty small particle. Dimensions, morphology, stability, and protein composition of the empty large particle are very similar to those of the capsids or empty heads of mature phage. The other type of particle (empty small particle) is very similar in dimensions and stability to the prehead, but differs in that it is composed of processed proteins (gp23, gp24, IpIII). Structural analysis has shown that the protein subunits of the empty small particles are arranged in an unexpanded type of lattice (11.2 to 11.3 nm), whereas the empty large particles have an expanded lattice (13 nm). The characterization of the empty small particle as being composed of cleaved proteins, but still unexpanded, shows that the expansion of the T4 head shell is not necessarily linked to the cleavage of the structural proteins. Images PMID:642076

  2. Studies on the Recombination Genes of Bacteriophage T4: Suppression of uvsX and uvsY Mutations by uvsW Mutations

    PubMed Central

    Yonesaki, Tetsuro; Minagawa, Teiichi

    1987-01-01

    Genes uvsW, uvsX and uvsY are dispensable for T4 growth but are implicated in recombination and in the repair of damaged DNA. We found that large-plaque mutants arose efficiently from small-plaque uvsX and uvsY mutants at 42° and were pseudorevertants containing a new mutation in uvsW. Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants. At 41° the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46, but at 30° it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants. Restored DNA synthesis at 41° was paralleled by the overproduction of single-stranded DNA and gene 32 protein. Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes. Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product. PMID:3549448

  3. Nanoscale bacteriophage biosensors beyond phage display

    PubMed Central

    Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

    2013-01-01

    Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology. PMID:24143096

  4. 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

  5. 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 ...

  6. Lysozymes in the animal kingdom

    Microsoft Academic Search

    Lien Callewaert; Chris W. Michiels

    2010-01-01

    Lysozymes (EC 3.2.1.17) are hydrolytic enzymes, characterized by their ability to cleave the ?-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer. In the animal kingdom, three major distinct lysozyme\\u000a types have been identified — the c-type (chicken or conventional type), the g-type (goose-type) and the i-type (invertebrate type) lysozyme. Examination of the phylogenetic

  7. Comparative studies of ?-Lactalbumin and lysozyme: Echidna lysozyme

    Microsoft Academic Search

    K. E. Hopper; H. A. McKenzie

    1974-01-01

    Summary a-Lactalbumin is a modifier protein for galactosyl transferase in the biosynthesis of lactose and lysozyme catalyses the cleavage of ß(1–4) glycosidic linkages. It is now generally believed that a-lactalbumin and lysozyme have diverged from a common ancestor. Comparative studies of these proteins from several species are reported here.

  8. 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...

  9. 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

  10. Identification and characterization of a novel phage-type like lysozyme from Manila clam, Ruditapes philippinarum.

    PubMed

    Ding, Jianfeng; Wang, Rui; Yang, Feng; Zhao, Liqiang; Qin, Yanjie; Zhang, Guofan; Yan, Xiwu

    2014-11-01

    A novel lysozyme gene (RpLysPh) with high similarity to the bacteriophage lysozymes was identified in Manila clam, Ruditapes philippinarum. The full length cDNA of RpLysPh is 828bp and contains a 462bp open reading frame (ORF) that codes for a 154 amino acid protein. Multiple sequence alignment analysis revealed that the three residues essential for catalytic activity in phage-type lysozyme (Glu(20), Asp(29), and Thr(35)) are conserved in RpLysPh. The comparison of the 3D models of RpLysPh and Coxiella burnetii lysozyme also suggested that the active sites involved in the binding of substrate have similar conformations. Phylogenetic analysis suggested that RpLysPh shares a similar origin with the bacterial phage-type lysozyme group. The highest level of expression of RpLysPh was observed in hemocytes, followed by mantle. Induction of RpLysPh expression was observed in gills in response to lipopolysaccharide (LPS), peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly(I:C)), and whole glucan particles (WGP) challenge. The recombinant protein of RpLysPh showed antibacterial activity against both Gram-positive and Gram-negative bacteria. PMID:24995730

  11. Bacteriophage Interference in Bacillus subtilis 168

    PubMed Central

    Yasbin, R. E.; Ganesan, A. T.; Young, F. E.

    1974-01-01

    Strains of Bacillus subtilis lysogenic for temperate bacteriophage SPO2 inhibit the development of bacteriophage ?1. After infection by bacteriophage ?1, DNA and RNA synthesis in the lysogenic host terminates, culminating in cell death. Bacteriophage SPO2 also prevents the production of bacteriophage ?105. Mechanisms for these two types of bacteriophage interference are discussed. PMID:4206943

  12. Biophysical aspects of lysozyme adduct with monocrotophos.

    PubMed

    Amaraneni, Sreenivasa Rao; Kumar, Sudhir; Gourinath, Samudrala

    2014-09-01

    The present study on in vitro formation and characterization of lysozyme adduct with monocrotophos (MP) evaluates the potential of lysozyme to be used as a sensitive biomarker to monitor exposure levels to the commonly used organophosphorus pesticide monocrotophos. Crystallization of lysozyme protein adduct with monocrotophos was also undertaken to understand the adduct formation mechanism at a molecular level. The binding of organophosphorus pesticides to lysozyme is one of the key steps in their mutagenicity. The formation and structural characterization of lysozyme adduct with monocrotophos was done using MALDI-TOFMS, fluorescence, UV/Vis spectroscopy, circular dichroism, and X-ray diffraction studies. We report the crystal structure of lysozyme adduct with monocrotophos at 1.9 Å. It crystallized in the P43 space group with two monomers in one asymmetric unit having one molecule of monocrotophos bound to each protein chain. The results proved that the fluorescence quenching of lysozyme by monocrotophos is due to binding of monocrotophos with a tryptophan residue of lysozyme. Monocrotophos interacts most strongly with the Trp-108 and Asp-52 of lysozyme. The interactions of the monocrotophos molecule with the lysozyme suggest the formation of a stable adduct. In addition, the alteration of lysozyme secondary structure in the presence of monocrotophos was confirmed by circular dichroism and fluorescence inhibition of lysozyme by increasing monocrotophos and UV/Vis spectrophotometry. The formation of lysozyme adduct with monocrotophos was confirmed by MALDI-TOFMS. PMID:24969463

  13. 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...

  14. Division M: Bacteriophage

    NSDL National Science Digital Library

    American Society for Microbiology. Division M.

    1998-01-01

    This website from the American Society for Microbiology provides current information on Bacteriophages ('Phages' for short), the tiny, microscopic viruses whose hosts are bacterial cells. The site is organized into five sections: general information, resources for teachers, images (micrographs, diagrams, etc.), upcoming meetings and events, and related links. Uncluttered but full of information, the site is an excellent resource for educators and students.

  15. Observing Lysozyme's Closing and Opening Motions by High-Resolution Single-Molecule Enzymology.

    PubMed

    Akhterov, Maxim V; Choi, Yongki; Olsen, Tivoli J; Sims, Patrick C; Iftikhar, Mariam; Gul, O Tolga; Corso, Brad L; Weiss, Gregory A; Collins, Philip G

    2015-06-19

    Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 ?s allowed the direct recording of lysozyme's opening and closing transitions. Unexpectedly, both motions required 37 ?s, on average. The distribution of transition durations was also independent of the enzyme's state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme's two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 ?s in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme's state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed. PMID:25763461

  16. Lysozyme: a model protein for amyloid research.

    PubMed

    Swaminathan, Rajaram; Ravi, Vijay Kumar; Kumar, Satish; Kumar, Mattaparthi Venkata Satish; Chandra, Nividh

    2011-01-01

    Ever since lysozyme was discovered by Fleming in 1922, this protein has emerged as a model for investigations on protein structure and function. Over the years, several high-resolution structures have yielded a wealth of structural data on this protein. Extensive studies on folding of lysozyme have shown how different regions of this protein dynamically interact with one another. Data is also available from numerous biotechnological studies wherein lysozyme has been employed as a model protein for recovering active recombinant protein from inclusion bodies using small molecules like l-arginine. A variety of conditions have been developed in vitro to induce fibrillation in hen lysozyme. They include (a) acidic pH at elevated temperature, (b) concentrated solutions of ethanol, (c) moderate concentrations of guanidinium hydrochloride at moderate temperature, and (d) alkaline pH at room temperature. This review aims to bring together similarities and differences in aggregation mechanisms, morphology of aggregates, and related issues that arise using the different conditions mentioned above to improve our understanding. The alkaline pH condition (pH 12.2), discovered and studied extensively in our lab, shall receive special attention. More than a decade ago, it was revealed that mutations in human lysozyme can cause accumulation of large quantities of amyloid in liver, kidney, and other regions of gastrointestinal tract. Understanding the mechanism of lysozyme aggregation will probably have therapeutic implications for the treatment of systemic nonneuropathic amyloidosis. Numerous studies have begun to focus attention on inhibition of lysozyme aggregation using antibody or small molecules. The enzymatic activity of lysozyme presents a convenient handle to quantify the native population of lysozyme in a sample where aggregation has been inhibited. The rich information available on lysozyme coupled with the multiple conditions that have been successful in inducing/inhibiting its aggregation in vitro makes lysozyme an ideal model protein to investigate amyloidogenesis. PMID:21846563

  17. RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities

    Microsoft Academic Search

    Tapas K. Sengupta; Johnthan Gordon; Eleanor K. Spicer

    2001-01-01

    The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of

  18. Lysozyme in chronic liver disease: a biochemical and histological study.

    PubMed Central

    Manifold, I H; Bishop, F M; Cloke, P; Triger, D R; Underwood, J C

    1982-01-01

    Serum lysozyme activities and semiquantitative analysis of tissue lysozyme distribution were studied in patients with primary biliary cirrhosis (PBC), chronic hepatitis (CH), miscellaneous liver diseases, and normal subjects. Serum lysozyme was significantly raised in PBC and CH. Portal venous blood has similar lysozyme activities to peripheral venous blood in a group of various liver diseases. Lysozyme-containing intralobular cells were decreased in all liver diseases studied but portal tract lysozyme was increased only in PBC and CH. Thus the increase in serum lysozyme in PBC and CH appears to originate from the portal inflammatory infiltrate, seen in these diseases. Images PMID:7050185

  19. THE MORTALITY OF BACTERIOPHAGE CONTAINING ASSIMILATED RADIOACTIVE PHOSPHORUS

    PubMed Central

    Hershey, A. D.; Kamen, M. D.; Kennedy, J. W.; Gest, H.

    1951-01-01

    The bacteriophage T4 containing assimilated radioactive phosphorus is inactivated at a rate proportional to the specific radioactivity of the constituent phosphorus. The beta radiation from the phosphorus makes a negligible contribution to this effect. The inactivation is therefore a direct consequence of the nuclear reaction, which kills the phage with an efficiency of about 1/12. Several phages related to T4 behave similarly. When radioactive phage is grown from a seed of non-radioactive phage, all of the phage progeny are subject to killing by radioactive decay. The phage is killed by beta radiation from P32 with an efficiency of about 1/100 per ionization within the particle volume. Bacteriophage T4 and its relatives contain about 500,000 atoms of phosphorus per infective particle. Virtually all this phosphorus is adsorbed to bacteria with the specificity characteristic of the infective particles, and none of it can be removed from the particles by the enzyme desoxyribonuclease. The phosphorus content per particle, together with the published data on analytical composition, indicates a particle diameter close to 110 mµ for the varieties of phage studied. PMID:14824499

  20. Acetylated lysozyme as impurity in lysozyme crystals: constant distribution coefficient

    NASA Astrophysics Data System (ADS)

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

    2001-11-01

    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 and B impurities added were 0.76, 0.38 and 0.1 mg/ml and 0.43, 0.22, 0.1 mg/ml, respectively. The HEWL concentration were 20, 30 and 40 mg/ml. The crystals grown in 18 experiments for each impurity concentration and supersaturation 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±0.13 for A and K=3.42±0.25 for B. According to definition of K by Eq. (1) in the text, the condition K=const is equivalent to a decrease of impurity amount in the crystal as the supersaturation increases. 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 the impurity adsorption and incorporation rates are proportional to the impurity concentration and that the growth rate is proportional to the concentration of crystallizing protein in solution. The frequency at which an impurity molecules irreversibly join the crystal was estimated to be 3 s -1, much higher than such frequency for regular crystal molecules 5×10 -2 s -1 at 30 mg/ml lysozyme concentration. Reasons for this inequality are discussed.

  1. The SPO1-related bacteriophages

    Microsoft Academic Search

    Jochen Klumpp; Rob Lavigne; Martin J. Loessner; Hans-Wolfgang Ackermann

    2010-01-01

    A large and diverse group of bacteriophages has been termed ‘SPO1-like viruses’. To date, molecular data and genome sequences\\u000a are available for Bacillus phage SPO1 and eight related phages infecting members of other bacterial genera. Many additional bacteriophages have been\\u000a described as SPO1-related, but very few data are available for most of them. We present an overview of putative ‘SPO1-like

  2. Effects of DNA-binding drugs on T4 DNA ligase.

    PubMed Central

    Montecucco, A; Pedrali-Noy, G; Spadari, S; Lestingi, M; Ciarrocchi, G

    1990-01-01

    A number of DNA intercalating and externally binding drugs have been found to inhibit nick sealing, cohesive and blunt end ligation, AMP-dependent DNA topoisomerization and EDTA-induced DNA nicking mediated by bacteriophage T4 DNA ligase. The inhibition seems to arise from drug-substrate interaction so that formation of active DNA-Mg2(+)-AMP-enzyme complex is impaired while assembled and active complexes are not disturbed by drug binding to the substrate. Images Fig. 2. Fig. 4. Fig. 5. PMID:2156493

  3. Bacteriophage behavioral ecology

    PubMed Central

    Hargreaves, Katherine R; Kropinski, Andrew M; Clokie, Martha RJ

    2014-01-01

    Bacteriophages have an essential gene kit that enables their invasion, replication, and production. In addition to this “core” genome, they can carry “accessory” genes that dramatically impact bacterial biology, and presumably boost their own success. The content of phage genomes continue to surprise us by revealing new ways that viruses impact bacterial biology. The genome of a Clostridium difficile myovirus, phiCDHM1, contains homologs of three bacterial accessory gene regulator (agr) genes. The agr system is a type of quorum sensing (QS), via which the phage may modify C. difficile interactions with its environment. Although their mechanism of action is unknown, mutants in bacterial versions of these genes impact sporulation and virulence. To explore how phage QS genes may influence C. difficile biology, we examine the main categories of bacterial behavior that phages have been shown to influence and discuss how interactions via QS could influence behavior at a wider level. PMID:25105060

  4. 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.

  5. Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase

    Microsoft Academic Search

    Craig Tuerk; Larry Gold

    1990-01-01

    High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with

  6. 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...

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

    PubMed

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

    1988-05-11

    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. PMID:3287337

  8. 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

  9. Composite cryogels for lysozyme purification.

    PubMed

    Baydemir, Gözde; Türko?lu, Emir Alper; Andaç, Müge; Perçin, I??k; Denizli, Adil

    2015-03-01

    Beads-embedded novel composite cryogel was synthesized to purify lysozyme (Lyz) from chicken egg white. The poly(hydroxyethyl methacrylate-N-methacryloyl-L-phenylalanine) (PHEMAPA) beads of smaller than 5 µm size were synthesized by suspension polymerization and then embedded into a poly(hydroxyethyl methacrylate) (PHEMA)-based cryogel column. The PHEMAPA bead-embedded cryogel (BEC) column was characterized by swelling tests, scanning electron microscopy (SEM), surface area measurements by the Brunauer-Emmett-Teller (BET) method, elemental analysis, and flow dynamics. The specific surface area of the PHEMAPA BEC was found as 41.2 m(2) /g using BET measurements. Lyz-binding experiments were performed using aqueous solutions in different conditions such as initial Lyz concentration, pH, flow rate, temperature, and NaCl concentration of an aqueous medium. The PHEMAPA BEC column could be used after 10 adsorption-desorption studies without any significant loss in adsorption capacity of Lyz. The PHEMAPA BEC column was used to purify Lyz from chicken egg white, and gel electrophoresis was used to estimate the purity of Lyz. The chromatographic application of the PHEMAPA BEC column was also performed using fast protein liquid chromatography. PMID:24923509

  10. Virulence reduction in bacteriophage resistant bacteria.

    PubMed

    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

  11. 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

  12. INDEPENDENT FUNCTIONS OF VIRAL PROTEIN AND NUCLEIC ACID IN GROWTH OF BACTERIOPHAGE

    Microsoft Academic Search

    A. D. Hershey; MARTHA CHASE

    1952-01-01

    The work of Doermaml (1948), Doermann and Dissosway (1949), and Anderson and Doermann (1952) has shown that bacteriophages T2, T3, and T4 multiply in the bacterial cell in a non-infective form. The same is true of the phage carried by certain lysogenic bacteria (Lwoff and Gutmann, 1950). Little else is known about the vegetative phase of these viruses. The experi-

  13. 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.

  14. Organic Cosolvents and Hen Egg White Lysozyme Folding

    E-print Network

    Luhua, Lai

    1 Organic Cosolvents and Hen Egg White Lysozyme Folding Bing Lai, Aoneng Cao, Luhua Lai* State Key of Chemistry, Peking University, BEIJING 100871, China Key words: lysozyme; organic cosolvent; circular on the influence of organic cosolvents on lysozyme folding have been reported. As most of the researches

  15. Ultrasound and Hypersound Speeds in Lysozyme Solutions

    NASA Astrophysics Data System (ADS)

    Schulte, Alfons; Pruner, Christian; Wilhelm, Emmerich; Asenbaum, Augustinus

    2011-03-01

    Ultrasound velocimetry and Brillouin spectroscopy provide information on the compressibility of proteins and the surrounding hydration layer. Employing both techniques we investigate the sound speeds at GHz (hypersound) and MHz (ultrasound) frequencies in lysozyme solutions (250 mg / ml, pH 7) and pure water over the temperature range from 275 K to 335 K. Compared to water the Brillouin peaks in the lysozyme solutions are shifted by about 400 MHz towards higher frequencies. This shift reflects the change in sound speed and is attributed to the influence of the compressibility of the protein and bound water in the hydration shell. In addition, we measure a dispersion of the sound velocity in the lysozyme solution. The higher sound speed at GHz frequencies, as measured by Brillouin scattering, may indicate additional relaxation processes as compared to pure bulk water, where no sound dispersion between ultrasound speed and hypersound speed is observed.

  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. 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...

  18. Immunogenicity Studies of Proteins Forming the T4 Phage Head Surface

    PubMed Central

    Miernikiewicz, Paulina; Piotrowicz, Agnieszka; Hodyra, Katarzyna; Owczarek, Barbara; Lecion, Dorota; Ka?mierczak, Zuzanna; Letarov, Andrey; Górski, Andrzej

    2014-01-01

    ABSTRACT Advances in phage therapy and novel applications of phages in biotechnology encourage interest in phage impact on human and animal immunity. Here we present comparative studies of immunogenic properties of T4 phage head surface proteins gp23*, gp24*, Hoc, and Soc, both as elements of the phage capsid and as isolated agents. Studies comprise evaluation of specific antibodies in the human population, analysis of the proteins' impact on the primary and secondary responses in mice, and the effect of specific antibodies on phage antibacterial activity in vitro and in vivo in mice. In humans, natural antibodies specific to T4-like phages were abundant (81% of investigated sera). Among those, significantly elevated levels of IgG antibodies only against major head protein (gp23*) were found, which probably reflected cross-reactions of T4 with antibodies induced by other T4-like phages. Both IgM and IgG antibodies were induced mostly by gp23* and Hoc, while weak (gp24*) and very weak (Soc) reactivities of other head proteins were noticed. Thus, T4 head proteins that markedly contribute to immunological memory to the phage are highly antigenic outer capsid protein (Hoc) and major capsid protein (gp23*). Specific anti-gp23* and anti-Hoc antibodies substantially decreased T4 phage activity in vitro and to some extent in vivo. Cooperating with antibodies, the immune complement system also contributed to annihilating phages. IMPORTANCE Current descriptions of phage immunogenicity and its biological consequences are still vague and incomplete; thus, the central problem of this work is timely and may have strong practical implications. Here is presented the very first description of the contribution of bacteriophage proteins to immunological memory of the phage. Understanding of interactions between phages and mammalian immunology may help in biotechnological adaptations of phages for therapeutic requirements as well as for better appreciation of phage ecology and their role in the biosphere. PMID:25142581

  19. Detection and quantification of lysozyme in champagne wines.

    PubMed

    Marchal, R; Chaboche, D; Marchal-Delahaut, L; Gerland, C; Gandon, J P; Jeandet, P

    2000-08-01

    We describe here techniques to detect and quantify lysozyme in Pinot noir and Chardonnay Champagne wines. Using a dot-blot technique, lysozyme antibodies were able to recognize their antigens even when the concentration of lysozyme in wine was 75 mg/L. SDS-PAGE was the second technique used. After Coomassie Brilliant Blue (CBB) staining or antibody immunostaining was performed, the wine originating from the lysozyme-treated must gave only one band corresponding to the lysozyme. It is then possible to precisely determine the concentration of lysozyme in a must or a wine by densitometric measurement of this band. The control wine gave no band with the CBB staining, such as with the immunostaining. The quantification of lysozyme with HPLC is another useable technique because the lysozyme elution time is largely superior to that of all of the wine compounds. In wines, losses of lysozyme were higher when the enzyme was added at one time to the must (-34% for the Pinot noir and -37% for the Chardonnay) than when lysozyme is added in 2-fold both in the must and in the wine (around -26% for the two wines). The lowest diminution is observed when lysozyme was added to the wine only (-18%) in comparison to the addition to the must at 300 mg/L (-43%). PMID:10956095

  20. STUDIES ON BACTERIOPHAGE

    PubMed Central

    Goebel, Walther F.

    1950-01-01

    By serological means it has been shown that E. coli B contains an antigen closely related to the protein-lipocarbohydrate complex of Phase II Sh. sonnei. Lysis of E. coli B by three of the T viruses, T3, T4, and T7, can be inhibited by the Phase II dysentery antigen. It has been suggested that the receptor of E. coli B with which these viruses combine is this newly described antigenic component. Two variants of the virus T3 have also been described, in stocks which have been treated with the Phase II antigen. One of these variants infects both Phase II Sh. sonnei and E. coli B, and the other infects only the latter microorganism; neither of the two variants is inhibited by concentrations of the Phase II antigen of 1 mg. per cc. The distinctive properties of the variants are not hereditary. PMID:14784535

  1. Protonation favors aggregation of lysozyme with SDS.

    PubMed

    Khan, Javed M; Chaturvedi, Sumit K; Rahman, Shah K; Ishtikhar, Mohd; Qadeer, Atiyatul; Ahmad, Ejaz; Khan, Rizwan H

    2014-04-21

    Different proteins have different amino acid sequences as well as conformations, and therefore different propensities to aggregate. Electrostatic interactions have an important role in the aggregation of proteins as revealed by our previous report (J. M. Khan et al., PLoS One, 2012, 7, e29694). In this study, we designed and executed experiments to gain knowledge of the role of charge variations on proteins during the events of protein aggregation with lysozyme as a model protein. To impart positive and negative charges to proteins, we incubated lysozyme at different pH values of below and above the pI (?11). Negatively charged SDS was used to 'antagonize' positive charges on lysozyme. We examined the effects of pH variations on SDS-induced amyloid fibril formation by lysozyme using methods such as far-UV circular dichroism, Rayleigh scattering, turbidity measurements, dye binding assays and dynamic light scattering. We found that sub-micellar concentrations of SDS (0.1 to 0.6 mM) induced amyloid fibril formation by lysozyme in the pH range of 10.0-1.0 and maximum aggregation was observed at pH 1.0. The morphology of aggregates was fibrillar in structure, as visualized by transmission electron microscopy. Isothermal titration calorimetry studies demonstrated that fibril formation is exothermic. To the best of our current understanding of the mechanism of aggregation, this study demonstrates the crucial role of electrostatic interactions during amyloid fibril formation. The model proposed here will help in designing molecules that can prevent or reverse the amyloid fibril formation or the aggregation. PMID:24647567

  2. Adherence of Streptococcus sanguis to hydroxyapatite coated with lysozyme and lysozyme-supplemented saliva.

    PubMed

    Tellefson, L M; Germaine, G R

    1986-03-01

    The adherence of [3H]thymidine-labeled Streptococcus sanguis strains to bare hydroxyapatite and to hydroxyapatite coated with a range of concentrations of lysozyme, poly-L-lysine, poly-L-glutamic acid, whole saliva supernatant, and combinations of some of the above was studied. Adherence of several strains of S. sanguis to bare hydroxyapatite and saliva-coated hydroxyapatite was compared. Saliva present as a pellicle on the hydroxyapatite inhibited adherence of some strains (903, M-5, 73X11) and stimulated that of others (S35, B-4, 66X49). Strains 903 and S35 were chosen for further study. Adherence of both strains was stimulated up to fivefold by the presence of adsorbed lysozyme or poly-L-lysine on the hydroxyapatite, whereas poly-L-glutamic acid inhibited adherence (80 to 95%). Adherence of strain S35 to hydroxyapatite coated with combinations of saliva and (i) lysozyme, (ii) poly-L-lysine, or (iii) poly-L-glutamic acid was unaffected compared with adherence to hydroxyapatite coated with saliva alone. In contrast, adherence of strain 903 to hydroxyapatite coated with combinations of saliva and either lysozyme or poly-L-lysine was inhibited up to ca. 90% compared with hydroxyapatite coated with saliva alone. Strain 903 was also unaffected by combinations of poly-L-glutamic acid and saliva on the hydroxyapatite. Adherent cells of both strains were completely (greater than 90%) eluted with high-ionic-strength buffer from either bare hydroxyapatite or hydroxyapatite coated with lysozyme alone. Adherent cells of strain S35 were only poorly eluted (25%) from hydroxyapatite coated with either saliva alone or saliva and lysozyme. Strain 903 elution from hydroxyapatite coated with either saliva alone or saliva and lysozyme was essentially complete. These observations were taken to indicate that the two test strains adhered to saliva-coated hydroxyapatite by different mechanisms. Protein-coated hydroxyapatite was shown not to be saturated under the conditions described here. Examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the variously supplemented salivary pellicles formed on the hydroxyapatite demonstrated that major changes in salivary protein composition did not occur when lysozyme, poly-L-lysine, or poly-L-glutamic acid was used to supplement saliva. Lysozyme-dependent aggregation of strain 903 was shown not to occur under the conditions of our experiments. We suggest that the basis for stimulation of adherence to hydroxyapatite coated only with lysozyme is an increase in the cationic surface area available for electrostatic adherence of the microorganisms.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2419251

  3. Complete Genome Sequence of vB_EcoM_112, a T-Even-Type Bacteriophage Specific for Escherichia coli O157:H7

    PubMed Central

    Coffey, Brid; Ross, R. Paul; O’Flynn, Gary; O’Sullivan, Orla; Casey, Aidan; Callanan, Michael; Coffey, Aidan

    2014-01-01

    Bacteriophage vB_EcoM_112 (formerly e11/2) is an Escherichia coli phage with specificity for the O157:H7 serotype. The vB_EcoM_112 genome sequence shares high degrees of similarity with the phage T4 genome sequence. PMID:25395625

  4. Modifying bacteriophage lambda with recombineering.

    PubMed

    Thomason, Lynn C; Oppenheim, Amos B; Court, Donald L

    2009-01-01

    Recombineering is a recently developed method of in vivo genetic engineering used in Escherichia coli and other Gram-negative bacteria. Recombineering can be used to create single-base changes, small and large deletions, and small insertions in phage lambda as well as in bacterial chromosomes, plasmids, and bacterial artificial chromosomes (BACS). This technique uses the bacteriophage lambda generalized recombination system, Red, to catalyze homologous recombination between linear DNA and a replicon using short homologies of 50 base pairs. With recombineering, single-stranded oligonucleotides or double-stranded PCR products can be used to directly modify the phage lambda genome in vivo. It may also be possible to modify the genomes of other bacteriophages with recombineering. PMID:19066825

  5. 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

  6. Genome and Proteome of Campylobacter jejuni Bacteriophage NCTC 12673?†

    PubMed Central

    Kropinski, Andrew M.; Arutyunov, Denis; Foss, Mary; Cunningham, Anna; Ding, Wen; Singh, Amit; Pavlov, Andrey R.; Henry, Matthew; Evoy, Stephane; Kelly, John; Szymanski, Christine M.

    2011-01-01

    Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms. PMID:21965409

  7. Lysozyme depolymerization of photo-activated chitosan adhesive films.

    PubMed

    Mawad, Damia; Warren, Charles; Barton, Mathew; Mahns, David; Morley, John; Pham, Binh T T; Pham, Nguyen T H; Kueh, Sindy; Lauto, Antonio

    2015-05-01

    Effective tissue bioadhesion of rose bengal-chitosan films can be achieved by photoactivation using a green laser. In this study, lysozyme was incorporated in these films to enhance the rate of depolymerization and assess the laser impact on lysozyme. The lysozyme loaded films exhibited a 21% mass loss after 4 weeks implantation in rats while control films (without lysozyme) had only 7% mass loss. Capillary electrophoresis-mass spectroscopy showed that chitosan degraded into monomers and oligomers of glucosamine and N-acetyl-glucosamine. Irradiation with laser did not affect the depolymerization of adhesive by lysozyme suggesting that the inclusion of lysozyme in the bioadhesive is a viable technique for tailoring the depolymerization. PMID:25659671

  8. The toxicity study on marine low-temperature lysozyme.

    PubMed

    Yan, Chun-ling; Ding, Bo-xiao; Lan, Xiao-ming; Guo, Shen-bo; Xie, Yan-ying; Wang, Chun-bo

    2008-02-01

    Marine low-temperature lysozyme is purified from a marine bacterium. The lysozyme can keep high activity at low-temperature and has broad-spectrum antibiotic reaction. This study was undertaken to investigate the major characteristics, acute and subchronic toxicity of marine low-temperature lysozyme. The relative molecular weight of this lysozyme was determined as approximate 16 kD; its optimum pH value and temperature towards Micrococcus lysodleikticus were pH 6.5 and 35 degrees C, respectively. The lysozyme activity was slightly enhanced by Zn(2+) and Cu(2+) and slightly inhibited by Mn(2+) and Ag(+). The lysozyme showed good compatibility to many common chemical agents such as EDTA (0.1%), KH(2)PO(4) (1.0%), etc. In experiments on acute toxicity, the drug was injected through the tail vein of mice, and intoxication symptoms and date of death were recorded. The 50% lethal dose (LD(50)) of Marine low-temperature lysozyme and 95%, 99% confidence interval (CI) was calculated. The subchronic study was designed to determine whether effects progressed with repeated Marine low-temperature lysozyme exposure. Wistar rats were tested by daily intragastric administration of Marine low-temperature lysozyme at the doses of 1.0; 0.5; 0.25 g/kg bw for 90 days. The LD(50) value of lysozyme was 4530 mg/kg bw; 90 days of Marine low-temperature lysozyme treatment at three doses, and there is no significant difference on blood biochemistry and organ index in drug treatment groups compared to saline treatment group. There is no affirmative pathologic change of all the observed organs in this study. The present results suggest that Marine low-temperature lysozyme can be safely used at the dose of experiment applied. PMID:17935850

  9. Hydration of lysozyme studied by Raman spectroscopy.

    PubMed

    Kocherbitov, Vitaly; Latynis, Jekaterina; Misiunas, Audrius; Barauskas, Justas; Niaura, Gediminas

    2013-05-01

    Hydration plays a fundamental role in maintaining the three-dimensional structure and function of proteins. In this study, Raman spectroscopy was used to probe the hydration induced structural changes at various sites of lysozyme under isothermal conditions in the range of water contents from 0 to 44 wt %. Raman hydration curves were constructed from detailed analysis of marker bands. Transition inflection points (w(m)) and onsets determined from the hydration curves have shown that structural changes start at 7-10 and end at about 35 wt % water. The onset of structural changes coincides with the onset of the broad glass transition earlier observed in this system. The increase of ?-helix content starts at very low concentrations of water with w(m) = 12 wt %. Monitoring the development of importance for enzymatic action hydrophobic clusters has revealed wm = 15 wt % and completion of the process at 25 wt %. The parameters of 621 cm(-1) (Phe) and 1448 cm(-1) (CH2 bending) modes were found to be sensitive to hydration, suggesting changes in organization of water molecules near the protein surface. The native structure of lysozyme was achieved at 35 wt % water where its content is high enough for filling the space between lysozyme molecules. PMID:23557185

  10. 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)...

  11. 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)...

  12. Evaluation of oriented lysozyme immobilized with monoclonal antibody

    NASA Astrophysics Data System (ADS)

    Aoyagi, Satoka; Okada, Keigo; Shigyo, Ayako; Man, Naoki; Karen, Akiya

    2008-12-01

    The orientation of a lysozyme immobilized with a monoclonal antibody was evaluated based on determination of the uppermost surface structure using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Specific peaks of the oriented lysozyme immobilized with monoclonal anti-lysozyme antibody were obtained in comparison with reference samples, non-oriented immobilized lysozyme and immobilized anti-lysozyme antibody. All samples were freeze-dried before TOF-SIMS measurement, and then each sample was measured using TOF-SIMS with a bismuth cluster ion source. TOF-SIMS spectra were analyzed to select peaks specific to the oriented immobilized lysozyme as well as to identify their chemical formula and ensemble of amino acids. The possible chemical formulae of the lysozyme fragments were then investigated with an element matching program and a residue matching program. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the lysozyme and its three-dimensional structure registered in the protein data bank. Finally, the fragment-ion-generating regions of the oriented immobilized lysozyme were determined based on the suggested residues and the three-dimensional structure.

  13. Electrostatic Contributions to T4 Lysozyme Stability: Solvent-Exposed Charges versus Semi-Buried Salt Bridges

    E-print Network

    Weston, Ken

    unfolding at extreme pH values, yet quantitative understanding of these roles has proven elusive due calculated by continuum electrostatics is very sensitive to the definition of the boundary between the high

  14. Force field impact and spin-probe modeling in molecular dynamics simulations of spin-labeled T4 lysozyme

    Microsoft Academic Search

    Ileana Stoica

    2005-01-01

    Several attempts have been made to compute electron paramagnetic resonance (EPR) spectra of biomolecules, using motional models or simulated trajectories to describe dynamics. Ideally, the simulated trajectories should capture “fast” (picosecond) snapshots of spin-probe rotations accurately, while being lengthy enough to ensure a proper Fourier integration of the time-domain signal. It is the interplay of the two criteria that poses

  15. Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family.

    PubMed

    Ito, Y; Yoshikawa, A; Hotani, T; Fukuda, S; Sugimura, K; Imoto, T

    1999-01-01

    Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N-terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme-like enzymatic features. PMID:9914527

  16. T4-Like Phage Bp7, a Potential Antimicrobial Agent for Controlling Drug-Resistant Escherichia coli in Chickens

    PubMed Central

    Zhang, Can; Li, Wenli; Liu, Wenhua; Zou, Ling; Yan, Chen; Lu, Kai

    2013-01-01

    Chicken-pathogenic Escherichia coli is severely endangering the poultry industry in China and worldwide, and antibiotic therapy is facing an increasing problem of antibiotic resistance. Bacteriophages can kill bacteria with no known activity in human or animal cells, making them an attractive alternative to antibiotics. In this study, we present the characteristics of a novel virulent bacteriophage, Bp7, specifically infecting pathogenic multidrug-resistant E. coli. Phage Bp7 was isolated from chicken feces. Bp7 belongs to the family Myoviridae, possessing an elongated icosahedral head and contractile sheathed tail. It has a 168-kb double-stranded DNA genome. For larger yields, its optimal multiplicity of infection (MOI) to infect E. coli was about 0.001. The latent period was 10 to 15 min, and the burst size was 90 PFU/infected cell. It was stable both at pH 5.0 to 10.0 and at 40°C or 50°C for at least 1 h. Bp7 could infect 46% of pathogenic clinical E. coli strains. Bp7 harbored 791 open reading frames (ORFs) and 263 possible genes. Among the 263 genes, 199 possessed amino acid sequence identities with ORFs of phage T4, 62 had identities with other T4-like phages, and only one lacked any database match. The genome of Bp7 manifested obvious division and rearrangement compared to phages T4, JS98, and IME08. Bp7 is a new member of the “T4-like” genus, family Myoviridae. Its wide host range, strong cell-killing activity, and high stability to pH make it an alternative to antimicrobials for controlling drug-resistant E. coli in chickens. PMID:23835183

  17. Bacteria screening, viability, and confirmation assays using bacteriophage-impedimetric/loop-mediated isothermal amplification dual-response biosensors.

    PubMed

    Tlili, Chaker; Sokullu, Esen; Safavieh, Mohammadali; Tolba, Mona; Ahmed, Minhaz Uddin; Zourob, Mohammed

    2013-05-21

    Here, we integrate two complementary detection strategies for the identification and quantification of Escherichia coli based on bacteriophage T4 as a natural bioreceptor for living bacteria cells. The first approach involves screening and viability assays, employing bacteriophage as the recognition element in label-free electrochemical impedance spectroscopy. The complementary approach is a confirmation by loop-mediated isothermal amplification (LAMP) to amplify specifically the E. coli Tuf gene after lysis of the bound E. coli cells, followed by detection using linear sweep voltammetry. Bacteriphage T4 was cross-linked, in the presence of 1,4-phenylene diisothiocyanate, on a cysteamine-modified gold electrode. The impedimetric biosensor exhibits specific and reproducible detection with sensitivity over the concentration range of 10(3)-10(9) cfu/mL, while the linear response of the LAMP approach was determined to be 10(2)-10(7) cfu/mL. The limit of detection (LOD) of 8 × 10(2) cfu/mL in less than 15 min and 10(2) cfu/mL within a response time of 40 min were achieved for the impedimetric and LAMP method, respectively. This work provides evidence that integration of the T4-bacteriophage-modified biosensor and LAMP can achieve screening, viability, and confirmation in less than 1 h. PMID:23510137

  18. 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...

  19. Influence of lysozyme treatments on champagne base wine foaming properties.

    PubMed

    Marchal, R; Chaboche, D; Douillard, R; Jeandet, P

    2002-03-13

    The objective of this study was to estimate the effect of lysozyme on the foaming characteristics of Champagne base wine. Lysozyme additions were made to the musts and also to the wines before and after bentonite or charcoal treatments, which remove endogenous proteins. Treatments with bentonite diminished foamability and foam stability of wines, whatever the dose (30 or 80 g/hL) and variety [Chardonnay, -28%; Pinot noir, -20% (at 30 g/hL)]. An addition of lysozyme in must raised Pinot noir wine foamability by 21%, whereas the difference is hardly perceptible for Chardonnay wine (+3%). Pinot noir and Chardonnay wines, originating from lysozyme-treated musts, in addition to bentonite treatment on the wine, presented higher foamability than wines treated only with bentonite. Lysozyme was removed (91-100%) by the bentonite treatment. Then, it was not responsible for the increase in foamability but seemed to have a protective effect on the wine proteins. When wines were initially treated with bentonite (150 g/hL) and then enriched with 80 g/hL lysozyme, this enzyme was not able to restore foaming properties. Treatments with charcoal always diminished foamability. The average increase in foamability due to an addition of lysozyme after charcoal treatment (80 g/hL) was 23%. Results showed a real positive effect of lysozyme on foam stability when wines have to be treated with charcoal (+25% and +56% for the Pinot noir wine and the Chardonnay wine, respectively, at 30 g/hL). PMID:11879014

  20. Dynamic clusters in highly concentrated lysozyme solutions

    NASA Astrophysics Data System (ADS)

    Hudson, S. D.; Godfrin, P. D.; Porcar, L.; Falus, P.; Hong, K.; Wagner, N. J.; Liu, Y.

    2014-03-01

    New biologic drugs need to be highly concentrated to have the required dosage for injection. Such high concentrations pose challenges for solution viscosity and stability. We therefore have studied the viscosity and dynamic clustering behavior of concentrated (up to 500 mg/mL) lysozyme solutions. Cluster dynamics are measured by neutron spin echo scattering experiments, which yield the mutual diffusivity. Viscosity is measured with a miniature capillary viscometer. While static scattering indicates cluster-like organization, the dynamic measurements show that these are momentary and do not survive local diffusion times. At high concentrations, they persist and diffusivity and viscosity dramatically increase.

  1. Biochemical and Genetic Characterization of Bacteriophage Holins 

    E-print Network

    To, Kam Ho

    2013-11-06

    Bacteriophages infect and kill bacterial cells. During the infection cycle, a phage attaches to the host cell surface, then ejects its DNA into the cytoplasm, where its progenies are subsequently assembled. The final step of the infection cycle...

  2. Comparison of the microbicidal and muramidase activities of mouse lysozyme M and P.

    PubMed Central

    Markart, Philipp; Faust, Nicole; Graf, Thomas; Na, Cheng-Lun; Weaver, Timothy E; Akinbi, Henry T

    2004-01-01

    Lysozyme is one of the most abundant antimicrobial proteins in the airspaces of the lung. Mice express two lysozyme genes, lysozyme M and P, but only the M enzyme is detected in abundance in lung tissues. Disruption of the lysozyme M locus significantly increased bacterial burden and mortality following intratracheal infection with a Gram-negative bacterium. Unexpectedly, significant lysozyme enzyme activity (muramidase activity) was detected in the airspaces of uninfected lysozyme M-/- mice, amounting to 25% of the activity in wild-type mice. Muramidase activity in lysozyme M-/- mice was associated with increased lysozyme P mRNA and protein in lung tissue and bronchoalveolar lavage fluid respectively. The muramidase activity of recombinant lysozyme P was less than that of recombinant M lysozyme. Recombinant P lysozyme was also less effective in killing selected Gram-negative bacteria, requiring higher concentrations than lysozyme M to achieve the same level of killing. The lower antimicrobial activity of P lysozyme, coupled with incomplete compensation by P lysozyme in lysozyme M-/- mice, probably accounts for the increased susceptibility of null mice to infection. Recombinant lysozyme M and P were equally effective in killing selected Gram-positive organisms. This outcome suggests that disruption of both M and P loci would significantly increase susceptibility to airway infections, particularly those associated with colonization by Gram-positive organisms. PMID:14977423

  3. Models of bacteriophage DNA packaging motors

    Microsoft Academic Search

    Philip Serwer

    2003-01-01

    An ATP-dependent motor drives a DNA genome into a bacteriophage capsid during morphogenesis of double-stranded DNA bacteriophages both in vivo and in vitro. The DNA molecule enters the capsid through a channel in the center of a symmetric protein ring called a connector. Mechanisms in two classes have been proposed for this motor: (1) An ATP-driven rotating connector pulls a

  4. Bacteriophage endolysins as novel antimicrobials

    PubMed Central

    Schmelcher, Mathias; Donovan, David M; Loessner, Martin J

    2013-01-01

    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 access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology. PMID:23030422

  5. Expression and prognostic significance of lysozyme in male breast cancer

    PubMed Central

    Serra, Carlos; Vizoso, Francisco; Alonso, Lorena; Rodríguez, Juan C; González, Luis O; Fernández, María; Lamelas, María L; Sánchez, Luis M; García-Muñiz, José L; Baltasar, Aniceto; Medrano, Justo

    2002-01-01

    Background Lysozyme, one of the major protein components of human milk that is also synthesized by a significant percentage of breast carcinomas, is associated with lesions that have a favorable outcome in female breast cancer. Here we evaluate the expression and prognostic value of lysozyme in male breast cancer (MBC). Methods Lysozyme expression was examined by immunohistochemical methods in a series of 60 MBC tissue sections and in 15 patients with gynecomastia. Staining was quantified using the HSCORE (histological score) system, which considers both the intensity and the percentage of cells staining at each intensity. Prognostic value of lysozyme was retrospectively evaluated by multivariate analysis taking into account conventional prognostic factors. Results Lysozyme immunostaining was negative in all cases of gynecomastia. A total of 27 of 60 MBC sections (45%) stained positively for this protein, but there were clear differences among them with regard to the intensity and percentage of stained cells. Statistical analysis showed that lysozyme HSCORE values in relation to age, tumor size, nodal status, histological grade, estrogen receptor status, metastasis and histological type did not increase the statistical significance. Univariate analysis confirmed that both nodal involvement and lysozyme values were significant predictors of short-term relapse-free survival. Multivariate analysis, according to Cox's regression model, also showed that nodal status and lysozyme levels were significant independent indicators of short-term relapse-free survival. Conclusion Tumor expression of lysozyme is associated with lesions that have an unfavorable outcome in male breast cancer. This milk protein may be a new prognostic factor in patients with breast cancer. PMID:12473177

  6. Partial Exclusion between T-Even Bacteriophages; an Incipient Genetic Isolation Mechanism

    PubMed Central

    Russell, Richard L.; Huskey, Robert J.

    1974-01-01

    Conditional lethal mutant systems developed in T-even bacteriophages T2, T4 and T6 have been used to study the partial exclusion which characterizes mixed infections of these phages. In bacteria mixedly infected with T2 and T4, the dominant phage (T4) acts against localized exclusion sensitivity determinants in the genome of the excluded phage (T2). These determinants are clustered near genes controlling early functions; the determinants themselves do not appear among the progeny, but markers located close to them appear infrequently, by recombination. The excluding action of T4 does not depend on the action of any gene so far identified by conditional lethal mutations, nor does it depend on differences in DNA glucosylation between infecting phages. Regardless of mechanism, the genetic consequence of this partial exclusion is to limit genetic exchange between T2 and T4 in the region of the genome controlling early functions, while retaining the capacity for extensive exchange in other regions; in short, partial exclusion constitutes a localized genetic isolating mechanism. Related forms of partial exclusion characterize mixed infections of other T-even phages, including those of some phages newly isolated from nature. PMID:4455560

  7. On-chip impedimetric detection of bacteriophages in dairy samples

    Microsoft Academic Search

    Cristina García-Aljaro; Xavier Muñoz-Berbel; Francisco J. Muñoz

    2009-01-01

    Bacteriophage infection of starter cultures constitutes a major problem in the dairy fermentation industry, which may bring about important economic losses. In this study, a rapid detection method of bacteriophages was developed based on analysis of impedance changes occurred upon infection of a host-biofilm established onto metal microelectrodes. Bacteriophage PhiX174 and Escherichia coli WG5 were chosen as models for bacteriophage

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

    SciTech Connect

    Mamontov, Eugene [ORNL] [ORNL; O'Neill, Hugh Michael [ORNL] [ORNL

    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.

  9. BACTERIOPHAGE: A SAFE AND NATURAL ALTERNATIVE TO ANTIMICROBIAL GROWTH PROMOTERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage are viruses that infect and kill bacteria. Bacteriophage do not infect either animal or plant cells making them a potentially safe alternative to antibiotics to both prevent and treat bacterial diseases in animals and humans. Bacteriophage lytic to a sero-type 02 strain of Escherichi...

  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, 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...

  12. 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...

  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. Osmotic pressure measurements of ovalbumin and lysozyme mixtures

    E-print Network

    McCarty, Bradley Wayne

    1984-01-01

    of Depa nt J. B. Natowitz May 1984 ABSTRACT Osmotic Pressure Measurements of Ovalbumin and Lysozyme Mixtures. (May 1984) Bradley Wayne McCarty, B. S, , Abilene Christian University Chairman of Advisory Committee: Dr. E. T. Adams, Jr. Ovalbumin...

  15. Penetration and fusion of phospholipid vesicles by lysozyme

    SciTech Connect

    Kim, J.; Kim, H. (Korea Advanced Institute of Science and Technology, Seoul)

    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.

  16. 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

  17. Strong and selective adsorption of lysozyme on graphene oxide.

    PubMed

    Li, Shanghao; Mulloor, Jerome J; Wang, Lingyu; Ji, Yiwen; Mulloor, Catherine J; Micic, Miodrag; Orbulescu, Jhony; Leblanc, Roger M

    2014-04-23

    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

  18. 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

  19. Bacteriophage Protein–Protein Interactions

    PubMed Central

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

    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

  20. Bacteriophages from the forestomachs of Australian marsupials.

    PubMed

    Klieve, A V

    1991-12-01

    Bacteriophages were observed in forestomach contents from three species of Australian macropodoid marsupials possessing a foregut fermentative digestion: the eastern grey kangaroo (Macropus giganteus), the eastern wallaroo (Macropus robustus robustus), and the rufous bettong (Aepyprymnus rufescens). Forty-six morphologically distinct phage types, representing the families Myoviridae, Siphoviridae, and Podoviridae, were identified. The range of forms varied between host species. The greatest diversity of phage types was found in forestomach contents of the wallaroo, and few phage types were recorded from the rufous bettongs. It is concluded that macropodoid marsupials, in common with their eutherian counterparts, possess diverse populations of bacteriophages in their fermentative forestomachs. PMID:1785938

  1. 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

  2. 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

  3. Bacteriophages with the ability to degrade uropathogenic Escherichia coli biofilms.

    PubMed

    Chibeu, Andrew; Lingohr, Erika J; Masson, Luke; Manges, Amee; Harel, Josée; Ackermann, Hans-W; Kropinski, Andrew M; Boerlin, Patrick

    2012-04-01

    Escherichia coli-associated urinary tract infections (UTIs) are among the most common bacterial infections in humans. UTIs are usually managed with antibiotic therapy, but over the years, antibiotic-resistant strains of uropathogenic E. coli (UPEC) have emerged. The formation of biofilms further complicates the treatment of these infections by making them resistant to killing by the host immune system as well as by antibiotics. This has encouraged research into therapy using bacteriophages (phages) as a supplement or substitute for antibiotics. In this study we characterized 253 UPEC in terms of their biofilm-forming capabilities, serotype, and antimicrobial resistance. Three phages were then isolated (vB_EcoP_ACG-C91, vB_EcoM_ACG-C40 and vB_EcoS_ACG-M12) which were able to lyse 80.5% of a subset (42) of the UPEC strains able to form biofilms. Correlation was established between phage sensitivity and specific serotypes of the UPEC strains. The phages' genome sequences were determined and resulted in classification of vB_EcoP_ACG-C91 as a SP6likevirus, vB_EcoM_ACG-C40 as a T4likevirus and vB_EcoS_ACG-M12 as T1likevirus. We assessed the ability of the three phages to eradicate the established biofilm of one of the UPEC strains used in the study. All phages significantly reduced the biofilm within 2-12 h of incubation. PMID:22590682

  4. Lysozyme gene expression by hemocytes of Pacific white shrimp, Litopenaeus vannamei, after injection with Vibrio

    E-print Network

    Burnett, Louis E.

    Lysozyme gene expression by hemocytes of Pacific white shrimp, Litopenaeus vannamei, after antibacterial protein produced by shrimp hemocytes, within tissues of Litopenaeus vannamei Boone in response rights reserved. Keywords: Litopenaeus vannamei; Shrimp; Hemocyte; Vibrio; Lysozyme; Gene expression

  5. Bacteriophage Host Range and Bacterial Resistance

    Microsoft Academic Search

    Paul Hyman; Stephen T. Abedon

    2010-01-01

    Host range describes the breadth of organisms a parasite is capable of infecting, with limits on host range stemming from parasite, host, or environmental characteristics. Parasites can adapt to overcome host or environmental limitations, while hosts can adapt to control the negative impact of parasites. We consider these adaptations as they occur among bacteriophages (phages) and their bacterial hosts, since

  6. Optimal bacteriophage mutation rates for phage therapy

    Microsoft Academic Search

    David T. Kysela; Paul E. Turner

    2007-01-01

    The mutability of bacteriophages offers a particular advantage in the treatment of bacterial infections not afforded by other antimicrobial therapies. When phage-resistant bacteria emerge, mutation may generate phage capable of exploiting and thus limiting population expansion among these emergent types. However, while mutation potentially generates beneficial variants, it also contributes to a genetic load of deleterious mutations. Here, we model

  7. Bacteriophages as surface and ground water tracers

    NASA Astrophysics Data System (ADS)

    Rossi, P.; Dörfliger, N.; Kennedy, K.; Müller, I.; Aragno, M.

    Bacteriophages are increasingly used as tracers for quantitative analysis in both hydrology and hydrogeology. The biological particles are neither toxic nor pathogenic for other living organisms as they penetrate only a specific bacterial host. They have many advantages over classical fluorescent tracers and offer the additional possibility of multi-point injection for tracer tests. Several years of research make them suitable for quantitative transport analysis and flow boundary delineation in both surface and ground waters, including karst, fractured and porous media aquifers. This article presents the effective application of bacteriophages based on their use in differing Swiss hydrological environments and compares their behaviour to conventional coloured dye or salt-type tracers. In surface water and karst aquifers, bacteriophages travel at about the same speed as the typically referenced fluorescent tracers (uranine, sulphurhodamine G extra). In aquifers of interstitial porosity, however, they appear to migrate more rapidly than fluorescent tracers, albeit with a significant reduction in their numbers within the porous media. This faster travel time implies that a modified rationale is needed for defining some ground water protection area boundaries. Further developments of other bacteriophages and their documentation as tracer methods should result in an accurate and efficient tracer tool that will be a proven alternative to conventional fluorescent dyes.

  8. An undergraduate laboratory activity demonstrating bacteriophage specificity.

    PubMed

    Allen, Mary E; Gyure, Ruth A

    2013-01-01

    Bacteriophage are among the most diverse and numerous microbes inhabiting our planet. Yet many laboratory activities fail to engage students in meaningful exploration of their diversity, unique characteristics, and abundance. In this curriculum activity students use a standard plaque assay to enumerate bacteriophage particles from a natural sample and use the scientific method to address questions about host specificity and diversity. A raw primary sewage sample is enriched for bacteriophage using hosts in the family Enterobacteriaceae. Students hypothesize about host specificity and use quantitative data (serial dilution and plaque assay) to test their hypotheses. Combined class data also help them answer questions about phage diversity. The exercise was field tested with a class of 47 students using pre- and posttests. For all learning outcomes posttest scores were higher than pretest scores at or below p = 0.01. Average individualized learning gain (G) was also calculated for each learning outcome. Students' use of scientific language in reference to bacteriophage and host interaction significantly improved (p = 0.002; G = 0.50). Improved means of expression helped students construct better hypotheses on phage host specificity (G = 0.31, p = 0.01) and to explain the plaque assay method (G = 0.33, p = 0.002). At the end of the exercise students also demonstrated improved knowledge and understanding of phage specificity as related to phage therapy in humans (p < 0.001; G = 51). PMID:23858357

  9. 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 ...

  10. Absence of equilibrium cluster phase in concentrated lysozyme solutions

    PubMed Central

    Shukla, Anuj; Mylonas, Efstratios; Di Cola, Emanuela; Finet, Stephanie; Timmins, Peter; Narayanan, Theyencheri; Svergun, Dmitri I.

    2008-01-01

    In colloidal systems, the interplay between the short range attraction and long-range repulsion can lead to a low density associated state consisting of clusters of individual particles. Recently, such an equilibrium cluster phase was also reported for concentrated solutions of lysozyme at low ionic strength and close to the physiological pH. Stradner et al. [(2004) Equilibrium cluster formation in concentrated protein solutions and colloids. Nature 432:492–495] found that the position of the low-angle interference peak in small-angle x-ray and neutron scattering (SAXS and SANS) patterns from lysozyme solutions was essentially independent of the protein concentration and attributed these unexpected results to the presence of equilibrium clusters. This work prompted a series of experimental and theoretical investigations, but also revealed some inconsistencies. We have repeated these experiments following the protein preparation protocols of Stradner et al. using several batches of lysozyme and exploring a broad range of concentrations, temperature and other conditions. Our measurements were done in multiple experimental sessions at three different high-resolution SAXS and SANS instruments. The low-ionic-strength lysozyme solutions displayed a clear shift in peak positions with concentration, incompatible with the presence of the cluster phase but consistent with the system of repulsively interacting individual lysozyme molecules. Within the decoupling approximation, the experimental data can be fitted using an effective interparticle interaction potential involving short-range attraction and long-range repulsion. PMID:18362340

  11. Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides.

    PubMed

    Laible, N J; Germaine, G R

    1985-06-01

    The basis of the bactericidal activity of human lysozyme against Streptococcus sanguis was studied. Experiments were designed to evaluate the role of lysozyme muramidase activity in its bactericidal potency. Inactivation of the muramidase activity of lysozyme was achieved by reduction of essential disulfides with dithiothreitol (DTT) or by incubation with the chitin oligosaccharides chitotriose and chitobiose. Muramidase-inactive lysozyme, prepared by reduction with DTT, was equal in bactericidal potency to native lysozyme. Solutions of native chicken egg white lysozyme and human lysozyme exhibited equal bactericidal potency yet differed ca. fourfold with respect to lytic (muramidase) activity. The above results suggested that the bactericidal activity of lysozyme is not dependent upon muramidase activity. Chitotriose and chitobiose were found to inhibit both lytic and bactericidal activities of lysozyme. The bactericidal activity of muramidase-inactive lysozyme (reduction with DTT) was also inhibited by chitotriose and chitobiose. Further investigations demonstrated that chitotriose and chitobiose were also potent inhibitors of the bactericidal activity of the cationic homopolypeptides poly-L-arginine and poly-D-lysine. These latter results suggested that the essential bactericidal property of lysozyme was its extreme cationic nature and that some bacterial endogenous activities, inhibitable by chitotriose and chitobiose, were essential for expression of the bactericidal activity of either native or muramidase-inactive lysozyme or of the cationic homopolypeptides. Experiments with Streptococcus faecalis whole cells, cell walls, and crude autolysin preparations implicated endogenous autolytic muramidases as the bacterial targets of chitotriose and chitobiose. The essentially identical responses of S. sanguis and S. faecalis to chitotriose in bactericidal assays with muramidase-inactive lysozyme and polylysine suggested that muramidase-like enzymes exist in S. sanguis and, furthermore, play an essential role in cationic protein-induced loss of viability of the oral microbe. PMID:3922894

  12. Organic cosolvents and hen egg white lysozyme folding Bing Lai, Aoneng Cao, Luhua Lai *

    E-print Network

    Luhua, Lai

    Organic cosolvents and hen egg white lysozyme folding Bing Lai, Aoneng Cao, Luhua Lai * State Key July 2000; accepted 3 August 2000 Abstract Studies on the influence of organic cosolvents on lysozyme soluble organic cosolvents on hen egg white lysozyme heat induced denaturation and refolding dynamics

  13. 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

  14. Larval and pupal induction and N-terminal amino acid sequence of lysozyme from Heliothis virescens

    Microsoft Academic Search

    Kyung Tae Chung; Donald D Ourth

    2000-01-01

    Fifth instar larvae and prepupae of Heliothis virescens (tobacco budworm) were injected with live Enterobacter cloacae and bled at different times after vaccination. Immune pupal hemolymph showed a 54 times increase in lysozyme activity when compared with normal larval hemolymph, and an 11 times increase of lysozyme activity when compared with immune larval hemolymph. Lysozyme activity of the normal pupal

  15. 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.

  16. Salt induced reduction of lysozyme adsorption at charged interfaces.

    PubMed

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

    2015-06-17

    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. PMID:25992483

  17. Nanopore analysis of amyloid fibrils formed by lysozyme aggregation.

    PubMed

    Martyushenko, Nikolay; Bell, Nicholas A W; Lamboll, Robin D; Keyser, Ulrich F

    2015-07-21

    The measurement of single particle size distributions of amyloid fibrils is crucial for determining mechanisms of growth and toxicity. Nanopore sensing is an attractive solution for this problem since it gives information on aggregates' shapes with relatively high throughput for a single particle technology. In this paper we study the translocation of lysozyme fibrils through quartz glass nanopores. We demonstrate that, under appropriate salt and pH conditions, lysozyme fibrils translocate through bare quartz nanopores without causing significant clogging. This enables us to measure statistics on tens of thousands of translocations of lysozyme fibrils with the same nanopore and track their development over a time course of aggregation spanning 24 h. Analysis of our events shows that the statistics are consistent with a simple bulk conductivity model for the passage of rods with a fixed cross sectional area through a conical glass nanopore. PMID:25994201

  18. Modeling Tetragonal Lysozyme Crystal Growth Rates

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar; Forsythe, Elizabeth L.; Pusey, Marc L.

    2003-01-01

    Tetragonal lysozyme 110 face crystal growth rates, measured over 5 orders of magnitude in range, can be described using a model where growth occurs by 2D nucleation on the crystal surface for solution supersaturations of c/c(sub eq) less than or equal to 7 +/- 2. Based upon the model, the step energy per unit length, beta was estimated to be approx. 5.3 +/- 0.4 x 10(exp -7) erg/mol-cm, which for a step height of 56 A corresponds to barrier of approx. 7 +/- 1 k(sub B)T at 300 K. For supersaturations of c/c(sub eq) > 8, the model emphasizing crystal growth by 2D nucleation not only could not predict, but also consistently overestimated, the highest observable crystal growth rates. Kinetic roughening is hypothesized to occur at a cross-over supersaturation of c/c(sub eq) > 8, where crystal growth is postulated to occur by a different process such as adsorption. Under this assumption, all growth rate data indicated that a kinetic roughening transition and subsequent crystal growth by adsorption for all solution conditions, varying in buffer pH, temperature and precipitant concentration, occurs for c/c(sub eq)(T, pH, NaCl) in the range between 5 and 10, with an energy barrier for adsorption estimated to be approx. 20 k(sub B)T at 300 K. Based upon these and other estimates, we determined the size of the critical surface nucleate, at the crossover supersaturation and higher concentrations, to range from 4 to 10 molecules.

  19. 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.

  20. 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

  1. 21 CFR 866.2050 - Staphylococcal typing bacteriophage.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2050 Staphylococcal typing bacteriophage. (a) Identification. A...

  2. 21 CFR 866.2050 - Staphylococcal typing bacteriophage.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2050 Staphylococcal typing bacteriophage. (a) Identification. A...

  3. Differential lytic response of enterococci associated with addition order of lysozyme and anions.

    PubMed

    Metcalf, R H; Deibel, R H

    1969-09-01

    Suspensions of Streptococcus faecium, prepared by washing with and resuspending in water, were lysed slowly if sodium chloride was added prior to lysozyme; however, if brief incubation with lysozyme was followed by addition of sodium chloride, lysis was immediate and extensive. Relatively lysozyme-resistant strains of S. faecalis could be lysed readily by adding lysozyme first. The primary addition of lysozyme apparently resulted in a "sensitized" cell with a damaged wall, as evidenced by N-acetylhexosamine release. Anionic detergents could replace sodium chloride in lysing these sensitized cells. The difference in activity associated with the order of addition probably involved a competition for reactive sites on the cell surface. PMID:5370273

  4. 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.

  5. Bacteriophage exclusion, a new defense system.

    PubMed

    Barrangou, Rodolphe; van der Oost, John

    2015-01-13

    The ability to withstand viral predation is critical for survival of most microbes. Accordingly, a plethora of phage resistance systems has been identified in bacterial genomes (Labrie et al, 2010), including restriction?modification systems (R?M) (Tock & Dryden, 2005), abortive infection (Abi) (Chopin et al, 2005), Argonaute?based interference (Swarts et al, 2014), as well as clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) adaptive immune system (CRISPR?Cas) (Barrangou & Marraffini, 2014; Van der Oost et al, 2014). Predictably, the dark matter of bacterial genomes contains a wealth of genetic gold. A study published in this issue of The EMBO Journal by Goldfarb et al (2015) unveils bacteriophage exclusion (BREX) as a novel, widespread bacteriophage resistance system that provides innate immunity against virulent and temperate phage in bacteria. PMID:25502457

  6. Bacteriophage P22 accessory recombination function

    Microsoft Academic Search

    Anthony R. Poteete; Anita C. Fenton; Arlene Semerjian

    1991-01-01

    The accessory recombination function (arf) gene of bacteriophage P22 is located immediately upstream of the essential recombination function (erf) gene. Three mutant alleles of arf were constructed and installed in P22 in place of the wild-type allele: an out-of-frame internal deletion, an in-frame internal deletion, and an amber mutation. The deletion mutant phages are partially defective in homologous recombination and

  7. HEMOLYSIN AND LYSOZYME PRODUCTION BY STAPHYLOCOCCI ISOLATED FROM

    E-print Network

    Paris-Sud XI, Université de

    of animal origin, especially in bovine mastitis, presence of beta hemo- lysin is considered to be veryHEMOLYSIN AND LYSOZYME PRODUCTION BY STAPHYLOCOCCI ISOLATED FROM BOVINE UDDERS G. ZIV R. RISENBERG of bovine udder origin. The alpha- beta-delta pattern was seen by more than 50 p. Ioo of the strains and all

  8. 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…

  9. Kinetic Roughening Transition and Energetics of Tetragonal Lysozyme Crystal Growth

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar; Forsythe, Elizabeth L.; Pusey, Marc L.

    2004-01-01

    Interpretation of lysozyme crystal growth rates using well-established physical theories enabled the discovery of a phenomenon possibly indicative of kinetic roughening. For example, lysozyme crystals grown above a critical supersaturation sigma, (where supersaturation sigma = ln c/c(sub eq), c = the protein concentration and c(sub eq) = the solubility concentration) exhibit microscopically rough surfaces due to the continuous addition of growth units anywhere on the surface of a crystal. The rate of crystal growth, V(sub c), for the continuous growth process is determined by the continuous flux of macromolecules onto a unit area of the crystal surface, a, from a distance, xi, per unit time due to diffusion, and a probability of attachment onto the crystal surface, expressed. Based upon models applied, the energetics of lysozyme crystal growth was determined. The magnitudes of the energy barriers of crystal growth for both the (110) and (101) faces of tetragonal lysozyme crystals are compared. Finally, evidence supportive of the kinetic roughening hypothesis is presented.

  10. 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

  11. Role of lysozyme inhibitors in the virulence of avian pathogenic Escherichia coli.

    PubMed

    Vanderkelen, Lise; Ons, Ellen; Van Herreweghe, Joris M; Callewaert, Lien; Goddeeris, Bruno M; Michiels, Chris W

    2012-01-01

    Lysozymes are key effectors of the animal innate immunity system that kill bacteria by hydrolyzing peptidoglycan, their major cell wall constituent. Recently, specific inhibitors of the three major lysozyme families occuring in the animal kingdom (c-, g- and i-type) have been discovered in Gram-negative bacteria, and it has been proposed that these may help bacteria to evade lysozyme mediated lysis during interaction with an animal host. Escherichia coli produces two inhibitors that are specific for c-type lysozyme (Ivy, Inhibitor of vertebrate lysozyme; MliC, membrane bound lysozyme inhibitor of c-type lysozyme), and one specific for g-type lysozyme (PliG, periplasmic lysozyme inhibitor of g-type lysozyme). Here, we investigated the role of these lysozyme inhibitors in virulence of Avian Pathogenic E. coli (APEC) using a serum resistance test and a subcutaneous chicken infection model. Knock-out of mliC caused a strong reduction in serum resistance and in in vivo virulence that could be fully restored by genetic complementation, whereas ivy and pliG could be knocked out without effect on serum resistance and virulence. This is the first in vivo evidence for the involvement of lysozyme inhibitors in bacterial virulence. Remarkably, the virulence of a ivy mliC double knock-out strain was restored to almost wild-type level, and this strain also had a substantial residual periplasmic lysozyme inhibitory activity that was higher than that of the single knock-out strains. This suggests the existence of an additional periplasmic lysozyme inhibitor in this strain, and indicates a regulatory interaction in the expression of the different inhibitors. PMID:23049900

  12. The EFTTRA-T4 experiment on americium transmutation

    Microsoft Academic Search

    R. J. M Konings; R. Conrad; G. Dassel; B. J Pijlgroms; J. Somers; E. Toscano

    2000-01-01

    In the EFTTRA-T4 experiment the irradiation behaviour of a target containing americium dispersed in MgAl2O4 was studied. Pellets containing 10–12 wt% 241Am were fabricated by the infiltration method. However, it was found that the americium, intended to be present as AmO2?x, formed a compound, probably AmAlO3, during sintering. The T4 target was irradiated in the High Flux Reactor (HFR) Petten

  13. Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7*

    PubMed Central

    Daudén, María I.; Martín-Benito, Jaime; Sánchez-Ferrero, Juan C.; Pulido-Cid, Mar; Valpuesta, José M.; Carrascosa, José L.

    2013-01-01

    During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7. PMID:23632014

  14. 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...

  15. 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...

  16. Genomic Analysis of Burkholderia And Rhodococcus equi Bacteriophages

    E-print Network

    Orchard II, Robert C.

    2011-08-04

    GENOMIC ANALYSIS OF Burkholderia AND Rhodococcus equi BACTERIOPHAGES Major: Microbiology April 2008 Submitted to the Office of Undergraduate Research Texas A&M University in partial fulfillment of the requirements... for the designation as UNDERGRADUATE RESEARCH SCHOLAR A Senior Scholars Thesis by ROBERT CHARLES ORCHARD II GENOMIC ANALYSIS OF Burkholderia AND Rhodococcus equi BACTERIOPHAGES Approved by: Research Advisor: Elizabeth Summer Associate Dean...

  17. Preparation and evaluation of lysozyme-loaded nanoparticles coated with poly-?-glutamic acid and chitosan.

    PubMed

    Liu, Yong; Sun, Yan; Xu, Yaoxing; Feng, Hai; Fu, Sida; Tang, Jiangwu; Liu, Wei; Sun, Dongchang; Jiang, Hua; Xu, Shaochun

    2013-08-01

    To improve the application of lysozymes, methods for coating lysozymes with poly-?-glutamic acid and chitosan were studied. Several lysozyme-loaded chitosan/poly-?-glutamic acid composite nanosystems for loading and controlling the release of lysozymes were established. The lysozyme loading content and efficiency of the different systems were examined. The antibacterial activity of the composite nanoparticles was also investigated. Results showed that when the lysozymes were coated with poly-?-glutamic acid and further rewrapped with chitosan, smooth spherical composite nanoparticles were obtained; the loading efficiency and loading content reached 76% and 40%, respectively. The lysozyme release in vitro was slow and presented a two-stage programmed release. Antibacterial testing in vitro indicated that lysozyme-loaded nanoparticles coated with poly-?-glutamic acid/chitosan had outstanding antibacterial activity. An obvious assembly of bacterial cells and composite nanoparticles was observed during co-incubation. Therefore, the poly-?-glutamic acid/chitosan composite coating broadened the antibacterial spectrum of the composite lysozyme nanoreagent, and presented satisfactory antibacterial effect. The lysozyme-loaded chitosan/poly-?-glutamic acid nanocoating system established in this research could provide reference for coating and controlled releasing of alkaline proteins. PMID:23628585

  18. Molecular interactions and residues involved in force generation in the T4 viral DNA packaging motor.

    PubMed

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

    2014-12-12

    Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations. PMID:25311860

  19. 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

  20. Bacteriophage Diversity in the North Sea

    PubMed Central

    Wichels, Antje; Biel, Stefan S.; Gelderblom, Hans R.; Brinkhoff, Thorsten; Muyzer, Gerard; Schütt, Christian

    1998-01-01

    In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (104 to 107 particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the ? subdivision of the class Proteobacteria. PMID:9797256

  1. 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.

  2. 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.

  3. Exploiting gut bacteriophages for human health.

    PubMed

    Dalmasso, Marion; Hill, Colin; Ross, R Paul

    2014-07-01

    The human gut contains approximately 10(15) bacteriophages (the 'phageome'), probably the richest concentration of biological entities on earth. Mining and exploiting these potential 'agents of change' is an attractive prospect. For many years, phages have been used to treat bacterial infections in humans and more recently have been approved to reduce pathogens in the food chain. Phages have also been studied as drug or vaccine delivery vectors to help treat and prevent diseases such as cancer and chronic neurodegenerative conditions. Individual phageomes vary depending on age and health, thus providing a useful biomarker of human health as well as suggesting potential interventions targeted at the gut microbiota. PMID:24656964

  4. Analysis of Two Lysozyme Genes and Antimicrobial Functions of Their Recombinant Proteins in Asian Seabass

    PubMed Central

    Fu, Gui Hong; Bai, Zhi Yi; Xia, Jun Hong; Liu, Feng; Liu, Peng; Yue, Gen Hua

    2013-01-01

    Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass (Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu50 and Asp67) and a “GSTDYGIFQINS” motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL) domain containing three conserved catalytic residues (Glu71, Asp84, Asp95) essential for catalytic activity. Real time quantitative PCR (qRT-PCR) revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs) in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases. PMID:24244553

  5. Simple and rapid procedure for the selective removal of lysozyme from human saliva.

    PubMed

    Germaine, G R; Tellefson, L M

    1979-12-01

    A simple and rapid method is described for the removal of lysozyme from human whole salivary supernatant. Saliva specimens were adsorbed with Micrococcus lysodeikticus. The saliva so treated was depleted of 95% of the lysozyme activity. Changes in total protein, lactoperoxidase, lactoferrin, immunoglobulin A, and the proportions of several anionic proteins were less than 10%. It is concluded that adsorption of saliva with M. lysodeikticus is a suitable procedure for the preparation of saliva that is selectively deficient in lysozyme. PMID:528061

  6. Amyloid fibrillation of hen egg-white lysozyme is inhibited by TCEP

    Microsoft Academic Search

    Steven S.-S. Wang; Kuan-Nan Liu; Yen-Chun Lu

    2009-01-01

    This work examines the inhibitory effect of TCEP on the in vitro fibrillation of hen lysozyme at pH 2. We demonstrate that the inhibition of hen lysozyme fibrillation by TCEP follows a dose-dependent manner. Our data show that the addition of TCEP prevents ?-to-? transition and promoted unfolding of lysozyme. Moreover, our findings suggested that the TCEP-induced attenuated fibrillation is

  7. Neutron structure of monoclinic lysozyme crystals produced in microgravity

    Microsoft Academic Search

    Joseph X Ho; Jean-Paul Declercq; Dean A. A Myles; Brenda S Wright; John R Ruble; Daniel C Carter

    2001-01-01

    Crystals of the monoclinic form of lysozyme (space group P21 with a=28.00Å, b=62.88Å, c=60.30Å, ?=90.68°) were grown under microgravity conditions on the Mir station and brought back to earth on the US Space Shuttle. Counter-diffusion methods developed specifically for application in microgravity have been utilized to produce several examples of macroscopic crystals. Large crystals are of great importance for neutron

  8. Sound velocity and dynamic elastic constants of lysozyme single crystals

    Microsoft Academic Search

    M. Tachibana; K. Kojima; R. Ikuyama; Y. Kobayashi; M. Ataka

    2000-01-01

    We have succeeded in the first measurement of the sound velocity of lysozyme single crystals using an ultrasonic pulse-echo method. The sound velocity along the [110] crystallographic direction in the tetragonal crystals is obtained to be 1817 m\\/s in the solution with pH 4.3 at 20°C. The corresponding elastic constant C11+C66 is evaluated to be approximately 3.99 GPa. Assuming the

  9. Magnetic molecularly imprinted nanoparticles for recognition of lysozyme

    Microsoft Academic Search

    Tao Jing; Hairong Du; Qing Dai; Huan Xia; Jiwei Niu; Qiaolin Hao; Surong Mei; Yikai Zhou

    2010-01-01

    Molecular imprinting is an attractive technique for preparing mimics of natural, biological receptors. Nevertheless, the imprinting of macromolecule remains a challenge due to their bulkiness and sensitivity to denaturation. In this work, we presented a method for preparing multifunctional lysozyme-imprinted nanoparticles (magnetic susceptibility, molecular recognition and environmental response). The magnetic susceptibility was imparted through the successful encapsulation of Fe3O4 nanoparticles.

  10. Influence of surface charge on lysozyme adsorption to ceria nanoparticles

    NASA Astrophysics Data System (ADS)

    Wang, Binghui; Wu, Peng; Yokel, Robert A.; Grulke, Eric A.

    2012-05-01

    Understanding mechanisms for forming protein coronas on nanomaterial surfaces is essential to designing drug delivery systems and designing and interpreting the results of nanomaterial toxicity tests. The study reports the adsorption behavior of a positively charged protein, lysozyme, on cerium dioxide (ceria) nanoparticles with three different surface charges. Adsorption isotherms were modeled with the Toth and Sips equations. Isotherm loading levels were compared to monolayer coverage estimate for 'side-on' and 'end-on' lysozyme orientations as well as random packing (jamming) and maximum packing limits. Evaluation of adsorption site energy distributions (generated using the model coefficients) suggested that the negatively charged ceria surface had a very broad site energy distribution and that its surface heterogeneity controls the adsorption process. By contrast, the adsorption of lysozyme on the positively charged nanoparticles appears to be influenced by lateral effects from adsorbed protein species. The results illustrate the importance of nanoparticle surface chemistry to protein adsorption. The modeling and site energy distribution evaluations may be useful for interpreting the formation of protein coronas on nanoparticles.

  11. 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.

  12. A novel electrochemical aptamer-antibody sandwich assay for lysozyme detection.

    PubMed

    Ocaña, Cristina; Hayat, Akhtar; Mishra, Rupesh; Vasilescu, Alina; Del Valle, Manel; Marty, Jean-Louis

    2015-06-21

    In this paper, we have reported a novel electrochemical aptamer-antibody based sandwich biosensor for the detection of lysozyme. In the sensing strategy, an anti-lysozyme aptamer was immobilized onto the carbon electrode surface by covalent binding via diazonium salt chemistry. After incubating with a target protein (lysozyme), a biotinylated antibody was used to complete the sandwich format. The subsequent additions of avidin-alkaline phosphatase as an enzyme label, and a 1-naphthyl phosphate substrate (1-NPP) allowed us to determine the concentration of lysozyme (Lys) via Differential Pulse Voltammetry (DPV) of the generated enzyme reaction product, 1-naphthol. Using this strategy, a wide detection range from 5 fM to 5 nM was obtained for a target lysozyme, with a detection limit of 4.3 fM. The control experiments were carried out by using bovine serum albumin (BSA), cytochrome c and casein. The results showed that the proposed biosensor had good specificity, stability and reproducibility for lysozyme analysis. In addition, the biosensor was applied for detecting lysozyme in spiked wine samples, and very good recovery rates were obtained in the range from 95.2 to 102.0% for lysozyme detection. This implies that the proposed sandwich biosensor is a promising analytical tool for the analysis of lysozyme in real samples. PMID:25905497

  13. 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

  14. 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

  15. Understanding Bacteriophage Specificity in Natural Microbial Communities

    PubMed Central

    Koskella, Britt; Meaden, Sean

    2013-01-01

    Studying the coevolutionary dynamics between bacteria and the bacteriophage viruses that infect them is critical to understanding both microbial diversity and ecosystem functioning. Phages can play a key role in shaping bacterial population dynamics and can significantly alter both intra- and inter-specific competition among bacterial hosts. Predicting how phages might influence community stability and apparent competition, however, requires an understanding of how bacteria-phage interaction networks evolve as a function of host diversity and community dynamics. Here, we first review the progress that has been made in understanding phage specificity, including the use of experimental evolution, we then introduce a new dataset on natural bacteriophages collected from the phyllosphere of horse chestnut trees, and finally we highlight that bacterial sensitivity to phage is rarely a binary trait and that this variation should be taken into account and reported. We emphasize that there is currently insufficient evidence to make broad generalizations about phage host range in natural populations, the limits of phage adaptation to novel hosts, or the implications of phage specificity in shaping microbial communities. However, the combination of experimental and genomic approaches with the study of natural communities will allow new insight to the evolution and impact of phage specificity within complex bacterial communities. PMID:23478639

  16. 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

  17. Experiments on the Inactivation of Bacteriophage by Radiations, and their Bearing on the Nature of Bacteriophage

    Microsoft Academic Search

    D. E. Lea; M. H. Salaman

    1946-01-01

    Three bacteriophages (S-13, C-36 and Staph-K) were irradiated by gamma -rays, X-rays and alpha -rays. The survival curves were exponential, and the effect of a given dose was independent of the exposure time. For any given phage the inactivation doses of the three radiations increased in the order gamma -rays, X-rays, alpha -rays, while for any given radiation the inactivation

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

    E-print Network

    Harrison, Stephen C.

    MOLECULAR ORGANIZATION OF VIRUS PARTICLES: IMPLICATIONS FOR ASSEMBLY Stephen C. Harrison Gibbs Laboratory of smalfer viruses and to suggest what they might imply for bacteriophage. ICOSAHEDRAL SHELLS Phage heads

  19. 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...

  20. Structural characterization of bacteriophage M13 solubilization by amphiphiles

    E-print Network

    Hemminga, Marcus A.

    Structural characterization of bacteriophage M13 solubilization by amphiphiles David Stopar 1 for phage integrity. All amphiphiles studied were capable of phage disruption. However, no significant phage disruption was detected below the critical micelle concentration of the amphiphile used. Based

  1. Limitations of a new free thyroxine assay (Amerlex free T4).

    PubMed

    Stockigt, J R; de Garis, M; Csicsmann, J; Barlow, J W; White, E L; Hurley, D M

    1981-09-01

    We have assessed a new method of free T4 measurement (Amerlex) which uses a novel unidentified T4-labelled analogue, said to be unreactive with T4 binding proteins in serum, together with an antibody that binds both analogue and T4. Free T4 is assessed by competition with analogue for antibody binding-sites. The test method has been compared with free T4 measured by equilibrium dialysis and with a technique using an immobilized T4 antibody. All methods gave the expected free T4 levels in normal, hyperthyroid and hypothyroid subjects and normal free T4 levels with high or low levels of T4 binding globulin. However, in autosomal dominant familial euthyroid T4-excess, where T4 is abnormally bound to albumin, the test method gave apparent high free T4 levels suggestive of hyperthyroidism. In a selected group of severely-ill euthyroid patients the new method gave apparent low free T4 levels. In view of these discrepancies, binding of labelled analogue was evaluated by dextran-charcoal separation of 4 degraees C. Familial euthyroid T4-excess sera showed greater analogue binding and samples with low prealbumin concentration showed less binding than did normal sera. Despite its validity with variations in TBG, it appears that Amerlex Free T4 is influenced by lower-affinity, high-capacity T4 binding sites in serum, so that apparent free T4 concentration may vary with changes in the concentration of such sites. PMID:6796307

  2. Lytic bacteriophages reduce Escherichia coli O157

    PubMed Central

    Ferguson, Sean; Roberts, Cheryl; Handy, Eric; Sharma, Manan

    2013-01-01

    The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 ?g/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm2). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. Immersion of lettuce in suspensions containing high concentrations of EcoShield™ (9.8 log PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm2) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce. PMID:23819106

  3. Influence of Infected Cell Growth State on Bacteriophage Reactivation Levels

    PubMed Central

    Kadavy, Dana R.; Shaffer, Julie J.; Lott, Susan E.; Wolf, Thomas A.; Bolton, Cathy E.; Gallimore, William H.; Martin, Eugene L.; Nickerson, Kenneth W.; Kokjohn, Tyler A.

    2000-01-01

    Reactivation of UV-C-inactivated Pseudomonas aeruginosa bacteriophages D3C3, F116, G101, and UNL-1 was quantified in host cells infected during the exponential phase, during the stationary phase, and after starvation (1 day, 1 and 5 weeks) under conditions designed to detect dark repair and photoreactivation. Our experiments revealed that while the photoreactivation capacity of stationary-phase or starved cells remained about the same as that of exponential-phase cells, in some cases their capacity to support dark repair of UV-inactivated bacteriophages increased over 10-fold. This enhanced reactivation capacity was correlated with the ca. 30-fold-greater UV-C resistance of P. aeruginosa host cells that were in the stationary phase or exposed to starvation conditions prior to irradiation. The dark repair capacity of P. aeruginosa cells that were infected while they were starved for prolonged periods depended on the bacteriophage examined. For bacteriophage D3C3 this dark repair capacity declined with prolonged starvation, while for bacteriophage G101 the dark repair capacity continued to increase when cells were starved for 24 h or 1 week prior to infection. For G101, the reactivation potentials were 16-, 18-, 10-, and 3-fold at starvation intervals of 1 day, 1 week, 5 weeks, and 1.5 years, respectively. Exclusive use of exponential-phase cells to quantify bacteriophage reactivation should detect only a fraction of the true phage reactivation potential. PMID:11097891

  4. Lysozyme gene expression by hemocytes of Pacific white shrimp, Litopenaeus vannamei, after injection with Vibrio

    Microsoft Academic Search

    Erin J. Burge; Daniel J. Madigan; Louis E. Burnett; Karen G. Burnett

    2007-01-01

    The purpose of this study was to quantify the gene expression of lysozyme, an important antibacterial protein produced by shrimp hemocytes, within tissues of Litopenaeus vannamei Boone in response to a pathogen challenge. We quantified lysozyme transcripts with a real-time PCR method and used these data, along with total hemocyte counts, to infer patterns of hemocyte trafficking during the immune

  5. Multiple Scale Reorganization of Electrostatic Complexes of PolyStyrene Sulfonate and Lysozyme

    Microsoft Academic Search

    Fabrice Cousin; Je?re?mie Gummel; Daniel Clemens; Isabelle Grillo; François Boué

    2010-01-01

    We report on a SANS investigation into the potential for these structural reorganization of complexes composed of lysozyme and small PSS chains of opposite charge if the physicochemical conditions of the solutions are changed after their formation. Mixtures of solutions of lysozyme and PSS with high matter content and with an introduced charge ratio [-]\\/[+]intro close to the electrostatic stoichiometry,

  6. Lysozyme-imprinted polymer synthesized using UV free-radical polymerization

    Microsoft Academic Search

    Shuang Yu; Ai-qin Luo; Dipti Biswal; J. Zach Hilt; David A. Puleo

    2010-01-01

    Molecular imprinting is a method to fabricate a polymeric material (molecularly imprinted polymer or MIP) capable of selectively recognizing template molecules. Molecular imprinting of small molecules has been studied widely. Less common, however, is the imprinting of biological macromolecules, including proteins, among which lysozyme is an important molecule in the food, pharmaceutical, and diagnostic sciences. In this study, lysozyme MIP

  7. Sensitive fluorescence detection of lysozyme using a tris(bipyridine)ruthenium(II) complex containing multiple cyclodextrins.

    PubMed

    Zhang, Fan; Zhao, Ying-Ying; Chen, Hong; Wang, Xiu-Hua; Chen, Qiong; He, Pin-Gang

    2015-04-18

    A new series of photoactive metallocyclodextrins with increased fluorescence intensity upon binding with ssDNAs/aptamers has been demonstrated to sensitively and selectively detect lysozyme. The detection mechanism relies on the formation of an aptamer-lysozyme complex, which leads to reduction of fluorescence intensity. PMID:25776220

  8. Fingerprinting the tertiary structure of electroadsorbed lysozyme at soft interfaces by electrostatic spray ionization mass spectrometry.

    PubMed

    Alvarez de Eulate, Eva; Qiao, Liang; Scanlon, Micheál D; Girault, Hubert H; Arrigan, Damien W M

    2014-10-14

    Lysozyme can be electrochemically detected after adsorption at an electrified gel-water interface. Ex situ characterization by electrostatic spray ionization mass spectrometry provides insights into the interfacial detection mechanism by allowing changes to the tertiary structure of electroadsorbed lysozyme to be fingerprinted for the first time. PMID:25156670

  9. 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…

  10. Disulfide-Intact and -Reduced Lysozyme in the Gas Phase: Conformations and Pathways of Folding and Unfolding

    E-print Network

    Clemmer, David E.

    have been studied in the gas phase using ion mobility mass spectrometry techniques. When solutions-intact and disulfide-reduced lysozyme ions in the gas phase using ion-mobility techniques.11-14 Lysozyme is made up, 1997X The conformations of gaseous lysozyme ions (+5 through +18) produced by electrospray ionization

  11. Molecular Dynamics of Solid-State Lysozyme as Affected by Glycerol and Water: A Neutron Scattering Study

    Microsoft Academic Search

    Amos M. Tsai; Dan A. Neumann; Leonard N. Bell

    2000-01-01

    Glycerol has been shown to lower the heat denaturation temperature (Tm) of dehydrated lysozyme while elevating the Tm of hydrated lysozyme (Bell, Hageman, and Muraoka, 1995. J. Pharm. Sci. 84:707–712). Here, we report an in situ elastic neutron scattering study of the effect of glycerol and hydration on the internal dynamics of lysozyme powder. Anharmonic motions associated with structural relaxation

  12. Isolation and characterization of bacteriophage BCJA1, a novel temperate bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii

    Microsoft Academic Search

    Ken F. Jarrell; Tatiana Vydykhan; Peter Lee; M. Dorothy Agnew; Nikhil A. Thomas

    1997-01-01

    The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described. The bacteriophage, designated BCJA1, is a member of the Siphoviridae family with a B1 morphology. It possesses\\u000a an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length. It had a buoyant\\u000a density of 1.518

  13. Bacteriophage lambda: Early pioneer and still relevant.

    PubMed

    Casjens, Sherwood R; Hendrix, Roger W

    2015-05-01

    Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid-1950s to mid-1980s was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its relatives has continued to give important new insights. In this review we give some relevant early history and describe recent developments in understanding the molecular biology of lambda's life cycle. PMID:25742714

  14. Autoregulation of bacteriophage P2 repressor.

    PubMed Central

    Saha, S; Lundqvist, B; Haggård-Ljungquist, E

    1987-01-01

    The immunity repressor of bacteriophage P2 regulates the two convergent promoters, Pe and Pc, located in the early control region. Pe is the early promoter which is negatively regulated by the repressor. It was found, by DNase I protection studies, that the P2 repressor enhances the binding of RNA polymerase to Pc. Furthermore, under in vivo conditions the transcription initiated at Pc, measured as chloramphenicol acetyl transferase gene expression, is low in the absence of repressor but is stimulated by low repressor levels. With increasing repressor concentrations transcription from the Pc promoter decreases. Thus, the P2 repressor both negatively and positively regulates its own promoter. Images Fig. 2. Fig. 3. Fig. 5. PMID:3034610

  15. Light Scattering and Phase Behavior of Lysozyme-Poly(Ethylene Glycol) Mixtures

    NASA Astrophysics Data System (ADS)

    Bloustine, J.; Virmani, T.; Thurston, G. M.; Fraden, S.

    2006-03-01

    Measurements of liquid-liquid phase transition temperatures (cloud points) of mixtures of a protein (lysozyme) and a polymer, poly(ethylene glycol) (PEG) show that the addition of low molecular weight PEG stabilizes the mixture whereas high molecular weight PEG was destabilizing. We demonstrate that this behavior is inconsistent with an entropic lysozyme-PEG depletion interaction and suggest that an energetic lysozyme-PEG attraction is responsible. In order to independently characterize the lysozyme-PEG interactions, light scattering experiments on the same mixtures were performed to measure second and third virial coefficients. These measurements indicate that PEG induces repulsion between lysozyme molecules, contrary to the depletion prediction. Furthermore, it is shown that third virial terms must be included in the mixture’s free energy in order to qualitatively capture our data.

  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. [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering; Prausnitz, John M. [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering

    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. Design and evaluation of a novel nanoparticulate-based formulation encapsulating a HIP complex of lysozyme

    PubMed Central

    Gaudana, Ripal; Gokulgandhi, Mitan; Khurana, Varun; Kwatra, Deep; Mitra, Ashim K.

    2015-01-01

    Formulation development of protein therapeutics using polymeric nanoparticles has found very little success in recent years. Major formulation challenges include rapid denaturation, susceptibility to lose bioactivity in presence of organic solvents and poor encapsulation in polymeric matrix. In the present study, we have prepared hydrophobic ion pairing (HIP) complex of lysozyme, a model protein, using dextran sulfate (DS) as a complexing polymer. We have optimized the process of formation and dissociation of HIP complex between lysozyme and DS. The effect of HIP complexation on enzymatic activity of lysozyme was also studied. Nanoparticles were prepared and characterized using spontaneous emulsion solvent diffusion method. Furthermore, we have also investigated release of lysozyme from nanoparticles along with its enzymatic activity. Results of this study indicate that nanoparticles can sustain the release of lysozyme without compromising its enzymatic activity. HIP complexation using a polymer may also be employed to formulate sustained release dosage forms of other macromolecules with enhanced encapsulation efficiency. PMID:23137392

  18. 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

  19. 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.

  20. Bacteriophages of Leuconostoc, Oenococcus, and Weissella

    PubMed Central

    Kot, Witold; Neve, Horst; Heller, Knut J.; Vogensen, Finn K.

    2014-01-01

    Leuconostoc (Ln.), Weissella, and Oenococcus form a group of related genera of lactic acid bacteria, which once all shared the name Leuconostoc. They are associated with plants, fermented vegetable products, raw milk, dairy products, meat, and fish. Most of industrially relevant Leuconostoc strains can be classified as either Ln. mesenteroides or Ln. pseudomesenteroides. They are important flavor producers in dairy fermentations and they initiate nearly all vegetable fermentations. Therefore, bacteriophages attacking Leuconostoc strains may negatively influence the production process. Bacteriophages attacking Leuconostoc strains were first reported in 1946. Since then, the majority of described Leuconostoc phages was isolated from either dairy products or fermented vegetable products. Both lytic and temperate phages of Leuconostoc were reported. Most of Leuconostoc phages examined using electron microscopy belong to the Siphoviridae family and differ in morphological details. Hybridization and comparative genomic studies of Leuconostoc phages suggest that they can be divided into several groups, however overall diversity of Leuconostoc phages is much lower as compared to, e.g., lactococcal phages. Several fully sequenced genomes of Leuconostoc phages have been deposited in public databases. Lytic phages of Leuconostoc can be divided into two host species-specific groups with similarly organized genomes that shared very low nucleotide similarity. Phages of dairy Leuconostoc have rather limited host-ranges. The receptor binding proteins of two lytic Ln. pseudomesenteroides phages have been identified. Molecular tools for detection of dairy Leuconostoc phages have been developed. The rather limited data on phages of Oenococcus and Weissella show that (i) lysogeny seems to be abundant in Oenococcus strains, and (ii) several phages infecting Weissella cibaria are also able to productively infect strains of other Weissella species and even strains of the genus Lactobacillus. PMID:24817864

  1. Models of the three-dimensional structures of echidna, horse, and pigeon lysozymes: Calcium-binding lysozymes and their relationship with ?-lactalbumins

    Microsoft Academic Search

    K. Ravi Acharya; David I. Stuart; David C. Phillips; Hugh A. McKenzie; Carmel G. Teahan

    1994-01-01

    Similarities in amino acid sequences, three-dimensional structures, and the exon-intron patterns of their genes have indicated thatc-type lysozymes anda-lactalbumins are homologous proteins, i.e., descended by divergent evolution from a common ancestor. Like thea-lactalbumins, echidna milk, horse milk, and pigeon eggwhite lysozymes all bind Ca(II). Models of their three-dimensional structures, based on their amino acid sequences and the known crystal structures

  2. 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.

  3. 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.

  4. 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 ...

  5. MAPPING EXPERIMENTS WITH r MUTANTS OF BACTERIOPHAGE

    E-print Network

    Klein, Stanley

    for crosses and preparation of phage stocks is given by STEINBERGand EDGAR(1962). The rI mutant, r48 r mutants of phage T4 fall into three clusters, each of which is essentially unlinked to the others. Since there is one cluster in each of the three so-calledlinkage groups, we will refer to the clusters

  6. Adsorption of lysozyme from human whole saliva by Streptococcus sanguis 903 and other oral microorganisms.

    PubMed

    Laible, N J; Germaine, G R

    1982-04-01

    Several strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus mitis, Actinomyces viscosus, and Actinomyces naeslundii plus fresh isolates of Streptococcus salivarius were surveyed for their abilities to deplete lysozyme from human-whole-saliva supernatant. Bacteria were incubated in saliva for 60 min at 37 degrees C and then removed by centrifugation, and the recovered supernatant solutions were assayed for lysozyme activity by using whole cells of Micrococcus lysodeikticus as the substrate. Mean lysozyme depletions by bacterial strains varied over a wide (eightfold) range. The greatest mean depletion of lysozyme (60 to 70%) was observed with S. sanguis (biotype I), serotype b of S. mutans, and the fresh S. salivarius isolates. The lowest mean depletion was noted with S. mitis (15%) and biotype II S. sanguis (ca. 30%). The remaining species and strains exhibited an intermediate degree of depletion. In studies with S. sanguis 903, lysozyme was depleted by normal or heated (90 degrees C, 30 min) bacteria and could be recovered from the organism. Furthermore, under appropriate conditions, lysozyme depletion by cells at 0 and 37 degrees C was very similar. On the basis of these observations, we concluded that depletion was due to the adsorption of lysozyme by the organism. With S. sanguis 903, lysozyme adsorption depended on the concentration of bacteria, time of incubation, and the ionic strength of the medium. The extent of adsorption, however, was independent of pH's of 3.9 to 8.3. When a low concentration of S. sanguis 903 was used, lysozyme adsorption reached saturation (4 mug of adsorbed lysozyme per 10(7) cells) at 20 mug of lysozyme added per ml. Salivary lysozyme adsorption by several other oral microorganisms (A. viscosus WVU 626 and WVU 627, S. sanguis 73x11, S. mutans BHT, and S. salivarius NG) was similar to that of S. sanguis 903 in sensitivity to ionic strength. Lysozyme adsorption by S. sanguis 903 from either a buffer solution or a saliva supernatant was more sensitive to ionic strength at 0 than at 37 degrees C. On the basis of results from experiments in saliva versus buffer, we concluded that saliva had no major effect on the extent of lysozyme adsorption by S. sanguis 903 other than providing a source of ionic strength. A comparison of pH and ionic strength effects on lysozyme adsorption by S. sanguis 903 with literature reports of lysozyme lysis of whole cells and hydrolysis of cell walls, peptidoglycan, and (GlcNAc)(4) suggested that adsorption by S. sanguis 903 was more dependent on electrostatic interactions than was lysozyme catalysis. The possibility is discussed that anionic bacterial surface components mediate lysozyme adsorption and temper the potential effects of lysozyme on the microorganisms. PMID:7076291

  7. Interaction between Bacteriophage DMS3 and Host CRISPR Region Inhibits Group Behaviors of Pseudomonas aeruginosa

    Microsoft Academic Search

    Michael E. Zegans; Jeffrey C. Wagner; Kyle C. Cady; Daniel M. Murphy; John H. Hammond; George A. O'Toole

    2009-01-01

    Bacteriophage infection has profound effects on bacterial biology. Clustered regular interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) genes are found in most archaea and many bacteria and have been reported to play a role in resistance to bacteriophage infection. We observed that lysogenic infection of Pseudomonas aeruginosa PA14 with bacteriophage DMS3 inhibits biofilm formation and swarming motility, both important

  8. research papers 1260 Simpson et al. Bacteriophage 929 headtail connector Acta Cryst. (2001). D57, 12601269

    E-print Network

    Tao, Yizhi Jane

    2001-01-01

    research papers 1260 Simpson et al. Bacteriophage 929 head±tail connector Acta Cryst. (2001). D57 determination of the head±tail connector of bacteriophage u29 Alan A. Simpson,a Petr G. Leiman,a Yizhi Tao of Crystallography Printed in Denmark ± all rights reserved The head±tail connector of bacteriophage 929 is composed

  9. 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.

  10. The effect of protein impurities on lysozyme crystal growth.

    PubMed

    Judge, R A; Forsythe, E L; Pusey, M L

    1998-09-20

    While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. To further examine the issue of purity in macromolecule crystallization, this study investigates the effect of the protein impurities, avidin, ovalbumin, and conalbumin at concentrations up to 50%, on the solubility, crystal face growth rates, and crystal purity of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the ¿110¿ and ¿101¿ lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit operation for protein purification. PMID:10099398

  11. 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.

  12. Intestinal absorption of lysozyme, an egg-white allergen, in rats: kinetics and effect of NSAIDs.

    PubMed

    Yokooji, Tomoharu; Hamura, Koh; Matsuo, Hiroaki

    2013-08-16

    The absorption pathway(s) of a representative food allergen, lysozyme, and the mechanisms of lysozyme absorption facilitated by non-steroidal anti-inflammatory drugs were examined by intestinal closed-loop and re-circulating perfusion methods in rats. The absorption rate of fluorescein isothiocyanate (FITC)-labeled lysozyme in the proximal intestine was higher than that for a marker of non-specific absorption, FD-10, and was suppressed by colchicine (endocytosis inhibitor). Aspirin increased the absorption of FITC-lysozyme in the proximal intestine with no effects on tissue accumulation. Diclofenac facilitated FITC-lysozyme absorption, but meloxicam and loxoprofen exerted no effects on absorption. Co-administration of misoprostol (synthetic prostaglandin-E1 analog) with aspirin significantly ameliorated the aspirin-facilitated absorption of FITC-lysozyme to the same level as that seen with controls. Thus, lysozyme absorption was mediated by endocytic and paracellular pathways in the proximal intestine, and was facilitated by aspirin and diclofenac after impairment of the paracellular pathway. Misoprostol may suppress the allergen absorption facilitated by aspirin. PMID:23872145

  13. Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni.

    PubMed

    Siringan, Patcharin; Connerton, Phillippa L; Cummings, Nicola J; Connerton, Ian F

    2014-01-01

    Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage. PMID:24671947

  14. Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni

    PubMed Central

    Siringan, Patcharin; Connerton, Phillippa L.; Cummings, Nicola J.; Connerton, Ian F.

    2014-01-01

    Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage. PMID:24671947

  15. [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

  16. Production of human lysozyme in biofilm reactor and optimization of growth parameters of Kluyveromyces lactis K7.

    PubMed

    Ercan, Duygu; Demirci, Ali

    2013-07-01

    Lysozyme (1,4-?-N-acetylmuramidase) is a lytic enzyme, which degrades the bacterial cell wall. Lysozyme has been of interest in medicine, cosmetics, and food industries because of its anti-bactericidal effect. Kluyveromyces lactis K7 is a genetically modified organism that expresses human lysozyme. There is a need to improve the human lysozyme production by K. lactis K7 to make the human lysozyme more affordable. Biofilm reactor provides high biomass by including a solid support, which microorganisms grow around and within. Therefore, the aim of this study was to produce the human lysozyme in biofilm reactor and optimize the growth conditions of K. lactis K7 for the human lysozyme production in biofilm reactor with plastic composite support (PCS). The PCS, which includes polypropylene, soybean hull, soybean flour, bovine albumin, and salts, was selected based on biofilm formation on PCS (CFU/g), human lysozyme production (U/ml), and absorption of lysozyme inside the support. To find the optimum combination of growth parameters, a three-factor Box-Behnken design of response surface method was used. The results suggested that the optimum conditions for biomass and lysozyme productions were different (27 °C, pH 6, 1.33 vvm for biomass production; 25 °C, pH 4, no aeration for lysozyme production). Then, different pH and aeration shift strategies were tested to increase the biomass at the first step and then secrete the lysozyme after the shift. As a result, the lysozyme production amount (141 U/ml) at 25 °C without pH and aeration control was significantly higher than the lysozyme amount at evaluated pH and aeration shift conditions (p?

  17. Bacteriophage Infection of Model Metal Reducing Bacteria

    NASA Astrophysics Data System (ADS)

    Weber, K. A.; Bender, K. S.; Gandhi, K.; Coates, J. D.

    2008-12-01

    Microbially-mediated metal reduction plays a significant role controlling contaminant mobility in aqueous, soil, and sedimentary environments. From among environmentally relevant microorganisms mediating metal reduction, Geobacter spp. have been identified as predominant metal-reducing bacteria under acetate- oxidizing conditions. Due to the significance of these bacteria in environmental systems, it is necessary to understand factors influencing their metabolic physiology. Examination of the annotated finished genome sequence of G. sulfurreducens PCA, G. uraniumreducens Rf4, G. metallireduceans GS-15 as well as a draft genome sequence of Geobacter sp. FRC-32 have identified gene sequences of putative bacteriophage origin. Presence of these sequences indicates that these bacteria are susceptible to phage infection. Polymerase chain reaction (PCR) primer sets designed tested for the presence of 12 of 25 annotated phage-like sequences in G. sulfurreducens PCA and 9 of 17 phage-like sequences in FRC- 32. The following genes were successfully amplified in G. sulfurreducens PCA: prophage type transcription regulator, phage-induced endonuclease, phage tail sheath, 2 phage tail proteins, phage protein D, phage base plate protein, phage-related DNA polymerase, integrase, phage transcriptional regulator, and Cro-like transcription regulator. Nine of the following sequences were present in FRC-32: 4 separate phage- related proteins, phage-related tail component, viron core protein, phage Mu protein, phage base plate, and phage tail sheath. In addition to the bioinformatics evidence, incubation of G. sulfurreducens PCA with 1 ?g mL-1 mytomycin C (mutagen stimulating prophage induction) during mid-log phase resulted in significant cell lysis relative to cultures that remained unamended. Cell lysis was concurrent with an increase in viral like particles enumerated using epifluorescent microscopy. In addition, samples collected following this lytic event (~44hours) were filtered through a 0.22 ? m sterile nylon filter, stained with phosphotungstic acid (PTA), and examined using transmission electron microscopy (TEM). TEM revealed the presence of viral like particles in the culture exposed to mytomycin C. Together these results suggest an active infection with a lysogenic bacteriophage in the model metal reducing bacteria, Geobacter spp., which could affect metabolic physiology and subsequently metal reduction in environmental systems.

  18. Unrelatedness of Temperate Bacillus subtilis Bacteriophages SPO2 and ?105

    PubMed Central

    Rutberg, Lars; Armentrout, Richard W.; Jonasson, Jon

    1972-01-01

    SPO2 and ?105 are temperate Bacillus subtilis bacteriophages which have been suggested to belong to a cluster of related bacteriophages. In the present work, we show that SPO2 does not complement any of the 11 essential genes known in ?105 and that the phages do not recombine. Deoxyribonucleic acid (DNA)-DNA hybridization shows less than 10% homology between SPO2 and ?105 DNA. DNA synthesis in ?105 shows a greater dependence on host functions than does SPO2 DNA synthesis. Growth of ?105 but not of SPO2 is inhibited by the uracil analogue 6-(p-hydroxyphenylazo)-uracil. Infection of a DNA polymerase-deficient strain of B. subtilis with SPO2 leads to an increase in DNA polymerase activity in crude extracts, whereas no such increase is found after infection of this strain with ?105. It is concluded that SPO2 and ?105 are unrelated bacteriophages. PMID:4623614

  19. M13 bacteriophage production for large-scale applications.

    PubMed

    Warner, Christopher M; Barker, Natalie; Lee, Seung-Wuk; Perkins, Edward J

    2014-10-01

    Bacteriophage materials have the potential to revolutionize medicine, energy production and storage, agriculture, solar cells, optics and many other fields. To fulfill these needs, this study examined critical process parameters during phage propagation to increase phage production capability. A representative scale-down system was created in tube spin reactors to allow parallel experimentation with single- and multi-variable analysis. Temperature, harvest time, media composition, feed regime, bacteriophage, and bacteria concentration were analyzed in the scale-down system. Temperature, media composition, and feeding regimens were found to affect phage production more than other factors. Temperature affected bacterial growth and phage production inversely. Multi-variate analysis identified an optimal parameter space which provided a significant improvement over the base line method. This method should be useful in scaled production of bacteriophage for biotechnology. PMID:24728964

  20. Marine Transducing Bacteriophage Attacking a Luminous Bacterium

    PubMed Central

    Keynan, Alex; Nealson, Kenneth; Sideropoulos, Henry; Hastings, J. W.

    1974-01-01

    The isolation and partial characterization of a marine bacteriophage attacking a strain of luminous bacteria is described, including some physical, biological, and genetic properties. It is a DNA phage of density of 1.52 with a long flexible tail and an apparently icosohedral head. With respect to stability in suspension, it has a rather specific requirement for the sodium ion in high concentration; it is further stabilized by the addition of calcium and magnesium ions. These same ions are likewise all required for both good plating efficiency and plaque uniformity. Although it goes through a typical lytic growth cycle (about 45 min), with a burst size of 100, and no stable lysogens have been isolated, it is nevertheless a transducing phage specifically for the tryptophan region, transducing several, but not all, independently isolated Trp? auxotrophs to protrophy. No other auxotrophs of a variety of amino acids were transduced by this phage to prototrophy. Phage infection does not change the normal expression of the luminescent system, and light remains at near normal levels until cell lysis occurs. Images PMID:16789143

  1. Bacteriophage Ecology in Commercial Sauerkraut Fermentations†

    PubMed Central

    Lu, Z.; Breidt, F.; Plengvidhya, V.; Fleming, H. P.

    2003-01-01

    Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages 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 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc, Weissella, and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations. PMID:12788716

  2. Bacteriophage ecology in commercial sauerkraut fermentations.

    PubMed

    Lu, Z; Breidt, F; Plengvidhya, V; Fleming, H P

    2003-06-01

    Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages 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 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc, Weissella, and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations. PMID:12788716

  3. The DNA ejection process in bacteriophage lambda

    NASA Astrophysics Data System (ADS)

    Grayson, Paul

    Bacteriophages have long served as model systems through which the nature of life may be explored. From a physical or mechanical point of view, phages are excellent examples of natural nanotechnology: they are nanometer-scale systems which depend critically on forces, pressures, velocities, and other fundamentally physical quantities for their biological functions. The study of the physical properties of phages has therefore provided an arena for application of physics to biology. In particular, recent studies of the motor responsible for packaging a phage gnome into a capsid showed a buildup of pressure within the capsid of tens of atmospheres. This thesis reports a combined theoretical and experimental study on various aspects of the genome ejection process, so that a comparison may be drawn with the packaging experiments. In particular, we examine various theoretical models of the forces within a phage capsid, deriving formulas both for the force driving genome ejection and for the velocity at which the genome is translocated into a host cell. We describe an experiment in which the force was measured as a function of the amount of genome within the phage capsid, and another where the genome ejection velocity was measured for single phages under the microscope. We make direct quantitative comparisons between the theory and experiments, stringently testing the extent to which we are able to model the genome ejection process.

  4. 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.

  5. T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates.

    PubMed

    Atencia, E A; Madrid, O; Günther Sillero, M A; Sillero, A

    1999-05-01

    T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5'-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P3) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm [alpha-32P] ATP, synthesis of labeled Ap4A was observed: ATP was acting as both donor (Km, microm) and acceptor (Km, mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with Km values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [alpha-32P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap4A) and 4 mm of the indicated nucleotides or P3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap4X (from XTP, 100); Ap4dG (from dGTP, 74); Ap4G (from GTP, 49); Ap4dC (from dCTP, 23); Ap4C (from CTP, 9); Ap3A (from ADP, 5); Ap4ddA, (from ddATP, 1); p4A (from P3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap4G: pCp > pAp > pA2'p. PMID:10215898

  6. Modeling the Growth Rates of Tetragonal Lysozyme Crystal Faces

    NASA Technical Reports Server (NTRS)

    Li, Meirong; Nadarajah, Arunan; Pusey, Marc L.

    1998-01-01

    The measured macroscopic growth rates of the (110) and (101) faces of tetragonal lysozyme show an unexpectedly complex dependence on the supersaturation. The growth rates decay asymptotically to zero when the supersaturation is lowered to zero and increase rapidly when the supersaturation is increased. When supersaturations are increased still further the growth rates attain a maximum before starting to decrease. However, growth of these crystals is known to proceed by the classical dislocation and 2D nucleation growth mechanisms. This anomaly can be explained if growth is assumed to occur not by monomer units but by lysozyme aggregates. Analysis of the molecular packing of these crystals revealed that they were constructed of strongly bonded 4(sub 3) helices, while weaker bonds were responsible for binding the helices to each other. It follows that during crystal growth the stronger bonds are formed before the weaker ones. Thus, the growth of these crystals could be viewed as a two step process: aggregate growth units corresponding to the 4(sub 3) helix are first formed in the bulk solution by stronger intermolecular bonds and then attached to the crystal face by weaker bonds on dislocation hillocks or 2D islands. This will lead to a distribution of aggregates in the solution with monomers and lower order aggregates being predominant at low supersaturations and higher order aggregates being predominant at high supersaturations. If the crystal grows mostly by higher order aggregates, such as tetramers and octamers, it would explain the anomalous dependence of the growth rates on the supersaturation. Besides the analysis of molecular packing, a comprehensive analysis of the measured (110) and (101) growth rates was also undertaken in this study. The distribution of aggregates in lysozyme nutrient solutions at various solution conditions were determined from reversible aggregation reactions at equilibrium. The supersaturation was defined for each aggregate species with respect to its concentration at saturation in order to apply growth rate models to this process. The measured growth rates were then compared with the predicted ones from several dislocation and 2D nucleation growth models, employing tetramer and octamer growth units in polydisperse solutions and monomer units in monodisperse solutions. For the (110) face, the calculations consistently showed that the measured growth rates followed the expected model relations with octamer growth units. For the (101) face, it is not possible to obtain a clear agreement between the predicted and measured growth rates for a single growth unit as done for the (110) face. However, the calculations do indicate that the average size of the growth unit is between a tetramer and an octamer. This suggests that tetramers, octamers and other intermediate size growth units all participate in the growth process for this face. These calculations show that it is possible to model the macroscopic protein crystal growth rates if the molecular level processes can be account for, particularly protein aggregation processes in the bulk solution. Our recent investigations of tetragonal lysozyme crystals employing high resolution atomic force microscopy scans have further confirmed the growth of these crystals by aggregate growth units corresponding to 4(sub 3) helices.

  7. Preferential solvation of lysozyme in water/ethanol mixtures

    NASA Astrophysics Data System (ADS)

    Ortore, Maria Grazia; Mariani, Paolo; Carsughi, Flavio; Cinelli, Stefania; Onori, Giuseppe; Teixeira, José; Spinozzi, Francesco

    2011-12-01

    We provide a quantitative description of the solvation properties of lysozyme in water/ethanol mixtures, which has been obtained by a simultaneous analysis of small-angle neutron scattering and differential scanning calorimetry experiments. All data sets were analyzed by an original method, which integrates the exchange equilibrium model between water and ethanol molecules at the protein surface and activity coefficients data of water/ethanol binary mixtures. As a result, the preferential binding of ethanol molecules at the protein surface was obtained for both native and thermal unfolded protein states. Excess solvation numbers reveal a critical point at ethanol molar fraction ?0.06, corresponding to the triggering of the hydrophobic clustering of alcohol molecules detected in water/ethanol binary mixtures.

  8. The Shake-and-Bake structure determination of triclinic lysozyme.

    PubMed

    Deacon, A M; Weeks, C M; Miller, R; Ealick, S E

    1998-08-01

    The crystal structure of triclinic lysozyme, comprised of 1,001 non-H protein atoms and approximately 200 bound water molecules, has been determined ab initio (using native data alone) by the "Shake-and-Bake" method by using the computer program SnB. This is the largest structure determined so far by the SnB program. Initial experiments, using default SnB parameters derived from studies of smaller molecules, were unsuccessful. In fact, such experiments produced electron density maps dominated by a single large peak. This problem was overcome by considering the choice of protocol used during the parameter-shift phase refinement. When each phase was subjected to a single shift of +/-157.5 degrees during each SnB cycle, an unusually high percentage of random trials (approximately 22%) yielded correct solutions within 750 cycles. This success rate is higher than that typically observed, even for much smaller structures. PMID:9689072

  9. The Shake-and-Bake structure determination of triclinic lysozyme

    PubMed Central

    Deacon, Ashley M.; Weeks, Charles M.; Miller, Russ; Ealick, Steven E.

    1998-01-01

    The crystal structure of triclinic lysozyme, comprised of 1,001 non-H protein atoms and ?200 bound water molecules, has been determined ab initio (using native data alone) by the “Shake-and-Bake” method by using the computer program SnB. This is the largest structure determined so far by the SnB program. Initial experiments, using default SnB parameters derived from studies of smaller molecules, were unsuccessful. In fact, such experiments produced electron density maps dominated by a single large peak. This problem was overcome by considering the choice of protocol used during the parameter-shift phase refinement. When each phase was subjected to a single shift of ±157.5° during each SnB cycle, an unusually high percentage of random trials (?22%) yielded correct solutions within 750 cycles. This success rate is higher than that typically observed, even for much smaller structures. PMID:9689072

  10. 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.

  11. Posttranscriptional lipopolysaccharide regulation of the lysozyme gene at processing of the primary transcript in myelomonocytic HD11 cells.

    PubMed

    Goethe, R; Phi-van, L

    1998-05-15

    Lysozyme is increasingly expressed in macrophages in inflammatory response to bacterial LPS. In this study, we investigated the mechanisms that control expression of the lysozyme gene in myelomonocytic HD11 cells activated by LPS. Nuclear run-on transcription assays showed that LPS caused a 15-fold increase in the transcription rate of the lysozyme gene. However, Northern analyses with lysozyme cDNA and intron sequences revealed that the LPS-induced increase in nuclear lysozyme transcripts greatly exceeded the increase in transcription rate. Furthermore, nuclear lysozyme transcripts in untreated cells with a t(1/2) of <10 min were more unstable than those accumulated in LPS-activated cells. We suggested, therefore, that the increased lysozyme expression following LPS treatment was largely due to a nuclear stabilization of the primary transcript. Interestingly, the increase in stability of the lysozyme primary transcript was accompanied by changes in nuclear processing including an increase in poly(A) tail length, which gradually shortened after entering the cytoplasm. The long lysozyme poly(A) tail, however, did not result in any increase in polysomal recruitment for translation or in stability of the cytoplasmic lysozyme mRNA. PMID:9590245

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Effect of lysozyme on glucose fermentation, cytoplasmic pH, and intracellular potassium concentrations in Streptococcus mutans 10449.

    PubMed

    Wang, Y B; Germaine, G R

    1991-02-01

    Several previous findings have suggested that the cationic nature of lysozyme is a major factor in its bactericidal activity. Since a number of cationic proteins or peptides have been reported to cause membrane damage in bacteria, we investigated the effect of lysozyme on glucose fermentation and intracellular pH and K+ in Streptococcus mutans under conditions in which lysis does not occur. Results showed that lysozyme and poly-D-lysine (PDL) cause inhibition of glucose fermentation at pH 5.5 in a dose-dependent manner. Human placental lysozyme and hen egg-white lysozyme exhibited similar inhibitory potency on glucose fermentation. Both lysozyme and PDL caused a marked acidification of the cytoplasm of S. mutans. However, when cytoplasmic pH was examined as a function of fermentation rate, the relationship was similar regardless of the presence or absence of lysozyme or PDL. Therefore, acidification of the cytoplasm appeared to not depend specifically on lysozyme or PDL. In contrast, the same relationship between the profound loss of intracellular K+, when fermenting cells were exposed to either lysozyme or PDL, and the fermentation rate was not exhibited in the controls. These results indicate that lysozyme and PDL specifically affected the ability of the cells to maintain intracellular K+. We concluded that lysozyme and PDL indeed perturb membrane function, perhaps in a selective manner. Furthermore, the similarity in action of lysozyme and the cationic homopolypeptide PDL supports the notion that the cationic property of lysozyme indeed plays a significant role in its antibacterial activity. PMID:1987080

  18. 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.

  19. 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.

  20. BSA and Lysozyme adsorption on homoionic montmorillonite: influence of the interlayer cation

    E-print Network

    Boyer, Edmond

    on Langmuir model isotherms, a strong influence of interlayer cation on maximum adsorbed amount and adsorption to describe proteins adsorption. Keywords : Protein adsorption, montmorillonite, Langmuir isotherm, homoionicBSA and Lysozyme adsorption on homoionic montmorillonite: influence of the interlayer cation

  1. Investigation by dielectric spectroscopy of domain motions in lysozyme: effect of solvent and binding of inhibitors

    NASA Astrophysics Data System (ADS)

    Bonincontro, A.; Onori, G.

    2004-11-01

    Recently, we have shown that ethylene-glycol, a co-solvent commonly used in studying protein folding, strongly affects the dielectric response of lysozyme. The data were coherently interpreted hypothesizing that glycol molecules wedges between and separates the two domains of lysozyme making them rotationally independent. In this Letter, we describe a new dielectric experiment on lysozyme in water/ethylene-glycol solutions in the presence of N-acetyl- D-glucosammine, a well known inhibitor for the protein. The results indicate that the interaction of ethylene-glycol with the protein is markedly influenced by NAG, in the sense that the saccharide preferentially links with lysozyme driving away the glycol molecules.

  2. Investigation of the interactions of lysozyme and trypsin with biphenol A using spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Wang, Yan-Qing; Chen, Ting-Ting; Zhang, Hong-Mei

    2010-03-01

    The interaction between bisphenol A (BPA) and lysozyme (or trypsin) was investigated by UV-vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques under physiological pH 7.40. BPA effectively quenched the intrinsic fluorescence of lysozyme and trypsin via static quenching. H-bonds and van der Waals interactions played a major role in stabilizing the BPA-proteinase complex. The distance r between donor and acceptor was obtained to be 1.65 and 2.26 nm for BPA-lysozyme and BPA-trypsin complexes, respectively. The effect of BPA on the conformation of lysozyme and trypsin was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra.

  3. 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.

  4. Le lysozyme dans le matriel et les scrtions digestives chez le lapin domestique

    E-print Network

    Boyer, Edmond

    for the bile and the pancreatic juice. It has been found to be absent from the digestive material of the rabbit intestinal and gastric fistulae showed high enzyme levels. Lysozyme is thus of probable plasmatic origin

  5. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 false Borate(1-), tris...kappa.O)hydro-, sodium, (T-4... § 721.1880 Borate(1-), tris...kappa.O)hydro-, sodium, (T-4...substance identified as borate(1-), tris...kappa.O)hydro-, sodium,...

  6. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...SUBSTANCES Significant New Uses for Specific Chemical Substances...acetato-.kappa.O)hydro-, sodium, (T-4)-. (a) Chemical substance and significant new uses subject to reporting. ...acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN...

  7. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...SUBSTANCES Significant New Uses for Specific Chemical Substances...acetato-.kappa.O)hydro-, sodium, (T-4)-. (a) Chemical substance and significant new uses subject to reporting. ...acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN...

  8. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...SUBSTANCES Significant New Uses for Specific Chemical Substances...acetato-.kappa.O)hydro-, sodium, (T-4)-. (a) Chemical substance and significant new uses subject to reporting. ...acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN...

  9. A stopped-flow fluorescence study of the native and modified lysozyme

    Microsoft Academic Search

    Khosrow Khalifeh; Bijan Ranjbar; Khosro Khajeh; Hossein Naderi-Manesh; Mehdi Sadeghi; Sara Gharavi

    2007-01-01

    The protein folding kinetics of hen egg white lysozyme (HEWL) was studied using experimental and bioinformatics tools. The\\u000a structure of the transition state in the unfolding pathway of lysozyme was determined with stopped-flow kinetics using intact\\u000a HEWL and its chemically modified derivative, in which six lysine residues have been modified. The overall consistency of ?-value\\u000a (? ? 1) indicates that

  10. Expression of lysozyme in the life history of the house fly (Musca domestica l.).

    PubMed

    Nayduch, Dana; Joyner, Chester

    2013-07-01

    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 because of its broad-spectrum activity against gram-positive and gram-negative bacteria as well as fungi. This study aimed to determine the temporal expression of lysozyme in the life history of house flies (from egg through adults) on both the mRNA and protein level, and to determine the tissue-specific expression of lysozyme in adult flies induced by feeding Staphylococcus aureus. From 30-min postoviposition through adulthood, all life history stages of the house fly express lysozyme on the mRNA level. In adult flies, lysozyme is expressed both locally in the alimentary canal and systemically in the fat body. Interestingly, we found that during the normal life history of flies, lysozyme protein was only detected in larval stages and older adults, likely because of ingestion of immune-stimulating levels of bacteria, not experienced during egg, pupa, and teneral adult stages. Constitutive expression on the mRNA level implies that this effector is a primary defense molecule in all stages of the house fly life history, and that a mechanism for posttranscriptional control of mature lysozyme enzyme expression may be present. Lysozyme active enzyme primarily serves both a digestive and defensive function in larval and adult flies, and may be a key player in the ability of Musca domestica L. to thrive in microbe-rich environments. PMID:23926784

  11. Lysozyme-mediated aggregation and lysis of the periodontal microorganism Capnocytophaga gingivalis 2010.

    PubMed Central

    Iacono, V J; Zove, S M; Grossbard, B L; Pollock, J J; Fine, D H; Greene, L S

    1985-01-01

    The ability of lysozyme to aggregate and lyse the gram-negative capnophilic periodontal microorganism Capnocytophaga gingivalis 2010 was monitored optically at 540 nm. Both hen egg white and chromatographically purified human lysozymes had significant but similar aggregation potentials for both logarithmic- and stationary-phase bacteria. In general, an increase in enzyme concentration resulted in a graded increase in both the initial and maximum changes in turbidity which occurred during the reaction period. The greatest change in turbidity occurred within the initial minutes of interaction of lysozyme and the cells, and the extent of aggregation paralleled a rapid depletion of lysozyme by the suspensions during the first minute of its incubation with the bacteria. Interestingly, the muramidase inhibitors N-acetyl-D-glucosamine and histamine did not block aggregation, whereas maleylation of lysozyme completely inhibited its aggregating ability. Demaleylation, however, restored aggregation activity comparable to the native enzyme, indicating that maleylated lysozyme retained its integrity and that aggregation was primarily dependent on charge. The addition of up to physiological concentrations of NaHCO3 and NaCl to cell aggregates resulted in varying degrees of deaggregation and lysis. Surprisingly, ultrastructural analysis of lysozyme-treated cells revealed morphological changes with or without the addition of salt. Damage appeared to occur at the blunted polar end of the cells where there was a large spherical outpouching bordered by a damaged cell envelope. Damaged cells uniformly contained dense granular cytoplasmic debris. In effect, the cationic enzyme lysed C. gingivalis 2010, which was not apparent in the spectrophotometric assay. The paradoxical finding that during bacterial aggregation there was lysis may be of significance to the further elucidation of lysozyme's antibacterial role in the gingival sulcus. Images PMID:3967924

  12. Molecular Basis of Resistance to Muramidase and Cationic Antimicrobial Peptide Activity of Lysozyme in Staphylococci

    PubMed Central

    Herbert, Silvia; Bera, Agnieszka; Nerz, Christiane; Kraus, Dirk; Peschel, Andreas; Goerke, Christiane; Meehl, Michael; Cheung, Ambrose; Götz, Friedrich

    2007-01-01

    It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide 107R-A-W-V-A-W-R-N-R115 (LP9), polymyxin B, or gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators (rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilm-negative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7% decrease in the content of esterified d-alanyl groups in teichoic acids. The oatA/dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid–bound d-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity (oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA/dltA or oatA/graRS double mutants are much more susceptible to lysozyme than each of the single mutants. PMID:17676995

  13. Characterisation of lysozyme activity in the in situ pellicle using a fluorimetric assay

    Microsoft Academic Search

    Christian Hannig; Bettina Spitzmüller; Matthias Hannig

    2009-01-01

    Lysozyme is among the most protective enzymes in the pellicle layer. The aim of the present study was to establish a precise\\u000a fluorimetric assay for determination and characterisation of lysozyme activity immobilised in the initial in situ formed pellicle.\\u000a For in situ pellicle formation, bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different\\u000a times

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

    Microsoft Academic Search

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

    2007-01-01

    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.

  15. Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid?

    Microsoft Academic Search

    M. Malisauskas; A. Darinskas; V. V. Zamotin; A. Gharibyan; I. A. Kostanyan; L. A. Morozova-Roche

    2006-01-01

    In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme.\\u000a We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57C.\\u000a These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by\\u000a atomic force microscopy.

  16. Novel surface-modified molecularly imprinted membrane prepared with iniferter for permselective separation of lysozyme

    Microsoft Academic Search

    Run-Run Chen; Lei Qin; Man Jia; Xi-Wen He; Wen-You Li

    2010-01-01

    A novel and efficient molecularly imprinted membrane (MIM) for the selective transport and separation of lysozyme was synthesized by the molecular imprinting technique. The layer of molecularly imprinted materials containing recognition sites for the lysozyme was formed on the poly(acrylontrile-co-N,N?-diethylaminodithiocarbamoylmethylstyrene) [P(AN-co-DTCS)] membrane, which was prepared via a phase inversion method and modified with photosensitive dithiocarbamate group for surface initiated living-radical

  17. Kinetics of folding of guanidine-denatured hen egg white lysozyme and carboxymethyl(Cys6,Cys127)-lysozyme: a stopped-flow absorbance and fluorescence study.

    PubMed

    Denton, M E; Rothwarf, D M; Scheraga, H A

    1994-09-20

    The folding kinetics of hen egg white lysozyme and of a three-disulfide derivative of lysozyme [carboxymethyl(Cys6,Cys127)-hen egg white lysozyme] have been studied by absorbance- and fluorescence-detected stopped-flow techniques. A "very-fast" phase with a time constant in the millisecond range has been observed by both absorbance and fluorescence when unfolded lysozyme in 4 M guanidine hydrochloride, 100 mM phosphate buffer, and pH 2.0 is refolded at 0.5 M guanidine hydrochloride, 100 mM phosphate, and pH 6.7. Data obtained from fluorescence-detected refolding studies show that a transient intermediate is formed during the very-fast refolding phase. This intermediate is characterized by substantial quenching of tryptophan fluorescence. In addition, analysis of the fluorescence data indicates the presence of an additional "burst" phase that occurs within the dead time of the instrument, < 3 ms. The very-fast phase is not observed during the refolding of the three-disulfide derivative. In addition, the three-disulfide derivative re-attains the final native folded conformation more rapidly than the unmodified protein over the range of temperatures studied (10-20 degrees C). We conclude that, not only does the presence of the disulfide bond between Cys6 and Cys127 slow down the overall folding process of lysozyme, but it also directs the folding of lysozyme through a pathway characterized by a non-native tertiary interaction(s). PMID:7727374

  18. 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

  19. Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts.

    PubMed Central

    Goodman, H; Pollock, J J; Katona, L I; Iacono, V J; Cho, M I; Thomas, E

    1981-01-01

    Streptococcus mutans BHT was grown in a synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. Kinetic experiments demonstrated that although lysozyme alone could not liberate deoxyribonucleic acid, cellular deoxyribonucleic acid was liberated from lysozyme-treated cells by addition of low concentrations of inorganic sodium salts. When the salts were tested for their ability to dislodge cell-bound tritiated lysozyme, the extent of the initial release of enzyme by individual anions correlated with the anion potency for deoxyribonucleic acid liberation (SCN- greater than ClO4- greater than I- greater than Br- greater than NO3- greater than Cl- greater than F-), although the total amount of lysozyme dislodged did not correspond directly with cell lysis. Differences in the effectiveness of anions (SCN-, HCO3-, Cl- and F-) in potentiating cell lysis could be enhanced or minimized by varying the lysozyme, anion, and bacterial cell concentrations. As the anion concentration was increased for each enzyme concentration and cell concentration, the lysis increased, in some cases markedly, until maximum levels of released deoxyribonucleic acid were attained. The maximum levels of lysis of SCN- and HCO3- were similar and were greater than those for Cl- and F-. In addition, the maximum levels were observed to increase for each of the anions as the concentration of lysozyme increased. Images PMID:7217017

  20. Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme

    PubMed Central

    Durek, Thomas; Torbeev, Vladimir Yu.; Kent, Stephen B. H.

    2007-01-01

    In this article, we report the total chemical synthesis of human lysozyme. Lysozyme serves as a widespread model system in various fields of biochemical research, including protein folding, enzyme catalysis, and amyloidogenesis. The 130-aa wild-type polypeptide chain of the human enzyme was assembled from four polypeptide segments by using native chemical ligation in a fully convergent fashion. Key to the assembly strategy is the application of the recently developed kinetically controlled ligation methodology, which provides efficient control over the ligation of two peptide ?thioesters to yield a unique product. This result enables the facile preparation of a 64-residue peptide ?thioester; this segment is joined by native chemical ligation to a 66-aa Cys peptide, to yield the target 130-aa polypeptide chain. The synthetic polypeptide chain was folded in vitro into a defined tertiary structure with concomitant formation of four disulfides, as shown by 2D TOCSY NMR spectroscopy. The structure of the synthetic human lysozyme was confirmed by high-resolution x-ray diffraction, giving the highest-resolution structure (1.04 ?) observed to date for this enzyme. Synthetic lysozyme was obtained in good yield and excellent purity and had full enzymatic activity. This facile and efficient convergent synthesis scheme will enable preparation of unique chemical analogs of the lysozyme molecule and will prove useful in numerous areas of lysozyme research in the future. PMID:17360367

  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. 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

  3. 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

  4. 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

  5. cDNA and amino acid sequences of rainbow trout ( Oncorhynchus mykiss ) lysozymes and their implications for the evolution of lysozyme and lactalbumin

    Microsoft Academic Search

    André Dautigny; Ellen M. Prager; Danièle Pham-Dinh; Jacqueline Jollès; Farzad Pakdel; Bjørn Grinde; Pierre Jollès

    1991-01-01

    Summary  The complete 129-amino-acid sequences of two rainbow trout lysozymes (I and II) isolated from kidney were established using\\u000a protein chemistry microtechniques. The two sequences differ only at position 86, I having aspartic acid and II having alanine.\\u000a A cDNA clone coding for rainbow trout lysozyme was isolated from a cDNA library made from liver mRNA. Sequencing of the cloned\\u000a cDNA

  6. 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

  7. Use of bacteriophages in controlling E. coli in leafy vegetables

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages are viruses that can infect and lys (kill) bacteria. These viruses are not harmful to humans and are present in the environment and many foods. Enterohemmorhagic E. coli (EHEC), like E. coli O157:H7, have been associated with contaminated bagged leafy green commodities. Outbreaks o...

  8. First Complete Genome Sequence of Two Staphylococcus epidermidis Bacteriophages

    Microsoft Academic Search

    Anu Daniel; Penelope E. Bonnen; Vincent A. Fischetti

    2007-01-01

    Staphylococcus epidermidis is an important opportunistic pathogen causing nosocomial infections and is often associated with infections in patients with implanted prosthetic devices. A number of virulence determinants have been identified in S. epidermidis, which are typically acquired through horizontal gene transfer. Due to the high recombination potential, bacteriophages play an important role in these transfer events. Knowledge of phage genome

  9. Characterization of the Bacteriophage Lambda Holin and Its Membrane Lesion 

    E-print Network

    Dewey, Jill Sayes

    2011-10-21

    Bacteriophage holins are a diverse group of proteins that are responsible for the spontaneous and specifically-timed triggering of host cell lysis. The best-studied holin, S105 of phage lambda, is known to form lesions, or “holes”, in the inner...

  10. Structural characterization of bacteriophage M13 solubilization by amphiphiles

    Microsoft Academic Search

    David Stopar; Ruud B. Spruijt; Cor J. A. M. Wolfs; Marcus A. Hemminga

    2002-01-01

    The structural properties of bacteriophage M13 during disassembly were studied in different membrane model systems, composed of a homologue series of the detergents sodium octyl sulfate, sodium decyl sulfate, and sodium dodecyl sulfate. The structural changes during phage disruption were monitored by spin-labeled electron spin resonance (ESR) and circular dichroism spectroscopy. For the purpose of ESR spectroscopy the major coat

  11. Evidence for a lineage of virulent bacteriophages that target Campylobacter

    E-print Network

    Timms, Andrew R; Cambray-Young, Joanna; Scott, Andrew E; Petty, Nicola K; Connerton, Phillippa L; Clarke, Louise; Seeger, Kathy; Quail, Michael A; Cummings, Nicola; Maskell, Duncan; Thomson, Nicholas R; Connerton, Ian F

    2010-03-30

    ). Strains were grown on blood agar base No. 2 (Oxoid) or Mueller-Hinton agar (Oxoid), supplemented with 5% defibrinated horse blood (Oxoid), for 24-48 h under microaerobic conditions at 42°C. Bacteriophage were propagated using the whole plate lysis method...

  12. 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

  13. Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages

    PubMed Central

    Yang, Hongyan; Makeyev, Eugene V.; Bamford, Dennis H.

    2001-01-01

    The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage ?6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3? termini from the ?6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from ?6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the ?6-related bacteriophages ?8 and ?13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from ?6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3?-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit. PMID:11602748

  14. Bacteriophage therapy: a revitalized therapy against bacterial infectious diseases

    Microsoft Academic Search

    Shigenobu Matsuzaki; Mohammad Rashel; Jumpei Uchiyama; Shingo Sakurai; Takako Ujihara; Masayuki Kuroda; Masahiko Ikeuchi; Toshikazu Tani; Mikiya Fujieda; Hiroshi Wakiguchi; Shosuke Imai

    2005-01-01

    Bacteriophage (phage) therapy involves using phages or their products as bioagents for the treatment or prophylaxis of bacterial infectious diseases. Much evidence in support of the effectiveness of phage therapy against bacterial infectious diseases has accumulated since 1980 from animal model studies conducted in Western countries. Reports indicate that appropriate administration of living phages can be used to treat lethal

  15. 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...

  16. 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...

  17. Alkylation of T7 bacteriophage blocks superinfection exclusion.

    PubMed Central

    Karska-Wysocki, B; Racine, J F; Mamet-Bratley, M D

    1982-01-01

    Alkylation of T7 bacteriophage by methyl methane sulfonate blocked superinfection exclusion. This blockage could be correlated with a delay in the synthesis of phage-specific proteins. Therefore we conclude that protein synthesis directed by the primary infecting phage is required for efficient exclusion of superinfecting phage particles. Images PMID:6754970

  18. Bacteriophage significantly reduces Listeria monocytogenes on raw salmon fillet tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have demonstrated the antilisterial activity of generally recognized as safe (GRAS) bacteriophage LISTEX P100 (phage P100) on the surface of raw salmon fillet tissue against Listeria monocytogenes serotypes 1/2a and 4b. In a broth model system, phage P100 completely inhibited L. monocytogenes gro...

  19. Elevated Abundance of Bacteriophage Infecting Bacteria in Soil

    Microsoft Academic Search

    Kevin E. Ashelford; Martin J. Day; John C. Fry

    2003-01-01

    Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean 1.5 107 g1), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here

  20. Lytic bacteriophages: Potential interventions against enteric bacterial pathogens on produce.

    PubMed

    Sharma, Manan

    2013-04-01

    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

  1. Isolation and characterization of 101-beta-lysozyme that possesses the beta-aspartyl sequence at aspartic acid-101.

    PubMed

    Yamada, H; Ueda, T; Kuroki, R; Fukumura, T; Yasukochi, T; Hirabayashi, T; Fujita, K; Imoto, T

    1985-12-31

    In the reaction of the intramolecular cross-linking between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-112 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-112 in 6 N HCl at 110 degrees C gave a considerable amount of beta-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the beta-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the beta-aspartyl sequence at Asp-101 (101-beta-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the beta-aspartyl sequence at Asp-101 (101-beta-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:4092046

  2. Study on the interactions between ginsenosides and lysozyme under acidic condition by ESI-MS and molecular docking

    NASA Astrophysics Data System (ADS)

    Qu, Chenling; Yu, Songcheng; Bai, Aixi; Wang, Jinshui

    2011-02-01

    In order to study the different effects of ginsenosides with similar structures, research on interactions between ginsenoside Rg 1, Re and lysozyme was carried out by electrospray ionization mass spectrometry (ESI-MS) and molecular docking. The 1:1 and 2:1 noncovalent complexes of ginsenosides and lysozyme were observed in the mass spectra and the dissociation constants for them were directly calculated based on peak intensities of lysozyme and its noncovalent complexes with ginsenosides. The results showed that the 1:1 complex of ginsenoside Rg 1 and lysozyme was more stable than that of ginsenoside Re and lysozyme. As the acidity increased, the stabilities of the 1:1 complexes of Rg 1, Re and lysozyme both decreased. Interestingly, as the acidity increased, the stability of the 2:1 complex of Rg 1 and lysozyme increased while that of Re decreased. From the result of molecular docking, ginsenosides interacted with the active sites of lysozyme. And the stability of the complexes could be affected by the conformation changes of lysozyme as acidity increased.

  3. Pathophysiology of rhinitis. Lactoferrin and lysozyme in nasal secretions.

    PubMed Central

    Raphael, G D; Jeney, E V; Baraniuk, J N; Kim, I; Meredith, S D; Kaliner, M A

    1989-01-01

    The antimicrobial proteins lactoferrin (Lf) and lysozyme (Ly) are invariably found in nasal secretions. To investigate the cellular sources and the secretory control of these nasal proteins in vivo, 34 adult subjects underwent nasal provocation tests with methacholine (MC), histamine (H), and gustatory stimuli. Nasal lavages were collected and analyzed for total protein (TP), albumin (Alb), Lf, and Ly. MC (25 mg), H (1 mg), and gustatory stimuli (spicy foods) all increased the concentrations of TP, Alb, Lf, and Ly. However, when each protein was assessed as a percentage of TP (i.e., Alb% = Alb/TP; Lf% = Lf/TP; Ly% = Ly/TP), MC and gustatory stimuli, which both induce glandular secretion, selectively augmented Lf% and Ly% without changing Alb%, while H, which primarily increases vascular permeability, increased Alb% without significantly affecting Lf% or Ly%. Gel electrophoresis and immunoblotting analysis of nasal secretions demonstrated both Lf and Ly in cholinergically induced secretions. Furthermore, histochemical analyses of nasal turbinate tissue revealed Lf and Ly colocalization within the serous cells of submucosal glands, providing evidence that both proteins are strictly glandular products within the nasal mucosa. Therefore, both Lf and Ly are produced and secreted from the glands, and their secretion may be pharmacologically regulated in attempts to improve host defenses. Images PMID:2681268

  4. Growth Modes and Energetics of 101 Face Lysozyme Crystal Growth

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar; Forsythe, Elizabeth L.; Pusey, L.

    2004-01-01

    From analyses of lysozyme 101 face growth rate data using a 2D nucleation model for layer-by-layer growth, we find the effective barrier for crystal growth to be gamma = 1.0 +/- 0.2 x 10(exp -13) erg/molecule. The magnitude of the effective barrier is 2.4 +/- 0.5 k(sub beta)T, at 22 C. We also find that beyond a critical solution supersaturation, sigma(sub c), crystal growth rates are more accurately described by a kinetic roughening hypothesis. Beyond sigma(sub c), crystals grow by the continuous addition of molecules anywhere on the crystal surface rather than layer-by-layer. The magnitude of the critical supersaturation (sigma(sub c), = 1.7 +/- 0.2) for a crossover from a layer-by-layer to continuous growth is found to be statistically independent of the solution conditions that vary with buffer pH, temperature or precipitant concentration. Using the experimentally determined values for gamma and sigma(sub c), we find the crystal growth unit to be comprised of 7 +/- 3 molecules. The energy barrier, E(sub c), for the continuous addition of the growth Units is 6.2 +/- 0.3 x 10(exp -13) erg/molecule or 15 +/1 1 k(sub beta)T at 22C.

  5. Interaction mechanism between berberine and the enzyme lysozyme

    NASA Astrophysics Data System (ADS)

    Cheng, Ling-Li; Wang, Mei; Wu, Ming-Hong; Yao, Si-De; Jiao, Zheng; Wang, Shi-Long

    2012-11-01

    In the present paper, the interaction between model protein lysozyme (Lys) and antitumorigenic berberine (BBR) was investigated by spectroscopic methods, for finding an efficient and safe photosensitizer with highly active transient products using in photodynamic therapy study. The fluorescence data shows that the binding of BBR could change the environment of the tryptophan (Trp) residues of Lys, and form a new complex. Static quenching is the main fluorescence quenching mechanism between Lys and BBR, and there is one binding site in Lys for BBR and the type of binding force between them was determined to be hydrophobic interaction. Furthermore, the possible interaction mechanism between BBR and Lys under the photoexcitation was studied by laser flash photolysis method, the results demonstrated that BBR neutral radicals (BBR(-H)•) react with Trp (K = 3.4 × 109 M-1 s-1) via electron transfer to give the radical cation (Trp/NH•+) and neutral radical of Trp (TrpN•). Additionally BBR selectively oxidize the Trp residues of Lys was also observed by comparing the transient absorption spectra of their reaction products. Through thermodynamic calculation, the reaction mechanisms between 3BBR? and Trp or Lys were determined to be electron transfer process.

  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. ATP-induced noncooperative thermal unfolding of hen lysozyme

    SciTech Connect

    Liu, Honglin; Yin, Peidong; He, Shengnan; Sun, Zhihu [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China)] [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China); Tao, Ye; Huang, Yan; Zhuang, Hao [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China)] [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Zhang, Guobin [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China)] [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China); Wei, Shiqiang, E-mail: sqwei@ustc.edu.cn [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China)] [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China)

    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.

  9. Triclinic lysozyme at 0.65 angstrom resolution.

    SciTech Connect

    Wang, J.; Dauter, M.; Alkire, R.; Joachimiak, A.; Dauter, Z.; Biosciences Division; SAIC-Frederick Inc.; National Cancer Inst.

    2007-01-01

    The crystal structure of triclinic hen egg-white lysozyme (HEWL) has been refined against diffraction data extending to 0.65 {angstrom} resolution measured at 100 K using synchrotron radiation. Refinement with anisotropic displacement parameters and with the removal of stereochemical restraints for the well ordered parts of the structure converged with a conventional R factor of 8.39% and an R{sub free} of 9.52%. The use of full-matrix refinement provided an estimate of the variances in the derived parameters. In addition to the 129-residue protein, a total of 170 water molecules, nine nitrate ions, one acetate ion and three ethylene glycol molecules were located in the electron-density map. Eight sections of the main chain and many side chains were modeled with alternate conformations. The occupancies of the water sites were refined and this step is meaningful when assessed by use of the free R factor. A detailed description and comparison of the structure are made with reference to the previously reported triclinic HEWL structures refined at 0.925 {angstrom} (at the low temperature of 120 K) and at 0.95 {angstrom} resolution (at room temperature).

  10. A Model for Tetragonal Lysozyme Crystal Nucleation and Growth

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

    Macromolecular crystallization is a complex process, involving a system that typically has 5 or more components (macromolecule, water, buffer + counter ion, and precipitant). Whereas small molecules have only a few contacts in the crystal lattice, macromolecules generally have 10's or even 100's of contacts between molecules. These can range from hydrogen bonds (direct or water-mediated), through van der Waals, hydrophobic, salt bridges, and ion-mediated contacts. The latter interactions are stronger and require some specificity in the molecular alignment, while the others are weaker, more prevalent, and more promiscuous, i.e., can be readily broken and reformed between other sites. Formation of a consistent, ordered, 3D structure may be difficult or impossible in the absence of any or presence of too many strong interactions. Further complicating the process is the inherent structural asymmetry of monomeric (single chain) macromolecules. The process of crystal nucleation and growth involves the ordered assembly of growth units into a defined 3D lattice. We suggest that for many macromolecules, particularly those that are monomeric, this involves a preliminary solution-phase assembly process into a growth unit having some symmetry prior to addition to the lattice, recapitulating the initial stages of the nucleation process. If this model is correct then fluids and crystal growth models assuming a strictly monodisperse nutrient solution need to be revised. This model has been developed from experimental evidence based upon face growth rate, AFM, and fluorescence energy transfer data for the nucleation and growth of tetragonal lysozyme crystals.

  11. Kinetic Roughening and Energetics of Tetragonal Lysozyme Crystal Growth

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar; Forsythe, Elizabeth L.; Pusey, Marc L.

    2003-01-01

    Lysozyme crystal growth rates over 5 orders of magnitude in range can be described using a layer-by-layer model where growth occurs by 2D nucleation on the crystal surface. Based upon the 2D nucleation model of layer growth, the effective barrier for growth was determined to be gamma = 1.3 plus or minus 0.3 x 10(exp -13) erg/molecule, corresponding to a barrier of 3.2 plus or minus 0.7 k(sub B)T, at 22 C. For solution supersaturation, In c/c(sub eq) greater than or equal to 1.9 plus or minus 0.2, the nucleation model would not predict or consistently estimate the highest observable crystal growth rates. As such, a kinetic roughening hypothesis where crystal growth occurs by a continuous mode was implemented for all growth rate data obtained above In c(sub r)/c(sub eq) greater than or equal to 2. That is, independent of the solution conditions that vary with either buffer pH, temperature or precipitant concentration, crystal growth occurs by the continuous addition of molecules anywhere on the crystal surface, above a roughening solution supersaturation. The energy barrier, E(sub c), for the continuous growth process is determined as 6.1 plus or minus 0.4 x 10(exp -13) erg/molecule or 15 plus or minus 1 k(sub B)T at 22 C.

  12. Preparation of lysozyme imprinted magnetic nanoparticles via surface graft copolymerization.

    PubMed

    Wang, Yanxia; Chai, Zhihua; Sun, Yingjuan; Gao, Ming; Fu, Guoqi

    2015-08-01

    Molecular imprinting as a facile and promising separation technique has received considerable attention because of their high selectivity for target molecules. In this study, we imprinted lysozyme (Lys) on the surface of core-shell magnetic nanoparticles via surface imprinting. The magnetic supports were functionalized with maleic acid and then coated with imprinted polymer layers. The structure and morphology of the resulting magnetic imprinted nanoparticles were characterized by transmission electron microscopy, scanning electron microscope, dynamic light scatting, vibrating sample magnetometer, and thermogravimetric analysis. Binding experiments were carried out to evaluate the properties of magnetic molecularly imprinted polymers (magnetic MIPs) and magnetic non-molecularly imprinted polymers (magnetic NIPs). The protein adsorption results showed that the magnetic MIPs had significant specific recognition toward the template protein and could be easily separated from solution by an external magnetic field. Moreover, the MIPs exhibited fast kinetics for the rebinding of the target protein due to the thin-imprinted layer and showed good reusability by four adsorption-desorption cycles. Therefore, the surface imprinting approach combined with magnetic nanoparticles provided an easy and fast method for the specific recognition of Lys. PMID:26073534

  13. 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.

  14. Longitudinal Study of Campylobacter jejuni Bacteriophages and Their Hosts from Broiler Chickens

    PubMed Central

    Connerton, P. L.; Loc Carrillo, C. M.; Swift, C.; Dillon, E.; Scott, A.; Rees, C. E. D.; Dodd, C. E. R.; Frost, J.; Connerton, I. F.

    2004-01-01

    A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance. PMID:15240258

  15. 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.

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

    PubMed

    Oliver, W T; Wells, J E; Maxwell, C V

    2014-11-01

    Lysozyme is a 1,4-?-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 26 d of age, blocked by litter and sex, and then randomly assigned to 1 of 24 pens in either a nursery room that had been fully disinfected or a nursery room left unclean since the previous group of pigs. Within a room, pigs were randomly assigned to either control diets (2 phase nursery regime), control diets + antibiotics (chlortetracycline/tiamulin hydrogen fumarate), or control diets + lysozyme (100 mg/kg diet). Pig weights and feed disappearance were measured and blood was collected on d 0, 14, and 28 of treatment. A group of 20 pigs were killed at 24 d of age for initial body composition analysis and 10 pigs of median weight were killed per diet room combination for body composition analysis after 28 d of treatment. Control + antibiotics and control + lysozyme-fed pigs grew at a faster rate for the 28-d study compared to control pigs (318 ± 14,320 ± 15 vs. 288 ± 15 g/d, respectively; P < 0.05), regardless of nursery environment (P > 0.05). The indirect immune challenge did not alter growth performance from d 0 to 14 of treatment but decreased ADG from d 14 to 28 of the study (415 ± 15 vs. 445 ± 13 g/d ;: P < 0.05). Feed intake was not altered by the nursery environment (P > 0.61) or dietary treatments (P > 0.10), but feed efficiency was worsened by the indirect immune challenge (P < 0.05) and improved by both control + antibiotics and control + lysozyme diets (P < 0.01). The immune challenge did not alter nutrient accretion (P > 0.25), but both control + antibiotics and control + lysozyme pigs had decreased accretion of whole-body lipid (P < 0.01) and increased accretion of protein (P < 0.09). Blood levels of tumor necrosis factor-? (TNF-?; P < 0.01), haptoglobin (P < 0.09), and C-reactive protein (CRP; P < 0.01) were higher due to the indirect immune challenge compared to pigs reared in the clean nursery (P < 0.05). In addition, pigs consuming antibiotics or lysozyme had lower TNF-?, haptoglobin, and CRP compared to control pigs, regardless of nursery environment (P < 0.04). Thus, lysozyme is a suitable alternative to antibiotics in swine nursery diets, and lysozyme ameliorates the effects of a chronic indirect immune challenge. PMID:25253813

  17. Ionic liquid-induced structural and activity changes in hen egg white lysozyme.

    PubMed

    Dang, Le-Ping; Fang, Wen-Zhi; Li, Yan; Wang, Qian; Xiao, Hua-Zhi; Wang, Zhan-Zhong

    2013-01-01

    Lysozyme crystals in the presence of 1-butyl-3-methylimidazolium tetrafluoroborate ([C(4)mim]BF(4)), 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl), 1-butyl-3-methylimidazolium bromide([C(4)mim]Br), and 1,3-dimethylimidazolium iodine([dmim]I) were prepared, and the influence of ionic liquids (ILs) on the structure and activity change of lysozyme was investigated. Fourier transform infrared spectroscopy revealed the major secondary structures of ?-helix and ?-sheet for lysozyme. It was interesting to note that increases of the band near 2,935 and 1,656 cm(-1) from Raman spectroscopy are attributed to the unfolding of lysozyme molecules. A shift in amide III from 1,230 to 1,270 cm(-1) in adding [dmim]I occurs, indicating a transformation from ?-sheet to random coil. With regard to adding [C(4)mim]BF(4), [C(4)mim]Cl, and [C(4)mim]Br, ?-helix and ?-sheet are the predominant structures for lysozyme. The activity study showed that the ILs used brought a positive effect. Especially, [dmim]I leads to a drastic increase in relative activity, and its value reaches 50 %. PMID:23179287

  18. 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

  19. A zinc complex of heparan sulfate destabilises lysozyme and alters its conformation

    SciTech Connect

    Hughes, Ashley J. [Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB (United Kingdom) [Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB (United Kingdom); Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire OX11 0DE (United Kingdom); Hussain, Rohanah [Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire OX11 0DE (United Kingdom)] [Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire OX11 0DE (United Kingdom); Cosentino, Cesare; Guerrini, Marco [Istituto di Chimica e Biochimica 'G. Ronzoni', Via G. Colombo 81, Milano 20133 (Italy)] [Istituto di Chimica e Biochimica 'G. Ronzoni', Via G. Colombo 81, Milano 20133 (Italy); Siligardi, Giuliano [Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire OX11 0DE (United Kingdom)] [Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire OX11 0DE (United Kingdom); Yates, Edwin A., E-mail: eayates@liv.ac.uk [Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB (United Kingdom); Rudd, Timothy R., E-mail: trudd@liv.ac.uk [Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB (United Kingdom); Istituto di Chimica e Biochimica 'G. Ronzoni', Via G. Colombo 81, Milano 20133 (Italy)

    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.

  20. Synthesis of HgS nanocrystals in the Lysozyme aqueous solution through biomimetic method

    NASA Astrophysics Data System (ADS)

    Zhang, Li; Yang, Guangrui; He, Guoxu; Wang, Li; Liu, Qiaoru; Zhang, Qiuxia; Qin, Dezhi

    2012-08-01

    In the present work, it is reported for Lysozyme-conjugated HgS nanocrystals with tunable sizes prepared at Lysozyme (Lyso) aqueous solutions by using biomimetic method. The obtained HgS nanoparticles with good dispersibility have been characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), high-resolution transmission microscopy (HRTEM) and energy-dispersive X-ray spectrum (EDS). The Lysozyme molecules can control nucleation and growth of HgS crystals by binding on the surface of nanocrystals to stabilize protein-capped nanoparticles. Quantum confinement effect of Lyso-conjugated HgS nanocrystals has been confirmed by UV-vis spectra. The nanoparticles exhibit a well-defined emission feature at about 470 nm. Fourier transform infrared (FT-IR) data are used to envisage the binding of nanoparticles with functional groups of Lysozyme. The results of circular dichroism (CD) spectra indicated that the formation of HgS nanocrystals can lead to conformational change of Lysozyme.

  1. Melting points of lysozyme and ribonuclease A crystals correlated with protein unfolding: a Raman spectroscopic study.

    PubMed

    Jacob, J; Krafft, C; Welfle, K; Welfle, H; Saenger, W

    1998-01-01

    The effects of a temperature increase on monoclinic and tetragonal lysozyme single crystals were investigated by polarizing microscopy, X-ray diffraction and laser Raman spectroscopy. To prevent dissolution, the mother liquor was removed, and the crystals were covered by the oil poly-(chlorotrifluoroethylene). Upon heating, their macroscopic shape was stable beyond 453 K but a change (or loss) of birefringence was observed around 352 and 367 K for the tetragonal and monoclinic crystal forms, respectively, which is associated with tighter packing and higher crystal forces in monoclinic lysozyme. Raman spectral changes in the amide I and amide III regions indicated denaturation of the protein within the crystalline environment at temperature where birefringence changes, and differences in the S-S band suggest that in monoclinic lysozyme, denaturation is accompanied with disruption of some S-S bonds. Comparison with thermal denaturation and gel formation (beta-aggregation) of lysozyme in solution indicates that intermolecular interactions are mainly involved in the stabilization of the denatured lysozyme crystals. The behavior of ribonuclease A is very different. This protein unfolds and refolds reversibly in solution and its crystals melt at the unfolding temperature at 333 K, i.e. loss of structure induces breakdown of crystal lattice and macroscopic shape. Although the crystal lattice of proteins is stabilized by only few intermolecular contacts, its breakdown with increasing temperature is primarily a result of thermal unfolding of the polypeptide chains. PMID:9761818

  2. Expression, purification, crystallization and preliminary X-ray diffraction analysis of a lactococcal bacteriophage small terminase subunit

    PubMed Central

    Ren, Bin; Pham, Tam M.; Surjadi, Regina; Robinson, Christine P.; Le, Thien-Kim; Chandry, P. Scott; Peat, Thomas S.; McKinstry, William J.

    2013-01-01

    Terminases are enzymes that are required for the insertion of a single viral genome into the interior of a viral procapsid by a process referred to as ‘encapsulation or packaging’. Many double-stranded DNA viruses such as bacteriophages T3, T4, T7, ? and SPP1, as well as herpes viruses, utilize terminase enzymes for this purpose. All the terminase enzymes described to date require two subunits, a small subunit referred to as TerS and a large subunit referred to as TerL, for in vivo activity. The TerS and TerL subunits interact with each other to form a functional hetero-oligomeric enzyme complex; however the stoichiometry and oligomeric state have not been determined. We have cloned, expressed and purified recombinant small terminase TerS from a 936 lactococcal bacteriophage strain ASCC454, initially isolated from a dairy factory. The terminase was crystallized using a combination of nanolitre sitting drops and vapour diffusion using sodium malonate as the precipitant, and crystallization optimized using standard vapour-diffusion hanging drops set up in the presence of a nitrogen atmosphere. The crystals belong to the P2 space group, with unit-cell parameters a = 73.93, b = 158.48, c = 74.23?Å, and diffract to 2.42?Å resolution using synchrotron radiation. A self-rotation function calculation revealed that the terminase oligomerizes into an octamer in the asymmetric unit, although size-exclusion chromatography suggests that it is possible for it to form an oligomer of up to 13 subunits. PMID:23519803

  3. Granulated lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology of 10-day-old 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 the efficacy of granulated lysozyme, compared to antibiotics, on growth performance, small intestinal morphology, and Campylobacter shedding in 10-d-old pigs. Forty-eight pigs ...

  4. Volume 108, number 1 FEBS LETTERS December 1979 ON THE BINDING OF N-ACETYLGLUCOSAMINE AND CHITOBIOSE TO HEN LYSOZYME

    E-print Network

    in the affinity of the enzyme for these compounds was noticed: at 40°C (physiological temperature for birds-ACETYLGLUCOSAMINE AND CHITOBIOSE TO HEN LYSOZYME IN THE SOLID STATE AT HIGH TEMPERATURE J. BERTHOU, A. LIFCHITZ, J. SAINT lysozyme (EC 3.2.1.17) a temperature-dependent transition in the solid state: the classical tetragonal

  5. Human lysozyme inhibits the in vitro aggregation of A? peptides, which in vivo are associated with Alzheimer's disease.

    PubMed

    Luo, Jinghui; Wärmländer, Sebastian K T S; Gräslund, Astrid; Abrahams, Jan Pieter

    2013-07-25

    Alzheimer's disease is a neurodegenerative disorder characterized by accumulation of A? peptide aggregates in the brain. Using ThT fluorescence assays, AFM imaging, NMR and CD spectroscopy, and MD modeling we show that lysozyme - a hydrolytic enzyme abundant in human secretions - completely inhibits the aggregation of A? peptides at equimolar lysozyme : A? peptide ratios. PMID:23764522

  6. Granulated lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology of 10-day-old 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 the effect of a purified granulated lysozyme, compared to antibiotics, on growth performance, small intestinal morphology, and Campylobacter shedding in 10-d-old pigs. Forty-...

  7. Molecular dynamics of solid-state lysozyme as affected by glycerol and water: a neutron scattering study.

    PubMed

    Tsai, A M; Neumann, D A; Bell, L N

    2000-11-01

    Glycerol has been shown to lower the heat denaturation temperature (T(m)) of dehydrated lysozyme while elevating the T(m) of hydrated lysozyme (. J. Pharm. Sci. 84:707-712). Here, we report an in situ elastic neutron scattering study of the effect of glycerol and hydration on the internal dynamics of lysozyme powder. Anharmonic motions associated with structural relaxation processes were not detected for dehydrated lysozyme in the temperature range of 40 to 450K. Dehydrated lysozyme was found to have the highest T(m) by. Upon the addition of glycerol or water, anharmonicity was recovered above a dynamic transition temperature (T(d)), which may contribute to the reduction of T(m) values for dehydrated lysozyme in the presence of glycerol. The greatest degree of anharmonicity, as well as the lowest T(d), was observed for lysozyme solvated with water. Hydrated lysozyme was also found to have the lowest T(m) by. In the regime above T(d), larger amounts of glycerol lead to a higher rate of change in anharmonic motions as a function of temperature, rendering the material more heat labile. Below T(d), where harmonic motions dominate, the addition of glycerol resulted in a lower amplitude of motions, correlating with a stabilizing effect of glycerol on the protein. PMID:11053145

  8. Molecular dynamics of solid-state lysozyme as affected by glycerol and water: a neutron scattering study.

    PubMed Central

    Tsai, A M; Neumann, D A; Bell, L N

    2000-01-01

    Glycerol has been shown to lower the heat denaturation temperature (T(m)) of dehydrated lysozyme while elevating the T(m) of hydrated lysozyme (. J. Pharm. Sci. 84:707-712). Here, we report an in situ elastic neutron scattering study of the effect of glycerol and hydration on the internal dynamics of lysozyme powder. Anharmonic motions associated with structural relaxation processes were not detected for dehydrated lysozyme in the temperature range of 40 to 450K. Dehydrated lysozyme was found to have the highest T(m) by. Upon the addition of glycerol or water, anharmonicity was recovered above a dynamic transition temperature (T(d)), which may contribute to the reduction of T(m) values for dehydrated lysozyme in the presence of glycerol. The greatest degree of anharmonicity, as well as the lowest T(d), was observed for lysozyme solvated with water. Hydrated lysozyme was also found to have the lowest T(m) by. In the regime above T(d), larger amounts of glycerol lead to a higher rate of change in anharmonic motions as a function of temperature, rendering the material more heat labile. Below T(d), where harmonic motions dominate, the addition of glycerol resulted in a lower amplitude of motions, correlating with a stabilizing effect of glycerol on the protein. PMID:11053145

  9. Do we need still more trials on T-4 and T-3 combination therapy in hypothyroidism?

    Microsoft Academic Search

    Wilmar M Wiersinga

    2009-01-01

    Approximately 10% of hypothyroid patients are dissatisfied with the outcome of levothyroxine replacement. It is unlikely that slight over- or under-treatment with thyroxine (T-4) explains remaining complaints Meta-analysis of randomized clinical trials shows no advantage of T-4\\/tri-iodothyronine (T-3) combination therapy over T-4 monotherapy However, each of these trials can be criticized, and none is perfect: most of them failed to

  10. Phamerator: a bioinformatic tool for comparative bacteriophage genomics

    PubMed Central

    2011-01-01

    Background Bacteriophage genomes have mosaic architectures and are replete with small open reading frames of unknown function, presenting challenges in their annotation, comparative analysis, and representation. Results We describe here a bioinformatic tool, Phamerator, that assorts protein-coding genes into phamilies of related sequences using pairwise comparisons to generate a database of gene relationships. This database is used to generate genome maps of multiple phages that incorporate nucleotide and amino acid sequence relationships, as well as genes containing conserved domains. Phamerator also generates phamily circle representations of gene phamilies, facilitating analysis of the different evolutionary histories of individual genes that migrate through phage populations by horizontal genetic exchange. Conclusions Phamerator represents a useful tool for comparative genomic analysis and comparative representations of bacteriophage genomes. PMID:21991981

  11. Bacteriophage capsids: Tough nanoshells with complex elastic properties

    PubMed Central

    Ivanovska, I. L.; de Pablo, P. J.; Ibarra, B.; Sgalari, G.; MacKintosh, F. C.; Carrascosa, J. L.; Schmidt, C. F.; Wuite, G. J. L.

    2004-01-01

    The shell of bacteriophages protects the viral DNA during host-to-host transfer and serves as a high-pressure container storing energy for DNA injection into a host bacterium. Here, we probe the mechanical properties of nanometer-sized bacteriophage ?29 shells by applying point forces. We show that empty shells withstand nanonewton forces while being indented up to 30% of their height. The elastic response varies across the surface, reflecting the arrangement of shell proteins. The measured Young's modulus (?1.8 GPa) is comparable with that of hard plastic. We also observe fatigue and breakage of capsids after probing them repetitively. These results illustrate the mechanoprotection that viral shells provide and also suggest design principles for nanotechnology. PMID:15133147

  12. 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

  13. Four lysozymes (one c-type and three g-type) in catfish are drastically but differentially induced after bacterial infection.

    PubMed

    Wang, Ruijia; Feng, Jianbin; Li, Chao; Liu, Shikai; Zhang, Yu; Liu, Zhanjiang

    2013-07-01

    Lysozyme is an important component of the innate immune system. In this study, four lysozyme genes including one c-type lysozyme and three g-type lysozymes were identified from channel catfish (Ictalurus punctatus). The lysozyme genes are highly conserved in their structural features as compared to those from other species. Phylogenetic analyses were conducted allowing annotation of these genes. Additional analyses using conserved syntenies allowed determination of orthologies for the c-type lysozyme. Phylogenetic analysis indicated that the g-type lysozyme may have gone through species-specific gene duplications leading to multiple copies in some teleost species. Channel catfish possessed three copies of the g-type lysozyme genes. Expression analysis revealed that the catfish lysozyme genes were expressed in a broad range of tissues. The highest levels of expression were found in head kidney, liver, spleen, and trunk kidney, compatible with the immune functions of these tissues/organs. The c-type and g-type lysozymes were drastically induced after bacterial infection, but exhibited large differences in the extent of induction and the tissue with the highest level of induction, with the g-type lysozyme being most highly induced in the head kidney whereas the other three lysozymes being most highly induced in the liver, suggesting their cooperative actions in the immune responses but difference in their detailed functions. PMID:23639933

  14. Structure and expression of the human and mouse T4 genes.

    PubMed Central

    Maddon, P J; Molineaux, S M; Maddon, D E; Zimmerman, K A; Godfrey, M; Alt, F W; Chess, L; Axel, R

    1987-01-01

    The T4 molecule may serve as a T-cell receptor recognizing molecules on the surface of specific target cells and also serves as the receptor for the human immunodeficiency virus. To define the mechanisms of interaction of T4 with the surface of antigen-presenting cells as well as with human immunodeficiency virus, we have further analyzed the sequence, structure, and expression of the human and mouse T4 genes. T4 consists of an extracellular segment comprised of a leader sequence followed by four tandem variable-joining (VJ)-like domains, a transmembrane domain, and a cytoplasmic segment. The structural domains of the T4 protein deduced from amino acid sequence are precisely reflected in the intron-exon organization of the gene. Analysis of the expression of the T4 gene indicates that T4 RNA is expressed not only in T lymphocytes, but in B cells, macrophages, and granulocytes. T4 is also expressed in a developmentally regulated manner in specific regions of the brain. It is, therefore, possible that T4 plays a more general role in mediating cell recognition events that are not restricted to the cellular immune response. Images PMID:3501122

  15. Structure and expression of the human and mouse T4 genes

    SciTech Connect

    Maddon, P.J.; Molineaux, S.M.; Maddon, D.F.; Zimmerman, K.A.; Godfrey, M.; Alt, F.W.; Chess, L.; Axel, R.

    1987-12-01

    The T4 molecule may serve as a T-cell receptor recognizing molecules on the surface of specific target cells and also serves as the receptor for the human immunodeficiency virus. To define the mechanisms of interaction of T4 with the surface of antigen-presenting cells as well as with human immunodeficiency virus, the authors have further analyzed the sequence, structure, and expression of the human and mouse T4 genes. T4 consists of an extracellular segment comprised of a leader sequence followed by four tandem variable-joining (VJ)-like domains, a transmembrane domain, and A cytoplasmic segment. The structural domains of the T4 protein deduced from amino acid sequence are precisely reflected in the intron-exon organization of the gene. Analysis of the expression of the T4 gene indicates that T4 RNA is expressed not only in T lymphocytes, but in B cells, macrophages, and granulocytes. T4 is also expressed in a developmentally regulated manner in specific regions of the brain. It is, therefore, possible that T4 plays a more general role in mediating cell recognition events that are not restricted to the cellular immune response.

  16. Isolation and Characterization of Bacteriophages from Fermenting Sauerkraut†

    PubMed Central

    Yoon, S. S.; Barrangou-Poueys, R.; Breidt, F.; Klaenhammer, T. R.; Fleming, H. P.

    2002-01-01

    This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family. PMID:11823247

  17. Isolation and characterization of bacteriophages from fermenting sauerkraut.

    PubMed

    Yoon, S S; Barrangou-Poueys, R; Breidt, F; Klaenhammer, T R; Fleming, H P

    2002-02-01

    This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family. PMID:11823247

  18. The influence of external factors on bacteriophages—review

    Microsoft Academic Search

    E. Jo?czyk; M. K?ak; R. Mi?dzybrodzki; A. Górski

    The ability of bacteriophages to survive under unfavorable conditions is highly diversified. We summarize the influence of\\u000a different external physical and chemical factors, such as temperature, acidity, and ions, on phage persistence. The relationships\\u000a between a phage’s morphology and its survival abilities suggested by some authors are also discussed. A better understanding\\u000a of the complex problem of phage sensitivity to

  19. Kinetic analysis of the weak affinity interaction between tris and lysozyme.

    PubMed

    Kang, Tengfei; Hao, Wenxin; Niu, Yu; Luo, Ziren; Jin, Gang

    2015-02-20

    The biosensor based on total internal reflection imaging ellipsometry (TIRIE), regarded as an automotive real-time research approach for biomolecular interaction, is introduced to analyze the kinetic process of the weak interaction between tris and lysozyme. The experiment is performed by delivering lysozyme solution diluted to different concentrations to the biosensor substrate interface immobilized with tris. By applying pseudo-first-order interaction kinetics model, we are able to obtain the kinetic parameters from fitting experimental data. The calculated association rate constant and dissociation rate constant of tris and lysozyme interaction are in 10(-2) mol(-1) s(-1) and 10(3)s(-1) magnitude, respectively. To further improve TIRIE's ability for kinetically characterizing biomolecular interaction, a theoretical method to deduce associate rate constant before experiment is proposed. PMID:25613863

  20. Expression, Purification, and Characteristic of Tibetan Sheep Breast Lysozyme Using Pichia pastoris Expression System.

    PubMed

    Li, Jianbo; Jiang, Mingfeng; Wang, Yong

    2014-04-01

    A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and 45°C. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification. PMID:25049990

  1. 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.

  2. 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.

  3. 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

  4. Novel Podoviridae Family Bacteriophage Infecting Weissella cibaria Isolated from Kimchi

    PubMed Central

    Holo, Helge; Jeon, Sang-Rok; Nes, Ingolf F.; Yoon, Sung-Sik

    2012-01-01

    The first complete genome sequence of a phage infecting Weissella cibaria (Weissella kimchii) is presented. The bacteriophage ?YS61 was isolated from kimchi, a Korean fermented vegetable dish. Bacteriophages are recognized as a serious problem in industrial fermentations; however, ?YS61 differed from many virulent phages associated with food fermentations since it was difficult to propagate and was very susceptible to resistance development. Sequence analysis revealed that ?YS61 resembles Podoviridae of the subfamily Picovirinae. Within the subfamily Picovirinae, the ?29-like phages have been extensively studied, and their terminal protein-primed DNA replication is well characterized. Our data strongly suggest that ?YS61 also replicates by a protein-primed mechanism. Weissella phage ?YS61 is, however, markedly different from members of the Picovirinae with respect to genome size and morphology. Picovirinae are characterized by small (approximately 20-kb) genomes which contrasts with the 33,594-bp genome of ?YS61. Based on electron microscopy analysis, ?YS61 was classified as a member of the Podoviridae of morphotype C2, similar to the ?29-like phages, but its capsid dimensions are significantly larger than those reported for these phages. The novelty of ?YS61 was also emphasized by the low number of open reading frames (ORFs) showing significant similarity to database sequences. We propose that the bacteriophage ?YS61 should represent a new subfamily within the family Podoviridae. PMID:22885743

  5. Molecular characterization of the genome of Bacillus subtilis (natto) bacteriophage PM1, a phage associated with disruption of food production

    Microsoft Academic Search

    Kenichi Umene; Satoko Oohashi; Fukiko Yamanaka; Atsushi Shiraishi

    2009-01-01

    “Natto”, regarded as a traditional food, is made by fermenting boiled soybeans with Bacillus subtilis (natto), which is a natto-producing strain related to B. subtilis. Natto production is disrupted by bacteriophage infection of B. subtilis (natto); thus, it is necessary to control bacteriophage infection. A bacteriophage of B. subtilis (natto), PM1, was isolated during interrupted natto production in a factory.

  6. 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 2011-07-01 false Escherichia coli O157:H7 specific bacteriophages...Tolerances § 180.1301 Escherichia coli O157:H7 specific bacteriophages...bacteriophages that are specific to Escherichia coli O157:H7,...

  7. Acta Cryst. (2000). D56, 11871190 Badasso et al. Headtail connector of bacteriophage 929 1187 crystallization papers

    E-print Network

    Tao, Yizhi Jane

    2000-01-01

    Acta Cryst. (2000). D56, 1187±1190 Badasso et al. Head±tail connector of bacteriophage 929 1187, crystallization and initial X-ray analysis of the head±tail connector of bacteriophage u29 Mohammed O. Badasso±tail connector of bacteriophage 929, an oligomer of gene product 10 (gp10), was crystallized into various forms

  8. 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 2014-07-01 false Escherichia coli O157:H7 specific bacteriophages...Tolerances § 180.1301 Escherichia coli O157:H7 specific bacteriophages...bacteriophages that are specific to Escherichia coli O157:H7,...

  9. 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 2013-07-01 false Escherichia coli O157:H7 specific bacteriophages...Tolerances § 180.1301 Escherichia coli O157:H7 specific bacteriophages...bacteriophages that are specific to Escherichia coli O157:H7,...

  10. Consumption of lysozyme-rich milk can alter microbial fecal populations.

    PubMed

    Maga, Elizabeth A; Desai, Prerak T; Weimer, Bart C; Dao, Nguyet; Kültz, Dietmar; Murray, James D

    2012-09-01

    Human milk contains antimicrobial factors such as lysozyme and lactoferrin that are thought to contribute to the development of an intestinal microbiota beneficial to host health. However, these factors are lacking in the milk of dairy animals. Here we report the establishment of an animal model to allow the dissection of the role of milk components in gut microbiota modulation and subsequent changes in overall and intestinal health. Using milk from transgenic goats expressing human lysozyme at 68%, the level found in human milk and young pigs as feeding subjects, the fecal microbiota was analyzed over time using 16S rRNA gene sequencing and the G2 Phylochip. The two methods yielded similar results, with the G2 Phylochip giving more comprehensive information by detecting more OTUs. Total community populations remained similar within the feeding groups, and community member diversity was changed significantly upon consumption of lysozyme milk. Levels of Firmicutes (Clostridia) declined whereas those of Bacteroidetes increased over time in response to the consumption of lysozyme-rich milk. The proportions of these major phyla were significantly different (P < 0.05) from the proportions seen with control-fed animals after 14 days of feeding. Within phyla, the abundance of bacteria associated with gut health (Bifidobacteriaceae and Lactobacillaceae) increased and the abundance of those associated with disease (Mycobacteriaceae, Streptococcaceae, Campylobacterales) decreased with consumption of lysozyme milk. This study demonstrated that a single component of the diet with bioactivity changed the gut microbiome composition. Additionally, this model enabled the direct examination of the impact of lysozyme on beneficial microbe enrichment versus detrimental microbe reduction in the gut microbiome community. PMID:22752159

  11. Adsorption of lysozyme on hyaluronic acid functionalized SBA-15 mesoporous silica: a possible bioadhesive depot system.

    PubMed

    Medda, Luca; Casula, Maria F; Monduzzi, Maura; Salis, Andrea

    2014-11-01

    Silica-based ordered mesoporous materials are very attractive matrices to prepare smart depot systems for several kinds of therapeutic agents. This work focuses on the well-known SBA-15 mesoporous silica and lysozyme, an antimicrobial protein. In order to improve the bioadhesion properties of SBA-15 particles, the effect of hyaluronic acid (HA) functionalization on lysozyme adsorption was investigated. SBA-15 samples having high (H-SBA) and low (L-SBA) levels of functionalization were analyzed during the three steps of the preparations: (1) introduction of the -NH2 groups to obtain the SBA-NH2 samples; (2) functionalization with HA to obtain the SBA-HA matrices; (3) adsorption of lysozyme. All silica matrices were characterized through N2-adsorption/desorption isotherms, small-angle X-ray scattering, transmission electron microscopy, thermogravimetric analysis, and Fourier transform infrared spectroscopy. The whole of the experimental data suggests that a high level of functionalization of the silica surface allows for a negligible lysozyme adsorption mainly due to unfavorable electrostatic interactions (H-SBA-NH2) or steric hindrance (H-SBA-HA). A low degree of functionalization of the silica surface brings about a very good performance toward lysozyme adsorption, being 71% (L-SBA-NH2) and 63% (L-SBA-HA) respectively, compared to that observed for original SBA-15. Finally, two different kinetic models--a "pseudo-second order" and a "intraparticle diffusion"--were compared to fit lysozyme adsorption data, the latter being more reliable than the former. PMID:25295387

  12. Disulfide-bond scrambling promotes amorphous aggregates in lysozyme and bovine serum albumin.

    PubMed

    Yang, Mu; Dutta, Colina; Tiwari, Ashutosh

    2015-03-12

    Disulfide bonds are naturally formed in more than 50% of amyloidogenic proteins, but the exact role of disulfide bonds in protein aggregation is still not well-understood. The intracellular reducing agents and/or improper use of antioxidants in extracellular environment can break proteins disulfide bonds, making them unstable and prone to misfolding and aggregation. In this study, we report the effect of disulfide-reducing agent dithiothreitol (DTT) on hen egg white lysozyme (lysozyme) and bovine serum albumin (BSA) aggregation at pH 7.2 and 37 °C. BSA and lysozyme proteins treated with disulfide-reducing agents form very distinct amorphous aggregates as observed by scanning electron microscope. However, proteins with intact disulfide bonds were stable and did not aggregate over time. BSA and lysozyme aggregates show unique but measurable differences in 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, suggesting a loose and flexible aggregate structure for lysozyme but a more compact aggregate structure for BSA. Scrambled disulfide-bonded protein aggregates were observed by nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for both proteins. Similar amorphous aggregates were also generated using a nonthiol-based reducing agent, tris(2-carboxyethyl)phosphine (TCEP), at pH 7.2 and 37 °C. In summary, formation of distinct amorphous aggregates by disulfide-reduced BSA and lysozyme suggests an alternate pathway for protein aggregation that may be relevant to several proteins. PMID:25689578

  13. 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.

  14. Quantifying Main Trends in Lysozyme Nucleation: The Effect of Precipitant Concentration and Impurities

    NASA Technical Reports Server (NTRS)

    Burke, Michael W.; Judge, Russell A.; Pusey, Marc L.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Full factorial experiment design incorporating multi-linear regression analysis of the experimental data allows the main trends and effects to be quickly identified while using only a limited number of experiments. These techniques were used to identify the effect of precipitant concentration and the presence of an impurity, the physiological lysozyme dimer, on the nucleation rate and crystal dimensions of the tetragonal form of chicken egg white lysozyme. Increasing precipitant concentration was found to decrease crystal numbers, the magnitude of this effect also depending on the supersaturation. The presence of the dimer generally increased nucleation. The crystal axial ratio decreased with increasing precipitant concentration independent of impurity.

  15. Rapid separation of lysozyme from chicken egg white by reductants and thermal treatment.

    PubMed

    Chang, H M; Yang, C C; Chang, Y C

    2000-02-01

    Reductants (0.1-2.0% ascorbic acid, cysteine, or cystine and 0.04-1. 0% beta-mercaptoethanol) were added to 5-fold diluted, salted duck egg whites (commercially and laboratory prepared) and fresh egg whites (chicken and duck), and subsequently the mixtures were heated at 70 degrees C for 1-10 min. The maximal recovery and purification fold of lysozyme obtained from fresh chicken egg whites added with 1. 0% ascorbic acid were 78% and 2.4, respectively. Storage tests showed that the obtained lyophilized lysozyme powder after dialysis was stable when refrigerated at 4 degrees C for 3 months. PMID:10691610

  16. Quantifying Main Trends in Lysozyme Nucleation: The Effects of Precipitant Concentration, Supersaturation and Impurities

    NASA Technical Reports Server (NTRS)

    Burke, Michael W.; Leardi, Riccardo; Judge, Russell A.; Pusey, Marc L.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    Full factorial experimental design incorporating multi-linear regression analysis of the experimental data allows quick identification of main trends and effects using a limited number of experiments. In this study these techniques were employed to identify the effect of precipitant concentration, supersaturation, and the presence of an impurity, the physiological lysozyme dimer, on the nucleation rate and crystal dimensions of the tetragonal forin of chicken egg white lysozyme. Decreasing precipitant concentration, increasing supers aturation, and increasing impurity, were found to increase crystal numbers. The crystal axial ratio decreased with increasing precipitant concentration, independent of impurity.

  17. CXCR4 Mediated Chemotaxis Is Regulated by 5T4 Oncofetal Glycoprotein in Mouse Embryonic Cells

    PubMed Central

    Southgate, Thomas D.; McGinn, Owen J.; Castro, Fernanda V.; Rutkowski, Andrzej J.; Al-Muftah, Mariam; Marinov, Georgi; Smethurst, Graeme J.; Shaw, David; Ward, Christopher M.; Miller, Crispin J.; Stern, Peter L.

    2010-01-01

    5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site. PMID:20376365

  18. Thyroxine softgel capsule in patients with gastric-related T4 malabsorption.

    PubMed

    Santaguida, Maria Giulia; Virili, Camilla; Del Duca, Susanna Carlotta; Cellini, Miriam; Gatto, Ilenia; Brusca, Nunzia; De Vito, Corrado; Gargano, Lucilla; Centanni, Marco

    2015-05-01

    The key role of an intact gastric acid secretion for subsequent intestinal T4 absorption is supported by an increased requirement of thyroxine in patients with gastric disorders. A better pH-related dissolution profile has been described in vitro for softgel T4 preparation than for T4 tablets. Our study was aimed at comparing softgel and tablet T4 requirements in patients with gastric disorders. A total of 37 patients with gastric-related T4 malabsorption were enrolled, but only 31 (28F/3M; median age = 50 years; median T4 dose = 2.04 ?g/kg/day) completed the study. All patients were in long-lasting treatment (>2 years) with the same dose of T4 tablets when treatment was switched to a lower dose of softgel T4 capsules (-17 %; p = 0.0002). Assessment of serum FT4 and TSH was carried out at baseline and after 3, 6, 12, and 18 months after the treatment switch. In more than 2/3 of patients (good-responders n = 21), despite the reduced dose of T4, median TSH values were similar at each time point (p = 0.3934) with no change in FT4 levels. In the remaining patients (poor-responders n = 10), TSH levels were significantly higher at each time point than at baseline (p < 0.0001). To note, in five of them intestinal comorbidity was subsequently detected. Comorbidity associated with poor-responders status was the only significant predictor in multivariate analysis (OR = 11.333). Doses of softgel T4 capsules lower than T4 tablet preparation are required to maintain the therapeutic goal in 2/3 of patients with impaired gastric acid secretion. PMID:25595886

  19. Structural studies of the bacteriophage lambda holin and M. tuberculosis secA translocase 

    E-print Network

    Savva, George Christos

    2009-05-15

    Double stranded DNA bacteriophages achieve release of phage progeny by disrupting the cell envelope of the host cell. This is accomplished by two phage-encoded proteins, the holin and the endolysin. In bacteriophage lambda, the S holin is a small...

  20. Complete Genome Sequence of the Salmonella enterica Serovar Paratyphi A Bacteriophage LSPA1 Isolated in China

    PubMed Central

    Mao, Pujia; Hong, Yu; Feng, Mengdie; Xu, Zeyang; Huang, Fen; Jing, Shenrong

    2015-01-01

    The bacteriophage LSPA1 was isolated from hospital sewage (Kunming, China), and lytic activity was demonstrated against the Salmonella enterica serovar Paratyphi A CMCC50973 strain. This bacteriophage has a 41,880-bp double-stranded DNA (dsDNA) genome encoding 58 coding sequences (CDSs) and belongs to the family Siphoviridae. PMID:25593244

  1. 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...

  2. 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...

  3. EVALUATION OF THE INFLUENCE OF BACTERIOPHAGE TITER ON THE TREATMENT OF COLIBACILLOSIS IN BROILER CHICKENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two studies were conducted to determine the efficacy of bacteriophage SPR02 and DAF6 at varying titers to treat colibacillosis in chickens. In Study 1 the treatments consisted of a control, i.m. injection of bacteriophage SPR02 or DAF6, E. coli airsac challenge, and E. coli challenge followed by tr...

  4. 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...

  5. [Generalized transduction of plasmid pKM101 by temperate bacteriophage ZF40 of Erwinia carotovora].

    PubMed

    Panshchina, A I; Tovkach, F I

    2007-01-01

    It was shown that temperate bacteriophage ZF40 of Erwinia carotovora can perform generalized transduction of plasmid pKM 101. The antibiotic-resistance marker transfer is coordinated with the fact of cyclic permutation of the phage genom. The presented results create preconditions for further use of bacteriophage ZF40 as a convenient instrument for genetic study of E. carotovora. PMID:18380179

  6. 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...

  7. Bacteriophage K for reduction of Staphylococcus aureusbiofilm on central venous catheter material

    PubMed Central

    Lungren, Matthew P; Christensen, Diana; Kankotia, Ravi; Falk, Irene; Paxton, Ben E; Kim, Charles Y

    2013-01-01

    The purpose of this project was to determine whether bacteriophage can reduce bacterial colonization and biofilm formation on central venous catheter material. Twenty silicone discs were inoculated for 24 h with broth culture of Methicillin sensitive staphylococcus aureus (0.5 McFarland standard). The inoculate was aspirated and discs placed into two equal groups for 24 h: (1) untreated controls; (2) bacteriophage treatment (staphylococcal bacteriophage K, propagated titer > 108). At the completion of the experiment discs were processed for quantitative culture. Statistical testing was performed using the rank sum test. Mean colony forming units (CFU) were significantly decreased in experimental compared with controls (control 6.3 × 105 CFU, experimental 6.7 × 101, P ? 0.0001). Application of bacteriophage to biofilm infected central venous catheter material significantly reduced bacterial colonization and biofilm presence. Our data suggests that bacteriophage treatment may be a feasible strategy for addressing central venous catheter staph aureus biofilm infections. PMID:24265979

  8. Evidence of a Bacterial Receptor for Lysozyme: Binding of Lysozyme to the Anti-? Factor RsiV Controls Activation of the ECF ? Factor ?V

    PubMed Central

    Sepúlveda, Carolina; Houtman, Jon C.; Forest, Katrina T.; Ellermeier, Craig D.

    2014-01-01

    ? factors endow RNA polymerase with promoter specificity in bacteria. Extra-Cytoplasmic Function (ECF) ? factors represent the largest and most diverse family of ? factors. Most ECF ? factors must be activated in response to an external signal. One mechanism of activation is the stepwise proteolytic destruction of an anti-? factor via Regulated Intramembrane Proteolysis (RIP). In most cases, the site-1 protease required to initiate the RIP process directly senses the signal. Here we report a new mechanism in which the anti-? factor rather than the site-1 protease is the sensor. We provide evidence suggesting that the anti-? factor RsiV is the bacterial receptor for the innate immune defense enzyme, lysozyme. The site-1 cleavage site is similar to the recognition site of signal peptidase and cleavage at this site is required for ?V activation in Bacillus subtilis. We reconstitute site-1 cleavage in vitro and demonstrate that it requires both signal peptidase and lysozyme. We demonstrate that the anti-? factor RsiV directly binds to lysozyme and muramidase activity is not required for ?V activation. We propose a model in which the binding of lysozyme to RsiV activates RsiV for signal peptidase cleavage at site-1, initiating proteolytic destruction of RsiV and activation of ?V. This suggests a novel mechanism in which conformational change in a substrate controls the cleavage susceptibility for signal peptidase. Thus, unlike other ECF ? factors which require regulated intramembrane proteolysis for activation, the sensor for ?V activation is not the site-1 protease but the anti-? factor. PMID:25275625

  9. AtlasT4SS: A curated database for type IV secretion systems

    PubMed Central

    2012-01-01

    Background The type IV secretion system (T4SS) can be classified as a large family of macromolecule transporter systems, divided into three recognized sub-families, according to the well-known functions. The major sub-family is the conjugation system, which allows transfer of genetic material, such as a nucleoprotein, via cell contact among bacteria. Also, the conjugation system can transfer genetic material from bacteria to eukaryotic cells; such is the case with the T-DNA transfer of Agrobacterium tumefaciens to host plant cells. The system of effector protein transport constitutes the second sub-family, and the third one corresponds to the DNA uptake/release system. Genome analyses have revealed numerous T4SS in Bacteria and Archaea. The purpose of this work was to organize, classify, and integrate the T4SS data into a single database, called AtlasT4SS - the first public database devoted exclusively to this prokaryotic secretion system. Description The AtlasT4SS is a manual curated database that describes a large number of proteins related to the type IV secretion system reported so far in Gram-negative and Gram-positive bacteria, as well as in Archaea. The database was created using the RDBMS MySQL and the Catalyst Framework based in the Perl programming language and using the Model-View-Controller (MVC) design pattern for Web. The current version holds a comprehensive collection of 1,617 T4SS proteins from 58 Bacteria (49 Gram-negative and 9 Gram-Positive), one Archaea and 11 plasmids. By applying the bi-directional best hit (BBH) relationship in pairwise genome comparison, it was possible to obtain a core set of 134 clusters of orthologous genes encoding T4SS proteins. Conclusions In our database we present one way of classifying orthologous groups of T4SSs in a hierarchical classification scheme with three levels. The first level comprises four classes that are based on the organization of genetic determinants, shared homologies, and evolutionary relationships: (i) F-T4SS, (ii) P-T4SS, (iii) I-T4SS, and (iv) GI-T4SS. The second level designates a specific well-known protein families otherwise an uncharacterized protein family. Finally, in the third level, each protein of an ortholog cluster is classified according to its involvement in a specific cellular process. AtlasT4SS database is open access and is available at http://www.t4ss.lncc.br. PMID:22876890

  10. 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

  11. 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.

  12. Involvement of PKA, PKC, and Ca2+ in LPS-activated expression of the chicken lysozyme gene.

    PubMed

    Regenhard, P; Goethe, R; Phi-van, L

    2001-04-01

    The lysozyme gene is activated in myelomonocytic HD11 cells in response to LPS. In this study, we described the involvement of LPS-activated signal transduction pathways in activation of the lysozyme gene. Pre-treatment of HD 11 cells with H-89, H-7, TMB-8, or KN-93 resulted in inhibition of the LPS-enhanced lysozyme expression, suggesting that PKA, PKC, and Ca2+-dependent protein kinases participate in the LPS activation. CaMKII seems to be required for the processing of lysozyme transcripts. TPA and calcium ionophore A23187, when separately added to HD11 cells, stimulated the lysozyme expression effectively, and forskolin was ineffective. It is interesting that simultaneous treatment of cells with forskolin and calcium ionophore A23187 resulted in a potentiated increase in lysozyme mRNA expression, indicating a synergistic cooperation of PKA and Ca2+. This synergistic effect of PKA and Ca2+ was observed on the expression of a stably integrated CAT construct, controlled by the lysozyme promoter and the -6.1-kb enhancer containing binding sites for C/EBP and NF-kappaB/Rel. Therefore, we discussed the role of C/EBPbeta(NF-M), CREB, and NF-kappaB/Rel as possible targets for phosphorylation mediated by PKA, PKC, and Ca2+. PMID:11310853

  13. Penetration of lysozyme and cytochrome C in lipid bilayer: fluorescent study.

    PubMed

    Zlatanov, Ivaylo; Popova, Antoaneta

    2011-07-01

    Lysozyme and cytochrome c (CytC) are well-investigated proteins. Their specific interactions with lipid membranes, however, keep surprising secrets. Lysozyme destroys bacterial membrane; CytC binds hydrophobically to alkyl chains of the membrane lipid tails, indicating that both proteins are able to interact directly with the inner membrane components, especially with the fatty acyl chains of membrane lipids. The degrees of integration, depth of localization in the hydrophobic interior of different types of model membranes, and the type of interaction of lysozyme and CytC with surrounding lipids were investigated by fluorescent spectroscopy. Three different fluorescent markers, located at approximately 6.5, 9, and 18 Å into the lipid bilayer, were used. In addition, liposomes were designed as electrically neutral or positively or negatively charged to unravel the importance of the net electrical charge for lipid/protein interaction. CytC penetrates deeper into the lipid bilayer in comparison with lysozyme, and data are discussed in the terms of Stern-Volmer quenching of fluorescence. PMID:21739361

  14. The model Lysozyme–PSSNa system for electrostatic complexation: Similarities and differences with complex coacervation

    Microsoft Academic Search

    F. Cousin; J. Gummel; S. Combet; F. Boué

    2011-01-01

    We review, based on structural information, the mechanisms involved when putting in contact two nano-objects of opposite electrical charge, in the case of one negatively charged polyion, and a compact charged one. The central case is mixtures of PSS, a strong flexible polyanion (the salt of a strong acid, and with high linear charge density), and Lysozyme, a globular protein

  15. Analysis of Core Region from Egg White Lysozyme Forming Amyloid Fibrils

    PubMed Central

    Tokunaga, Yuhei; Sakakibara, Yukako; Kamada, Yoshiki; Watanabe, Kei-ichi; Sugimoto, Yasushi

    2013-01-01

    Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having ?-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme. PMID:23459392

  16. 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.

  17. Analysis of the Native Structure, Stability and Aggregation of Biotinylated Human Lysozyme

    PubMed Central

    Ahn, Minkoo; De Genst, Erwin; Kaminski Schierle, Gabriele S.; Erdelyi, Miklos; Kaminski, Clemens F.; Dobson, Christopher M.; Kumita, Janet R.

    2012-01-01

    Fibril formation by mutational variants of human lysozyme is associated with a fatal form of hereditary non-neuropathic systemic amyloidosis. Defining the mechanistic details of lysozyme aggregation is of crucial importance for understanding the origin and progression of this disease and related misfolding conditions. In this study, we show that a biotin moiety can be introduced site-specifically at Lys33 of human lysozyme. We demonstrate, using biophysical techniques, that the structure and stability of the native-state of the protein are not detectably altered by this modification, and that the ability to form amyloid fibrils is unchanged. By taking advantage of biotin-avidin interactions, we show that super-resolution fluorescence microscopy can generate detailed images of the mature fibrils. This methodology can readily enable the introduction of additional probes into the protein, thereby providing the means through which to understand, in detail, the nature of the aggregation process of lysozyme and its variants under a variety of conditions. PMID:23166837

  18. A near-native state on the slow refolding pathway of hen lysozyme.

    PubMed Central

    Kulkarni, S. K.; Ashcroft, A. E.; Carey, M.; Masselos, D.; Robinson, C. V.; Radford, S. E.

    1999-01-01

    The refolding of four disulfide lysozyme (at pH 5.2, 20 degrees C) involves parallel pathways, which have been proposed to merge at a near-native state. This species contains stable structure in the alpha- and beta-domains but lacks a functional active site. Although previous experiments have demonstrated that the near-native state is populated on the fast refolding pathway, its relevance to slow refolding molecules could not be directly determined from previous experiments. In this paper, we describe experiments that investigate the effect of added salts on the refolding pathway of lysozyme at pH 5.2, 20 degrees C. We show, using stopped flow tryptophan fluorescence, inhibitor binding, and circular dichroism (CD), that the rate of formation of native lysozyme on the slow refolding track is significantly reduced in solutions of high ionic strength in a manner dependent on the position of the anion in the Hofmeister series. By contrast, the rate of evolution of hydrogen exchange (HX) protection monitored by electrospray ionization mass spectrometry (ESI MS) is unchanged under the refolding conditions studied. The data show, therefore, that at high ionic strengths beta-domain stabilization and native state formation on the slow refolding pathway become kinetically decoupled such that the near-native state becomes significantly populated. Thus, by changing the energy landscape with the addition of salts new insights into the relevance of intermediate states in lysozyme refolding are revealed. PMID:10210181

  19. Changes in Lysozyme Flexibility upon Mutation Are Frequent, Large and Long-Ranged

    PubMed Central

    Verma, Deeptak; Jacobs, Donald J.; Livesay, Dennis R.

    2012-01-01

    We investigate changes in human c-type lysozyme flexibility upon mutation via a Distance Constraint Model, which gives a statistical mechanical treatment of network rigidity. Specifically, two dynamical metrics are tracked. Changes in flexibility index quantify differences within backbone flexibility, whereas changes in the cooperativity correlation quantify differences within pairwise mechanical couplings. Regardless of metric, the same general conclusions are drawn. That is, small structural perturbations introduced by single point mutations have a frequent and pronounced affect on lysozyme flexibility that can extend over long distances. Specifically, an appreciable change occurs in backbone flexibility for 48% of the residues, and a change in cooperativity occurs in 42% of residue pairs. The average distance from mutation to a site with a change in flexibility is 17–20 Å. Interestingly, the frequency and scale of the changes within single point mutant structures are generally larger than those observed in the hen egg white lysozyme (HEWL) ortholog, which shares 61% sequence identity with human lysozyme. For example, point mutations often lead to substantial flexibility increases within the ?-subdomain, which is consistent with experimental results indicating that it is the nucleation site for amyloid formation. However, ?-subdomain flexibility within the human and HEWL orthologs is more similar despite the lowered sequence identity. These results suggest compensating mutations in HEWL reestablish desired properties. PMID:22396637

  20. Two Reliable and Inexpensive Lysozyme Assays for Teaching Enzymology and Microbiology

    NSDL National Science Digital Library

    John A. Snyder (Furman University; )

    1981-06-07

    This exercise will allow students to actively learn about enzyme assays by performing two types that have relevance to the students' interests, radial diffusion assay and spectrophotometry, and by using an enzyme, lysozyme, that is easily understandable and is applicable to their studies outside of this lab.

  1. 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.

  2. 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...

  3. Ultrasound assisted morphological control of mesoporous silica with improved lysozyme adsorption.

    PubMed

    Sun, Shumin; Wang, Shen; Wang, Peiyuan; Wu, Qiong; Fang, Shaoming

    2015-03-01

    The morphological control of mesoporous silica without any additives has attracted much attention. Discrete rod-like and string-of-beads like mesoporous SBA-15 can be achieved under ultrasound irradiation without changing the composition of synthesis system. The smaller particles of SBA-15 showed improved lysozyme immobilization capacity and higher adsorption rate over conventional rope-like SBA-15. PMID:25150609

  4. A novel matrix for hydrophobic interaction chromatography and its application in lysozyme adsorption.

    PubMed

    Gedikli, Mehmet; Ceylan, ?eyda; Erzengin, Mahmut; Odaba??, Mehmet

    2014-01-01

    A novel 1-naphthylamine (NA) coupled poly(2-hydroxyethyl methacrylate-co-N-methacryloyl-(L)-histidine methyl ester) [NA-PHEMAH] supermacroporous monolithic hydrophobic cryogel was prepared via covalent coupling of NA to PHEMAH for adsorption of lysozyme from aqueous solution. Firstly, PHEMAH monolithic cryogel was prepared by radical cryocopolymerization of HEMA with MAH as a functional comonomer and N,N'-methylene-bisacrylamide (MBAAm) as a crosslinker directly in a plastic syringe, and then NA molecules were covalently attached to the imidazole rings of MAH groups of the polymeric structure. The prepared, NA-PHEMAH, supermacroporous monolithic hydrophobic cryogel was characterized by scanning electron microscopy (SEM). The effects of initial lysozyme concentration, pH, salt type, temperature and flow rate on the adsorption efficiency of monolithic hydrophobic cryogel were studied in a column system. The maximum amount of lysozyme adsorption from aqueous solution in phosphate buffer was 86.1 mg/g polymer at pH 8.0 with a flow rate of 1 mL/min. It was observed that lysozyme could be repeatedly adsorbed and desorbed with the NA-PHEMAH monolithic hydrophobic cryogel without significant loss of the adsorption capacity. PMID:25298972

  5. Investigation of lysozyme adsorption performance of Cu(2+)-attached PHEMA beads embedded cryogel membranes.

    PubMed

    Cömert, Seyda Ceylan; Odaba??, Mehmet

    2014-01-01

    For the purification of large molecules, cryogels are an alternative stationary phase to particle based media. But, due to the drawbacks of cryogels (i.e., low surface area) and particle sorbents (i.e., pressure drop in fixed beds), in recent years, the use of hybrid cryogels has greatly increased. In this study, a novel hybrid cryogel column was synthesized for the purification of lysozyme from aqueous solutions and hen egg white (HEW). Firstly, poly(2-hydroxyethyl methacrylate) beads (2?m size) were prepared, and after iminodiacetic acid (IDA) immobilization, Cu(2+) ions were attached via the IDA chelating groups. These arranged Cu(2+)-attached PHEMA beads (Cu(2+)-ABs) were located into PHEMA based cryogel in order to prepare Cu(2+)-ABs embedded supermacroporous hybrid cryogel (Cu(2+)-ABsEHC) column. The specific surface area of the hybrid cryogel was found as 95m(2)/g by using BET. The amount of IDA on beads was found as 875?mol IDA/g. It was approached to the optimum adsorption levels at initial lysozyme concentration of 4mg/mL and pH8.0 as 874.9mg/g beads. The purity of lysozyme adsorbed from HEW was studied by SDS-PAGE with a purity of 86.4%. It is demonstrated that this column is a potential separation medium for purification of lysozyme from HEW. PMID:24268226

  6. 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,…

  7. Amino acids and glycine ethyl ester as new crystallization reagents for lysozyme

    PubMed Central

    Ito, Len; Shiraki, Kentaro; Yamaguchi, Hiroshi

    2010-01-01

    Several amino acids and their derivatives are prominent additives in the field of protein chemistry. This study reports the use of charged amino acids and glycine ethyl ester as precipitants in protein crystallization, using hen egg-white lysozyme (HEWL) as a model. A discussion of the crystallization of HEWL using these reagents as precipitating agents is given. PMID:20516616

  8. Lysozyme-mediated biomineralization of titanium-tungsten oxide hybrid nanoparticles with high photocatalytic activity.

    PubMed

    Kim, Jung Kyu; Jang, Ji-ryang; Choi, Noori; Hong, Dahyun; Nam, Chang-Hoon; Yoo, Pil J; Park, Jong Hyeok; Choe, Woo-Seok

    2014-10-21

    Titanium-tungsten oxide composites with greatly enhanced photocatalytic activity were synthesized by lysozyme-mediated biomineralization. It was shown for the first time that simple control of the onset of biomineralization could enable fine tuning of the composition and crystallinity of the composites to determine their photocatalytic performance. PMID:25188309

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

    PubMed

    Oliver, W T; Wells, J E

    2013-07-01

    Lysozyme is a 1,4-?-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, 96 females) were weaned from the sow at 24 d of age, blocked by BW and gender, and then assigned to 1 of 24 pens (4 pigs/pen). Each block was randomly assigned 1 of 3 dietary treatments for 28 d: control (two 14-d phases), control + antibiotics (carbadox/copper sulfate), or control + lysozyme (100 mg/kg diet). Pigs were weighed and blood sampled on d 0, 14, and 28 of treatment. Blood was analyzed for plasma urea nitrogen (PUN) and IgA. At 28 d, pigs were killed, and samples of jejunum and ileum were collected and fixed for intestinal morphology measurements. An additional jejunum sample was taken from the 12 pigs with the median BW per treatment to determine transepithelial electrical resistance (TER). Pigs consuming antibiotics or lysozyme grew at a faster rate than control pigs (0.433 ± 0.009 and 0.421 ± 0.008 vs. 0.398 ± 0.008 kg/d, respectively; P < 0.03), which resulted in heavier ending BW (20.00 ± 0.31, 19.8 ± 0.29, and 18.83 ± 0.32 kg, respectively; P < 0.03). Feed intake was not different (P > 0.48), but G:F was improved in pigs consuming antibiotics or lysozyme (0.756 ± 0.014, 0.750 ± 0.021, and 0.695 ± 0.019 kg/kg; P < 0.05). Immunoglobulin A (P < 0.03) and PUN (P < 0.01) increased during the experiment, regardless of dietary treatment (P > 0.48). Dietary treatment did not affect TER (P > 0.37), but gilts had lower TER compared with barrows (P < 0.04). No differences in villi height or crypt depth were observed in the ileum (P > 0.53). However, jejunum villi height was increased and crypt depth was decreased in pigs consuming antibiotics or lysozyme (P < 0.001), resulting in an increased villi height:crypt depth of 72% (P < 0.001). Thus, we concluded that lysozyme is a suitable alternative to carbadox/copper sulfate diets fed to pigs weaned from the sow at 24 d of age. PMID:23572262

  10. Utilization of atmospheric ammonia by an extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4.

    PubMed

    Yoshida, Nobuyuki; Inaba, Satoshi; Takagi, Hiroshi

    2014-01-01

    Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth and requires CO2 for its growth. In this report, nitrogen sources for the oligotrophic growth of N9T-4 were examined. As is true for most other bacteria, N9T-4 preferred ammonium salt to nitrate as the nitrogen source on an inorganic minimum medium without carbon sources. Interestingly, N9T-4 could also grow on the minimal medium solidified by agarose or silica gel without carbon and nitrogen sources, suggesting that this bacterium is also oligotrophic for nitrogen. We can rule out the possibility of diazotrophic growth of this bacterium, because nitrogenase activity was not detected in the cells and the putative gene encoding nitrogenase was not found in N9T-4 genome. DNA microarray analysis revealed that one of the ammonium transporter genes (amtB) was strongly upregulated 40-50 fold higher under oligotrophic conditions than under heterotrophic conditions. Disruption of amtB led to a growth defect under nitrogen-limiting conditions. Furthermore, additional ammonia vapor enhanced the growth of N9T-4 on the minimum medium without nitrogen sources in a closed culture system. These results suggest that N9T-4 utilizes the trace amount of atmospheric ammonia as the nitrogen source. PMID:23849805

  11. Lysozyme-like activity in eggs and in some tissues of land snails Helix aspersa maxima and Achatina achatina.

    PubMed

    Fio?ka, Marta J; Witkowski, Andrzej

    2004-01-01

    Antibacterial lysozyme-like activity against Micrococcus luteus in eggs and some tissues of snails Helix aspers maxima and Achatina achatina was detected in a turbidimetric standard assay. The bacteriolytic activity in Helix aspersa maxima was higher than in Achatina achatina. After the application of the bioautography technique, several lytic zones of Micrococcus luteus were observed in both studied species. Electrophoresis in denaturing conditions followed by immunodetection of lysozyme using EWL antibodies indicated the presence of several lysozyme forms in the tested snails. PMID:19058565

  12. The Natural Antimicrobial Enzyme Lysozyme is Up-Regulated in Gastrointestinal Inflammatory Conditions

    PubMed Central

    Rubio, Carlos A.

    2014-01-01

    The cells that line the mucosa of the human gastrointestinal tract (GI, that is, oral cavity, oesophagus, stomach, small intestine, large intestine, and rectum) are constantly challenged by adverse micro-environmental factors, such as different pH, enzymes, and bacterial flora. With exception of the oral cavity, these microenvironments also contain remnant cocktails of secreted enzymes and bacteria from upper organs along the tract. The density of the GI bacteria varies, from 103/mL near the gastric outlet, to 1010/mL at the ileocecal valve, to 1011 to 1012/mL in the colon. The total microbial population (ca. 1014) exceeds the total number of cells in the tract. It is, therefore, remarkable that despite the prima facie inauspicious mixture of harmful secretions and bacteria, the normal GI mucosa retains a healthy state of cell renewal. To counteract the hostile microenvironment, the GI epithelia react by speeding cell exfoliation (the GI mucosa has a turnover time of two to three days), by increasing peristalsis, by eliminating bacteria through secretion of plasma cell-immunoglobulins and by increasing production of natural antibacterial compounds, such as defensin-5 and lysozyme. Only recently, lysozyme was found up-regulated in Barrett’s oesophagitis, chronic gastritis, gluten-induced atrophic duodenitis (coeliac disease), collagenous colitis, lymphocytic colitis, and Crohn’s colitis. This up-regulation is a response directed to the special types of bacteria recently detected in these diseases. The aim of lysozyme up-regulation is to protect individual mucosal segments to chronic inflammation. The molecular mechanisms connected to the crosstalk between the intraluminal bacterial flora and the production of lysozyme released by the GI mucosae, are discussed. Bacterial resistance continues to exhaust our supply of commercial antibiotics. The potential use of lysozyme to treat infectious diseases is receiving much attention. PMID:25437608

  13. 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

  14. Plasma membrane proteomics identifies biomarkers associated with MMSET overexpression in T(4;14) multiple myeloma.

    PubMed

    Xie, Zhigang; Gunaratne, Jayantha; Cheong, Lip Lee; Liu, Shaw Cheng; Koh, Tze Loong; Huang, Gaofeng; Blackstock, Walter P; Chng, Wee Joo

    2013-07-01

    Multiple myeloma (MM) is characterized by recurrent chromosomal translocations. MMSET, identified by its fusion to the IgH locus in t(4;14) MM, is universally overexpressed in t(4;14) MM. In order to identify cell surface biomarkers associated with t(4;14) MM for small molecule or antibody based therapies, we knocked down MMSET expression with shRNA and generated a cell line pair from KMS11, a t(4;14) MM cell line. We used quantitative mass spectrometry to identify plasma membrane proteins associated with MMSET overexpression. Using this approach, 50 cell surface proteins were identified as differentially expressed between KMS11 and KMS11/shMMSET. Western blot and flow cytometry analysis indicated SLAMF7 was over-expressed in t(4;14) MM cell lines and down-regulated by MMSET shRNAs. SLAMF7 expression was also confirmed in primary t(4;14) MM samples by flow cytometry analysis. Quantitative RT-PCR and ChIP analysis indicated MMSET might regulate the transcription level of SLAMF7 and be an important functional element for SLAMF7 promoter activity. Furthermore, SLAMF7 shRNA could induce G1 arrest or apoptosis and reduce clonogenetic capacity in t(4;14) MM cells. Overall, these results illustrated SLAMF7 might be a novel cell surface protein associated with t(4;14) MM. It is potential to develop t(4;14) MM targeted therapy by SLAMF7 antibody mediated drug delivery. PMID:23900284

  15. TSH-Based Protocol, Tablet Instability, and Absorption Effects on L-T4 Bioequivalence

    PubMed Central

    DiStefano, Joseph J.

    2009-01-01

    Background FDA Guidance for pharmacokinetic (PK) testing of levothyroxine (L-T4) for interbrand bioequivalence has evolved recently. Concerns remain about efficacy and safety of the current protocol, based on PK analysis following supraphysiological L-T4 dosing in euthyroid volunteers, and recent recalls due to intrabrand manufacturing problems also suggest need for further refinement. We examine these interrelated issues quantitatively, using simulated what-if scenarios testing efficacy of a TSH-based protocol and tablet stability and absorption, to enhance precision of L-T4 bioequivalence methods. Methods We use an updated simulation model of human thyroid hormone regulation quantified and validated from data that span a wide range of normal and abnormal thyroid system function. Bioequivalence: We explored a TSH-based protocol, using normal replacement dosing in simulated thyroidectomized patients, switching brands after 8 weeks of full replacement dosing. We simulated effects of tablet potency differences and intestinal absorption differences on predicted plasma TSH, T4, and triiodothyronine (T3) dynamics. Stability: We simulated effects of potency decay and lot-by-lot differences in realistic scenarios, using actual tablet potency data spanning 2 years, comparing the recently reduced 95–105% FDA-approved potency range with the original 90–110% range. Results A simulated decrease as small as 10–15% in L-T4 or its absorption generated TSH concentrations outside the bioequivalence target range (0.5–2.5?mU/L TSH), whereas T3 and T4 plasma levels were maintained normal. For a 25% reduction, steady-state TSH changed 300% (from 1.5 to 6?mU/L) compared with <25% for both T4 and T3 (both within their reference ranges). Stability: TSH, T4, and T3 remained within normal ranges for most potency decay scenarios, but tablets of the same dose strength and brand were not bioequivalent between lots and between fresh and near-expired tablets. Conclusions A pharmacodynamic TSH-measurement bioequivalence protocol, using normal L-T4 replacement dosing in athyreotic volunteers, is likely to be more sensitive and safer than current FDA Guidance based on T4 PK. The tightened 95–105% allowable potency range for L-T4 tablets is a significant improvement, but otherwise acceptable potency differences (whether due to potency decay or lot-by-lot inconsistencies) may be problematic for some patients, for example, those undergoing high-dose L-T4 therapy for cancer. PMID:19191742

  16. Bacteriophage ? Cro Mutations: Effects on Activity and Intracellular Degradation

    NASA Astrophysics Data System (ADS)

    Pakula, Andrew A.; Young, Vincent B.; Sauer, Robert T.

    1986-12-01

    Following random mutagenesis of the bacteriophage ? cro gene, we have isolated missense mutations that affect approximately half of the 66 residue positions of Cro. About two-thirds of the mutations change residues involved in the maintenance of Cro structure and stability. The corresponding mutant proteins are severely degraded in the cell but often have specific activities near that of wild-type Cro. The remaining mutations affect residues involved in DNA binding. These mutant proteins are present at moderately reduced intracellular levels, but their specific activities are much lower than that of wild type.

  17. The lysis genes of bacteriophage 21: structure and function 

    E-print Network

    Bonovich, Maria Teresa

    1991-01-01

    that carried a 1 S-g+ (figure 42 0. 700 0. 600 0. 500 0. 400 0. 300 0. 200 0. 100 0 30 60 90 120 150 180 210 Figure 14. Growth curve of p21S, pTZ and 1 S-(p2lS). The effect of complementation on lysis in the S~~ clones was tested in a lysis profile... AND METHODS. . . . 17 Strains. Phages. . . . . 17 . . . 17 Plasmids Bacteriophage 21 induction. Construction of the Xhy21 phage Subcloning of the lysis region of phage 21. . M13 cloning and dideoxy sequencing. Phagemid cloning and sequencing. Sequencing...

  18. Bacteriophage-derived vectors for targeted cancer gene therapy.

    PubMed

    Pranjol, Md Zahidul Islam; Hajitou, Amin

    2015-01-01

    Cancer gene therapy expanded and reached its pinnacle in research in the last decade. Both viral and non-viral vectors have entered clinical trials, and significant successes have been achieved. However, a systemic administration of a vector, illustrating safe, efficient, and targeted gene delivery to solid tumors has proven to be a major challenge. In this review, we summarize the current progress and challenges in the targeted gene therapy of cancer. Moreover, we highlight the recent developments of bacteriophage-derived vectors and their contributions in targeting cancer with therapeutic genes following systemic administration. PMID:25606974

  19. Selection for mutations in the PR promoter of bacteriophage lambda.

    PubMed Central

    Brown, S; Ferm, J; Woody, S; Gussin, G

    1990-01-01

    Insertion of DNA containing PR, the early rightward promoter of bacteriophage lambda, is lethal to M13-derived vectors when the promoter directs transcription (using the '+' strand as template) toward the M13 origin of replication (ori). Lethality can be relieved by mutation of PR, repression of the promoter by the lambda cl repressor, or by insertion of a strong transcription terminator between PR and ori. We have used selection for plaque formation in the absence of repressor to isolate 14 different mutations at 8 sites in PR. This method of isolating promoter mutants in vivo is applicable generally to strong promoters whose activity is regulated either positively or negatively. Images PMID:2146590

  20. A novel formulation of L-thyroxine (L-T4) reduces the problem of L-T4 malabsorption by coffee observed with traditional tablet formulations.

    PubMed

    Vita, Roberto; Saraceno, Giovanna; Trimarchi, Francesco; Benvenga, Salvatore

    2013-02-01

    The purpose of this work is to evaluate if the coffee-associated malabsorption of tablet levothyroxine (L-T4) is reduced by soft gel capsule. We recruited 8 patients with coffee-associated L-T4 malabsorption including one hypothyroid patient. For 6 months, the patients were switched to the capsule maintaining the L-T4 daily dose. Patients took the capsule with water, having coffee 1 h later (proper habit, PH) on days 1-90, or with coffee ? 5 min later (improper habit, IH) on days 91-180. After 6 months, 2 patients volunteered for an acute loading test of 600 ?g L-T4 (capsule) ingested with water (PH) or with coffee (IH). In the single hypothyroid patient, the post-switch TSH ranged 0.06-0.16 mU/L (PH) versus 5.8-22.4 mU/L pre-switch (PH) and 0.025-0.29 mU/L (IH) versus 26-34 mU/L pre-switch (IH). In the other 7 patients, post-switch TSH was 0.41 ± 0.46 (PH) versus 0.28 ± 0.20 pre-switch (PH) (P = 0.61) and 0.34 ± 0.30 (IH) versus 1.23 ± 1.47 pre-switch (IH) (P < 0.001). Importantly, TSH levels in PH versus IH habit did not differ post-switch (P = 0.90), but they did pre-switch (P < 0.0001). The proportions of post-switch TSH levels <0.10 mU/L with PH (33.3 %) or with IH (33.3 %) were borderline significantly greater than the corresponding pre-switch levels with PH (10.3 %) (P = 0.088) or with IH (0 %) (P = 0.0096). In the two volunteers, the L-T4 loading test showed that coffee influenced L-T4 pharmacokinetics minimally. Soft gel capsules can be used in patients who are unable/unwilling to change their IH of taking L-T4. PMID:22932947

  1. Kinetics of thyroxine (T(4)) and triiodothyronine (T(3)) transport in the isolated rat heart.

    PubMed

    Rosic, M; Pantovic, S; Lucic, A; Ribarac-Stepic, N; Andjelkovic, I

    2001-01-01

    The dynamics and kinetics of thyroid hormone transport in the isolated rat heart were examined using the modified unidirectional paired tracer dilution method. The uptake of (125)I-thyroxine ((125)I-T(4)) and (125)I-triiodothyronine ((125)I-T(3)) from the extracellular space into heart cells was measured relative to the extracellular space marker (3)H-mannitol. The thyroid hormone maximal uptake was 54.4 % for (125)I-T(4) and 52.15 % for (125)I-T(3). The thyroid hormone net uptake was 25.69 % for (125)I-T(4) and 25.49 % for (125)I-T(3). Backflux from the intracellular space was 53.17 % for (125)I-T(4) and 61.59 % for (125)I-T(3). In the presence of unlabelled thyroid hormones, (125)I-T(4) and (125)I-T(3) maximal uptakes were reduced from 10.1 to 59.74 % and from 34.6 to 65.3 %, respectively, depending on the concentration of the unlabelled hormone, suggesting a saturable mechanism of the thyroid hormone uptake by the heart cells, with K(m(T4))= 105.46 microM and the maximal rate of (125)I-thyroid hormone flux from the extracellular space to heart cells (V(max(T4))) = 177.84 nM min(-1) for (125)I-T(4) uptake, and K(m(T3)) = 80.0 microM and V(max(T3)) = 118.5 nM min(-1) for (125)I-T(3) uptake. Experimental Physiology (2001) 86.1, 13-18. PMID:11429614

  2. Factors that predict early treatment failure for patients with locally advanced (T4) breast cancer

    Microsoft Academic Search

    E Montagna; V Bagnardi; N Rotmensz; J Rodriguez; P Veronesi; A Luini; M Intra; E Scarano; A Cardillo; R Torrisi; G Viale; A Goldhirsch; M Colleoni

    2008-01-01

    Locally advanced breast cancer (LABC) is associated with dire prognosis despite progress in multimodal treatments. We evaluated several clinical and pathological features of patients with either noninflammatory (NIBC, cT4a-c) or inflammatory (IBC, cT4d) breast cancer to identify subset groups of patients with high risk of early treatment failure. Clinical and pathological features of 248 patients with LABC, who were treated

  3. Alteration of bacteriophage attachment capacity by near-UV irradiation

    SciTech Connect

    Hartman, P.S.; Einsenstark, A.

    1982-08-01

    Near-UV (NUV) (300 to 400 nm) and far-UV (FUV) (254 nm) radiations damage bacteriophage by different mechanisms. Host cell reactivation, Weigle reactivation, and multiplicity reactivation were observed upon FUV, but not upon NUV irradiation. Also, the number of his+ recombinants increased with P22 bacteriophage transduction in Salmonella typhimurium after FUV, but not after NUV irradiation. This loss of reactivation and recombination after NUV irradiation was not necessarily due to host incapability to repair phage damage. Instead, the phage genome failed to enter the host cell after NUV irradiation. In the case of NUV-irradiated T7 phage, this was determined by genetic crosses with amber mutants, which demonstrated that either all or none of a T7 genome entered the Escherichia coli cell after NUV treatment. Further studies with radioactively labeled phage indicated that irradiated phage failed to adsorb to host cells. This damage by NUV was compared with the protein-DNA cross-link observed previously, when phage particles were irradiated with NUV in the presence of H/sub 2/O/sub 2/. H/sub 2/O/sub 2/ (in nonlethal concentration) acts synergistically with NUV so that equivalent phage inactivation is achieved by much lower irradiation doses.

  4. Alteration of bacteriophage attachment capacity by near-uv irradiation

    SciTech Connect

    Hartman, P.S. (Univ. of Minnesota, St. Paul); Eisenstark, A.

    1982-08-01

    Near-uv (NUV) (300 to 400 nm) and far-uv (FUV) (254 nm) radiations damage bacteriophage by different mechanisms. Host cell reactivation, Weigle reactivation, and multiplicity reactivation were observed upon FUV, but not upon NUV irradiation. Also, the number of his/sup +/ recombinants increased with P22 bacteriophage transduction in Salmonella typhimurium after FUV, but not after NUV irradiation. This loss of reactivation and recombination after NUV irradiation was not necessarily due to host incapability to repair phage damage. Instead, the phage genome failed to enter the host cell after NUV irradiation. In the case of NUV-irradiated T7 phage, this was determined by genetic crosses with amber mutants, which demonstrated that either ''all'' or ''none'' of a T7 genome entered the Escherichia coli cell after NUV treatment. Further studies with radioactively labeled phage indicated that irradiated phage failed to adsorb to host cells. This damage by NUV was compared with the protein-DNA cross-link observed previously, when phage particles were irradiated with NUV in the presence of H/sub 2/O/sub 2/. H/sub 2/O/sub 2/ (in nonlethal concentration) acts synergistically with NUV so that equivalent phage inactivation is achieved by much lower irradiation doses.

  5. Heavy ion induced double strand breaks in bacteria and bacteriophages

    NASA Astrophysics Data System (ADS)

    Micke, U.; Schäfer, M.; Anton, A.; Horneck, G.; Bücker, H.

    DNA damage induced by heavy ions in bacterial cells and bacteriophages such as Bacillus subtilis, E. coli and Bacteriophage Tl were investigated by analyzing the double strand breaks in the chromosomal DNA. This kind of lesion is considered as one of the main reasons for lethal events. To analyze double strand breaks in long molecules of DNA - up to some Mbp in length - the technique of pulse field agarose gel electrophoresis has been used. This allows the detection of one double strand break per genome. Cell lysis and DNA isolation were performed in small agarose blocks directly. This procedure secured minimum DNA destruction by shearing forces. After running a gel, the DNA was stained with ethidium bromide. The light intensity of ethidium bromide fluorescence for both the outcoming (running) DNA and the remaining intact DNA were measured by scanning. The mean number of double strand breaks was calculated by determining the quotient of these intensities. Strand break induction after heavy ion and X-ray irradiation was compared.

  6. Recombineering: A powerful tool for modification of bacteriophage genomes.

    PubMed

    Marinelli, Laura J; Hatfull, Graham F; Piuri, Mariana

    2012-01-01

    Recombineering, a recently developed technique for efficient genetic manipulation of bacteria, is facilitated by phage-derived recombination proteins and has the advantage of using DNA substrates with short regions of homology. This system was first developed in E. coli but has since been adapted for use in other bacteria. It is now widely used in a number of different systems for a variety of purposes, and the construction of chromosomal gene knockouts, deletions, insertions, point mutations, as well as in vivo cloning, mutagenesis of bacterial artificial chromosomes and phasmids, and the construction of genomic libraries has been reported. However, these methods also can be effectively applied to the genetic modification of bacteriophage genomes, in both their prophage and lytically growing states. The ever-growing collection of fully sequenced bacteriophages raises more questions than they answer, including the unknown functions of vast numbers of genes with no known homologs and of unknown function. Recombineering of phage genomes is central to addressing these questions, enabling the simple construction of mutants, determination of gene essentiality, and elucidation of gene function. In turn, advances in our understanding of phage genomics should present similar recombineering tools for dissecting a multitude of other genetically naïve bacterial systems. PMID:22666652

  7. Bacteriophage cocktail significantly reduces Escherichia coli O157

    PubMed Central

    Carter, Chandi D.; Parks, Adam; Abuladze, Tamar; Li, Manrong; Woolston, Joelle; Magnone, Joshua; Senecal, Andre; Kropinski, Andrew M.; Sulakvelidze, Alexander

    2012-01-01

    Foods contaminated with Escherichia coli O157:H7 cause more than 63,000 foodborne illnesses in the United States every year, resulting in a significant economic impact on medical costs and product liabilities. Efforts to reduce contamination with E. coli O157:H7 have largely focused on washing, application of various antibacterial chemicals, and gamma-irradiation, each of which has practical and environmental drawbacks. A relatively recent, environmentally-friendly approach proposed for eliminating or significantly reducing E. coli O157:H7 contamination of foods is the use of lytic bacteriophages as biocontrol agents. We found that EcoShield™, a commercially available preparation composed of three lytic bacteriophages specific for E. coli O157:H7, significantly (p < 0.05) reduced the levels of the bacterium in experimentally contaminated beef by ? 94% and in lettuce by 87% after a five minute contact time. The reduced levels of bacteria were maintained for at least one week at refrigerated temperatures. However, the one-time application of EcoShield™ did not protect the foods from recontamination with E. coli O157:H7. Our results demonstrate that EcoShield™ is effective in significantly reducing contamination of beef and lettuce with E. coli O157:H7, but does not protect against potential later contamination due to, for example, unsanitary handling of the foods post processing. PMID:23275869

  8. Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg-white lysozyme

    Microsoft Academic Search

    Surajit Bhattacharjya; Padmanabhan Balaram

    1997-01-01

    A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H\\/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The

  9. Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses

    Microsoft Academic Search

    Hui Li; Maria-Giovanna Parisi; Mylène Toubiana; Matteo Cammarata; Philippe Roch

    2008-01-01

    The aim of the present study was to evaluate the expression of the Mytilus galloprovincialis lysozyme gene in different in vivo stress situations, including injection of bacteria Vibrio splendidus LGP32, Vibrio anguillarum or Micrococcus lysodeikticus, as well as heat shock at 30°C and cold stress at 5°C. Injection of V. splendidus LGP32 resulted in: (i) a general down-regulation of lysozyme

  10. Eep Confers Lysozyme Resistance to Enterococcus faecalis via the Activation of the Extracytoplasmic Function Sigma Factor SigV

    PubMed Central

    Varahan, Sriram; Iyer, Vijayalakshmi S.; Moore, William T.

    2013-01-01

    Enterococcus faecalis is a commensal bacterium found in the gastrointestinal tract of most mammals, including humans, and is one of the leading causes of nosocomial infections. One of the hallmarks of E. faecalis pathogenesis is its unusual ability to tolerate high concentrations of lysozyme, which is an important innate immune component of the host. Previous studies have shown that the presence of lysozyme leads to the activation of SigV, an extracytoplasmic function (ECF) sigma factor in E. faecalis, and that the deletion of sigV increases the susceptibility of the bacterium toward lysozyme. Here, we describe the contribution of Eep, a membrane-bound zinc metalloprotease, to the activation of SigV under lysozyme stress by its effects on the stability of the anti-sigma factor RsiV. We demonstrate that the ?eep mutant phenocopies the ?sigV mutant in lysozyme, heat, ethanol, and acid stress susceptibility. We also show, using an immunoblot analysis, that in an eep deletion mutant, the anti-sigma factor RsiV is only partially degraded after lysozyme exposure, suggesting that RsiV is processed by unknown protease(s) prior to the action of Eep. An additional observation is that the deletion of rsiV, which results in constitutive SigV expression, leads to chaining of cells, suggesting that SigV might be involved in regulating cell wall-modifying enzymes important in cell wall turnover. We also demonstrate that, in the absence of eep or sigV, enterococci bind significantly more lysozyme, providing a plausible explanation for the increased sensitivity of these mutants toward lysozyme. PMID:23645601

  11. DNA A-tract bending in three dimensions: Solving the dA4T4 vs. dT4A4 conundrum

    PubMed Central

    Stefl, Richard; Wu, Haihong; Ravindranathan, Sapna; Sklená?, Vladimír; Feigon, Juli

    2004-01-01

    DNA A-tracts have been defined as four or more consecutive A·T base pairs without a TpA step. When inserted in phase with the DNA helical repeat, bending is manifested macroscopically as anomalous migration on polyacrylamide gels, first observed >20 years ago. An unsolved conundrum is why DNA containing in-phase A-tract repeats of A4T4 are bent, whereas T4A4 is straight. We have determined the solution structures of the DNA duplexes formed by d(GCAAAATTTTGC) [A4T4] and d(CGTTTTAAAACG) [T4A4] with \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\pagestyle{empty} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{NH}}_{4}^{+}\\end{equation*}\\end{document} counterions by using NMR spectroscopy, including refinement with residual dipolar couplings. Analysis of the structures shows that the ApT step has a large negative roll, resulting in a local bend toward the minor groove, whereas the TpA step has a positive roll and locally bends toward the major groove. For A4T4, this bend is nearly in phase with bends at the two A-tract junctions, resulting in an overall bend toward the minor groove of the A-tract, whereas for T4A4, the bends oppose each other, resulting in a relatively straight helix. NMR-based structural modeling of d(CAAAATTTTG)15 and d(GTTTTAAAAC)15 reveals that the former forms a left-handed superhelix with a diameter of ?110 Å and pitch of 80 Å, similar to DNA in the nucleosome, whereas the latter has a gentle writhe with a pitch of >250 Å and diameter of ?50 Å. Results of gel electrophoretic mobility studies are consistent with the higher-order structure of the DNA and furthermore depend on the nature of the monovalent cation present in the running buffer. PMID:14739342

  12. Preparation of anionic polyelectrolyte modified magnetic nanoparticles for rapid and efficient separation of lysozyme from egg white.

    PubMed

    Chen, Jia; Lin, Yuexin; Jia, Li

    2015-04-01

    Poly(sodium 4-styrenesulfonate) modified magnetic nanoparticles (PSS-MNPs) were successfully synthesized and characterized by transmission electron microscopy, scanning electron microscopy, zeta potential, vibrating sample magnetometry, and Fourier-transform infrared spectrometry. The PSS-MNPs were found to enable effective separation of lysozyme from egg white. The impacts of solution pH, ionic strength, and contact time on the adsorption process were investigated. The adsorption kinetic data were well fitted using a pseudo-second-order kinetic model and the adsorption equilibrium can be reached in 3 min. The adsorption isotherm data could be well described by the Langmuir equation. The maximum adsorption capacity of PSS-MNPs for lysozyme was calculated to be 476.2 mg g(-1) according to the Langmuir adsorption isotherm. The fast and efficient adsorption of lysozyme by PSS-MNPs was mainly based on electrostatic interactions between them. The adsorbed lysozyme can be eluted using 20mM phosphate buffer (pH 7.0) containing 1.0M NaCl with a recovery of 96%. The extracted lysozyme from egg white demonstrated high purity, retaining about 90.7% of total lysozyme activity. PMID:25728660

  13. A label-free fluorescence detection strategy for lysozyme assay using CuInS? quantum dots.

    PubMed

    Liu, Siyu; Na, Weidan; Pang, Shu; Shi, Fanping; Su, Xingguang

    2014-06-21

    We have developed a simple and efficient fluorescence detection system for lysozyme utilizing I-III-VI type Cu-In-S ternary quantum dots (CuInS2 QDs) as the probe. Water-soluble near-infrared CuInS2 QDs capped with 3-mercaptopropionic acid were directly synthesized by using a hydrothermal method. Poly(dimethyl diallyl ammonium chloride), as a cationic polyelectrolyte, could bind to 3-mercaptopropionic acid-capped CuInS2 QDs via electrostatic interactions that would lead to fluorescence quenching of CuInS2 QDs. And the addition of a lysozyme aptamer could effectively turn on the quenched fluorescence due to the stronger interaction between the cationic polyelectrolyte and negatively charged aptamer. The addition of lysozyme would result in fluorescence quenching of CuInS2 QDs again due to the specific binding between lysozyme and the lysozyme aptamer. Therefore, this label-free fluorescence system could be applied to selectively detect lysozyme. PMID:24763820

  14. Molecular modelling of (A4T4NN)n and (T4A4NN)n: sequence elements responsible for curvature.

    PubMed Central

    Sanghani, S R; Zakrzewska, K; Harvey, S C; Lavery, R

    1996-01-01

    The molecular modelling program JUMNA has been used to investigate the origins of the strikingly different curvature of the two sequences, (A4T4NN)n and (T4A4NN)n. Gel electrophoresis and cyclisation studies have shown that only the former of these two sequences is significantly curved. By developing novel superhelical symmetry constraints we were able to study the energetic and structural aspects of polymeric DNA having a controlled curvature. The results obtained (which do not take into account specific hydration effects) correlate well with the experimental data and offer a molecular level explanation of curvature. Although curvature is found to be initiated by specific dinucleotide junctions, deformations spread to surrounding dinucleotide steps and, moreover, sequence effects beyond the dinucleotide level are observed. PMID:8649979

  15. 76 FR 66187 - Bacteriophage of Clavibacter Michiganensis Subspecies Michiganensis; Exemption From the...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-26

    ...pork sausage, chicken, oysters, cheese, mushrooms...with submission of valid studies from the public literature...Ref. 3). Other studies showed that bacteriophage...Direct electron microscopy study on the morphological...abundant and diverse in oysters (Crassostrea...

  16. M13 Bacteriophage-Based Self-Assembly Structures and Their Functional Capabilities

    PubMed Central

    Moon, Jong-Sik; Kim, Won-Geun; Kim, Chuntae; Park, Geun-Tae; Heo, Jeong; Yoo, So Y; Oh, Jin-Woo

    2015-01-01

    Controlling the assembly of basic structural building blocks in a systematic and orderly fashion is an emerging issue in various areas of science and engineering such as physics, chemistry, material science, biological engineering, and electrical engineering. The self-assembly technique, among many other kinds of ordering techniques, has several unique advantages and the M13 bacteriophage can be utilized as part of this technique. The M13 bacteriophage (Phage) can easily be modified genetically and chemically to demonstrate specific functions. This allows for its use as a template to determine the homogeneous distribution and percolated network structures of inorganic nanostructures under ambient conditions. Inexpensive and environmentally friendly synthesis can be achieved by using the M13 bacteriophage as a novel functional building block. Here, we discuss recent advances in the application of M13 bacteriophage self-assembly structures and the future of this technology.

  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

    ...requirement of a tolerance is established for residues of Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3,...

  18. 40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...requirement of a tolerance is established for residues of Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3,...

  19. Characterization of six Leuconostoc fallax bacteriophages isolated from an industrial sauerkraut fermentation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Six bacteriophages active against Leuconostoc fallax strains were isolated from industrial sauerkraut fermentation brines. These phages were characterized as to host range, morphology, structural proteins, and genome fingerprint. They were exclusively lytic against the species L. fallax and had diff...

  20. Bacterial sensing of bacteriophages in communities: the search for the Rosetta stone.

    PubMed

    Debarbieux, Laurent

    2014-08-01

    Billions of years of evolution have resulted in microbial viruses and their hosts communicating in such a way that neither of these antagonists can dominate the other definitively. Studies of the molecular mechanisms underlying this dialog, initially in bacteriophages, rapidly identified several of the ways in which bacteria resist bacteriophage infections and bacteriophages defeat bacterial defenses. From an ecological perspective, recent data have raised many questions about the dynamic interactions between bacteria and bacteriophages, the densities of which, in complex microbial populations, are only beginning to be investigated. The next challenge will be determining how the dialog between microbial viruses and their hosts modulates complex ecosystems, such as those found in healthy humans or infected patients. PMID:24952283

  1. Lysozyme-promoted association of protein I molecules in the outer membrane of Escherichia coli.

    PubMed Central

    Chopra, I; Howe, G B; Ball, P R

    1977-01-01

    Incubation of whole envelopes prepared from sonically oscillated Escherichia coli K-12 cultures with lysozyme in vitro resulted in the appearance of a protein species with an apparent molecular weight double that of outer membrane protein I. Similar dimers were also detected in purified outer membranes and whole envelopes from lysozyme-induced spheroplasts of E. coli K-12. This was confirmed by two-dimensional electrophoresis in which the dimers were resolved in the second dimension to run as single polypeptides of protein I. Formation of dimers was correlated with peptidoglycan degradation, but the ability of protein I molecules to associate may vary between strains of E. coli, since dimers were found only in outer membranes from E. coli W7. We suggest that extensive degradation of peptidoglycan leads to nonspecific formation of protein I aggregates, but that these aggregates do not occur in vivo. Images PMID:334740

  2. Susceptibility of Rickettsia monacensis and Rickettsia peacockii to Cecropin A, Ceratotoxin A, and lysozyme.

    PubMed

    Baldridge, Gerald D; Kurtti, Timothy J; Munderloh, Ulrike G

    2005-10-01

    Ticks host obligate intracellular bacteria that range from benign symbiotes to virulent human pathogens. The effects on those bacteria of antimicrobial peptides (AMPs) involved in arthropod innate immunity to microbial infections are largely unknown. We evaluated effects of AMPs and a c-type lysozyme on host cell-free suspensions of the tick symbiotes Rickettsia monacensis and Rickettsia peacockii with stain-based infectivity and viability assays. Cecropin A at a concentration of 8 muM: had a lethal effect on both rickettsiae while ceratotoxin A was approximately 20-fold less effective. Toxicity of both AMPs was synergized by lysozyme, an enzyme expressed by ticks. Lactoferrin, a transferrin, had no effect on R. monacensis at up to 110 microM. The rickettsiae were less sensitive to the AMPs than is typical of bacteria that grow extracellularly. Our assays may be useful in the study of AMP activity against other obligate intracellular bacteria. PMID:16132458

  3. Transition from monomeric phase to dynamic cluster phase in lysozyme protein solutions

    NASA Astrophysics Data System (ADS)

    Liu, Yun; Falus, Peter; Porcar, Lionel; Fratini, Emiliano; Chen, Wei-Ren; Faraone, Antonio; Hong, Kunlun; Baglioni, Piero

    2013-03-01

    Intermediate range order (IRO) has been recently observed in lysozyme solution that is caused by a combination of a short-range attraction and long-range repulsion. At very high concentration, there is observed cluster formation in lysozyme solutions that is one type of IRO structures. Here, we investigate the temperature effect on the dynamic cluster formation and identify the transition concentration from a monomeric protein phase to a cluster phase. The normalized short-time self-diffusion coefficient is not affected by changing attraction strength at the concentration of about 10% mass fraction, indicating that the system is still dominated by monomeric protein phase. However, at high concentrations, the average self-diffusion coefficient is sensitive to the change of short-range attraction strength, which is interpreted due to the growth of the size of dynamic clusters in solution. The transition concentration from dominating monomeric phase to dynamic cluster phase is estimated to be around 14 % mass fraction.

  4. The Solubility of Orthorhombic Lysozyme Crystals Obtained at High pH

    SciTech Connect

    Aldabaibeh, Naser; Jones, Matthew J.; Myerson, Allan S.; Ulrich, Joachim; (IIT); (Martin-Luther)

    2009-07-06

    The high pH region of the phase diagram of lysozyme with NaCl as a precipitant was determined. In this region of the phase diagram, lysozyme crystallizes in one of two different orthorhombic modifications, the low and high temperature orthorhombic modifications. The solubility of two modifications was measured at different temperatures, pH values, and NaCl concentrations. Both modifications show a similar dependence on the solution conditions where solubility increases with temperature and decreases with pH and NaCl concentration. The transition temperature between the two modifications was determined from the solubility curves and was shown to increase with pH and NaCl concentration. At pH values close to the isoelectric point (pH 11), the transition temperature becomes independent of NaCl concentration.

  5. Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2,4-pentanediol

    PubMed Central

    Stauber, Mark; Jakoncic, Jean; Berger, Jacob; Karp, Jerome M.; Axelbaum, Ariel; Sastow, Dahniel; Buldyrev, Sergey V.; Hrnjez, Bruce J.; Asherie, Neer

    2015-01-01

    Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization. PMID:25760593

  6. Vitro packaging of bacteriophage T7 DNA requires ATP. [Escherichia Coli

    SciTech Connect

    Masker, W.E.

    1982-07-01

    Removal of nucleoside triphosphates from extracts prepared from bacteriophage T7-infected Escherichia coli results in a stringent requirement for added ATP to form infective phage particles by in vitro packaging of bacteriophage T7 DNA. Optimal packaging efficiency was achieved at a concentration of about 1.25 mM. Other nucleoside triphosphates could be substituted for ATP, but none of the common nucleoside triphosphates was as effective as ATP in promoting in vitro encapsulation.

  7. Isolation and Efficacy Evaluation of Virulent Bacteriophages Specific to Fish Pathogenic Bacterium, Flavobacterium columnare

    Microsoft Academic Search

    Yogendra Prasad; Aparna D. Kumar

    2010-01-01

    Prasad, Y., Aparna, Kumar, D. 2010. Isolation and efficacy evaluation of virulent bacteriophages specific to fish pathogenic bacterium, Flavobacterium columnare. J. Appl. Anim. Res., 38: 169–172.To develop phage therapy in freshwater aquaculture against antibiotic resistant bacterial pathogen, nine lytic bacteriophages (FCP1—FCP9) specific to fifteen isolates of Flavobacterium columnare (FC) were isolated from water and bottom sediments of pond, river and

  8. Electrospray versus Nebulization for Aerosolization and Filter Testing with Bacteriophage Particles

    Microsoft Academic Search

    Robert M. Eninger; Christopher J. Hogan Jr; Pratim Biswas; Atin Adhikari; Tiina Reponen; Sergey A. Grinshpun

    2009-01-01

    Aerosolization of bacteriophage MS2 virions by nebulization and charge-reduced electrospray were compared during testing of three filter media. Sample swatches were taken from a surgical mask, N95 filtering-facepiece respirator (FFR), and N100 FFR for use in repeated, short-duration (15 min) penetration tests with bacteriophage MS2 aerosolized by nebulizer and electrospray. Evaluated were (1) the virus suspension preparation protocol, (2) resulting

  9. Unfolding and aggregation of lysozyme: A thermodynamic and kinetic study by FTIR spectroscopy

    Microsoft Academic Search

    Paola Sassi; Alessandra Giugliarelli; Marco Paolantoni; Assunta Morresi; Giuseppe Onori

    2011-01-01

    The unfolding of hen egg-white lysozyme dissolved both in D2O and CH3CH2OD\\/D2O was studied by Fourier Transform Infrared (FTIR) absorption spectroscopy at different protein concentrations. A detailed description of the local and global rearrangement of the secondary structure upon a temperature increase, in the range 295 to 365K, was obtained through the analysis of the amide I band. Thermodynamic parameters

  10. Conformational changes in the unfolding process of lysozyme in water and ethanol\\/water solutions

    Microsoft Academic Search

    Paola Sassi; Giuseppe Onori; Alessandra Giugliarelli; Marco Paolantoni; Stefania Cinelli; Assunta Morresi

    2011-01-01

    In the present study, the thermal unfolding of lysozyme in D2O and D2O\\/CH3CH2OD solutions is analyzed by means of FTIR spectroscopy. A detailed investigation of the conformational rearrangement of the protein upon heating is achieved by inspection of the amide bands. The effects of partial and total deuteration on amide groups are also investigated for they suggest important information about

  11. Protein Interactions in Solution Characterized by Light and Neutron Scattering: Comparison of Lysozyme and Chymotrypsinogen

    Microsoft Academic Search

    O. D. Velev; E. W. Kaler; A. M. Lenhoff

    1998-01-01

    The effects of pH and electrolyte concentration on protein-protein interactions in lysozyme and chymotrypsinogen solutions were investigated by static light scattering (SLS) and small-angle neutron scattering (SANS). Very good agreement between the values of the virial coefficients measured by SLS and SANS was obtained without use of adjustable parameters. At low electrolyte concentration, the virial coefficients depend strongly on pH

  12. Acute exercise induces gastrointestinal leakage of allergen in lysozyme-sensitized mice

    Microsoft Academic Search

    Hiromi Yano; Yasuko Kato; Tsukasa Matsuda

    2002-01-01

    .   Food-dependent exercise-induced anaphylaxis (FDEIAn) is a leading cause of physical allergies. However, the mechanisms involved\\u000a in the development of FDEIAn are not yet clearly understood. In this study, to investigate the leakage of allergen from the\\u000a gastrointestinal tract into the circulation, lysozyme (LYZ)-sensitized B10.A mice, which have been shown to exhibit an especially\\u000a high concentration of plasma antigen-specific immunoglobulin-E

  13. Effects of dithiothreitol on the amyloid fibrillogenesis of hen egg-white lysozyme

    Microsoft Academic Search

    Steven S.-S. WangKuan-Nan; Kuan-Nan Liu; Bo-Wei Wang

    2010-01-01

    At least 25 human proteins can fold abnormally to form pathological deposits that are associated with several degenerative\\u000a diseases. Despite extensive investigation on amyloid fibrillation, the detailed molecular mechanisms remain rather elusive\\u000a and there are currently no effective cures for treating these amyloid diseases. The present study examined the effects of\\u000a dithiothreitol on the fibrillation of hen egg-white lysozyme (HEWL).

  14. Influence of lysozyme, yeast, azoxystrobin, and myclobutanil on fungal diseases of cucumbers grown hydroponically

    Microsoft Academic Search

    R. Utkhede; C. Bogdanoff

    2003-01-01

    The influence of hen egg white lysozyme (HEWL) on root rot caused by Pythium aphanidermatum, and of HEWL, yeast, azoxystrobin, and myclobutanil on stem canker caused by Botrytis cinerea and gummy stem blight caused by Didymella bryoniae in cucumber grown under near-commercial greenhouse conditions was studied. HEWL treatments applied as a root drench at 5g\\/l every 3 weeks gave a

  15. Growth rate measurements of lysozyme crystals under microgravity conditions by laser interferometry.

    PubMed

    Yoshizaki, Izumi; Tsukamoto, Katsuo; Yamazaki, Tomoya; Murayama, Kenta; Oshi, Kentaro; Fukuyama, Seijiro; Shimaoka, Taro; Suzuki, Yoshihisa; Tachibana, Masaru

    2013-10-01

    The growth rate vs. supersaturation of a lysozyme crystal was successfully measured in situ together with the crystal surface observation and the concentration measurements onboard the International Space Station. A Michelson-type interferometer and a Mach-Zehnder interferometer were, respectively, employed for real-time growth rate measurements and concentration field measurements. The hardware development, sample preparation, operation, and analysis methods are described. PMID:24182119

  16. HPLC analysis of ?-irradiation-induced products of tryptophan in peptides and lysozyme

    Microsoft Academic Search

    Barbara van Wickern; Thomas Simat; Hans Steinhart

    1997-01-01

    The products of ?-irradiation of tripeptides (AWA, LWL, LWM) and lysozyme were determined by HPLC and UV\\/fluorescence detection.\\u000a A fast and simple one-step hydrolysis with pronase E (30?–?60 min, 40°C) was developed to release the radiation products,\\u000a without damage, from the peptide chain. N-Formylkynurenine (NFK), oxindolylalanine (OIA), 4-, 5-, 6- and 7-hydroxytryptophan were the main products of irradiation\\u000a of peptides

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

    Microsoft Academic Search

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

    2011-01-01

    BackgroundThere 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 FindingsWe have produced 17 healthy cloned cattle expressing recombinant human

  18. Importance of the CMAP Correction to the CHARMM22 Protein Force Field: Dynamics of Hen Lysozyme

    Microsoft Academic Search

    Matthias Buck; Sabine Bouguet-Bonnet; Richard W. Pastor; Alexander D. MacKerell Jr.

    2006-01-01

    The recently developed CMAP correction to the CHARMM22 force field (C22) is evaluated from 25ns molecular dynamics simulations on hen lysozyme. Substantial deviations from experimental backbone root mean-square fluctuations and N-H NMR order parameters obtained in the C22 trajectories (especially in the loops) are eliminated by the CMAP correction. Thus, the C22\\/CMAP force field yields improved dynamical and structural properties

  19. Effects of Urea and Trimethylamine N-Oxide (TMAO) on the Interactions of Lysozyme in Solution

    Microsoft Academic Search

    Marc Niebuhr; Michel H. J. Koch

    2005-01-01

    The effect of two physiological cosolutes (urea and trimethylamine-N-oxide) and of KCl on the intermolecular interactions in concentrated lysozyme solutions were studied by synchrotron radiation small angle x-ray scattering. The evolution of the structure factors as a function of cosolute and\\/or salt concentration was modeled using pair potentials following an approach recently described in the literature. It was found that

  20. Three stages of lysozyme thermal stabilization by high and medium charge density anions.

    PubMed

    Bye, Jordan W; Falconer, Robert J

    2014-04-24

    Addition of high and medium charge density anions (phosphate, sulfate, and chloride) to lysozyme in pure water demonstrates three stages for stabilization of the protein structure. The first two stages have a minor impact on lysozyme stability and are probably associated with direct interaction of the ions with charged and partial charges on the protein's surface. There is a clear transition between the second and third stages; in the case of sodium chloride, disodium sulfate and disodium hydrogen phosphate this is at 550, 210, and 120 mM, respectively. Stabilization of lysozyme can be explained by the free energy required to hydrate the protein as it unfolds. At low ion concentrations, the protein's hydration layer is at equilibrium with the bulk water. After the transition, bulk water is depleted and the protein is competing for water with the ions. With competition for water between the protein and the ions at higher salt concentrations, the free energy required to hydrate the interior of the protein rises and it is this that stabilizes the protein structure. PMID:24684707

  1. Pressure-Accelerated Dissociation of Amyloid Fibrils in Wild-Type Hen Lysozyme

    PubMed Central

    Shah, Buddha R.; Maeno, Akihiro; Matsuo, Hiroshi; Tachibana, Hideki; Akasaka, Kazuyuki

    2012-01-01

    The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml?1 fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (kobs = 0.00193 min?1 at 0.1 MPa, 0.0348 min?1 at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ?450 MPa, we determined the activation volume ?V0‡ = ?32.9 ± 1.7 ml mol(monomer)?1 and the activation compressibility ??‡ = ?0.0075 ± 0.0006 ml mol(monomer)?1 bar?1 for the dissociation reaction. The negative ?V0‡ and ??‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril. PMID:22225805

  2. The Effect of Salt on Self-Assembled Actin-Lysozyme Complexes

    PubMed Central

    Guáqueta, Camilo; Sanders, Lori K.; Wong, Gerard C. L.; Luijten, Erik

    2006-01-01

    We present a combined experimental and computational study of the bundling of F-actin filaments induced by lysozyme proteins. Synchrotron small-angle x-ray scattering results show that these bundles consist of close-packed columnar complexes in which the actin is held together by incommensurate, one-dimensional arrays of lysozyme macroions. Molecular dynamics simulations of a coarse-grained model confirm the arrangement of the lysozyme and the stability of this structure. In addition, we find that these complexes remain stable even in the presence of significant concentrations of monovalent salt. The simulations show that this arises from partitioning of the salt between the aqueous and the condensed phases. The osmotic pressure resulting from the excess concentration of the salt in the aqueous phase balances the osmotic pressure increase in the bundle. These results are relevant for a variety of biological and biomedical problems in which electrostatic complexation between anionic polyelectrolytes and cationic globular proteins takes place, such as the pathological self-assembly of endogenous antibiotic polypeptides and inflammatory polymers in cystic fibrosis. PMID:16565060

  3. Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies.

    PubMed

    Knubovets, T; Osterhout, J J; Klibanov, A M

    1999-04-20

    The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.) PMID:10099601

  4. Production of Transgenic-Cloned Pigs Expressing Large Quantities of Recombinant Human Lysozyme in Milk

    PubMed Central

    Shang, Shengzhe; Wu, Fangfang; Wen, Xiao; Li, Zhiyuan; Li, Yan; Hu, Xiaoxiang; Zhao, Yaofeng; Li, Qiuyan; Li, Ning

    2015-01-01

    Human lysozyme is a natural non-specific immune factor in human milk that plays an important role in the defense of breastfed infants against pathogen infection. Although lysozyme is abundant in human milk, there is only trace quantities in pig milk. Here, we successfully generated transgenic cloned pigs with the expression vector pBAC-hLF-hLZ-Neo and their first generation hybrids (F1). The highest concentration of recombinant human lysozyme (rhLZ) with in vitro bioactivity was 2759.6 ± 265.0 mg/L in the milk of F0 sows. Compared with wild-type milk, rhLZ milk inhibited growth of Escherichia coli K88 during the exponential growth phase. Moreover, rhLZ in milk from transgenic sows was directly absorbed by the intestine of piglets with no observable anaphylactic reaction. Our strategy may provide a powerful tool for large-scale production of this important human protein in pigs to improve resistance to pathogen infection. PMID:25955256

  5. Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis.

    PubMed

    Mariño, Laura; Pauwels, Kris; Casasnovas, Rodrigo; Sanchis, Pilar; Vilanova, Bartolomé; Muñoz, Francisco; Donoso, Josefa; Adrover, Miquel

    2015-01-01

    Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors. PMID:26169912

  6. Comparison of two codon optimization strategies enhancing recombinant Sus scrofa lysozyme production in Pichia pastoris.

    PubMed

    Zhu, D; Cai, G; Wu, D; Lu, J

    2015-01-01

    Lysozyme has played an important role in animal feed additive industry, food additive industry and biological engineering. For improving expression efficiency of recombinant lysozyme from Sus scrofa, two genes respectively designed by the most used codon optimization strategies, "one amino acid one codon" and "codon randomization", were synthesized and expressed in Pichia pastoris X—33. At shaking flask level, Sus scrofa lysozyme (SSL) under two conditions had a highest activity of 153.33±10.41 and 538.33±15.18 U/mL after a 5 days induction of 1% methanol, with secreted protein concentration 80.03±1.94 and 239.60±4.16 mg/L, respectively. Compared with the original SSL gene, the expression of optimized SSL gene by the second strategy showed a 2.6 fold higher level, while the first method had no obvious improvement in production. In total secreted protein, the proportions of recombinant SSL encoded by the original gene, first method optimized gene and the second—strategy optimized one were 75.06±0.25%, 74.56±0.14% and 79.00±0.14%, respectively, with the same molecular weight about 18 kDa, optimum acidity pH 6.0 and optimum temperature 35degC. PMID:26025401

  7. Complexation of lysozyme with adsorbed PtBS-b-SCPI block polyelectrolyte micelles on silver surface.

    PubMed

    Papagiannopoulos, Aristeidis; Christoulaki, Anastasia; Spiliopoulos, Nikolaos; Vradis, Alexandros; Toprakcioglu, Chris; Pispas, Stergios

    2015-01-20

    We present a study of the interaction of the positively charged model protein lysozyme with the negatively charged amphiphilic diblock polyelectrolyte micelles of poly(tert-butylstyrene-b-sodium (sulfamate/carboxylate)isoprene) (PtBS-b-SCPI) on the silver/water interface. The adsorption kinetics are monitored by surface plasmon resonance, and the surface morphology is probed by atomic force microscopy. The micellar adsorption is described by stretched-exponential kinetics, and the micellar layer morphology shows that the micelles do not lose their integrity upon adsorption. The complexation of lysozyme with the adsorbed micellar layers depends on the micelles arrangement and density in the underlying layer, and lysozyme follows the local morphology of the underlying roughness. When the micellar adsorbed amount is small, the layers show low capacity in protein complexation and low resistance in loading. When the micellar adsorbed amount is high, the situation is reversed. The adsorbed layers both with or without added protein are found to be irreversibly adsorbed on the Ag surface. PMID:25525817

  8. FTIR, ESI-MS, VT-NMR and SANS study of trehalose thermal stabilization of lysozyme.

    PubMed

    Barreca, D; Laganà, G; Magazù, S; Migliardo, F; Gattuso, G; Bellocco, E

    2014-02-01

    Protein aggregation is often associated with conformational and structural changes of secondary structure elements that may lead to exposure of some specific residues. Data obtained in our experimental work indicate that trehalose (1.0M) effectively prevent thermal inactivation and aggregation of lysozyme. In fact, following heat treatment, lysozyme generates insoluble aggregates which are almost completely absent in the samples incubated in the presence of the disaccharide. The experimental approach consists in studying FTIR spectra of intrinsic chromophores and VT-NMR measurements on lysozyme water mixtures in the presence of trehalose. FTIR measurements suggest that in the presence of 1.0 M of trehalose there is a clear decrease in the loss of ?-helix structure and in the formation of intermolecularly aggregated structures. Electrospray ionization mass spectrometry (ESI-MS) was employed to characterize protein structural transition, highlighting as trehalose remarkably influenced solvent accessibility to the amide peptide backbone upon heat treatment, consequentially decreasing local protein environment changes. Complementary informations are also obtained by UV-vis spectroscopy measurements, Congo Red binding and activity determinations. PMID:24291767

  9. Molecular Dynamics Analysis of Lysozyme Protein in Ethanol-Water Mixed Solvent Environment

    NASA Astrophysics Data System (ADS)

    Ochije, Henry Ikechukwu

    Effect of protein-solvent interaction on the protein structure is widely studied using both experimental and computational techniques. Despite such extensive studies molecular level understanding of proteins and some simple solvents is still not fully understood. This work focuses on detailed molecular dynamics simulations to study of solvent effect on lysozyme protein, using water, alcohol and different concentrations of water-alcohol mixtures as solvents. The lysozyme protein structure in water, alcohol and alcohol-water mixture (0-12% alcohol) was studied using GROMACS molecular dynamics simulation code. Compared to water environment, the lysozome structure showed remarkable changes in solvents with increasing alcohol concentration. In particular, significant changes were observed in the protein secondary structure involving alpha helices. The influence of alcohol on the lysozyme protein was investigated by studying thermodynamic and structural properties. With increasing ethanol concentration we observed a systematic increase in total energy, enthalpy, root mean square deviation (RMSD), and radius of gyration. a polynomial interpolation approach. Using the resulting polynomial equation, we could determine above quantities for any intermediate alcohol percentage. In order to validate this approach, we selected an intermediate ethanol percentage and carried out full MD simulation. The results from MD simulation were in reasonably good agreement with that obtained using polynomial approach. Hence, the polynomial approach based method proposed here eliminates the need for computationally intensive full MD analysis for the concentrations within the range (0-12%) studied in this work.

  10. Crystallization of insulin and lysozyme under reduced convection condition in a large gradient magnetic field

    NASA Astrophysics Data System (ADS)

    Yin, D. C.; Wakayama, N. I.; Fujiwara, M.; Harata, K.; Xue, X. P.; Fu, Z. X.; Zhang, S. W.; Shang, P.; Tanimoto, Y.

    The crystallization of protein from solution is governed by the process of transport phenomenon Any reason affecting the process of solute transport will impose effects on the crystallization process thus further affects the crystal quality Recent advancement in superconducting magnet technology makes it possible to provide a low cost long-time durable low effective gravity environment for the control of convection which is similar to the environment in the space As an ideal means to damp natural convection in a non-conductive solution on the Earth it may find applications in the field of protein crystallization In this presentation the authors investigated the crystallization of orthorhombic lysozyme crystals tetragonal lysozyme crystals and insulin crystals in a large gradient magnetic field Three effective gravity levels were used milli-gravity around 0G normal gravity 1G and hypergravity 1 8G Comparisons of the crystal quality obtained inside and outside the magnetic field showed that both the magnetic field and the effective gravity could affect the crystal quality But the effect also depends on the crystal and protein type For lysozyme crystals in tetragonal form the magnetic field and effective gravity showed no obvious effect on the quality whereas for the crystals in orthorhombic form both the magnetic field and effective gravity improved the crystal quality For insulin crystal which is highly symmetrical magnetic field and effective gravity showed no strong effect on the crystal quality It is well known that

  11. The effect of demulsifiers on lysozyme extraction from hen egg white using reverse micelles.

    PubMed

    Jarudilokkul, S; Paulsen, E; Stuckey, D C

    2000-01-01

    The liquid-liquid extraction of protein from buffered aqueous phases using reverse micelles (RM) has been extensively researched from a fundamental point of view. However, very little effort has been expended at scaling up this process for the extraction of real fermentation broth. When real broths are used with reverse micellar phases there are major problems with emulsion formation. In this study the effect of a variety of demulsifiers on lysozyme extraction was evaluated in terms of their influence on the separating properties of the emulsion, water content (Wo), and, extraction yield and kinetics from both buffer and hen egg white. In addition, the use of a low shear contactor (a Graesser or 'raining bucket') was assessed in terms of its suitability as a RM contactor. It was found that most of the demulsifiers reduced the settling time of the emulsion, and enhanced the yield and kinetics of lysozyme extraction from hen egg white. It was hypothesised that this was due to the demulsifier displacing the lysozyme from the interface and preventing the protein unfolding and precipitating. This effect was found to depend on both the generic type of demulsifier, and its concentration. PMID:10892541

  12. Anisotropy of the Coulomb Interaction between Folded Proteins: Consequences for Mesoscopic Aggregation of Lysozyme

    PubMed Central

    Chan, Ho Yin; Lankevich, Vladimir; Vekilov, Peter G.; Lubchenko, Vassiliy

    2012-01-01

    Toward quantitative description of protein aggregation, we develop a computationally efficient method to evaluate the potential of mean force between two folded protein molecules that allows for complete sampling of their mutual orientation. Our model is valid at moderate ionic strengths and accounts for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. We apply the model to the protein lysozyme, whose solutions exhibit both mesoscopic clusters of protein-rich liquid and liquid-liquid separation; the former requires that protein form complexes with typical lifetimes of approximately milliseconds. We find the electrostatic repulsion is typically lower than the prediction of the Derjaguin-Landau-Verwey-Overbeek theory. The Coulomb interaction in the lowest-energy docking configuration is nonrepulsive, despite the high positive charge on the molecules. Typical docking configurations barely involve protonation or deprotonation of surface residues. The obtained potential of mean force between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation. PMID:22768950

  13. Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis

    PubMed Central

    Mariño, Laura; Pauwels, Kris; Casasnovas, Rodrigo; Sanchis, Pilar; Vilanova, Bartolomé; Muñoz, Francisco; Donoso, Josefa; Adrover, Miquel

    2015-01-01

    Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors. PMID:26169912

  14. Crystal Growth of Hen Egg-White Lysozyme (HEWL) under Various Gravity Conditions

    NASA Astrophysics Data System (ADS)

    Pan, Weichun; Xu, Jin; Tsukamoto, Katsuo; Koizumi, Masako; Yamazaki, Tomoya; Zhou, Ru; Li, Ang; Fu, Yuying

    2013-08-01

    Motivated by the enhancement of protein quality under microgravity condition, the behaviors of crystal growth under various gravity conditions have been monitored via Foton Satellite and parabolic flight. We found that the normal growth rate and the step velocity would be enhanced only at high protein concentration. Although the difference of diffusion between monomer lysozyme molecule and main impurity species in HWEL dimer may be able to explain this enhancement in long period at high protein concentration, it is not valid at low lysozyme concentration and it can't explain the results obtained by parabolic flight, in which microgravity condition maintained only about 20 s. In order to compromise this contradiction, cluster, universal existing in protein solution, has been picked up. The dynamic light scattering technique figured out dimer is served as the seed for cluster formation. Due to its large size, cluster keeps still under microgravity. Via this mechanism, the purification of lysozyme above crystal surface has been achieved. We also found the two supergravity (˜1.5 g) periods immediately before and after microgravity period have different effects on the step velocity. The pre-MG period depresses the step velocity while the post-MG enhances it. This odd phenomenon ascribes to two factors: (1) the flow rate modification and (2) the purity of protein solution immediate above crystal surface.

  15. PHEMA based composite cryogels with loaded hydrophobic beads for lysozyme purification.

    PubMed

    Türkmen, Deniz; Denizli, Adil

    2014-11-01

    The purpose of this study is to synthesize megaporous cryogel loaded with hydrophobic affinity beads which can be utilized for the purification of lysozyme from chicken egg-white. N-methacryloyl-(L)-tryptophan methylester (MATrp) was used as the hydrophobic ligand. In the first step, poly(glycidyl methacrylate-N-methacryloyl-(L)-tryptophan methyl-ester) [PGMATrp] beads (2.2 ?m in diameter) were produced by dispersion polymerization. In the second step, the poly(2-hydroxyethyl methacrylate) (PHEMA) cryogel loaded with PGMATrp beads [PHEMA/PGMATrp composite cryogel] was polymerized initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) at -12°C. Lysozyme adsorption capacity of the PHEMA/PGMATrp composite cryogel (332.7 mg/g polymer) was improved significantly due to the loading of PGMATrp beads into the cryogel structure. The hydrophobic MATrp comonomer played a vital role in this binding mechanism. The PHEMA/PGMATrp composite cryogel could be used many times without decreasing the lysozyme adsorption amount significantly. The main advantage of the PHEMA/PGMATrp composite cryogel is the high adsorption capacity. PMID:25454758

  16. Dissection of the locus control function located on the chicken lysozyme gene domain in transgenic mice.

    PubMed Central

    Bonifer, C; Yannoutsos, N; Krüger, G; Grosveld, F; Sippel, A E

    1994-01-01

    The entire chicken lysozyme gene locus including all known cis-regulatory sequences and the 5' and 3' matrix attachment sites defining the borders of the DNase I sensitive chromatin domain, is expressed at a high level and independent of its chromosomal position in macrophages of transgenic mice. It was concluded that the lysozyme gene locus carries a locus control function. We analysed several cis-regulatory deletion mutants to investigate their influence on tissue specificity and level of expression. Position independent expression of the gene is lost whenever one of the upstream tissue specific enhancer regions is deleted, although tissue specific expression is usually retained. Deletion of the domain border fragments has no influence on copy number dependency of expression. However, without these regions an increased incidence of ectopic expression is observed. This suggests that the domain border fragments may help to suppress transgene expression in inappropriate tissues. We conclude, that position independent expression of the lysozyme gene is not controlled by a single specific region of the locus but is the result of the concerted action of several tissue specific upstream regulatory DNA elements with the promoter. Images PMID:7937146

  17. Rodlike Complexes of a Polyelectrolyte (Hyaluronan) and a Protein (Lysozyme) observed by SANS

    E-print Network

    Boué, François; Cousin, Fabrice; Grillo, Isabelle; Morfin, Isabelle; 10.1021/bm100861g

    2012-01-01

    We study by Small Angle Neutron Scattering (SANS) the structure of Hyaluronan -Lysozyme complexes. Hyaluronan (HA) is a polysaccharide of 9 nm intrinsic persistence length that bears one negative charge per disaccharide monomer (Mmol = 401.3 g/mol); two molecular weights, Mw = 6000 and 500 000 Da were used. The pH was adjusted at 4.7 and 7.4 so that lysozyme has a global charge of +10 and + 8 respectively. The lysozyme concentration was varied from 3 to 40 g/L, at constant HA concentration (10 g/L). At low protein concentration, samples are monophasic and SANS experiments reveal only fluctuations of concentration although, at high protein concentration, clusters are observed by SANS in the dense phase of the diphasic samples. In between, close to the onset of the phase separation, a distinct original scattering is observed. It is characteristic of a rod-like shape, which could characterize "single" complexes involving one or a few polymer chains. For the large molecular weight (500 000) the rodlike rigid doma...

  18. Characterization of a T7Like Lytic Bacteriophage of Klebsiella pneumoniae B5055: A Potential Therapeutic Agent

    Microsoft Academic Search

    Vivek Verma; Kusum Harjai; Sanjay Chhibber

    2009-01-01

    Characterization of bacteriophages to be used prophylactically or therapeutically is mandatory, as use of uncharacterized\\u000a bacteriophages is considered as one of the major reasons of failure of phage therapy in preantibiotic era. In the present\\u000a study, one lytic bacteriophage, KPO1K2, specific for Klebsiella pneumoniae B5055, with broad host range was selected for characterization. As shown by TEM, morphologically KPO1K2 possessed

  19. Effects of 2'-O-methyl nucleotide on ligation capability of T4 DNA ligase.

    PubMed

    Zhao, Bin; Tong, Zhaoxue; Zhao, Guojie; Mu, Runqing; Shang, Hong; Guan, Yifu

    2014-09-01

    To further understand the ligation mechanism, effects of 2'-O-methyl nucleotide (2'-OMeN) on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5'- and 3'-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ± 4.0%, respectively. Substitutions at both 5'- and 3'-ends decreased the ligation efficiency by 76.6% ± 1.3%. Corresponding kinetic parameters, Vmax, Km, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2'-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5'- and 3'-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3'-end impairs the ligation efficiency more profoundly than that at 5'-end. Different concentrations of Mg(2+), Ca(2+), K(+), Na(+), and ATP were also demonstrated to affect the T4 DNA ligase activity. These results enriched our knowledge about the effects of 2'-OMeN substitutions on the T4 DNA ligase. PMID:25022752

  20. Genotypic and Phenotypic Diversity among Induced, stx2-Carrying Bacteriophages from Environmental Escherichia coli Strains?

    PubMed Central

    García-Aljaro, Cristina; Muniesa, Maite; Jofre, Juan; Blanch, Anicet R.

    2009-01-01

    Shiga toxin 2 (stx2) gene-carrying bacteriophages have been shown to convert Escherichia coli strains to Shiga toxin-producing Escherichia coli (STEC). In this study, 79 E. coli strains belonging to 35 serotypes isolated from wastewaters of both human and animal origin were examined for the presence of stx2-carrying bacteriophages in their genomes. The lytic cycle of the bacteriophages was induced by mitomycin, and the bacteriophage fraction was isolated and used for morphological and genetic characterization. The induced bacteriophages showed morphological diversity, as well as restriction fragment length polymorphism variation, in the different strains belonging to different serotypes. The ability to infect new hosts was highly variable, although most of the induced phages infected Shigella sonnei host strain 866. In summary, in spite of carrying either the same or different stx2 variants and in spite of the fact that they were isolated from strains belonging to the same or different serotypes, the induced bacteriophages were highly variable. The high level of diversity and the great infectious capacity of these phages could enhance the spread of the stx2 gene and variants of this gene among different bacterial populations in environments to which humans may be exposed. PMID:19011056

  1. Antibacterial activity of hen egg white lysozyme modified by heat and enzymatic treatments against oenological lactic acid bacteria and acetic acid bacteria.

    PubMed

    Carrillo, W; García-Ruiz, A; Recio, I; Moreno-Arribas, M V

    2014-10-01

    The antimicrobial activity of heat-denatured and hydrolyzed hen egg white lysozyme against oenological lactic acid and acetic acid bacteria was investigated. The lysozyme was denatured by heating, and native and heat-denatured lysozymes were hydrolyzed by pepsin. The lytic activity against Micrococcus lysodeikticus of heat-denatured lysozyme decreased with the temperature of the heat treatment, whereas the hydrolyzed lysozyme had no enzymatic activity. Heat-denatured and hydrolyzed lysozyme preparations showed antimicrobial activity against acetic acid bacteria. Lysozyme heated at 90°C exerted potent activity against Acetobacter aceti CIAL-106 and Gluconobacter oxydans CIAL-107 with concentrations required to obtain 50% inhibition of growth (IC50) of 0.089 and 0.013 mg/ml, respectively. This preparation also demonstrated activity against Lactobacillus casei CIAL-52 and Oenococcus oeni CIAL-91 (IC50, 1.37 and 0.45 mg/ml, respectively). The two hydrolysates from native and heat-denatured lysozyme were active against O. oeni CIAL-96 (IC50, 2.77 and 0.3 mg/ml, respectively). The results obtained suggest that thermal and enzymatic treatments increase the antibacterial spectrum of hen egg white lysozyme in relation to oenological microorganisms. PMID:25285490

  2. The genome and proteome of a Campylobacter coli bacteriophage vB_CcoM-IBB_35 reveal unusual features

    E-print Network

    Carvalho, Carla M.

    Background Campylobacter is the leading cause of foodborne diseases worldwide. Bacteriophages (phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and thus ...

  3. Differing Populations of Endemic Bacteriophages in Cattle Shedding High and Low Numbers of Escherichia coli O157:H7 Bacteria in Feces

    PubMed Central

    Hallewell, J.; Niu, Y. D.; Munns, K.; McAllister, T. A.; Johnson, R. P.; Ackermann, H.-W.; Thomas, J. E.

    2014-01-01

    The objectives of this study were to identify endemic bacteriophages (phages) in the feedlot environment and determine relationships of these phages to Escherichia coli O157:H7 from cattle shedding high and low numbers of naturally occurring E. coli O157:H7. Angus crossbred steers were purchased from a southern Alberta (Canada) feedlot where cattle excreting ?104 CFU · g?1 of E. coli O157:H7 in feces at a single time point were identified as supershedders (SS; n = 6), and cattle excreting <104 CFU · g?1 of feces were identified as low shedders (LS; n = 5). Fecal pats or fecal grabs were collected daily from individual cattle for 5 weeks. E. coli O157:H7 in feces was detected by immunomagnetic separation and enumerated by direct plating, and phages were isolated using short- and overnight-enrichment methods. The total prevalence of E. coli O157:H7 isolated from feces was 14.4% and did not differ between LS and SS (P = 0.972). The total prevalence of phages was higher in the LS group (20.9%) than in the SS group (8.3%; P = 0.01). Based on genome size estimated by pulsed-field gel electrophoresis and morphology determined by transmission electron microscopy, T4- and O1-like phages of Myoviridae and T1-like phage of Siphoviridae were isolated. Compared to T1- and O1-like phages, T4-like phages exhibited a broad host range and strong lytic capability when targeting E. coli O157:H7. Moreover, the T4-like phages were more frequently isolated from feces of LS than SS, suggesting that endemic phages may impact the shedding dynamics of E. coli O157:H7 in cattle. PMID:24747892

  4. Genetic map of the Staphylococcal bacteriophage phi11.

    PubMed Central

    Kretschmer, P J; Egan, J B

    1975-01-01

    Ten sus mutants of the staphylococcal bacteriophage phi 11, each a representative from a different complementation group, have been used in three-factor cross experiments. The results of these crosses indicate a circular genetic map for phi 11. Functional studies of the mutants have been limited to electron microscopic examinations of lysates after prophage induction (or infection). One gene is an early gene, five genes are concerned with tail formation, and three are concerned with head formation. The tenth gene is possibly a head gene. The contribution by phi 11 to the genomic content of the plasmid-phage hybrid phi 11 de has been investigated. Phi 11 de contains most of the late genes and appears to be missing a continuous phi 11 segment that includes the early gene flanked by two late genes. Images PMID:125802

  5. An electron microscopic study of bacteriophages from marine waters

    NASA Astrophysics Data System (ADS)

    Frank, Hermann; Moebus, Karlheinz

    1987-12-01

    The morphology of 75 bacteriophage strains isolated from water samples collected in the North Sea or in the northern Atlantic was studied by electron microscopy. Only tailed phages were observed (bradley groups A, B, and C). According to structural similarities, the strains are ascribed to 12 groups, 5 of which comprise types of marine phages not reported before. Four of these 5 groups include phage types that have not been detected from any other source. Among the phages isolated from northern Atlantic water a high incidence was observed of strains the particles of which have long appendages. Certain types of the northern Atlantic phages investigated were derived only from samples collected either east or west of the Azores. This finding agrees with former observations pointing to the existence of different populations of closely related bacteria east and west, respectively, of the northern Mid-Atlantic Ridge.

  6. Bacteriophage-mediated spread of bacterial virulence genes.

    PubMed

    Penadés, José R; Chen, John; Quiles-Puchalt, Nuria; Carpena, Nuria; Novick, Richard P

    2015-02-01

    Bacteriophages are types of viruses that infect bacteria. They are the most abundant and diverse entities in the biosphere, and influence the evolution of most bacterial species by promoting gene transfer, sometimes in unexpected ways. Although pac-type phages can randomly package and transfer bacterial DNA by a process called generalized transduction, some mobile genetic elements have developed elegant and sophisticated strategies to hijack the phage DNA-packaging machinery for their own transfer. Moreover, phage-like particles (gene transfer agents) have also evolved, that can package random pieces of the producing cell's genome. The purpose of this review is to give an overview of some of the various ways by which phages and phage-like particles can transfer bacterial genes, driving bacterial evolution and promoting the emergence of novel pathogens. PMID:25528295

  7. Complete Genomic Sequence of Bacteriophage Felix O1†

    PubMed Central

    Whichard, Jean M.; Weigt, Lee A.; Borris, Douglas J.; Li, Ling Ling; Zhang, Qing; Kapur, Vivek; Pierson, F. William; Lingohr, Erika J.; She, Yi-Min; Kropinski, Andrew M.; Sriranganathan, Nammalwar

    2010-01-01

    Bacteriophage O1 is a Myoviridae A1 group member used historically for identifying Salmonella. Sequencing revealed a single, linear, 86,155-base-pair genome with 39% average G+C content, 131 open reading frames, and 22 tRNAs. Closest protein homologs occur in Erwinia amylovora phage ?Ea21-4 and Escherichia coli phage wV8. Proteomic analysis indentified structural proteins: Gp23, Gp36 (major tail protein), Gp49, Gp53, Gp54, Gp55, Gp57, Gp58 (major capsid protein), Gp59, Gp63, Gp64, Gp67, Gp68, Gp69, Gp73, Gp74 and Gp77 (tail fiber). Based on phage-host codon differences, 7 tRNAs could affect translation rate during infection. Introns, holin-lysin cassettes, bacterial toxin homologs and host RNA polymerase-modifying genes were absent. PMID:21994654

  8. Capstan friction model for DNA ejection from bacteriophages

    PubMed Central

    Ghosal, Sandip

    2013-01-01

    Bacteriophages infect cells by attaching to the outer membrane and injecting their DNA into the cell. The phage DNA is then transcribed by the cell’s transcription machinery. A number of physical mechanisms by which DNA can be translocated from the phage capsid into the cell have been identified. A fast ejection driven by the elastic and electrostatic potential energy of the compacted DNA within the viral capsid appears to be used by most phages, at least to initiate infection. In recent in vitro experiments, the speed of DNA translocation from a ? phage capsid has been measured as a function of ejected length over the entire duration of the event. Here a mechanical model is proposed that is able to explain the observed dependence of exit velocity on ejected length, and that is also consistent with the accepted picture of the geometric arrangement of DNA within the viral capsid. PMID:23368388

  9. Capstan friction model for DNA ejection from bacteriophages.

    PubMed

    Ghosal, Sandip

    2012-12-14

    Bacteriophages infect cells by attaching to the outer membrane and injecting their DNA into the cell. The phage DNA is then transcribed by the cell's transcription machinery. A number of physical mechanisms by which DNA can be translocated from the phage capsid into the cell have been identified. A fast ejection driven by the elastic and electrostatic potential energy of the compacted DNA within the viral capsid appears to be used by most phages, at least to initiate infection. In recent in vitro experiments, the speed of DNA translocation from a ? phage capsid has been measured as a function of ejected length over the entire duration of the event. Here, a mechanical model is proposed that is able to explain the observed dependence of exit velocity on ejected length, and that is also consistent with the accepted picture of the geometric arrangement of DNA within the viral capsid. PMID:23368388

  10. Capstan Friction Model for DNA Ejection from Bacteriophages

    NASA Astrophysics Data System (ADS)

    Ghosal, Sandip

    2012-12-01

    Bacteriophages infect cells by attaching to the outer membrane and injecting their DNA into the cell. The phage DNA is then transcribed by the cell’s transcription machinery. A number of physical mechanisms by which DNA can be translocated from the phage capsid into the cell have been identified. A fast ejection driven by the elastic and electrostatic potential energy of the compacted DNA within the viral capsid appears to be used by most phages, at least to initiate infection. In recent in vitro experiments, the speed of DNA translocation from a ? phage capsid has been measured as a function of ejected length over the entire duration of the event. Here, a mechanical model is proposed that is able to explain the observed dependence of exit velocity on ejected length, and that is also consistent with the accepted picture of the geometric arrangement of DNA within the viral capsid.

  11. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning.

    PubMed

    Blattner, F R; Williams, B G; Blechl, A E; Denniston-Thompson, K; Faber, H E; Furlong, L; Grunwald, D J; Kiefer, D O; Moore, D D; Schumm, J W; Sheldon, E L; Smithies, O

    1977-04-01

    The Charon lambda bacteriophages have been developed as vectors for cloning. Their construction incorporates mutations that make them simple to use and also greatly increases their safety for the biological containment of cloned recombinant DNA. Three of the Charon vector phages, 3A, 4A, and 16A, have been certified for use as EK2 vector-host systems, when propagated in bulk in a special bacterial host, DP50SupF. We present here some of the data on which the safety of these systems was evaluated. DNA fragments ranging in size from 0 to 2.2 X 10(4) base pairs can be cloned in these EK2 Charon phages. PMID:847462

  12. RNA secondary structures of the bacteriophage phi6 packaging regions.

    PubMed Central

    Pirttimaa, M J; Bamford, D H

    2000-01-01

    Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements. PMID:10864045

  13. Targeting glioblastoma via intranasal administration of Ff bacteriophages

    PubMed Central

    Dor-On, Eyal; Solomon, Beka

    2015-01-01

    Bacteriophages (phages) are ubiquitous viruses that control the growth and diversity of bacteria. Although they have no tropism to mammalian cells, accumulated evidence suggests that phages are not neutral to the mammalian macro-host and can promote immunomodulatory and anti-tumorigenic activities. Here we demonstrate that Ff phages that do not display any proteins or peptides could inhibit the growth of subcutaneous glioblastoma tumors in mice and that this activity is mediated in part by lipopolysaccharide molecules attached to their virion. Using the intranasal route, a non-invasive approach to deliver therapeutics directly to the CNS, we further show that phages rapidly accumulate in the brains of mice and could attenuate progression of orthotopic glioblastoma. Taken together, this study provides new insight into phages non-bacterial activities and demonstrates the feasibility of delivering Ff phages intranasally to treat brain malignancies. PMID:26074908

  14. Bacteriophage-Derived Peptidase CHAPK Eliminates and Prevents Staphylococcal Biofilms

    PubMed Central

    Fenton, Mark; Keary, Ruth; McAuliffe, Olivia; Ross, R. Paul; O'Mahony, Jim; Coffey, Aidan

    2013-01-01

    New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4?h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors. PMID:23431312

  15. Dynamical types of bacteria and bacteriophages interaction: shielding by debris.

    PubMed

    Aviram, Ira; Rabinovitch, Avinoam

    2008-03-01

    The dynamics of the bacteria and bacteriophages interaction process was explored in depth, and laid on a firm basis through simulation and analysis. A modified Campbell model of phage-bacteria interaction was used to simulate three interacting species: bacteria, phages and bacterial debris, and their time behavior in terms of three parameters, in selected range values: the phages burst size (beta), the dissolution rate of the bacterial debris (q), and the lysing time delay (tau). Six types of dynamical behavior were identified, occupying various zones in the two-dimensional (2D) plane (q, beta) for several values of tau, allowing the determination of the region where shielding by bacterial debris is in effect. The problem of the occurrence of stability transitions between stable and unstable dynamical regimes was also addressed analytically, and compared with simulation results. PMID:18160076

  16. A novel minisequencing single-nucleotide polymorphism marker of the lysozyme gene detects high hatchability of Tsaiya ducks (Anas platyrhynchos).

    PubMed

    Huang, Hsiu-Lin; Cheng, Yu-Shin

    2014-11-01

    Lysozyme, one of the major albumen antimicrobials, can break down the polysaccharide walls of a broad spectrum of bacteria. This study presents a novel lysozyme marker of high hatchability in the form of minisequencing single-nucleotide polymorphisms (SNPs). Recently, lysozyme was identified by complementary DNA microarray analysis as one of several differentially expressed genes noted to influence hatchability and recognized as a marker candidate for animal marker-assisted selection. Higher levels (P < 0.05) of lysozyme mRNA (via real-time polymerase chain reaction analysis) and protein (in Western blotting results) were found to be associated with a high-hatchability phenotype. In the preliminary sequence analysis of this study, TsLy1-1 and TsLy1-2 primer pairs, designed according to the lysozyme sequence, were used to amplify small-scale genomic DNA samples from animals in two extreme groups of hatchability. Sequence analysis of the amplified 763-bp DNA products clearly showed that AA and GG genotypes of SNP g.390A > G were from the ducks of the low- and high-hatchability groups, respectively. The SNP g.390A > G also created a new specificity protein 1 transcription factor binding site in the lysozyme gene. Primer pairs of TsLy2-1 and TsLy2-2 then probed the amplified 763-bp DNA products to produce a shorter fragment for easier minisequencing analysis to divide 114 ducks into GG, GA, and AA genotypes. The GG ducks had the highest hatchability, representing that a new lysozyme SNP marker of good hatchability performance can be used for the purpose of marker-assisted selection in Tsaiya ducks. PMID:25179861

  17. DNA Packaging Motor Assembly Intermediate of Bacteriophage ?29

    PubMed Central

    Koti, Jaya S.; Morais, Marc C.; Rajagopal, Raj; Owen, Barbara A. L.; McMurray, Cynthia T.; Anderson, Dwight L.

    2008-01-01

    Unraveling the structure and assembly of the DNA packaging ATPases of the tailed, double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here the bacteriophage ?29 packaging ATPase, gene product gp16 (gp16), was over-expressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the ?29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent upon the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18–20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone, and therefore the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway. PMID:18674782

  18. Breast-conserving surgery for T3\\/T4 breast cancer: an analysis of 196 patients

    Microsoft Academic Search

    Florian Fitzal; Otto Riedl; Lisa Wutzl; Wolfgang Draxler; Margaretha Rudas; Ursula Pluschnig; Leonore Handl-Zeller; Peter Dubsky; Thomas Bachleitner-Hofmann; Günther Steger; Raimund Jakesz; Michael Gnant

    2007-01-01

    Introduction  Breast conservation therapy (BCT) increases quality of life and self-esteem of breast cancer patients. In special cancer centers\\u000a up to 90% of patients are treated with BCT. T3\\/T4 breast cancer is one of the few contraindications for BCT. However, retrospective\\u000a data suggest that BCT may be eligible in selected cases of T3\\/T4 breast cancer.\\u000a \\u000a \\u000a \\u000a Method  We analyzed retrospectively 196 breast cancer

  19. Prognostic Value of Subclassification Using MRI in the T4 Classification Nasopharyngeal Carcinoma Intensity-Modulated Radiotherapy Treatment

    SciTech Connect

    Chen Lei [State Key Laboratory of Oncology in South China, Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou (China)] [State Key Laboratory of Oncology in South China, Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou (China); Liu Lizhi [State Key Laboratory of Oncology in South China, Imaging Diagnosis and Interventional Center, Sun Yat-sen University Cancer Center, Guangzhou (China)] [State Key Laboratory of Oncology in South China, Imaging Diagnosis and Interventional Center, Sun Yat-sen University Cancer Center, Guangzhou (China); Chen Mo; Li Wenfei; Yin Wenjing [State Key Laboratory of Oncology in South China, Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou (China)] [State Key Laboratory of Oncology in South China, Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou (China); Lin Aihua [Department of Medical Statistics and Epidemiology, School of Public Health, Sun Yat-sen University, Guangzhou (China)] [Department of Medical Statistics and Epidemiology, School of Public Health, Sun Yat-sen University, Guangzhou (China); Sun Ying [State Key Laboratory of Oncology in South China, Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou (China)] [State Key Laboratory of Oncology in South China, Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou (China); Li Li [State Key Laboratory of Oncology in South China, Imaging Diagnosis and Interventional Center, Sun Yat-sen University Cancer Center, Guangzhou (China)] [State Key Laboratory of Oncology in South China, Imaging Diagnosis and Interventional Center, Sun Yat-sen University Cancer Center, Guangzhou (China); Ma Jun, E-mail: majun2@mail.sysu.edu.cn [State Key Laboratory of Oncology in South China, Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou (China)

    2012-09-01

    Purpose: To subclassify patients with the T4 classification nasopharyngeal carcinoma (NPC), according to the seventh edition of the American Joint Committee on Cancer staging system, using magnetic resonance imaging (MRI), and to evaluate the prognostic value of subclassification after intensity-modulated radiotherapy (IMRT). Methods and Materials: A total of 140 patients who underwent MRI and were subsequently histologically diagnosed with nondisseminated classification T4 NPC received IMRT as their primary treatment and were included in this retrospective study. T4 patients were subclassified into two grades: T4a was defined as a primary nasopharyngeal tumor with involvement of the masticator space only; and T4b was defined as involvement of the intracranial region, cranial nerves, and/or orbit. Results: The 5-year overall survival (OS) rate and distant metastasis-free survival (DMFS) rate for T4a patients (82.5% and 87.0%, respectively), were significantly higher than for T4b patients (62.6% and 66.8%; p = 0.033 and p = 0.036, respectively). The T4a/b subclassification was an independent prognostic factor for OS (hazard ratio = 2.331, p = 0.032) and DMFS (hazard ratio = 2.602, p = 0.034), and had no significant effect on local relapse-free survival. Conclusions: Subclassification of T4 patients, as T4a or T4b, using MRI according to the site of invasion, has prognostic value for the outcomes of IMRT treatment in NPC.

  20. The HsiB1C1 (TssB-TssC) complex of the Pseudomonas aeruginosa type VI secretion system forms a bacteriophage tail sheathlike structure.

    PubMed

    Lossi, Nadine S; Manoli, Eleni; Förster, Andreas; Dajani, Rana; Pape, Tillmann; Freemont, Paul; Filloux, Alain

    2013-03-15

    Protein secretion systems in Gram-negative bacteria evolved into a variety of molecular nanomachines. They are related to cell envelope complexes, which are involved in assembly of surface appendages or transport of solutes. They are classified as types, the most recent addition being the type VI secretion system (T6SS). The T6SS displays similarities to bacteriophage tail, which drives DNA injection into bacteria. The Hcp protein is related to the T4 bacteriophage tail tube protein gp19, whereas VgrG proteins structurally resemble the gp27/gp5 puncturing device of the phage. The tube and spike of the phage are pushed through the bacterial envelope upon contraction of a tail sheath composed of gp18. In Vibrio cholerae it was proposed that VipA and VipB assemble into a tail sheathlike structure. Here we confirm these previous data by showing that HsiB1 and HsiC1 of the Pseudomonas aeruginosa H1-T6SS assemble into tubules resulting from stacking of cogwheel-like structures showing predominantly 12-fold symmetry. The internal diameter of the cogwheels is ~100 ?, which is large enough to accommodate an Hcp tube whose external diameter has been reported to be 85 ?. The N-terminal 212 residues of HsiC1 are sufficient to form a stable complex with HsiB1, but the C terminus of HsiC1 is essential for the formation of the tubelike structure. Bioinformatics analysis suggests that HsiC1 displays similarities to gp18-like proteins in its C-terminal region. In conclusion, we provide further structural and mechanistic insights into the T6SS and show that a phage sheathlike structure is likely to be a conserved element across all T6SSs. PMID:23341461