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1

Systematic mutation of bacteriophage T4 lysozyme  

Microsoft Academic Search

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber

Dale Rennell; Suzanne E. Bouvier; Larry W. Hardy; Anthony R. Poteete

1991-01-01

2

Control of Bacteriophage T4 Tail Lysozyme Activity During the Infection Process  

SciTech Connect

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

Kanamaru, Shuji; Ishiwata, Yasutaka; Suzuki, Toshiharu; Rossmann, Michael G.; Arisaka, Fumio (Tokyo I); (Purdue)

2010-07-19

3

In vitro Synthesis of Bacteriophage Lysozyme  

Microsoft Academic Search

Active lysozyme of bacteriophage T4 has been synthesized in a cell free system programmed by RNA from cells infected with the phage. The appearance of this lysozyme activity has the characteristics of de novo synthesis.

W. Salser; R. F. Gesteland; A. Bolle

1967-01-01

4

Assignment of the backbone sup 1 H and sup 15 N NMR resonances of bacteriophage T4 lysozyme  

SciTech Connect

The proton and nitrogen ({sup 15}NH-H{sup {alpha}}-H{sup {beta}}) resonances of bacteriophage T4 lysozyme were assigned by {sup 15}N-aided {sup 1}H NMR. The assignments were directed from the backbone amide {sup 1}H-{sup 15}N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly {sup 15}N enriched protein serving as the master template for this work. The main-chain amide {sup 1}H-{sup 15}N resonances and H{sup {alpha}} resonances were resolved and classified into 18 amino acid types by using HMQC and {sup 15}N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of {alpha}-{sup 15}N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H{sub 2}O and D{sub 2}O buffers, from which the H{sup {beta}} resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in {sup 15}N-edited NOESY spectra of the selectively {sup 15}N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the {sup 15}NH-H{sup {alpha}}-H{sup {beta}} spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from {sup 1}H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.

McIntosh, L.P.; Dahlquist, F.W. (Univ. of Oregon, Eugene (USA)); Wand, A.J. (Fox Chase Cancer Center, Philadelphia, PA (USA)); Lowry, D.F.; Redfield, A.G. (Brandeis Univ., Waltham, MA (USA))

1990-07-10

5

Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins  

SciTech Connect

A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on /sup 15/N (or /sup 13/C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-/sup 15/N (or /sup 13/C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from /sup 15/N-bonded protons in the backbone and sidechains of uniformly /sup 15/N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-/sup 15/N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of /sup 15/N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple /sup 15/N and /sup 13/C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl(/sup 15/N)alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.

McIntosh, L.P.; Griffey, R.H.; Muchmore, D.C.; Nielson, C.P.; Redfield, A.G.; Dahlquist, F.W.

1987-03-01

6

Multicomponent anthrax toxin display and delivery using bacteriophage T4  

Microsoft Academic Search

We describe a multicomponent antigen display and delivery system using bacteriophage T4. Two dispensable outer capsid proteins, Hoc (highly antigenic outer capsid protein, 155 copies) and Soc (small outer capsid protein, 810 copies), decorate phage T4 capsid. These proteins bind to the symmetrically localized capsid sites, which appear following prohead assembly and expansion. We hypothesized that multiple antigens fused to

Sathish B. Shivachandra; Qin Li; Kristina K. Peachman; Gary R. Matyas; Stephen H. Leppla; Carl R. Alving; Mangala Rao; Venigalla B. Rao

2007-01-01

7

A mutant T4 lysozyme displays five different crystal conformations  

Microsoft Academic Search

PHAGE T4 lysozyme consists of two domains between which is formed the active-site cleft of the enzyme1,2. The crystallographically determined thermal displacement parameters for the protein2 suggested that the amino terminal of the two domains undergoes 'hinge-bending' motion about an axis passing through the waist of the molecule. Such conformational mobility may be important in allowing access of substrates to

H. R. Faber; B. W. Matthews

1990-01-01

8

T4 bacteriophage as a phage display platform.  

PubMed

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

Gamkrelidze, Mariam; D?browska, Krystyna

2014-07-01

9

Regulation of a Bacteriophage T4 Late Gene, soc, Which Maps in an Early Region  

PubMed Central

We have sequenced and analyzed the expression of an early region of the bacteriophage T4 genome that surprisingly contains a late gene, soc. soc is oriented in the same direction as early genes, like the T4 lysozyme gene. Northern hybridization of early and late T4 RNA, using cloned T4 restriction fragments as probes, identified two long early transcripts and a short late transcript, all containing the soc-coding sequence. Thus, soc is transcribed both early and late. It is, however, translated only late. The inhibition of soc translation from the long early transcripts can be explained by formation of a hairpin in the RNA that sequesters the soc ribosome-binding site. The transcript initiated at the late promoter cannot form this hairpin and is, therefore, translated. PMID:6693022

Macdonald, Paul M.; Kutter, Elizabeth; Mosig, Gisela

1984-01-01

10

REDOR NMR Characterization of DNA Packaging in Bacteriophage T4  

PubMed Central

Bacteriophage T4 is a large-tailed E. coli virus whose capsid is 120 × 86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 minutes during viral infection. We have isolated 50-mg quantities of uniform 15N and [?-15N]lysine-labeled bacteriophage T4. We have also introduced 15NH4+ into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo- and cryoprotected lyophilized T4 using 15N{31P} and 31P{15N} rotational-echo double resonance. The results of these experiments have shown that: (i) packaged DNA is in an unperturbed duplex B-form conformation; (ii) the DNA phosphate negative charge is balanced by lysyl amines (3.2%), polyamines (5.8%), and monovalent cations (40%); and (iii) 11% of lysyl amines, 40% of –NH2 groups of polyamines, and 80% of monovalent cations within the lyophilized T4 capsid, are involved in the DNA charge balance. The NMR evidence suggests that DNA enters the T4 capsid in a charge-unbalanced state. We propose that electrostatic interactions may provide free energy to supplement the nanomotor-driven T4 DNA packaging. PMID:18703073

Yu, Tsyr-Yan; Schaefer, Jacob

2008-01-01

11

A Restriction Map of the Bacteriophage T4 Genome  

PubMed Central

Summary We report a detailed restriction map of the bacteriophage T4 genome and the alignment of this map with the genetic map. The sites cut by the enzymes BglII, XhoI, KpnI, SalI, PstI, EcoRI and HindIII have been localized. Several novel approaches including two-dimensional (double restriction) electrophoretic separations were used. PMID:6258018

O'Farrell, Patrick H.; Kutter, Elizabeth; Nakanishi, Mikiye

2010-01-01

12

Model for DNA packaging into bacteriophage T4 heads.  

PubMed Central

The mechanism of DNA packaging into bacteriophage T4 heads in vivo was investigated by glucosylation of hydroxymethylcytosine residues in a conditionally glucose-deficient host. Cytoplasmic DNA associated with partially packaged ts49 heads can be fully glucosylated, whereas DNA already packaged into these heads is shown to be resistant to glucosylation. After temperature shift and completion of arrested packaging into the reversible temperature-sensitive ts49 head, the structure of the DNA in the mature ts49 phage was investigated by restriction enzyme digestion, autoradiography, and other techniques. Such mature DNA appears to be fully glucosylated along part of its length and nonglucosylated on the remainder. Its structure suggests that the DNA is run into the head linearly and unidirectionally from one mature end and that there is little sequence specificity in that portion of the T4 DNA which first enters the capsid. This technique should be useful in investigation of the three-dimensional structure of first- and last-packaged DNA within the head; preliminary studies including autoradiography of osmotically shocked phage suggest that the DNA which first enters the head is deposited toward the center of the capsid and that the end of the DNA which first enters the head exits first upon injection. In conjunction with studies of the structure of condensed DNA, the positions and functions of T4 capsid proteins in DNA packaging, and the order of T4 packaging functions [Earnshaw and Harrison, Nature (London) 268:598-602, 1977; Hsiao and Black, Proc. Natl. Acad. Sci. U.S.A. 74:3652-3656, 1977; Müller-Salamin et al., J. Virol. 24:121-134, 1977; Richards et al., J. Mol. Biol. 78:255-259, 1973], the features described above suggest the following model: the first DNA end is fixed to the proximal apex of the head at p20 and the DNA is then pumped into the head enzymatically by proteins (p20 + p17) which induce torsion in the DNA molecule. Images PMID:364076

Black, L W; Silverman, D J

1978-01-01

13

Genetic identification of cloned fragments of bacteriophage T4 DNA and complementation by some clones containing early T4 genes  

Microsoft Academic Search

Bacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56,denA anddenB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA withEcoRI have been cloned using the vector plasmid pCR1.

Tom Mattson; Griet Van Houwe; Antoinette Bolle; Gerald Selzer; Richard Epstein

1977-01-01

14

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

PubMed

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

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

2010-08-01

15

Deciphering Lysis and its Regulation in Bacteriophage T4  

E-print Network

...................................... 195 68 Conservation of residues at the surface of sT.................................................. 200 69 Confocal microscopy of E. coli cells expressing t-gfp .................................... 202 70 Model for T4 lysis... ...................................... 195 68 Conservation of residues at the surface of sT.................................................. 200 69 Confocal microscopy of E. coli cells expressing t-gfp .................................... 202 70 Model for T4 lysis...

Moussa, Samir

2012-10-19

16

T4-lysozyme fusion for the production of human formyl peptide receptors for structural determination.  

PubMed

T4-lysozyme (T4L) fusion was introduced in the intracellular loop of a G protein-coupled receptor (GPCR) of human formyl peptide receptor 3 (FPR3), and the ability of T4L fusion to be used in the production of human FPR3 for structural determination was evaluated in this work. The T4L variant of human FPR3 termed FPR3-T4L was expressed in stable tetracycline-inducible HEK293 cells. A systematic detergent screening showed that fos-choline-14 was the optimal detergent to solubilize and subsequently purify FPR3-T4L from HEK293 cells. Immunoaffinity purification in combination with gel filtration was employed to purify the T4L-fused receptor to high homogeneity. The final yield of the human FPR3-T4L monomer from 2 g of cells was 0.2 mg. Circular dichroism spectroscopy indicated that the receptor adopted a correct secondary structure after purification, while ligand binding measurement indicated that the receptor was functional. Thus, the presence of T4L fusion did not evidently disturb the expression in HEK293 cells, proper folding, and functionality of human FPR3. Our study of evaluating T4L fusion for the recombinant production of human formyl peptide receptor would facilitate ongoing efforts in the structural characterization of GPCRs. PMID:24407945

Wang, Xiaoqiang; Cui, Ying; Wang, Jiqian

2014-03-01

17

Bacteriophage T4 DNA Topoisomerase Mediates Illegitimate Recombination in vitro  

NASA Astrophysics Data System (ADS)

We have found that purified T4 DNA topoisomerase promotes recombination between two phage ? DNA molecules in an in vitro system. In this cross, T4 DNA topoisomerase alone is able to catalyze the recombination and produce a linear monomer recombinant DNA that can be packaged in vitro. ATP is not required for this recombination, while oxolinic acid stimulates it. The recombinant DNA molecules contain duplications or deletions, and the crossovers take place between nonhomologous and nonspecific sequences of ? DNA. Therefore, the recombination mediated by the T4 DNA topoisomerase is an illegitimate recombination that is similar to that mediated by Escherichia coli DNA gyrase. A model was proposed previously in which DNA gyrase molecules that are bound to DNA associate with each other and lead to the exchange of DNA strands through the exchange of DNA gyrase subunits. This model is also applicable to the recombination mediated by T4 DNA topoisomerase.

Ikeda, Hideo

1986-02-01

18

Early intermediates in bacteriophage T4 DNA replication and recombination.  

PubMed Central

We investigated, by density gradients and subsequent electron microscopy, vegetative T4 DNA after single or multiple infection of Escherichia coli with wild-type T4. Our results can be summarized as follows. (i) After single infection (i.e., when early intermolecular recombination could not occur), most, if not all, T4 DNA molecules initiated the first round of replication with a single loop. (ii) After multiple infection, recombinational intermediates containing label from both parents first appeared as early as 1 min after the onset of replication, long before all parental DNA molecules had finished their first round and before secondary replication was detectable. (iii) At the same time, in multiple infections only, complex, highly branched concatemeric T4 DNA first appeared. (iv) Molecules in which two loops or several branches were arranged in tandem were only found after multiple infections. (v) Secondary loops within primary loops were seen after both single and multiple infections, but they were rare and many appeared off center. Thus, recombination in wild-type T4-infected cells occurred very early, and the generation of multiple tandem loops or branches in vegetative T4 DNA depended on recombination. These results are consistent with the previous finding (A. Luder and G. Mosig, Proc. Natl. Acad. Sci. U.S.A. 79:1101-1105, 1982) that most secondary growing points of T4 are not initiated from origin sequences but from recombinational intermediates. By these and previous results, the various DNA molecules that we observed are most readily explained as intermediates in DNA replication and recombination according to a model proposed earlier to explain various other aspects of T4 DNA metabolism (Mosig et al., p. 277-295, in D. Ray, ed., The Initiation of DNA Replication, Academic Press, Inc., New York, 1981). Images PMID:6834472

Dannenberg, R; Mosig, G

1983-01-01

19

Protein Flexibility and Adaptability Seen in 25 Crystal Forms of T4 Lysozyme  

Microsoft Academic Search

The structures of various mutants of T4 lysozyme have been determined in 25 non-isomorphous crystal forms. This provides an unusually diverse data base to compare the structures and dynamics of a closely related set of proteins in different crystal packing environments.In general, the more tightly packed crystals diffract better than those that are highly hydrated although the wild-type crystal form

Xue-jun Zhang; Joan A. Wozniak; Brian W. Matthews

1995-01-01

20

An Introduction to the Bacteriophage T4 Virus.  

NSDL National Science Digital Library

This is a site concerning the characteristics of the T4 virus and how it infects their unsuspecting hosts. The page speaks about how viruses straddle the definition of life, along with a basic description of what viruses are, and how they infect/replicate in host organisms.

Projects, Dform:

21

T4 Bacteriophage Gene 32: A Structural Protein in the Replication and Recombination of DNA  

Microsoft Academic Search

A new type of protein essential for DNA replication and genetic recombination has been isolated from T4 bacteriophage-infected cells of E. coli. This protein binds cooperatively to single-stranded DNA, and it catalyses DNA denaturation and renaturation in physiological conditions in vitro.

Bruce M. Alberts; Linda Frey

1970-01-01

22

Catalytic mechanism of bacteriophage t4 rad50 ATP hydrolysis.  

PubMed

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH-rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites. PMID:25137526

Herdendorf, Timothy J; Nelson, Scott W

2014-09-01

23

Probing Single-Molecule T4 Lysozyme Conformational Dynamics by Intramolecular Fluorescence Energy Transfer  

SciTech Connect

We demonstrate the use of single-molecule spectroscopy to study enzyme conformational motions of T4 lysozyme under hydrolysis reaction of the polysaccharide walls of E. Coli B cells.By attaching a donoracceptor pair of dye molecules site-specifically to noninterfering sites on the enzyme, the hinge-bending motions of the enzyme are measured by monitoring the donor-acceptor emission intensity as a function of time. The overall enzymatic reaction rate constants are found to vary widely from molecule to molecule. The dominant contribution to this static inhomogeneity is attributed to enzyme searching for reactive sites on the substrate.

Chen, Yu; Hu, Dehong; Vorpagel, Erich R.; Lu, H PETER.

2003-07-16

24

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

Microsoft Academic Search

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

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

2011-01-01

25

Bacteriophage T4 Development in Escherichia coli is Growth Rate Dependent  

Microsoft Academic Search

Three independent parameters (eclipse and latent periods, and rate of ripening during the rise period) are essential and sufficient to describe bacteriophage development in its bacterial host. A general model to describe the classical “one-step growth” experiment [Rabinovitch et al. (1999a) J. Bacteriol.181, 1687–1683] allowed their calculations from experimental results obtained with T4 in Escherichia coli B\\/r under different growth

AVINOAM RABINOVITCH; Itzhak Fishovw; HILLA HADAS; MONICA EINAV; ARIEH ZARITSKY

2002-01-01

26

Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA  

Microsoft Academic Search

A series of experiments designed to characterize the impact of UV irradiation (260 nm) on 5-bromodeoxyuridine-labeled (heavy) T4 bacteriophage, both before and after infection of Escherichia coli has been carried out. The results have led us to propose a model for a novel mechanism of host-mediated repair synthesis, in which excision of UV-damaged areas is followed by initiation of replication,

L. L. Restifo; H. H. Vogelbacker; T. Madara; S. K. Ling; A. W. Kozinski

1983-01-01

27

Formation of the prohead core of bacteriophage T4 in vivo.  

PubMed Central

Formation of the prohead core of bacteriophage T4 was not dependent on shell assembly. In mutant infections, where the production or assembly of active shell protein was not possible, naked core structures were formed. The particles were generally attached to the bacterial inner membrane and possessed defined prolate dimensions. The intracellular yield varied between 15 and 71% of a corresponding prohead yield and was dependent on the temperature of incubation. The products of genes 21 and 22 were found to be essential for in vivo core formation, whereas those of genes 20, 23, 24, 31, and 40, as well as the internal proteins I to III, were dispensable. Images PMID:6366245

Traub, F; Maeder, M

1984-01-01

28

Kinetic factors and form determination of the head of bacteriophage T4.  

PubMed Central

The form of the bacteriophage T4 prehead is described by its icosahedral symmetry, its diameter, and its length. We show how each of these parameters is regulated during prehead formation and ascribe specific form-determining functions to the prehead proteins. The major protein of the head shell can assemble in several different forms. The structure produced in vivo depends on the rate of synthesis of the major protein relative to the rates of synthesis of minor shell proteins and the major core protein. From our observations, we propose a model for form determination of the prehead and suggest a pathway for the evolution of its prolate shape. Images PMID:279906

Showe, M K; Onorato, L

1978-01-01

29

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

E-print Network

-Buried Salt Bridges Feng Dong and Huan-Xiang Zhou Department of Physics, Drexel University, Philadelphia titration of the H31-D70 semi-buried salt bridge on the stability of T4 lysozyme. Instead of the widely used to protein stability, semi-buried salt bridges can provide significant stabilization. INTRODUCTION

Weston, Ken

30

Bacteriophage T4 late transcription from plasmid templates is enhanced by negative supercoiling.  

PubMed Central

Concurrent viral replication is normally required to activate bacteriophage T4 late promoters; replication is thought to provide a template structure which is competent for late transcription. Transcription from plasmid-borne T4 late promoters, however, is independent of replication in vivo and in vitro. In this work, we have shown that, when the late gene 23 promoter is located on a plasmid, its utilization in vivo depends upon the ability of host DNA gyrase to maintain some degree of negative superhelicity. This suggests that an alternative pathway exists for activation of late promoters: DNA which is under sufficient negative torsional stress is already competent for late transcription. We also describe a method for isolating ternary complexes of plasmid DNA, RNA polymerase, and nascent RNA which have initiated transcription in vivo. The topoisomer distribution of such ternary complexes prepared from T4-infected cells showed that, late in infection, transcriptional activity resides primarily in the subset of the plasmid population with the most negatively supercoiled topoisomers. However, the overall transcriptional pattern in these ternary complexes indicated that both vector and T4 sequences are actively transcribed. Much of this transcriptional activity could be independent of gp55, the T4-specific RNA polymerase-binding protein that confers late promoter recognition. Images PMID:2830234

Albright, L M; Kassavetis, G A; Geiduschek, E P

1988-01-01

31

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants  

PubMed Central

The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering. PMID:24167295

Lopez, Carlos J.; Yang, Zhongyu; Altenbach, Christian; Hubbell, Wayne L.

2013-01-01

32

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants.  

PubMed

The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering. PMID:24167295

López, Carlos J; Yang, Zhongyu; Altenbach, Christian; Hubbell, Wayne L

2013-11-12

33

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant  

PubMed Central

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers)1–3. However, a rigorous understanding of the relation between a protein’s structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules4 and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the ‘invisible’, excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta7 model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein’s function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states. PMID:21857680

Bouvignies, Guillaume; Vallurupalli, Pramodh; Hansen, D. Flemming; Correia, Bruno E.; Lange, Oliver; Bah, Alaji; Vernon, Robert M.; Dahlquist, Frederick W.; Baker, David; Kay, Lewis E.

2013-01-01

34

Localization of minor protein components of the head of bacteriophage T4.  

PubMed Central

The bacteriophage T4 capsid contains a number of minor proteins that are required for head assembly but whose detailed function and position in the head are unknown. We have found that by systematically varying the conditions of extraction, some of these minor proteins can be removed while the main capsid structure is left substantially intact. Electron microscopic examination of the residual capsids showed that the extraction of the product of gene 20 is correlated with the loss of a plug that distinguishes one vertex position (presumably the tail attachment site) from the others. Extraction of the product of gene 24 is correlated with the loss of the other 11 (nonproximal) vertexes of the capsid. We further show that antibody to P24 binds specifically to the nonproximal vertexes of both T4 preheads and T4 phages. On the basis of our findings, we suggest that P20 is located at or near the tail attachment site of the capsid, whereas P24 forms the 11 nonproximal vertexes of preheads and P24 forms the nonproximal vertexes of the mature head. Images PMID:904022

Muller-Salamin, L; Onorato, L; Showe, M K

1977-01-01

35

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

SciTech Connect

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

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

2009-07-22

36

Autoinhibition of Bacteriophage T4 Mre11 by Its C-terminal Domain.  

PubMed

Mre11 and Rad50 form a stable complex (MR) and work cooperatively in repairing DNA double strand breaks. In the bacteriophage T4, Rad50 (gene product 46) enhances the nuclease activity of Mre11 (gene product 47), and Mre11 and DNA in combination stimulate the ATPase activity of Rad50. The structural basis for the cross-activation of the MR complex has been elusive. Various crystal structures of the MR complex display limited protein-protein interfaces that mainly exist between the C terminus of Mre11 and the coiled-coil domain of Rad50. To test the role of the C-terminal Rad50 binding domain (RBD) in Mre11 activation, we constructed a series of C-terminal deletions and mutations in bacteriophage T4 Mre11. Deletion of the RBD in Mre11 eliminates Rad50 binding but only has moderate effect on its intrinsic nuclease activity; however, the additional deletion of the highly acidic flexible linker that lies between RBD and the main body of Mre11 increases the nuclease activity of Mre11 by 20-fold. Replacement of the acidic residues in the flexible linker with alanine elevates the Mre11 activity to the level of the MR complex when combined with deletion of RBD. Nuclease activity kinetics indicate that Rad50 association and deletion of the C terminus of Mre11 both enhance DNA substrate binding. Additionally, a short peptide that contains the flexible linker and RBD of Mre11 acts as an inhibitor of Mre11 nuclease activity. These results support a model where the Mre11 RBD and linker domain act as an autoinhibitory domain when not in complex with Rad50. Complex formation with Rad50 alleviates this inhibition due to the tight association of the RBD and the Rad50 coiled-coil. PMID:25077970

Gao, Yang; Nelson, Scott W

2014-09-19

37

Mutagenic Effect of Temperature-Sensitive Mutants of Gene 42 (dCMP Hydroxymethylase) of Bacteriophage T4  

PubMed Central

Certain temperature-sensitive mutants of gene 42 of bacteriophage T4 increase the reversion rates of some rII mutants in the same genome by about 4 to 10 times. This effect was usually found at 34 C, an intermediate permissive temperature, but not at 28 C. PMID:4579822

Chiu, Che-Shen; Greenberg, G. Robert

1973-01-01

38

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

SciTech Connect

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.

Rao, V.B.

1997-09-01

39

The introduction of strain and its effects on the structure and stability of T4 lysozyme.  

PubMed

In order to try to better understand the role played by strain in the structure and stability of a protein a series of "small-to-large" mutations was made within the core of T4 lysozyme. Three different alanine residues, one involved in backbone contacts, one in side-chain contacts, and the third adjacent to a small cavity, were each replaced with subsets of the larger residues, Val, Leu, Ile, Met, Phe and Trp. As expected, the protein is progressively destabilized as the size of the introduced side-chain becomes larger. There does, however, seem to be a limit to the destabilization, suggesting that a protein of a given size may be capable of maintaining only a certain amount of strain. The changes in stability vary greatly from site to site. Substitution of larger residues for both Ala42 and Ala98 substantially destabilize the protein, even though the primary contacts in one case are predominantly with side-chain atoms and in the other with backbone. The results suggest that it is neither practical nor meaningful to try to separate the effects of introduced strain on side-chains from the effects on the backbone. Substitutions at Ala129 are much less destabilizing than at sites 42 or 98. This is most easily understood in terms of the pre-existing cavity, which provides partial space to accommodate the introduced side-chains. Crystal structures were obtained for a number of the mutants. These show that the changes in structure to accommodate the introduced side-chains usually consist of essentially rigid-body displacements of groups of linked atoms, achieved through relatively small changes in torsion angles. On rare occasions, a side-chain close to the site of substitution may change to a different rotamer. When such rotomer changes occur, they permit the structure to dissipate strain by a response that is plastic rather than elastic. In one case, a surface loop moves 1.2 A, not in direct response to a mutation, but in an interaction mediated via an intermolecular contact. It illustrates how the structure of a protein can be modified by crystal contacts. PMID:10623513

Liu, R; Baase, W A; Matthews, B W

2000-01-01

40

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

SciTech Connect

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

Hu, Dehong; Lu, H PETER.

2004-07-01

41

Genetic analysis of the bacteriophage T4-encoded cochaperonin Gp31.  

PubMed Central

Previous genetic and biochemical analyses have established that the bacteriophage T4-encoded Gp31 is a cochaperonin that interacts with Escherichia coli's GroEL to ensure the timely and accurate folding of Gp23, the bacteriophage-encoded major capsid protein. The heptameric Gp31 cochaperonin, like the E. coli GroES cochaperonin, interacts with GroEL primarily through its unstructured mobile loop segment. Upon binding to GroEL, the mobile loop adopts a structured, beta-hairpin turn. In this article, we present extensive genetic data that strongly substantiate and extend these biochemical studies. These studies begin with the isolation of mutations in gene 31 based on the ability to plaque on groEL44 mutant bacteria, whose mutant product interacts weakly with Gp31. Our genetic system is unique because it also allows for the direct selection of revertants of such gene 31 mutations, based on their ability to plaque on groEL515 mutant bacteria. Interestingly, all of these revertants are pseudorevertants because the original 31 mutation is maintained. In addition, we show that the classical tsA70 mutation in gene 31 changes a conserved hydrophobic residue in the mobile loop to a hydrophilic one. Pseudorevertants of tsA70, which enable growth at the restrictive temperatures, acquire the same mutation previously shown to allow plaque formation on groEL44 mutant bacteria. Our genetic analyses highlight the crucial importance of all three highly conserved hydrophobic residues of the mobile loop of Gp31 in the productive interaction with GroEL. PMID:10430575

Richardson, A; Georgopoulos, C

1999-01-01

42

A mutation in the gene for polynucleotide kinase of bacteriophage T4 K10 affects mRNA processing.  

PubMed

The bacteriophage T4 insertion-substitution (I/S) vector system has become one of the most important tools for the introduction of site-directed mutations into the T4 genome. In this study, we show that the I/S phage T4 K10 carries two point mutations within the gene for polynucleotide kinase pseT, resulting in amino acid substitutions G14D and R229H. The G14D mutation impairs 5'-kinase activity in vivo as well as in vitro and leads to diminished processing at secondary sites of several RegB-cleaved transcripts. PMID:23948816

Strazdait?-Žielien?, Živil?; Zajan?kauskait?, Aurelija; Kalinien?, Laura; Meškys, Rolandas; Truncait?, Lidija

2014-02-01

43

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

PubMed

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

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

2014-03-01

44

Multiple mechanisms for degradation of bacteriophage T4 soc mRNA.  

PubMed Central

The dmd gene of bacteriophage T4 is required for regulation of mRNA stability in a stage-dependent manner during infection. When this gene is mutated, late genes are globally silenced because of rapid degradation of mRNAs. To investigate the mechanism of such mRNA degradation, we analyzed the late gene soc transcripts. The degradation of soc mRNA was remarkably stabilized when its ability to be translated was impaired; either disruption of translation initiation signals or elimination of termination codons was effective in stabilization of soc mRNA and removal of elongation modestly stabilized it. Even in the absence of translation, however, the residual activity was still significant. These results suggested that the degradation of soc transcripts was promoted by two different mechanisms; one is dependent on translation and the other independent of translation. We found several cleavages introduced into soc RNA specifically when the dmd gene was mutated; some of them could be linked to polypeptide chain elongation and termination, suggesting the correlation with ribosomal action, and the others were independent of translation. PMID:11805040

Kai, Toshie; Yonesaki, Tetsuro

2002-01-01

45

Destabilization of Bacteriophage T4 Mrnas by a Mutation of Gene 61.5  

PubMed Central

We identified a novel gene of bacteriophage T4, gene 61.5, which appears to be involved in protein synthesis late in infection. Northern blot analysis revealed that a mutant of 61.5 accumulated truncated transcripts of representative late genes. Using a double mutant of genes 61.5 and 55, which prevents transcription of late genes, we demonstrate that even transcripts of middle genes, while full-length when initially expressed, are similarly truncated at later stages of infection. These results indicate that the abnormality in transcript length occurs late in infection, regardless of whether the transcript derives from a middle or a late gene. Primer-extension analysis revealed that the 5' ends of the late gene 23 transcripts that accumulated in gene 61.5 mutant-infected cells were located at internal discrete sites as well as at the expected transcription start site. Moreover, the decay rates of full-length transcripts from genes uvsY or 45 were more than twofold faster in the absence of a functional gene 61.5. These results suggest that mutation of gene 61.5 activates endonucleolytic cleavage of middle and late transcripts, probably by RNase M. PMID:8878669

Kai, T.; Selick, H. E.; Yonesaki, T.

1996-01-01

46

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

PubMed Central

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

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

47

Survey of major capsid genes ( g23) of T4-type bacteriophages in rice fields in Northeast China  

Microsoft Academic Search

We surveyed the capsid genes (g23) of T4-type bacteriophages in DNA extracted from fifteen rice field soils in Northeast China using primers MZIA1bis and MZIA6. Denaturing gradient gel electrophoresis (DGGE) was performed to separate PCR-amplified g23 products. In total, 53 DGGE bands were identified as g23 clones, nine of which belonged to a novel, Northeast China-specific group. In addition, four

Guanghua Wang; Jian Jin; Susumu Asakawa; Makoto Kimura

2009-01-01

48

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

PubMed

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

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

2013-07-01

49

Structural refinement from restrained-ensemble simulations based on EPR/DEER data: application to T4 lysozyme.  

PubMed

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 parametrized 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

Islam, Shahidul M; Stein, Richard A; McHaourab, Hassane S; Roux, Benoît

2013-05-01

50

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

PubMed Central

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

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

2013-01-01

51

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

E-print Network

membrane lesion formed by T allows E to gain access to the cell wall. T4 exhibits lysis inhibition (LIN), a phenomenon in which a second T4 infection occurs ? 3 min after primary infection results a delay in lysis. Mutations that abolish LIN mapped...

Tran, Tram Anh Thi

2009-05-15

52

An ORFan No More: The Bacteriophage T4 39.2 Gene Product, NwgI, Modulates GroEL Chaperone Function  

PubMed Central

Bacteriophages are the most abundant biological entities in our biosphere, characterized by their hyperplasticity, mosaic composition, and the many unknown functions (ORFans) encoded by their immense genetic repertoire. These genes are potentially maintained by the bacteriophage to allow efficient propagation on hosts encountered in nature. To test this hypothesis, we devised a selection to identify bacteriophage-encoded gene(s) that modulate the host Escherichia coli GroEL/GroES chaperone machine, which is essential for the folding of certain host and bacteriophage proteins. As a result, we identified the bacteriophage RB69 gene 39.2, of previously unknown function and showed that homologs of 39.2 in bacteriophages T4, RB43, and RB49 similarly modulate GroEL/GroES. Production of wild-type bacteriophage T4 Gp39.2, a 58-amino-acid protein, (a) enables diverse bacteriophages to plaque on the otherwise nonpermissive groES or groEL mutant hosts in an allele-specific manner, (b) suppresses the temperature-sensitive phenotype of both groES and groEL mutants, (c) suppresses the defective UV-induced PolV function (UmuCD) of the groEL44 mutant, and (d) is lethal to the host when overproduced. Finally, as proof of principle that Gp39.2 is essential for bacteriophage growth on certain bacterial hosts, we constructed a T4 39.2 deletion strain and showed that, unlike the isogenic wild-type parent, it is incapable of propagating on certain groEL mutant hosts. We propose a model of how Gp39.2 modulates GroES/GroEL function. PMID:22234860

Ang, Debbie; Georgopoulos, Costa

2012-01-01

53

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

SciTech Connect

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.

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

2011-09-16

54

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

PubMed Central

The alc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. We examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D+ 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+ or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D+ 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-32P]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. It thus appears likely that gpalc inhibits transcript elongation on cytosine-containing DNA by interacting with actively transcribing core polymerase as a complex with the enzyme and cytosine-rich stretches of the template. Images PMID:2185231

Drivdahl, R H; Kutter, E M

1990-01-01

55

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

NASA Astrophysics Data System (ADS)

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.

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

56

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages  

SciTech Connect

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a 'small terminase' and a 'large terminase' component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.

Sun, Siyang; Gao, Song; Kondabagil, Kiran; Xiang, Ye; Rossmann, Michael G.; Rao, Venigalla B. (CUA); (Purdue)

2012-04-04

57

Length and shape variants of the bacteriophage T4 head: mutations in the scaffolding core genes 68 and 22.  

PubMed Central

The shape and size of the bacteriophage T4 head are dependent on genes that determine the scaffolding core and the shell of the prohead. Mutants of the shell proteins affect mainly the head length. Two recently identified genes (genes 67 and 68) and one already known gene (gene 22), whose products are scaffold constituents, have been investigated. Different types of mutants were shown to strongly influence the proportion of aberrantly shaped particles. By model building, these shape variants could be represented as polyhedral bodies derived from icosahedra, through outgrowths along different polyhedral axes. The normal, prolate particle is obtained by elongation along a fivefold axis. The mutations of the three core genes (genes 67, 68, and 22) affect the width mainly by lateral outgrowths of the prolate particle, although small and large isometric particles are also found. Many of the aberrant particles are multitailed, suggesting a correlation between tail attachment sites and shape. Images PMID:3292792

Keller, B; Dubochet, J; Adrian, M; Maeder, M; Wurtz, M; Kellenberger, E

1988-01-01

58

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

PubMed

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

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

2014-10-21

59

Bacteriophage T4D Gene 42 Mutants Exhibit a Defective Genetic Exclusion Phenotype.  

National Technical Information Service (NTIS)

Genetic exclusion in phage T4 is the prime responsibility of the imm and sp genes. The map region containing imm does not allow sufficient coding space to encode for proteins the size reported for the imm gp. After assaying 30 mutants of the genes adjacen...

J. W. Obringer

1991-01-01

60

Bacteriophage T4 Development Depends on the Physiology of its Host Escherichia Coli  

Microsoft Academic Search

Several parameters of phage T4 adsorption to and growth in Escherichia coli B\\/r were determined. All changed monotonously with the bacterial growth rate (l), which was modified by nutritional conditions. Adsorption rate was faster at higher l values, positively correlated to cell size, and increased by pretreatment with low penicillin (Pn) concentrations; it was directly proportional to total cellular surface

Hilla Hadas; Monica Einav; Itzhak Fishov; Arieh Zaritsky

1997-01-01

61

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

PubMed Central

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

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

2011-01-01

62

Using the rate of respiration to monitor events in the infection of Escherichia coli cultures by bacteriophage T4.  

PubMed

The growing interest in applications of bacteriophages creates a need for improvements in the production processes. Continuous monitoring of the phage production is an essential aspect of any control strategy and, at present, there is no completely satisfactory option. The approach presented here uses IR-spectrometry to continuously measure the rate of respiration (CO(2) released) of Escherichia coli infected by phage T4 at various multiplicities of infection (MOI). Within the trends in these data, or in other aspects of the rate of respiration, it was possible to reliably and reproducibly identify five features that reflected specific events in the infection process. These included two events in the host cell apparent growth rate and events in the magnitude of the host cell density, in the measurement of OD(600) or in the specific rate of respiration. All of these correlations were within 95% confidence showing that they are suitable for the monitoring and control of E. coli populations infected by phage T4. This method is reliable, cheap, and can be operated in-line and in real time. PMID:20039436

Sauvageau, Dominic; Allain, Béatrice; Cooper, David G

2010-01-01

63

Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA  

NASA Astrophysics Data System (ADS)

Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork.

Shokri, Leila; Rouzina, Ioulia; Williams, Mark C.

2009-06-01

64

Functional Analysis of the Highly Antigenic Outer Capsid Protein, Hoc, a Virus Decoration Protein from T4-like Bacteriophages  

PubMed Central

Summary Bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein, Hoc. The Hoc molecule (40 kDa) is present at the center of each hexameric capsomer and provides a good platform for surface display of pathogen antigens. Biochemical and modeling studies show that Hoc consists of a string of four domains, three immunoglobulin (Ig)-like and one non-Ig domain at the C-terminus. Biochemical data suggest that the Hoc protein has two functional modules, a capsid binding module containing domains 1 and 4 and a solvent-exposed module containing domains 2 and 3. This model is consistent with the dumbbell-shaped cryo-EM density of Hoc observed in the reconstruction of the T4 capsid. Mutagenesis localized the capsid binding site to the C-terminal 25 amino acids, which are predicted to form two ?-strands flanking a capsid binding loop. Mutations in the loop residues, ESRNG, abolished capsid binding, suggesting that these residues might interact with the major capsid protein, gp23*. With the conserved capsid binding module forming a foothold on the virus and the solvent-exposed module able to adapt to bind to a variety of surfaces, Hoc probably provides survival advantages to the phage, such as increasing the virus concentration near the host, efficient dispersion of the virus, and exposing the tail for more efficient contact with the host cell surface prior to infection. PMID:20497329

Sathaliyawala, Taheri; Islam, Mohammad Z.; Li, Qin; Fokine, Andrei; Rossmann, Michael G.; Rao, Venigalla B.

2010-01-01

65

Phylogenetic diversity and assemblage of major capsid genes (g23) of T4-type bacteriophages in paddy field soils during rice growth season in Northeast China  

Microsoft Academic Search

Although bacteriophages (phages) are ubiquitous and the most abundant biological entities on Earth, the genetic information on their diversity and community composition in natural environments, particularly in soils, is limited. This study elucidated the diversity and composition of T4-type phages by analyzing partial major capsid gene (g23) sequences in DNA extracts from five paddy field soils in Northeast China during

Junjie Liu; Guanghua Wang; Qiang Wang; Judong Liu; Jian Jin; Xiaobing Liu

2012-01-01

66

The Structure of the Receptor-binding Domain of the Bacteriophage T4 Short Tail Fibre Reveals a Knitted Trimeric Metal-binding Fold  

Microsoft Academic Search

Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres. After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipo-polysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube.

Ellen Thomassen; Gerrit Gielen; Michael Schütz; Guy Schoehn; Jan Pieter Abrahams; Stefan Miller; Mark J. van Raaij

2003-01-01

67

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

SciTech Connect

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

Cheng, X.; Pflugrath, J.W. [W.M. Keck Structural Biology Lab., Cold Spring Harbor, NY (United States); Zhang, X.; Studier F.W. [Brookhaven National Lab., Upton, NY (United States)

1994-04-26

68

The roles of the bacteriophage T4 r genes in lysis inhibition and fine-structure genetics: a new perspective.  

PubMed Central

Seldom has the study of a set of genes contributed more to our understanding of molecular genetics than has the characterization of the rapid-lysis genes of bacteriophage T4. For example, T4 rII mutants were used to define gene structure and mutagen effects at the molecular level and to help unravel the genetic code. The large-plaque morphology of these mutants reflects a block in expressing lysis inhibition (LIN), the ability to delay lysis for several hours in response to sensing external related phages attacking the cell, which is a unique and highly adaptive attribute of the T4 family of phages. However, surprisingly little is known about the mechanism of LIN, or how the various r genes affect its expression. Here, we review the extensive old literature about the r genes and the lysis process and try to sort out the major players affecting lysis inhibition. We confirm that superinfection can induce lysis inhibition even while infected cells are lysing, suggesting that the signal response is virtually instantaneous and thus probably the result of post-translational regulation. We identify the rI gene as ORF tk.-2, based on sequence analysis of canonical rI mutants. The rI gene encodes a peptide of 97 amino acids (Mr = 11.1 kD; pI = 4.8) that probably is secreted into the periplasmic space. This gene is widely conserved among T-even phage. We then present a model for LIN, postulating that rI is largely responsible for regulating the gpt holin protein in response to superinfection. The evidence suggests that the rIIA and B genes are not directly involved in lysis inhibition; rather, when they are absent, an alternate pathway for lysis develops which depends on the presence of genes from any of several possible prophages and is not sensitive to lysis inhibition. PMID:9560373

Paddison, P; Abedon, S T; Dressman, H K; Gailbreath, K; Tracy, J; Mosser, E; Neitzel, J; Guttman, B; Kutter, E

1998-01-01

69

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

SciTech Connect

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

Ogura, Toshihiko, E-mail: t-ogura@aist.go.jp

2009-03-06

70

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

SciTech Connect

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

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

2009-07-21

71

Destabilizing effect of proline substitutions in two helical regions of T4 lysozyme: leucine 66 to proline and leucine 91 to proline.  

PubMed Central

A class of temperature-sensitive (ts) mutants of T4 lysozyme with reduced activity at 30 degrees C and no activity at 43 degrees C has been selected. These mutants, designated "tight" ts mutants, differ from most other T4 lysozyme mutants that are active at 43 degrees C, but only manifest their ts lesion by a reduced halo size around phage plaques after exposure of the growth plates to chloroform vapors. For example, in the series of T4 lysozyme mutants at position 157, the original randomly selected mutant, T1571, is the least stable of the series, yet, apart from the halo assay and subsequent in vitro protein stability measurements, this mutant is indistinguishable from wild type (WT) even at 43 degrees C. Two mutants were identified: L91P and L66P. Both insert proline residues into alpha-helical regions of the WT protein structure. The stabilities (delta delta G) as determined by urea denaturation are 8.2 kcal/mol for L91P and 7.1 kcal/mol for L66P. CD spectra indicate that no major conformational changes have occurred in the mutant structures. The structures of the mutants were modeled with a 40-ps molecular dynamics simulation using explicit solvent. For L91P, the reduction of stability appears to be due to an unsatisfied hydrogen bond in the alpha-helix and to a new buried cavity. For L66P, the reduction of stability appears to be due to a disruption of the interdomain alpha-helix, at least two unsatisfied hydrogen bonds, and a newly formed solvent-filled pocket that protrudes into the hydrophobic core, possibly reducing the stabilizing contribution of a partially buried intrachain salt bridge. PMID:8845764

Gray, T. M.; Arnoys, E. J.; Blankespoor, S.; Born, T.; Jagar, R.; Everman, R.; Plowman, D.; Stair, A.; Zhang, D.

1996-01-01

72

Cpl-7, a Lysozyme Encoded by a Pneumococcal Bacteriophage with a Novel Cell Wall-binding Motif*  

PubMed Central

Bacteriophage endolysins include a group of new antibacterials reluctant to development of resistance. We present here the first structural study of the Cpl-7 endolysin, encoded by pneumococcal bacteriophage Cp-7. It contains an N-terminal catalytic module (CM) belonging to the GH25 family of glycosyl hydrolases and a C-terminal region encompassing three identical repeats of 42 amino acids (CW_7 repeats). These repeats are unrelated to choline-targeting motifs present in other cell wall hydrolases produced by Streptococcus pneumoniae and its bacteriophages, and are responsible for the protein attachment to the cell wall. By combining different biophysical techniques and molecular modeling, a three-dimensional model of the overall protein structure is proposed, consistent with circular dichroism and sequence-based secondary structure prediction, small angle x-ray scattering data, and Cpl-7 hydrodynamic behavior. Cpl-7 is an ?115-? long molecule with two well differentiated regions, corresponding to the CM and the cell wall binding region (CWBR), arranged in a lateral disposition. The CM displays the (??)5?3 barrel topology characteristic of the GH25 family, and the impact of sequence differences with the CM of the Cpl-1 lysozyme in substrate binding is discussed. The CWBR is organized in three tandemly assembled three-helical bundles whose dispositions remind us of a super-helical structure. Its approximate dimensions are 60 × 20 × 20 ? and presents a concave face that might constitute the functional region involved in bacterial surface recognition. The distribution of CW_7 repeats in the sequences deposited in the Entrez Database have been examined, and the results drastically expanded the antimicrobial potential of the Cpl-7 endolysin. PMID:20720016

Bustamante, Noemí; Campillo, Nuria E.; García, Ernesto; Gallego, Cristina; Pera, Benet; Diakun, Gregory P.; Sáiz, José Luis; García, Pedro; Díaz, J. Fernando; Menéndez, Margarita

2010-01-01

73

Molecular analysis of the major capsid genes ( g23 ) of T4-type bacteriophages in an upland black soil in Northeast China  

Microsoft Academic Search

Bacteriophages (phages) are the most abundant biological entities on the planet and are important as the greatest genomic\\u000a reservoirs in both marine and terrestrial environments. In this study, we analysed T4-type phage communities in an upland\\u000a black soil by monitoring g23 clones in DNA extracted from seasonal soil samples with no fertilizer, chemical fertilizers, chemical fertilizers plus manure,\\u000a and natural

Guanghua Wang; Zhenhua Yu; Junjie Liu; Jian Jin; Xiaobing Liu; Makoto Kimura

2011-01-01

74

Bacteriophage T4 can produce progeny virions in extremely slowly growing Escherichia coli host: comparison of a mathematical model with the experimental data.  

PubMed

Development of bacteriophage T4 depends on the physiological state of its host cell. Based on previous studies performed under laboratory conditions with different media determining various growth rates of Escherichia coli, a mathematical model was developed which suggested that phage T4 development cannot proceed efficiently in bacteria growing with a doubling time longer than 160 min. Contrary to this prediction, using a chemostat culture system allowing for culturing E. coli at different growth rates without changes in the medium composition, we found that T4 can yield progeny in host cells growing with a doubling time as long as 21 h. Our results indicate that the actual limiting growth rate of the host culture for the development of phage T4 is about 0.033 h(-1) , corresponding to the doubling time of about 21 h. PMID:24386916

Golec, Piotr; Karczewska-Golec, Joanna; ?o?, Marcin; W?grzyn, Grzegorz

2014-02-01

75

Bacteriophage  

NSDL National Science Digital Library

These online lecture notes define bacteriophage and review the composition and structure of bacteriophage, infection of host cells, and phage multiplication cycles (lytic and lysogenic). The notes include several supplements such as animations, images and illustrations, a downloadable movie and a Microbe Radio broadcast. The link at the bottom of the page also directs users to a list of correlating PowerPoint presentations.

Mayer, Gene; Medicine, University O.

76

Analysis of five presumptive protein-coding sequences clustered between the primosome genes, 41 and 61, of bacteriophages T4, T2, and T6.  

PubMed Central

In bacteriophage T4, there is a strong tendency for genes that encode interacting proteins to be clustered on the chromosome. There is 1.6 kb of DNA between the DNA helicase (gene 41) and the DNA primase (gene 61) genes of this virus. The DNA sequence of this region suggests that it contains five genes, designated as open reading frames (ORFs) 61.1 to 61.5, predicted to encode proteins ranging in size from 5.94 to 22.88 kDa. Are these ORFs actually genes? As one test, we compared the DNA sequence of this region in bacteriophages T2, T4, and T6 and found that ORFs 61.1, 61.3, 61.4, and 61.5 are highly conserved among the three closely related viruses. In contrast, ORF 61.2 is conserved between phages T4 and T6 yet is absent from phage T2, where it is replaced by another ORF, T2 ORF 61.2, which is not found in the T4 and T6 genomes. As a second, independent test for coding sequences, we calculated the codon base position preferences for all ORFs in this region that could encode proteins that contain at least 30 amino acids. Both the T4/T6 and T2 versions of ORF 61.2, as well as the other ORFs, have codon base position preferences that are indistinguishable from those of known T4 genes (coefficients of 0.81 to 0.94); the six other possible ORFs of at least 90 bp in this region are ruled out as genes by this test (coefficients less than zero). Thus, both evolutionary conservation and codon usage patterns lead us to conclude that ORFs 61.1 to 61.5 represent important protein-coding sequences for this family of bacteriophages. Because they are located between the genes that encode the two interacting proteins of the T4 primosome (DNA helicase plus DNA primase), one or more may function in DNA replication by modulating primosome function. Images PMID:8383243

Selick, H E; Stormo, G D; Dyson, R L; Alberts, B M

1993-01-01

77

Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase  

SciTech Connect

The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined. UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant. T4 denV phage were equally sensitive when plated on wild-type E. coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase. A uvrA mutant of E. coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed. Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation. We were unable to construct a uvrA xth nfo triple mutant. In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.

Saporito, S.M.; Gedenk, M.; Cunningham, R.P.

1989-05-01

78

Changes in major capsid genes ( g23 ) of T4-type bacteriophages with soil depth in two Japanese rice fields  

Microsoft Academic Search

Although microbial communities in soil are well known to change with soil depth, the changes in viral communities with soil\\u000a depth have not been documented. This study examined the soil depth profiles of T4-type phage communities in two Japanese rice\\u000a fields from g23 clones in soil DNA extracts to a depth of 1 m. T4-type phage communities changed with soil depth,

Guanghua Wang; Jun Murase; Katsutoshi Taki; Yoshinori Ohashi; Nanako Yoshikawa; Susumu Asakawa; Makoto Kimura

2009-01-01

79

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

PubMed

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

Chang, P L; Yen, T F

1984-03-01

80

Genetic studies on capsid-length determination in bacteriophage T4. II. Genetic evidence that specific protein-protein interactions are involved.  

PubMed Central

A bacteriophage T4 mutation (ptg19-80c) located in gene 23, which encodes the major structural protein of the T4 capsid, results in the production of capsids of abnormal length. Mutations outside gene 23 which partially suppress ptg19-80c have been described in the accompanying paper (D. H. Doherty, J. Virol. 43:641-654, 1982). Characterization of these suppressors was extended. A complementation test suggested that the suppressors were in genes 22 and 24. These genes coded for the major component of the morphogenetic core of the capsid precursor and the vertex protein of the capsid, respectively. The suppressor mutations were found to have no obvious phenotype in the absence of ptg19-80c. Suppression was shown to be allele specific: other ptg mutations at different sites in gene 23 were not suppressed by the suppressors of ptg19-80c. These results indicated that specific interactions among the three proteins gp22, gp23, and gp24 may play a role in the regulation of T4 capsid-length determination. Current models for capsid-length determination are considered in the light of these results. PMID:7109035

Doherty, D H

1982-01-01

81

Structural Determinants of Nitroxide Motion in Spin-Labeled Proteins: Solvent-Exposed Sites in Helix B of T4 Lysozyme  

SciTech Connect

Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.

Guo,Z.; Cascio, D.; Hideg, K.; Hubbell, W.

2008-01-01

82

Structural Determinants of Nitroxide Motion in Spin-labeled Proteins: Tertiary Contact and Solvent-inaccessible Sties in Helix G of T4 Lysozyme  

SciTech Connect

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.

Guo,Z.; Cascio, D.; Hideg, K.; Kalai, T.; Hubbell, W.

2007-01-01

83

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

PubMed Central

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

Guo, Zhefeng; Cascio, Duilio; Hideg, Kalman; Kalai, Tamas; Hubbell, Wayne L.

2007-01-01

84

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

PubMed Central

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

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

2013-01-01

85

pH-Induced denaturation of proteins: A single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme  

SciTech Connect

The energetics of a salt bridge formed between the side chains of aspartic acid 70 (Asp70) and histidine 31 (His31) of T4 lysozyme have been examined by nuclear magnetic resonance techniques. The pK{sub a} values of the residues in the native state are perturbed from their values in the unfolded protein such that His31 has a pK{sub a} value of 9.1 in the native state and 6.8 in the unfolded state at 10{degree}C in moderate salt. Similarly, the aspartate pK{sub a} is shifted to a value of about 0.5 in the native state from its value of 3.5-4.0 in the unfolded state. These shifts in pK{sub a} show that the salt bridge is stabilized 3-5 kcal/mol. These observations and consideration of the thermodynamic coupling of protonation state to folding of proteins suggest a mechanism of acid denaturation in which the unfolded state is progressively stabilized by protonation of its acid residues as pH is lowered below pH 4. The unfolded state is stabilized only if acidic groups in the folded state have lower pK{sub a} values than in the unfolded state. When the pH is sufficiently low, the acid groups of both the native and unfolded states are fully protonated, and the apparent unfolding equilibrium constant becomes pH independent. Similar arguments apply to base-induced unfolding. These observations suggest that the electrostatic contribution of each ionizable group to the stability of the folded state can be directly assessed by simply measuring its apparent pK{sub a} by NMR or other methods.

Anderson, D.E.; Becktel, W.J.; Dahlquist, F.W. (Univ. of Oregon, Eugene (USA))

1990-03-06

86

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

PubMed Central

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

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

87

RNA Initiation with Dinucleoside Monophosphates during Transcription of Bacteriophage T4 DNA with RNA Polymerase of Escherichia coli  

PubMed Central

The effects of dinucleoside monophosphates on the transcription of phage T4 DNA by E. coli RNA polymerase have been examined at various concentrations of the sigma subunit and extremely low concentration of ribonucleoside triphosphate. The following conclusions were reached: (i) Labeled specific dinucleoside monophosphates are incorporated as chain initiators. (ii) When the ratio of sigma factor to core enzyme is small, there is a general stimulation by most 5?-guanosyl dinucleoside monophosphates. (iii) When the ratio is increased or holoenzyme is present, ApU, CpA, UpA, and GpU are the most effective stimulators. (iv) At high concentrations of sigma factor, only certain adenosine-containing dinucleoside monophosphates (ApU, CpA, UpA, and ApA) stimulate the reaction. (v) Competition hybridization studies indicate that the RNAs stimulated by dinucleoside monophosphates (ApU, CpA, UpA, and GpU) are of the T4 “early” type. (vi) Studies involving both combinations of stimulatory dinucleoside monophosphates and competitive effects of these compounds on chain initiation by ATP and GTP suggest that the stimulatory dinucleoside monophosphates act as chain initiators and may recognize part of a continuous sequence in a promoter region. Studies based on the incorporation of 3H-labeled stimulatory dinucleoside monophosphates support the above conclusions. PMID:4568732

Hoffman, David J.; Niyogi, Salil K.

1973-01-01

88

P15 and P3, the Tail Completion Proteins of Bacteriophage T4, Both Form Hexameric Rings  

PubMed Central

Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed. PMID:12591887

Zhao, Li; Kanamaru, Shuji; Chaidirek, Chatree'chalerm; Arisaka, Fumio

2003-01-01

89

Polyamines in the Synthesis of Bacteriophage Deoxyribonucleic Acid II. Requirement for Polyamines in T4 Infection of a Polyamine Auxotroph  

PubMed Central

Polyamine depletion produced by exogenous arginine in Escherichia coliK-12 cultures defective in agmatine ureohydrolase activity resulted in a marked inhibition of the rates of growth and nucleic acid synthesis. Addition of putrescine or spermidine to such depleted cultures restored the control rate of growth and nucleic acid accumulation. The omission of lysine resulted in a further decrease in the rates of growth and nucleic acid synthesis in polyamine-depleted cells. The addition of exogenous cadaverine increased the rates of growth and ribonucleic acid synthesis to those observed in lysine-supplemented cultures, suggesting that lysine or a derivative of lysine serves a function similar to cadaverine. Addition of lysine to polyamine-depleted cultures at neutral pH results in the synthesis of cadaverine and a new spermidine analogue, both containing lysine carbon. This new metabolite has been isolated and identified as N-3-aminopropyl-1, 5-diaminopentane. T4D infection of the polyamine-depleted mutant resulted in a very low rate of DNA synthesis and phage maturation. The addition of putrescine or spermidine 15 min before infection restored phage DNA synthesis and phage maturation to control rates, i.e., rates observed in infected cells grown in the absence of arginine. Images PMID:4552550

Dion, Arnold S.; Cohen, Seymour S.

1972-01-01

90

Genetic control of capsid length in bacteriophage T4: DNA sequence analysis of petite and petite/giant mutants.  

PubMed Central

The T4 gene 23 product (gp23) encodes the major structural protein of the mature capsid. Mutations in this gene have been described which disrupt the normal length-determining mechanism (A.H. Doermann, F.A. Eiserling, and L. Boehner, J. Virol. 12:374-385, 1973). Mutants which produce high levels of petite and giant phage (ptg) are restricted to three tight clusters in gene 23 (A.H. Doermann, A. Pao, and P. Jackson, J. Virol. 61:2823-2827, 1987). Twenty-six of these ptg mutations were cloned, and their DNA sequence alterations were determined. Each member of this set of ptg mutants arose from a single mutation, and the set defined 10 different sites at which ptg mutations can occur in gene 23. Two petite (pt) mutations in gene 23 (pt21-34 and ptE920g), which produce high frequencies of petite particles but no giants, were also sequenced. Both pt21-34 and ptE920g were shown to include multiple mutations. The phenotypes attributed to both pt and ptg mutations are discussed relative to the mechanism of capsid morphogenesis. A site-directed mutation (SD-1E) was created at the ptgNg191 site, and its phenotypic consequences were examined. Plaque morphology revertants arising from a gene 23 mutant derivative of pt21-34 and from SD-1E were isolated. A preliminary mapping of the mutation(s) responsible for their revertant phenotypes suggested that both intra- and extragenic suppressors of the petite phenotype can be isolated by this method. PMID:3612953

Mooney, D T; Stockard, J; Parker, M L; Doermann, A H

1987-01-01

91

The Effect of Bacteriophage T4 and PB-1 Infection with Tobramycin on the Survival of Escherichia coli and Pseudomonas aeruginosa Biofilms.  

E-print Network

??Populations of bacterial cells growing as biofilms demonstrate greater resistance to antibiotics compared to planktonic cells. Consequently, there is renewed interest in bacteriophage therapy as… (more)

Coulter, Lindsey Blythe

2012-01-01

92

Structural basis for the nucleic acid binding cooperativity of bacteriophage T4 gene 32 protein: the (Lys/Arg)3(Ser/Thr)2 (LAST) motif.  

PubMed

To identify the functional residues of the N-terminal B region of bacteriophage T4 gene 32 protein involved in its cooperative binding to single-stranded nucleic acids, a process dependent on homotypic protein-protein interaction, we have studied the interaction of the protein with synthetic peptides containing different portions of this domain. Gel-permeation chromatography showed that a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan)-labeled fluorescent peptide corresponding to the first 17 residues of gene 32 protein formed a complex with whole protein. The fluorescence was blue-shifted 14 nm upon interaction with intact protein, and somewhat less so (7-11 nm) with cleavage products of the protein lacking B domains. The intrinsic tryptophan fluorescence of whole and truncated protein was quenched by this peptide and by the nonderivatized peptide. The peptide bound tightly to truncated protein at both 0.015 and 0.44 M Na+, with a stoichiometry of 1:1. Similar tryptophan quenching or acrylodan blue shifts were obtained with peptides corresponding to residues 1-9 and 3-8, but not residues 1-4, 5-9, or 5-17, indicating that the essential amino acids are contained within positions 3-8, Lys-Arg-Lys-Ser-Thr-Ala. Several other DNA binding proteins contain a LAST motif with documented involvement of these residues in nucleic acid interaction. The amino acid and coding sequence of residues 110-114, a region proposed to be involved in nucleic acid binding, is virtually identical to that of residues 3-7. Based on these observations, we have formulated a model for the cooperative interactions of gene 32 protein with single-stranded nucleic acids. PMID:1736285

Casas-Finet, J R; Fischer, K R; Karpel, R L

1992-02-01

93

Structural basis for the nucleic acid binding cooperativity of bacteriophage T4 gene 32 protein: the (Lys/Arg)3(Ser/Thr)2 (LAST) motif.  

PubMed Central

To identify the functional residues of the N-terminal B region of bacteriophage T4 gene 32 protein involved in its cooperative binding to single-stranded nucleic acids, a process dependent on homotypic protein-protein interaction, we have studied the interaction of the protein with synthetic peptides containing different portions of this domain. Gel-permeation chromatography showed that a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan)-labeled fluorescent peptide corresponding to the first 17 residues of gene 32 protein formed a complex with whole protein. The fluorescence was blue-shifted 14 nm upon interaction with intact protein, and somewhat less so (7-11 nm) with cleavage products of the protein lacking B domains. The intrinsic tryptophan fluorescence of whole and truncated protein was quenched by this peptide and by the nonderivatized peptide. The peptide bound tightly to truncated protein at both 0.015 and 0.44 M Na+, with a stoichiometry of 1:1. Similar tryptophan quenching or acrylodan blue shifts were obtained with peptides corresponding to residues 1-9 and 3-8, but not residues 1-4, 5-9, or 5-17, indicating that the essential amino acids are contained within positions 3-8, Lys-Arg-Lys-Ser-Thr-Ala. Several other DNA binding proteins contain a LAST motif with documented involvement of these residues in nucleic acid interaction. The amino acid and coding sequence of residues 110-114, a region proposed to be involved in nucleic acid binding, is virtually identical to that of residues 3-7. Based on these observations, we have formulated a model for the cooperative interactions of gene 32 protein with single-stranded nucleic acids. PMID:1736285

Casas-Finet, J R; Fischer, K R; Karpel, R L

1992-01-01

94

Assembly of the Small Outer Capsid Protein, Soc, on Bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid  

PubMed Central

Summary Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a “triplex” protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N- and C-termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N- and C-termini facing the two- and three-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology. PMID:17544446

Li, Qin; Shivachandra, Sathish B.; Zhang, Zhihong; Rao, Venigalla B.

2007-01-01

95

Photocatalytic antimicrobial activity of thin surface films of TiO 2 , CuO and TiO 2 \\/CuO dual layers on Escherichia coli and bacteriophage T4  

Microsoft Academic Search

TiO2-coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with\\u000a thin films of TiO2, CuO and hybrid CuO\\/TiO2 prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO2 prepared by a sol–gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of\\u000a viruses. The chemical oxidising activity was also

Iram B. Ditta; Alex Steele; Christopher Liptrot; Julie Tobin; Helen Tyler; Heather M. Yates; David W. Sheel; Howard A. Foster

2008-01-01

96

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

PubMed Central

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

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

2014-01-01

97

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

PubMed

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

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

2012-02-17

98

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

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.

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

99

The non-enzymatic microbicidal activity of lysozymes  

Microsoft Academic Search

T4 lysozyme was thought to destroy bacteria by its muramidase activity. However, we demonstrate here that amphipathic helix stretches in the C-terminus of T4 lysozyme mediate its bactericidal and fungistatic activities. In heat-denatured T4 lysozyme, the enzymatic activity is completely abolished but unexpectedly, the antimicrobial functions remain preserved. Small synthetic peptides corresponding to amphipathic C-terminal domains of T4 lysozyme show

Petra Porsch; Andreas Mahn; Olaf Brinkmann; Werner Gieffers

1999-01-01

100

Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.  

PubMed Central

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated. PMID:8880907

Ren, Z. J.; Lewis, G. K.; Wingfield, P. T.; Locke, E. G.; Steven, A. C.; Black, L. W.

1996-01-01

101

The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of ?70 Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between ?70 and Core  

PubMed Central

The N-terminal region (region 1.1) of ?70, the primary ? subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of ?70 regions 2 and 4. Region 1.1 prevents the interaction of free ?70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of ?70-dependent transcription from promoters that require an interaction between ?70 region 4 and the ?35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with ?70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that ?70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter PuvsX. However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with ?70 that lacks region 1.1 is less stable than polymerase with full-length ?70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free ?70 and then the AsiA/?70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free ?70 plus core, yielding more free ?70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free ?70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free ?70 arises through an indirect effect. PMID:16452409

Hinton, Deborah M.; Vuthoori, Srilatha; Mulamba, Rebecca

2006-01-01

102

Genetic studies on capsid-length determination in bacteriophage T4. I. Isolation and partial characterization of second-site revertants of a gene 23 mutation affecting capsid length.  

PubMed Central

The T4 mutation ptg19-80 affects the mechanism of capsid-length determination. It is located in gene 23, which encodes the major structural protein of the capsid. The mutation results in the production of abnormal-length capsids in high frequencies. This paper describes the isolation and partial characterization of second-site revertants of ptg19-80. In the course of their analysis, it was discovered that ptg19-80 is itself a double mutation consisting of a gene 23 mutation (ptg19-80c), which causes the morphogenetic defect, and a suppressor mutation located near the lysozyme gene. Phenotypic characterization of nine pseudo-wild-type revertants of this double-mutation revealed that these revertants all produced lower frequencies of abnormal capsids than did ptg19-80. Seven of these revertants were shown to contain two suppressor mutations, one mapping in or near gene 22 and done mapping in or near gene 24. Both mutations were required for suppression. These suppressors displayed no discernible phenotype in the absence of ptg19-80c. Images PMID:7109034

Doherty, D H

1982-01-01

103

Protein Engineering with Biosynthesized Libraries from Bordetella bronchiseptica Bacteriophage  

PubMed Central

Phage display offers a powerful approach to engineer protein affinity. A naturally occurring analog to phage display, the Bordetella bronchiseptica bacteriophage (BP) employs a highly variable protein termed the major tropism determinant (Mtd) to recognize its dynamic host. Propagation of BP provides a self-made phage library (SMPL) with vast numbers of phage particles, each displaying a single Mtd variant. We report applying the diversity of the BP-SMPL to access a tyrosine-rich library of Mtd variants. Expression of the SMPL-engineered Mtd variant as a GST-bound fusion protein demonstrated specific binding to the target T4 lysozyme with dissociation constants in the sub-micromolar range. The results guide future experiments with SMPLs applied to protein engineering. PMID:23409008

Yuan, Tom Z.; Overstreet, Cathie M.; Moody, Issa S.; Weiss, Gregory A.

2013-01-01

104

Protein engineering with biosynthesized libraries from Bordetella bronchiseptica bacteriophage.  

PubMed

Phage display offers a powerful approach to engineer protein affinity. A naturally occurring analog to phage display, the Bordetella bronchiseptica bacteriophage (BP) employs a highly variable protein termed the major tropism determinant (Mtd) to recognize its dynamic host. Propagation of BP provides a self-made phage library (SMPL) with vast numbers of phage particles, each displaying a single Mtd variant. We report applying the diversity of the BP-SMPL to access a tyrosine-rich library of Mtd variants. Expression of the SMPL-engineered Mtd variant as a GST-bound fusion protein demonstrated specific binding to the target T4 lysozyme with dissociation constants in the sub-micromolar range. The results guide future experiments with SMPLs applied to protein engineering. PMID:23409008

Yuan, Tom Z; Overstreet, Cathie M; Moody, Issa S; Weiss, Gregory A

2013-01-01

105

Single Molecule Recordings of Lysozyme Activity  

PubMed Central

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

Choi, Yongki; Weiss, Gregory A.

2013-01-01

106

Lysozyme Crystal  

NASA Technical Reports Server (NTRS)

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

2004-01-01

107

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

PubMed Central

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

Jucovic, M.; Poteete, A. R.

1998-01-01

108

The bacteriophage T4 inhibitor and coactivator AsiA inhibits Escherichia coli RNA Polymerase more rapidly in the absence of sigma70 region 1.1: evidence that region 1.1 stabilizes the interaction between sigma70 and core.  

PubMed

The N-terminal region (region 1.1) of sigma70, the primary sigma subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of sigma70 regions 2 and 4. Region 1.1 prevents the interaction of free sigma70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of sigma70-dependent transcription from promoters that require an interaction between sigma70 region 4 and the -35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with sigma70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that sigma70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P(uvsX). However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with sigma70 that lacks region 1.1 is less stable than polymerase with full-length sigma70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free sigma70 and then the AsiA/sigma70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free sigma70 plus core, yielding more free sigma70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free sigma70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free sigma70 arises through an indirect effect. PMID:16452409

Hinton, Deborah M; Vuthoori, Srilatha; Mulamba, Rebecca

2006-02-01

109

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 ...

110

Nanoscale bacteriophage biosensors beyond phage display  

PubMed Central

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

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

2013-01-01

111

Lytic bacteriophages  

PubMed Central

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

Sharma, Manan

2013-01-01

112

t4 Workshop Report  

PubMed Central

Summary In October 2010, a group of experts met as part of the transatlantic think tank for toxicology (t4) to exchange ideas about the current status and future of safety testing of nanomaterials. At present, there is no widely accepted path forward to assure appropriate and effective hazard identification for engineered nanomaterials. The group discussed needs for characterization of nanomaterials and identified testing protocols that incorporate the use of innovative alternative whole models such as zebrafish or C. elegans, as well as in vitro or alternative methods to examine specific functional pathways and modes of action. The group proposed elements of a potential testing scheme for nanomaterials that works towards an integrated testing strategy, incorporating the goals of the NRC report Toxicity Testing in the 21st Century: A Vision and a Strategy by focusing on pathways of toxic response, and utilizing an evidence-based strategy for developing the knowledge base for safety assessment. Finally, the group recommended that a reliable, open, curated database be developed that interfaces with existing databases to enable sharing of information. PMID:21993959

Silbergeld, Ellen K.; Contreras, Elizabeth Q.; Hartung, Thomas; Hirsch, Cordula; Hogberg, Helena; Jachak, Ashish C.; Jordan, William; Landsiedel, Robert; Morris, Jeffery; Patri, Anil; Pounds, Joel G.; de Vizcaya Ruiz, Andrea; Shvedova, Anna; Tanguay, Robert; Tatarazako, Norihasa; van Vliet, Erwin; Walker, Nigel J.; Wiesner, Mark; Wilcox, Neil; Zurlo, Joanne

2014-01-01

113

Template reporter bacteriophage platform and multiple bacterial detection assays based thereon  

NASA Technical Reports Server (NTRS)

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

Goodridge, Lawrence (Inventor)

2007-01-01

114

Rate of Lysozyme Crystallization  

NASA Astrophysics Data System (ADS)

We have observed the following: Free solution measurements of the electrophoretic mobility of hen egg-white lysozyme crystals grown in aqueous NaCl at 10 deg C at pH values between 3.6 and 5.7 demonstrate that the crystals are positively charged.(J.K. Baird, A.M. Holmes, and J.C. Clunie, Bull.Am.Phys.Soc. 41, 620 (1996)) (2) When the decaying concentration of uncrystallized lysozyme in the growth solution is monitored as a function of time, the log of the half-life decreases linearly with the square-root of the ionic strength. (3) Acid-base titration shows that lysozyme molecules in solution exist as highly charged cations.(R. Roxby and C. Tanford, Biochemistry 10, 3348 (1971)) These three observations combine to suggest that lysozyme crystallizes by addition of lysozyme cations to positively charged crystal nuclei and that the rate is accelerated by the presence of strong electrolytes.

Baird, J. K.; Clunie, J. C.

1997-03-01

115

Three functions of bacteriophage P1 involved in cell lysis.  

PubMed Central

Amber and deletion mutants were used to assign functions in cell lysis to three late genes of bacteriophage P1. Two of these genes, lydA and lydB of the dar operon, are 330 and 444 bp in length, respectively, with the stop codon of lydA overlapping the start codon of lydB. The third, gene 17, is 558 bp in length and is located in an otherwise uncharacterized operon. A search with the predicted amino acid sequence of LydA for secondary motifs revealed a holin protein-like structure. Comparison of the deduced amino acid sequence of gene 17 with sequences of proteins in the SwissProt database revealed homologies with the proteins of the T4 lysozyme family. The sequence of lydB is novel and exhibited no known extended homology. To study the effect of gp17, LydA, and LydB in vivo, their genes were cloned in a single operon under the control of the inducible T7 promoter, resulting in plasmid pAW1440. A second plasmid, pAW1442, is identical to pAW1440 but has lydB deleted. Induction of the T7 promoter resulted in a rapid lysis of cells harboring pAW1442. In contrast, cells harboring pAW1440 revealed only a small decrease in optical density at 600 nm compared with cells harboring vector alone. The rapid lysis phenotype in the absence of active LydB suggests that this novel protein might be an antagonist of the holin LydA. PMID:8576044

Schmidt, C; Velleman, M; Arber, W

1996-01-01

116

Microneedle-mediated transdermal bacteriophage delivery  

PubMed Central

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

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

2012-01-01

117

Bacteriophages Infecting Propionibacterium acnes  

PubMed Central

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

2013-01-01

118

What's new in lysozyme research?  

Microsoft Academic Search

Summary The present review is focused on the main achievements realized in the lysozyme research field since the meeting held in 1972 to commemorate the fiftieth anniversary of the discovery of this enzyme. Despite of extensive structural, physico-chemical, crystallographic, genetic, immunological and evolutionary studies devoted to lysozymes, their biological role is still not exactly known.

Pierre Jollès; Jacqueline Jollès

1984-01-01

119

Surface-immobilization of chromatographically purified bacteriophages for the optimized capture of bacteria  

PubMed Central

Bacteriophages offer interesting alternatives to antibodies for the specific capture and detection of pathogenic bacteria onto biosensing surfaces. Procedures for the optimal chemical immobilization of lytic bacteriophages onto surfaces are presented. More specifically, the removal of lysate contaminants from bacteriophage suspensions by size exclusion chromatography significantly increases the resultant planar surface density of immobilized bacteriophages. E. coli T4 and Salmonella enterica serovar Typhimurium P22 phage systems seem to undergo highly heterogeneous adsorption to the surface, possibly explaining the observed phage clustering at higher surface densities. The T4 phage and its E. coli host were initially employed as a model system where we discovered an optimal planar surface density of phages for best bacterial capture: 18.9 ± 0.8 phages/?m2 capturing 18.0 ± 0.3 bacteria/100 ?m2. Phage surface clustering ultimately limits the T4 phage-immobilized surface’s ability to specifically capture its host bacteria. Nevertheless, this is to our knowledge the largest surface capture density of E. coli reported using intact T4 bacteriophages. Two additional purified bacteriophage systems (P22 and Campylobacter jejuni phage NCTC 12673) were then similarly studied for their ability to capture their corresponding host bacteria (Salmonella enterica serovar Typhimurium and Campylobacter jejuni respectively) on a surface. PMID:22666653

Naidoo, Ravendra; Singh, Amit; Arya, Sunil K.; Beadle, Bernadette; Glass, Nick; Tanha, Jamshid; Szymanski, Christine M.; Evoy, Stephane

2012-01-01

120

Structure of the small outer capsid protein, Soc: a clamp for stabilizing capsids of T4-like phages.  

PubMed

Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a "glue" between neighboring hexameric capsomers, forming a "cage" that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 A resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids. PMID:19835886

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

2010-01-29

121

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

PubMed Central

Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9 kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as “glue” between neighboring hexameric capsomers, forming a “cage” that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 Å resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryo-electron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids. PMID:19835886

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

2009-01-01

122

IQENS from hydrated lysozyme  

NASA Astrophysics Data System (ADS)

We present quasi-elastic incoherent neutron scattering data on wet lysozyme powders at water contents (grams of water/grams of protein) of h=0.20 and 0.45, corresponding to hydration levels below and above the full monolayer coverage of the protein surface. The data were collected under H2O and D2O hydration condition; using the IRIS spectrometer with a resolution of 11 ?eV (full-width). From spectra for the D2O hydrated sample we obtain information on the protein dynamics. These spectra are used to separate from the H2O hydrated sample the contribution due to the hydration water. We show that at low hydration the dynamics of water is very slow and on the same time scale as that of the protein dynamics, while at higher hydration the water molecules show a faster dynamics. The results support the idea that there are clusters of hydration water.

Wanderlingh, U. N.; Giordano, R.; Kagunya, W. W.

1998-04-01

123

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

PubMed

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

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

2014-11-01

124

A bioluminescence-based assay for enumeration of lytic bacteriophage.  

PubMed

A bioluminescence-based assay for enumeration of lytic bacteriophage was developed. The assay consists of a bioluminescent Escherichia coli as the host bacterium, the lytic bacteriophage T4 and an automated luminometer measuring luminescence over time. The assay is based on the decrease in luminescence as the bioluminescent host cells are lysed by T4. The T4 concentration, bioluminescent E. coli concentration, phage suspension medium, and temperature (25 degrees C and 37 degrees C) were varied. There was a strong negative correlation between bioluminescence intensities and T4 phage concentrations at both room temperature (R(2)=0.993) and 37 degrees C (R(2)=0.970). Phage was detected more rapidly at 37 degrees C than at 25 degrees C. The detection limit was also lower when the assay was performed at 37 degrees C with a minimum detection level of 2.4 log CFU/ml compared to 3.4 log CFU/ml for 25 degrees C. The assay was used to determine thermal inactivation using T4 phages heated at 70 degrees C for 0 to 30 min, and phage concentrations were determined using the bioluminescence assay and a standard plaque assay. There was no significant difference between the two enumeration methods (P>0.01). This study suggests the bioluminescence-based assay can be used as an alternative for quantitatively monitoring phage infectivity, instead of conventional standard plaque assays. PMID:19628012

Kim, S; Schuler, B; Terekhov, A; Auer, J; Mauer, L J; Perry, L; Applegate, B

2009-10-01

125

IMMUNOCHEMICAL STUDIES ON THE HUMAN SKIN LYSOZYME  

Microsoft Academic Search

Rabbit antiserum was prepared against crystalline hen egg white and human milk lysozymes. The immunological cross-reactions among egg white lysozyme and human lysozymes (skin, milk, leucocyte and crude tears) were studied by the qualitative gel diffusion precipitation technique and quantitative neutralization test using anti-E.W.L. Serum and anti-H.M.L. Serum. Immunological identity of the human skin lysozyme with both the human milk

Hideoki Ogawa; Hiroaki Miyazaki

1972-01-01

126

Division M: Bacteriophage  

NSDL National Science Digital Library

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.

2007-07-23

127

Method for Produce Sanitation using Bacteriophages.  

National Technical Information Service (NTIS)

A method for produce sanitation using bacteriophages is disclosed. According to one embodiment of the present invention, the method includes the steps of (1) providing at least one bacteriophage; and (2) applying the bacteriophage to the produce. The prod...

A. Sulakvelidze, J. G. Morris, Z. Alavidze, G. R. Pasternack, T. C. Brown

2003-01-01

128

Kinetic Traps in Lysozyme Folding  

Microsoft Academic Search

Folding of lysozyme from hen egg white was investigated by using interrupted refolding experiments. This method makes use of a high energy barrier between the native state and transient folding intermediates, and, in contrast to conventional optical techniques, it enables one to specifically monitor the amount of native molecules during protein folding. The results show that under strongly native conditions

Thomas Kiefhaber

1995-01-01

129

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

ERIC Educational Resources Information Center

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

Szeberenyi, Jozsef

2008-01-01

130

Cytoplasmic bacteriophage display system  

DOEpatents

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

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

1998-06-16

131

Bacteriophage-Based Biosensors  

Microsoft Academic Search

\\u000a Bacteriophages, or phages for short, are viruses that infect bacteria and are considered the main regulators of microbial\\u000a balance on Earth. These viruses are extremely specific, and their long-term survivability and ability to reproduce quickly\\u000a in suitable hosts play a major role in the preservation of a dynamic equilibrium amid the diverse variety of bacterial species\\u000a in the Earth’s ecosystem.

Mohammed Zourob; Steven Ripp

132

Structural aberrations in T-even bacteriophage. IX. Effect of mixed infection on the production of giant bacteriophage.  

PubMed Central

To date, the production of T-even bacteriophage with giant heads has been achieved in two ways: (i) by use of canavanine-arginine treatment of Escherichia coli B cultures infected by wild-type bacteriophage (Cummings and Bolin, Bacteriol. Rev. 40:314-359, 1976; Cummings et al., Virology 54:245-261, 1973), which give a size distribution of giants that is phage specific (Cummings et al., Virology 54:245-261, 1973); and (ii) by infection with certain missense mutants of T4D gene 23 (Doermann et al., J. Virol. 12:374-385, 1973; ICN-UCLA Symposium on Molecular Biology, p. 243-285, 1973) or temperature-sensitive mutants of gene 24 (Aebi et al., J. Supramol. Struct. 2:253-275, 1974; Biljenga et al., J. Mol. Biol. 103:469-498, 1976). We now report the effect of mixed infection with several mutants of T4D on both the production and the size of giant bacteriophage. We found that gene 24 mutant is a critical partner for the production of giants. Infection using T4.24 mutants together with either T4.23 mutants, T4B+ or T6+ led to the formation of giants with heads 10- to 14-fold longer than normal-length heads. Infection with amber 24-bypass 24 double mutants of T4D led to the production of giants when gene 23 mutant was used to co-infect. Addition of canavanine to the co-infected cultures could alter the size distribution of giants, depending on which phage were used to coinfect. Gene 22 mutants had a modifying effect on these results. In the absence of canavanine co-infection with gene 22 mutants prevented the production of giants, and in the presence of canavanine giants of 1.5 to 5 head lengths were found. We have interpreted these results to mean that critical concentrations of gene products 22, 23, and 24 interact to control head length in T-even bacteriophage. Images PMID:864836

Cummings, D J; Chapman, V A; DeLong, S S

1977-01-01

133

Scientist prepare Lysozyme Protein Crystal  

NASA Technical Reports Server (NTRS)

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

1996-01-01

134

A noncovalent switch for lysozyme.  

PubMed

A new concept for the external control of protein activity is presented and demonstrated on the example of an artificial Lysozyme switch. Radical copolymerization of selected methacrylamide-based comonomer units tailored for amino acid residues surrounding the active site furnishes polymeric protein hosts that are able to inhibit enzymatic activity in a highly efficient dose-dependent manner (IC50 approximately 1.0 equiv approximately 0.7 microM). All binding site types on the polymer work cooperatively, using electrostatic attraction, hydrophobic forces, and substrate mimicry. In a native gel electrophoresis, the well-defined 2:1 complex (polymer/protein) migrates to the anode. Even at 250 mM NaCl, a 10-fold polymer excess is able to shut down bacterial cell wall degradation completely. A kinetic investigation points to a competitive mechanism (Lineweaver-Burk plots). CD spectra of pure Lysozyme and its polymer complex are indistinguishable; together with a total lack of preincubation time for maximum inhibition, experimental evidence is thus produced for a preserved tertiary enzyme structure-no denaturation occurs. Addition of the superior complexing agent polyarginine to the enzyme-polymer complex mildly detaches the inhibitor from the protein surface and leads to 90% recovery of enzymatic activity. Thus, Lysozyme could be turned off, on, and off again by consecutive addition of the polymeric inhibitor, polyarginine, and polymer again. PMID:18052064

Wenck, Kirstin; Koch, Sebastian; Renner, Christian; Sun, Wei; Schrader, Thomas

2007-12-26

135

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2012 CFR

... (a) Identification. A lysozyme (muramidase) test system is a device intended to measure the activity of the bacteriolytic enzyme lysozyme (muramidase) in serum, plasma, leukocytes, and urine. Lysozyme measurements are used...

2012-04-01

136

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2013 CFR

... (a) Identification. A lysozyme (muramidase) test system is a device intended to measure the activity of the bacteriolytic enzyme lysozyme (muramidase) in serum, plasma, leukocytes, and urine. Lysozyme measurements are used...

2013-04-01

137

Control of gene function in baceriophage T4. II. Synthes of messenger ribonucleiccid and proei after interrupting deoxyribonucleic acid replication and glucosylation.  

PubMed

Replication of T4 deoxyribonucleic acid (DNA) is known to be required for the onset of transcription of late T4 genes. Once late gene transcription has been initiated, DNA replication is no longer required for maintaining synthesis of late or early T4 messenger ribonucleic acid (mRNA). Late phage proteins (lysozyme and tail fibers) continue to be produced at constant rates after interrupting T4 DNA synthesis. The ability of the host cell to glucosylate the T4 progeny DNA has no demonstrable influence on the rates at which T4 mRNA and late proteins are synthesized after the interruption of DNA synthesis. To explain the requirement of T4 DNA replication for the onset of late gene transcription, we suggest that T4 DNA in a nascent state is mandatory for the initial late gene transcription, or perhaps for late gene transcription throughout the lytic cycle. T4 DNA in a nascent state could be segregated from the bulk of the replicating DNA, used only as template for RNA synthesis, and prevented from being modified by methylation, glucosylation, or maturation processes. The fact that no, or very little, nonglucosylated T4 DNA is extractable from T4LB3-infected CR63 after arresting DNA synthesis does not rule out this possibility. PMID:4910356

Sauerbier, W; räutigam, A R

1970-02-01

138

Thyroxine (T4) release from thyroglobulin and its T4-containing peptide by thyroid thiol proteases.  

PubMed

Two thiol proteases, TP-1 and TP-2, were purified 500- and 400-fold, respectively, from hog thyroid lysosomal extracts and examined for their activities as releasers of T4 1) from hog thyroglobulin, 2) a fragment (mol wt, approximately 15,000) prepared therefrom, and 3) a T4-containing peptide (19 amino acid residues) derived from the fragment, by measuring the liberation of free T4 with reversed phase HPLC. TP-1 released 8%, 55%, and 95% of the bound T4 present in thyroglobulin, the fragment, and the peptide, respectively, after incubation for 8 h. TP-2, on the other hand, released only 2% of the bound T4 in thyroglobulin and liberated no T4 from the fragment and the peptide. The release of T4 from thyroglobulin by the action of TP-1 was increased approximately 2-fold by the addition of TP-2. This synergistic effect suggested that TP-1 can release T4 from thyroglobulin much more efficiently after the protein has been partially degraded by the action of TP-2, which has only a poor T4-releasing activity. It also suggested that the T4-releasing activity of TP-1 markedly increases as the size of protein substrate is decreased. The amino acid composition of the T4-containing peptide was identical with that previously isolated from bovine thyroglobulin, and this peptide sequence has been shown to be derived from the NH2-terminal portion of the thyroglobulin. This suggests that TP-1 can release T4 at least from the NH2-terminal region of thyroglobulin. PMID:3918854

Nakagawa, H; Ohtaki, S

1985-04-01

139

Precursors of the T4 internal peptides.  

PubMed Central

The precursors of the two T4 internal peptides have been identified by in vitro cleavage of individual phage proteins eluted from sodium dodecyl sulfate-acrylamide gels. The precursor of internal peptide VII is p22, the product of T4 gene 22 and an essential component of the morphogenic core. The precursor of peptide II is a protein with a molecular weight of approximately 13,000, whose gene has yet to be defined by mutation. A newly detected protein of approximately 15,000 molecular weight is found to be cleaved and is, therefore, likely to be a component of precursor head structures. Images PMID:864834

Kurtz, M B; Champe, S P

1977-01-01

140

Morphogenesis of the T4 tail and tail fibers  

PubMed Central

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

2010-01-01

141

Genomic polymorphism in the T-even bacteriophages.  

PubMed Central

We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4. Images PMID:8076614

Repoila, F; Tetart, F; Bouet, J Y; Krisch, H M

1994-01-01

142

Bacteriophages of methanotrophic bacteria  

SciTech Connect

Bacteriophages of methanotrophic bacteria have been found in 16 out of 88 studied samples (underground waters, pond water, soil, gas and oil installation waters, fermentor cultural fluids, bacterial paste, and rumen of cattle) taken in different geographic zones of the Soviet Union. Altogether, 23 phage strains were isolated. By fine structure, the phages were divided into two types (with very short or long noncontractile tails); by host range and serological properties, they fell into three types. All phages had guanine- and cytosine-rich double-stranded deoxyribonucleic acid consisting of common nitrogen bases. By all of the above-mentioned properties, all phages within each of the groups were completely identical to one another, but differed from phages of other groups.

Tyutikow, F.M. (All-Union Research Inst. for Genetics and Selection of Industrial Microorganisms, Moscow, USSR); Bespalova, I.A.; Rebentish, B.A.; Aleksandrushkina, N.N.; Krivisky, A.S.

1980-10-01

143

Bacteriophages of Erwinia amylovora.  

PubMed

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and PODOVIRIDAE: PMID:12676693

Gill, J J; Svircev, A M; Smith, R; Castle, A J

2003-04-01

144

Lysozyme Activity of High-Leucocyte-Count Milk and the Effect of Heat and Potassium Dichromate on Lysozyme Activity1  

Microsoft Academic Search

No support could be found for the hypothesis that mastitis, as evidenced by increased leucocyte counts in milk, is accompanied by an increase in lysozyme activity. The presence of potassium di- chromate in the preserved milk samples did not seem to affect lysozyme activity. The sensitivity of lysozyme to heat was demonstrated. All lysozyme isolates and purifications were made with

Gary L. Weaver; Manfred Kroger

1978-01-01

145

Lysozyme  

NASA Technical Reports Server (NTRS)

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

2004-01-01

146

Controlling Salmonella in Poultry using Bacteriophages  

E-print Network

for new pathogen control strategies in the poultry industry. Scientific research has focused on the use of bacteriophages as therapeutic agents for humans and animals; however, limited studies have been conducted on bacteriophage application on food safety...

Sanchez Pena, Ana

2012-10-19

147

ORF334 in Vibrio phage KVP40 plays the role of gp27 in T4 phage to form a heterohexameric complex.  

PubMed

KVP40 is a T4-related phage, composed of 386 open reading frames (ORFs), that has a broad host range. Here, we overexpressed, purified, and biophysically characterized two of the proteins encoded in the KVP40 genome, namely, gp5 and ORF334. Homology-based comparison between KVP40 and its better-characterized sister phage, T4, was used to estimate the two KVP40 proteins' functions. KVP40 gp5 shared significant homology with T4 gp5 in the N- and C-terminal domains. Unlike T4 gp5, KVP40 gp5 lacked the internal lysozyme domain. Like T4 gp5, KVP40 gp5 was found to form a homotrimer in solution. In stark contrast, KVP40 ORF334 shared no significant homology with any known proteins from T4-related phages. KVP40 ORF334 was found to form a heterohexamer with KVP40 gp5 in solution in a fashion nearly identical to the interaction between the T4 gp5 and gp27 proteins. Electron microscope image analysis of the KVP40 gp5-ORF334 complex indicated that it had dimensions very similar to those of the T4 gp5-gp27 structure. On the basis of our biophysical characterization, along with positional genome information, we propose that ORF334 is the ortholog of T4 gp27 and that it plays the role of a linker between gp5 and the phage baseplate. PMID:18326574

Nemoto, Mai; Mio, Kazuhiro; Kanamaru, Shuji; Arisaka, Fumio

2008-05-01

148

Sperm Specific Lysozyme-Like Proteins.  

National Technical Information Service (NTIS)

The present invention is directed to a family of testis specific proteins (SLLPs) that share high sequence identity to lysozyme-C proteins. The application encompasses compositions comprising the SLLP proteins, antibodies specific for the SLLP polypeptide...

A. Mandal, J. C. Herr, L. C. Digilio, M. B. Herrero

2004-01-01

149

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

SciTech Connect

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

Gajewski, Stefan; Webb, Michael R.; Galkin, Vitold; Egelman, Edward H.; Kreuzer, Kenneth N.; White, Stephen W. (Duke); (UV); (SJCH)

2012-07-11

150

The genome sequence and proteome of bacteriophage ?CPV1 virulent for Clostridium perfringens.  

PubMed

Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents and ?CPV1, a virulent bacteriophage, was classified in the family Podoviridae. The purified virus had an icosahedral head and collar of approximately 42nm and 23nm in diameter, respectively, with a structurally complex tail of 37nm lengthwise and a basal plate of 30nm. The ?CPV1 double-stranded DNA genome was 16,747 base pairs with a GC composition of 30.5%. Twenty-two open reading frames (ORFs) coding for putative peptides containing 30 or more amino acid residues were identified and analyzed in the genome. Amino acid sequences of the predicted proteins from the ?CPV1 genome ORFs were compared with those from the NCBI database and potential functions of 12 proteins were predicted by sequence homology. Three putative proteins were similar to hypothetical proteins with unknown functions, whereas seven proteins did not have similarity with any known bacteriophage or bacterial proteins. Identified ORFs formed at least four genomic clusters that accounted for predicted proteins involved with replication of the viral DNA, its folding, production of structural components and lytic properties. One bacteriophage genome encoded lysin was predicted to share homology with N-acetylmuramoyl-l-alanine amidases and a second structural lysin was predicted to be a lysozyme-endopeptidase. These enzymes digest peptidoglycan of the bacterial cell wall and could be considered potential therapeutics to control C. perfringens. PMID:21144870

Volozhantsev, Nikolay V; Verevkin, Vladimir V; Bannov, Vasily A; Krasilnikova, Valentina M; Myakinina, Vera P; Zhilenkov, Eugeni L; Svetoch, Edward A; Stern, Norman J; Oakley, Brian B; Seal, Bruce S

2011-02-01

151

Lysogenic bacteriophage isolated from acidophilium  

DOEpatents

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.

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

1992-01-01

152

A New Family of Lysozyme Inhibitors Contributing to Lysozyme Tolerance in Gram-Negative Bacteria  

Microsoft Academic Search

Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel

Lien Callewaert; Abram Aertsen; Daphne Deckers; Kristof G. A. Vanoirbeek; Lise Vanderkelen; Joris M. Van Herreweghe; Barbara Masschalck; Dorothy Nakimbugwe; Johan Robben; Chris W. Michiels

2008-01-01

153

The amino acid sequence of wood duck lysozyme.  

PubMed

The amino acid sequence of wood duck (Aix sponsa) lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had the highest similarity to duck III lysozyme with four amino acid substitutions, and had eighteen amino acid substitutions from chicken lysozyme. The valine at position 75 was newly detected in chicken-type lysozymes. In the active site, Tyr34 and Glu57 were found at subsites F and D, respectively, when compared with chicken lysozyme. PMID:10052146

Araki, T; Torikata, T

1999-01-01

154

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

SciTech Connect

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

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

2009-06-30

155

Enhanced antimicrobial activity of engineered human lysozyme  

PubMed Central

Lysozymes contain a disproportionately large fraction of cationic residues, and are thereby attracted towards the negatively charged surface of bacterial targets. Importantly, this conserved biophysical property may inhibit lysozyme antibacterial function during acute and chronic infections. A mouse model of acute pulmonary Pseudomonas aeruginosa infection demonstrated that anionic biopolymers accumulate to high concentrations in the infected lung, and the presence of these species correlates with decreased endogenous lysozyme activity. To develop antibacterial enzymes designed specifically to be used as antimicrobial agents in the infected airway, the electrostatic potential of human lysozyme (hLYS) was remodeled by protein engineering. A novel, high throughput screen was implemented to functionally interrogate combinatorial libraries of charge engineered hLYS proteins, and variants with improved bactericidal activity were isolated and characterized in detail. These studies illustrate a general mechanism by which polyanions inhibit lysozyme function, and they are the first direct demonstration that decreasing hLYS's net cationic character improves its antibacterial activity in the presence of disease-associated biopolymers. In addition to avoiding electrostatic sequestration, at least one charge engineered variant also kills bacteria more rapidly in the absence of inhibitory biopolymers; this observation supports a novel hypothesis that tuning the cellular affinity of peptidoglycan hydrolases may be a general strategy for improving kinetics of bacterial killing. PMID:20604527

Scanlon, Thomas C.; Teneback, Charlotte C.; Gill, Avinash; Bement, Jenna L.; Weiner, Joshua A.; Lamppa, John W.; Leclair, Laurie W.; Griswold, Karl E.

2010-01-01

156

Lysozyme activity in the plaice ( Pleuronectes platessa L.)  

Microsoft Academic Search

Zusammenfassung Die Untersuchung der Lysozymverteilung im Gewebe des Knochenfischs,Pleuronectes platessa L., ergab, dass das Plasma-Lysozym von Leukozyten abstammt, das Hautschleim-Lysozym dagegen von spezifischen Epidermiszellen.

Thelma C. Fletcher; Ann White

1973-01-01

157

X-ray topography of a lysozyme crystal  

SciTech Connect

X-ray topography methods were employed to identify defects in lysozyme crystals. White-beam and monochromatic topographs of lysozyme crystals obtained at the National Synchrotron Light Source are presented.

Stojanoff,V.; Siddons, D.

1996-01-01

158

Bacteriophage-Resistant Mutants of Pasteurella Pestis and Their Properties.  

National Technical Information Service (NTIS)

An inquiry was made into spontaneous mutation from the bacteriophage-sensitivity to bacteriophage-resistance in Past. pestis of different virulence. According to preliminary data, mutants resistant to bacteriophage appeared irrespective of the degree of t...

A. I. Volosivetz

1965-01-01

159

Effect of alcohols on aqueous lysozyme-lysozyme interactions from static light-scattering measurements.  

PubMed

Alcohols have been widely used as protein denaturants, precipitants and crystallization reagents. We have studied the effect of alcohols on aqueous hen-egg lysozyme self-interactions by measuring the osmotic second virial coefficient (B22) using static light scattering. Addition of alcohols increases B22, indicating stronger protein-protein repulsion or weaker attraction. For the monohydric alcohols used in this study (methanol, ethanol, 1-propanol, n-butanol, iso-butanol and trifluoroethanol), B22 for lysozyme reaches a common plateau at approximately 5% (v/v) alcohol, while glycerol increases B22 more than monohydric alcohols. For a 0.05 M NaCl hen-egg lysozyme solution at pH 7, B22 increases from 2.4 x 10(-4) to 4.7 x 10(-4) ml mol/g2 upon addition of monohydric alcohols and to 5.8 x 10(-4) ml mol/g2 upon addition of glycerol. We describe the alcohol effect using a simple model that supplements the DLVO theory with an additional alcohol-dependent term representing orientation-averaged hydrophobic interactions. In this model, the increased lysozyme repulsive forces in the presence of monohydric alcohols are interpreted in terms of adsorption of alcohol molecules on hydrophobic sites on the protein surface. This adsorption reduces attractive hydrophobic protein-protein interactions. A thicker lysozyme hydration layer in aqueous glycerol solution can explain the glycerol-increased lysozyme-lysozyme repulsion. PMID:14967244

Liu, Wei; Bratko, Dusan; Prausnitz, John M; Blanch, Harvey W

2004-02-15

160

Coacervates of lysozyme and ?-casein.  

PubMed

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

Anema, Skelte G; de Kruif, C G Kees

2013-05-15

161

Equilibrium Clusters in Concentrated Lysozyme Protein Solutions  

E-print Network

We have studied the structure of salt-free lysozyme at 293 K and pH 7.8 using molecular simulations and experimental SAXS effective potentials between proteins at three volume fractions, 0.012, 0.033, and 0.12. We found that the structure of lysozyme near physiological conditions strongly depends on the volume fraction of proteins. The studied lysozyme solutions are dominated by monomers only for <0.012; for the strong dilution 70% of proteins are in a form of monomers. For 0.033 only 20% of proteins do not belong to a cluster. The clusters are mainly elongated. For 0.12 almost no individual particles exits, and branched, irregular clusters of large extent appear. Our simulation study provides new insight into the formation of equilibrium clusters in charged protein solutions near physiological conditions.

Kowalczyk, P; Gauden, P A; Terzyk, A P

2011-01-01

162

Equilibrium Clusters in Concentrated Lysozyme Protein Solutions  

E-print Network

We have studied the structure of salt-free lysozyme at 293 K and pH 7.8 using molecular simulations and experimental SAXS effective potentials between proteins at three volume fractions, 0.012, 0.033, and 0.12. We found that the structure of lysozyme near physiological conditions strongly depends on the volume fraction of proteins. The studied lysozyme solutions are dominated by monomers only for <0.012; for the strong dilution 70% of proteins are in a form of monomers. For 0.033 only 20% of proteins do not belong to a cluster. The clusters are mainly elongated. For 0.12 almost no individual particles exits, and branched, irregular clusters of large extent appear. Our simulation study provides new insight into the formation of equilibrium clusters in charged protein solutions near physiological conditions.

P. Kowalczyk; A. Ciach; P. A. Gauden; A. P. Terzyk

2011-06-03

163

The cytochemical localization of lysozyme in Paneth cell granules  

Microsoft Academic Search

Synopsis  The breakdown products resulting from the hydrolysis of chitin by lysozyme stain with Alcian Blue. A method based upon this observation has been developed for the histochemical demonstration of lysozyme activity. The application of this method to the jejunal crypts of several animal species indicates that Paneth cell granules contain lysozyme. The binding of the hydrolysis products with Alcian Blue

Y. Ghoos; G. Vantrappen

1971-01-01

164

Expression of natural antimicrobial human lysozyme in rice grains  

Microsoft Academic Search

In the present study, we explored the expression of human lysozyme in maturing rice grains. Particle bombardment-mediated transformation was utilized to deliver the codon-optimized structural gene for human lysozyme to the callus of rice cultivar Taipei 309. Lysozyme expression is controlled by the promoter and signal peptide sequence for rice storage protein Glutelin 1. A total of 33 fertile plants

Jianmin Huang; Somen Nandi; Liying Wu; Dorice Yalda; Glenn Bartley; Raymond Rodriguez; Bo Lonnerdal; Ning Huang

2002-01-01

165

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

SciTech Connect

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

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

2009-08-28

166

Thermophysical properties of lysozyme (protein) solutions  

NASA Technical Reports Server (NTRS)

Thermophysical properties of protein solutions composed of the lysozyme crystals with a 0.1 M sodium acetate and 5 percent NaCl solution as the buffer (pH = 4.0) are determined. The properties being measured include specific heat, thermal conductivity, dynamic viscosity, and surface tension. The protein concentrations are varied. Thermal diffusivity is calculated using the measured results. The purpose of the research is to measure thermophysical properties of lysozyme solutions which would serve as the data bank for controlling and modeling the crystal growth process on earth as well as in space.

Liu, Jiaching; Yang, Wen-Jei

1992-01-01

167

Growth kinetics of tetragonal lysozyme crystals  

NASA Technical Reports Server (NTRS)

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

Pusey, M.; Naumann, R.

1986-01-01

168

Classification of Myoviridae bacteriophages using protein sequence similarity  

PubMed Central

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

2009-01-01

169

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

PubMed Central

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

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

2014-01-01

170

sup 1 H NMR studies of human lysozyme: Spectral assignment and comparison with hen lysozyme  

SciTech Connect

Complete main-chain (NH and {alpha}CH){sup 1}H NMR assignments are reported for the 130 residues of human lysozyme, along with extensive assignments for side-chain protons. Analysis of 2-D NOESY experiments shows that the regions of secondary structure for human lysozyme in solution are essentially identical with those found previously in a similar study of hen lysozyme and are in close accord with the structure of the protein reported previously from x-ray diffraction studies in the crystalline state. Comparison of the chemical shifts, spin-spin coupling constants, and hydrogen exchange behavior are also consistent with closely similar structures for the two proteins in solution. In a number of cases specific differences in the NMR parameters between hen and human lysozymes can be correlated with specific differences observed in the crystal structures.

Redfield, C.; Dobson, C.M. (Univ. of Oxford (England))

1990-08-07

171

T-4G Simulator and T-4 Ground Training Devices in USAF Undergraduate Pilot Training.  

ERIC Educational Resources Information Center

The objective of the project was to investigate the utility of using an A/F37A-T4G T-37 flight simulator within the context of Air Force undergraduate pilot training. Twenty-one subjects, selected from three undergraduate pilot training classes, were given contact flight training in a TP4G/EPT simulator before going to T-37 aircraft for further…

Woodruff, Robert R.; Smith, James F.

172

Monolithic molecularly imprinted cryogel for lysozyme recognition.  

PubMed

The application of molecularly imprinted polymers in the selective adsorption of macromolecules such as proteins by monolithic protein-imprinted columns requires a macroporous structure, which can be provided by cryogelation at low temperature in which the formation of ice crystals gives a porous structure to the molecularly imprinted polymer. In this study, we applied this technique to synthesize lysozyme-imprinted polyacrylamide cryogels containing 8% w/v of total monomers and 0.3% w/v of lysozyme. The synthesized cryogel was sponge-like and elastic with very fast swelling and reshaping properties, showing a swelling ratio of 24.5 ± 3 and gel fraction yield of about 72%. It showed an imprinting effect of 1.58 and a separation factor of 1.37 for cytochrome c as the competing protein. Adsorption studies on the cryogel revealed that it follows the Langmuir isotherm, with a maximum theoretical adsorption capacity of 36.3 mg lysozyme per gram of cryogel. Additionally, it was shown that a salt-free rebinding solution at low flow rate and pH = 7.0 is favorable for lysozyme rebinding. This kind of monolithic column promises a wide range of application in separation of various biomolecules due to its preparation simplicity, good rebinding characteristics, and macroporosity. PMID:25115847

Rabieizadeh, Mohammadmahdi; Kashefimofrad, Seyed Mohammadreza; Naeimpoor, Fereshteh

2014-10-01

173

The Antimicrobial Peptide Lysozyme Is Induced after Multiple Trauma  

PubMed Central

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

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

2014-01-01

174

Recombinant Expression and Purification of T4 Phage Hoc, Soc, gp23, gp24 Proteins in Native Conformations with Stability Studies  

PubMed Central

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro. PMID:22808021

Miernikiewicz, Paulina; Owczarek, Barbara; Piotrowicz, Agnieszka; Boczkowska, Barbara; Rzewucka, Kamila; Figura, Grzegorz; Letarov, Andrey; Kulikov, Eugene; Kopciuch, Agnieszka; Switala-Jelen, Kinga; Oslizlo, Anna; Hodyra, Katarzyna; Gubernator, Jerzy; Dabrowska, Krystyna

2012-01-01

175

Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies.  

PubMed

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro. PMID:22808021

Miernikiewicz, Paulina; Owczarek, Barbara; Piotrowicz, Agnieszka; Boczkowska, Barbara; Rzewucka, Kamila; Figura, Grzegorz; Letarov, Andrey; Kulikov, Eugene; Kopciuch, Agnieszka; Swita?a-Jele?, Kinga; O?liz?o, Anna; Hodyra, Katarzyna; Gubernator, Jerzy; D?browska, Krystyna

2012-01-01

176

Preparation of lysozyme molecularly imprinted polymers and purification of lysozyme from egg white.  

PubMed

Molecular imprinting as a promising and facile separation technique has received much attention because of its high selectivity for target molecules. In this study, lysozyme molecularly imprinted polymers (Lys-MIPs) were successfully prepared by the entrapment method with lysozyme as the template molecule, acrylamide as the functional monomer and N,N-methylenebisacrylamide as the cross-linker. The removal of the template lysozyme from the molecularly imprinted polymers was investigated in detail by two methods. The synthesized Lys-MIPs were characterized by scanning electron microscopy and Fourier transform-infrared, and the adsorption capacity, selectivity and reproducibility of the Lys-MIPs were also evaluated. The maximum adsorption capacity reached 94.8 mg/g, which is twice that of nonmolecularly imprinted polymers, and satisfactory selectivity and reproducibility were achieved. Using the Lys-MIP column, lysozyme could be separated completely from egg white, with purity close to 100% and mass recovery of 98.2%. This illustrated that the synthesized Lys-MIPs had high specific recognition and selectivity to the template lysozyme when they were applied to a mixture of protein standards and a real sample. PMID:24861763

Wang, Xuejiao; Dong, Shaohua; Bai, Quan

2014-06-01

177

Elasticity and Strength of Biomacromolecular Crystals: Lysozyme  

NASA Technical Reports Server (NTRS)

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

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

2003-01-01

178

Dynamics of Lysozyme in Trehalose solutions  

NASA Astrophysics Data System (ADS)

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

Ghatty, Pavan; Uberbacher, Edward C.

2008-03-01

179

Light scattering and phase behavior of Lysozyme-PEG mixtures  

E-print Network

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 depletion interaction between lysozyme and PEG and suggest that an energetic attraction between lysozyme and PEG 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 cloud point and light scattering data. The light scattering results were consistent with the cloud point measurements and indicate that attractions do exist between lysozyme and PEG.

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

2005-07-20

180

Growth and dissolution kinetics of tetragonal lysozyme  

NASA Technical Reports Server (NTRS)

The growth and dissolution kinetics of lysozyme in a 25 ml solution bridge inside a closed growth cell was investigated. It was found that, under all growth conditions, the growth habit forming (110) and (101) faces grew through layer spreading with different growth rate dependence on supersaturation/temperature. On the other hand, (100) faces which formed only at low temperatures underwent a thermal roughening transition around 12 C.

Monaco, L. A.; Rosenberger, F.

1993-01-01

181

THz characterization of lysozyme at different conformations  

NASA Astrophysics Data System (ADS)

This work demonstrates application of Fourier Transform Infrared Spectroscopy (FTIR) technique in the low terahertz frequency range of 10-25 cm-1 to discriminate between different protein conformations and evaluate possible application of THz spectroscopy for monitoring of protein folding-unfolding process. A specific procedure developed earlier for unfolding lysozyme by salt (KSCN) precipitation and refolding the lysozyme molecules by removing of KSCN and dissolving in sodium acetate was used to prepare three different forms of lysozyme. In addition, two standard procedures were used to prepare samples in unfolded conformation: denaturation at high temperature ~95° C followed by fast freezing, and dissolution in 6 M guanidine. Thin, air dried protein films were characterized as well as material in the form of gel. Spectra reveal resonance features in transmission which represent vibrational modes in the protein samples. A great variability of spectral features for the different conformational states showed the sensitivity of vibrational frequencies to the three dimensional structure of proteins. The results obtained on liquid (gel) samples indicate that THz transmission spectroscopy can be used for monitoring folding-unfolding process in a realistic, aqueous environment.

Globus, Tatiana; Khromova, Tatyana; Lobo, Rebecca; Woolard, Dwight; Swami, Nathan; Fernandez, Erik

2005-05-01

182

Structure of the Bacteriophage [phi]KZ Lytic Transglycosylase gp144  

SciTech Connect

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

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

2008-04-02

183

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2011 CFR

...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)...

2011-04-01

184

Disruption of the Bacteriophage T4 Mre11 Dimer Interface Reveals a Two-state Mechanism for Exonuclease Activity*  

PubMed Central

The Mre11-Rad50 (MR) complex is a central player in DNA repair and is implicated in the processing of DNA ends caused by double strand breaks. Recent crystal structures of the MR complex suggest that several conformational rearrangements occur during its ATP hydrolysis cycle. A comparison of the Mre11 dimer interface from these structures suggests that the interface is dynamic in nature and may adopt several different arrangements. To probe the functional significance of the Mre11 dimer interface, we have generated and characterized a dimer disruption Mre11 mutant (L101D-Mre11). Although L101D-Mre11 binds to Rad50 and dsDNA with affinity comparable with the wild-type enzyme, it does not activate the ATP hydrolysis activity of Rad50, suggesting that the allosteric communication between Mre11 and Rad50 has been interrupted. Additionally, the dsDNA exonuclease activity of the L101D-MR complex has been reduced by 10-fold under conditions where processive exonuclease activity is required. However, we unexpectedly found that under steady state conditions, the nuclease activity of the L101D-MR complex is significantly greater than that of the wild-type complex. Based on steady state and single-turnover nuclease assays, we have assigned the rate-determining step of the steady state nuclease reaction to be the productive assembly of the complex at the dsDNA end. Together, our data suggest that the Mre11 dimer interface adopts at least two different states during the exonuclease reaction. PMID:22798142

Albrecht, Dustin W.; Herdendorf, Timothy J.; Nelson, Scott W.

2012-01-01

185

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

PubMed Central

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

Seal, Bruce S.

2014-01-01

186

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

PubMed

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

Seal, Bruce S

2013-02-01

187

Arthrobacter globiformis and its bacteriophage in soil  

NASA Technical Reports Server (NTRS)

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

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

1974-01-01

188

Taking bacteriophage therapy seriously: a moral argument.  

PubMed

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

Verbeken, Gilbert; Huys, Isabelle; Pirnay, Jean-Paul; Jennes, Serge; Chanishvili, Nina; Scheres, Jacques; Górski, Andrzej; De Vos, Daniel; Ceulemans, Carl

2014-01-01

189

Taking Bacteriophage Therapy Seriously: A Moral Argument  

PubMed Central

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

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

2014-01-01

190

Bacteriophage endolysins as novel antimicrobials  

PubMed Central

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

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

2013-01-01

191

Bacteriophage typing of Corynebacterium diphtheriae.  

PubMed

Bacteriophage types of over 3,000 strains of Corynebacterium diphtheriae isolated in Canada have been determined. The typing scheme used involved the use of nine phages. Results indicated that phage types correlate with biotypes to a large degree. Corynecin types were also determined for a limited number of cultures, and results indicated that the indicator strains presently available are unsuitable for the typing of C. diphtheriae strains isolated in North America. The distribution of phage types is similar throughout Canada, and the types present correspond to types reported from other countries. Phage typing could be of value in the study of circumscribed outbreaks and in epidemiological surveillance of types over long periods. PMID:410896

Toshach, S; Valentine, A; Sigurdson, S

1977-11-01

192

Temperature micro-Raman study of lysozyme crystals  

E-print Network

We have performed for the first time micro-Raman studies of lysozyme crystals for the temperatures lower than the room one (in the range 282-300K). Anomalous temperature dependence of the positions of Raman scattering bands has been detected and discussed, which corresponds to the crystalline lysozyme structure in the vicinity of T=285K.

Yu. Vasylkiv; M. Polomska; Yu. Nastishin; R. Vlokh

2007-06-28

193

Mouse Lysozyme M Gene: Isolation, Characterization, and Expression Studies  

Microsoft Academic Search

We have isolated and characterized both cDNA and genomic DNA of the mouse lysozyme M gene. Derivation of the amino acid sequence from the nucleotide sequences revealed six positions in the carboxyl terminus that differ from partial sequences previously published. The differential detection of specific mRNAs from the closely related lysozyme M and P genes has revealed different but overlapping

Michael Cross; Inge Mangelsdorf; Angela Wedel; Rainer Renkawitz

1988-01-01

194

Intralysosomal digestion of lysozyme in renal proximal tubule cells  

Microsoft Academic Search

Intralysosomal digestion of lysozyme in renal proximal tubule cells. This investigation was carried out to determine the ability of lysosomes in intact proximal tubule cells to digest absorbed protein. Lysozyme labeled with 125I was injected i.v. into rats and renal cortical slices removed after one hour. The slices were incubated in vitro for up to two hours. The location of

Erik Ilsø Christensen; Arvid B Maunsbach

1974-01-01

195

Purification of Lysozyme by Intrinsically Shielded Hydrogel Beads  

NASA Astrophysics Data System (ADS)

Macro-sized intrinsically shielded hydrogel beads have been prepared from BSA and CM-dextran grafted with CB using a technique based on freeze-thawing gelation method. The size of the beads lies in around 500 ?m. Isothemal titration calorimetry (ITC) showed that the relative binding affinities of the lysozyme for CB, compared with BSA, at pH 3.0 was stronger than that at pH 7.4. They were employed for the affinity separation of lysozyme using chromatography column. Their adsorption capacity for lysozyme at pH 3.0 is higher than that at pH 9. In a binary mixture of lysozyme and ovalbumin, the beads showed very high selectivity toward lysozyme. Lysozyme of very high purity (> 93%) was obtained from a mixture of lysozyme and ovalbumin, and 85% from egg white solution. The results indicate that the macro-sized bead can be used for the separation, purification, and recovery of lysozyme in a chromatograph column.

Li, Cong; Zhang, R.; Wang, L.; Bowyer, A.; Eisenthal, R.; Shen, Yehua; Hubble, J.

2013-07-01

196

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

NASA Technical Reports Server (NTRS)

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

Pusey, Marc L.

1992-01-01

197

Adaptive functional diversification of lysozyme in insectivorous bats.  

PubMed

The role of gene duplication in generating new genes and novel functions is well recognized and is exemplified by the digestion-related protein lysozyme. In ruminants, duplicated chicken-type lysozymes facilitate the degradation of symbiotic bacteria in the foregut. Chicken-type lysozyme has also been reported to show chitinase-like activity, yet no study has examined the molecular evolution of lysozymes in species that specialize on eating insects. Insectivorous bats number over 900 species, and lysozyme expression in the mouths of some of these species is associated with the ingestion of insect cuticle, suggesting a chitinase role. Here, we show that chicken-type lysozyme has undergone multiple duplication events in a major family of insect-eating bats (Vespertilionidae) and that new duplicates have undergone molecular adaptation. Examination of duplicates from two insectivorous bats-Pipistrellus abramus and Scotophilus kuhlii-indicated that the new copy was highly expressed in the tongue, whereas the other one was less tissue-specific. Functional assays applied to pipistrelle lysozymes confirmed that, of the two copies, the tongue duplicate was more efficient at breaking down glycol chitin, a chitin derivative. These results suggest that the evolution of lysozymes in vespertilionid bats has likely been driven in part by natural selection for insectivory. PMID:25135943

Liu, Yang; He, Guimei; Xu, Huihui; Han, Xiuqun; Jones, Gareth; Rossiter, Stephen J; Zhang, Shuyi

2014-11-01

198

Amino acid sequences of stomach and nonstomach lysozymes of ruminants  

Microsoft Academic Search

Summary Complete amino acid sequences are presented for lysozymesc from camel and goat stomachs and compared to sequences of other lysozymesc. Tree analysis suggests that the rate of amino acid replacement went up as soon as lysozyme was recruited for the stomach function in early ruminants. The two lysozymes from goat stomach are the products of a gene duplication that

Jacqueline Jollès; Ellen M. Prager; Emad S. Alnemri; Pierre Jollès; Ibrahim M. Ibrahimi; Allan C. Wilson

1990-01-01

199

Tetragonal Lysozyme, From Monomer to Crystal  

NASA Technical Reports Server (NTRS)

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

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

2002-01-01

200

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

PubMed

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

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

2005-09-30

201

IN VITRO SYNTHESIS AND SECRETION OF LYSOZYME BY MONONUCLEAR PHAGOCYTES  

PubMed Central

Pure cultures of three types of mononuclear phagocytes—mouse peritoneal macrophages, unstimulated or after thioglycollate stimulation, and human monocytes—synthesize and secrete large amounts of lysozyme in vitro. The macrophage lysozyme is indistinguishable from authentic lysozyme in its ability to lyse M. lysodeikticus, inhibition by specific antisera, a similar size of 14,000 and cationic charge. Lysozyme secretion in culture is characterized by a large net increase in total lysozyme, 4–20-fold in 3 h, 75–95% of which is in the medium, and its continued extracellular accumulation over at least 2 wk in culture. Lysozyme is the major 14C-labeled protein secreted into the medium by both unstimulated and thioglycollate-stimulated macrophages and the 0.75–1 µg produced per 1 x 106 cells/day represents 0.5–2.5% of the total cell protein. Lysozyme is a cell-specific marker for mononuclear phagocytes and the PMN, which contains preformed enzyme, since it is absent in lymphoid cells and a variety of fibroblast and epithelioid cell lines. Lysozyme production is also a useful measure of mononuclear phagocyte cell number. The rate of lysozyme production and secretion is remarkably constant for all cell types under a variety of culture conditions. Production by the mouse macrophage increases threefold on the 2nd day in culture and then remains linear with time. Production is optimal at a relatively low serum concentration, but can be maintained, in the absence of serum, in lactalbumin hydrolysate or, at a reduced level in basal media. The production and secretion of lysozyme are independent of the production of macrophage acid hydrolases. Net increase and secretion of lysozyme occur under conditions where acid hydrolases like N-acetyl ?-glucosaminidase, ?-glucuronidase, ?-galactosidase, and cathepsin D are neither accumulated nor secreted. Massive phagocytosis of latex particles has no effect on lysozyme production and secretion. Lysozyme production can be rapidly inhibited by treatment with cycloheximide (0.4 µg/ml) whereas inhibition of its production by colchicine (10–6 M) occurs only after a lag period of more than 8 h, and is probably due to a secondary effect. These results show that mouse macrophages provide a simple in vitro system to measure lysozyme secretion and its control. These studies also indicate the possible importance of mononuclear phagocytes in the secretion of a variety of biologically active products and in the modification of their environment. PMID:4825244

Gordon, Salmon; Todd, Jean; Cohn, Z. A.

1974-01-01

202

Acanthamoeba T4 genotype associated with keratitis infections in Tunisia.  

PubMed

Acanthamoeba keratitis (AK) is a sight-threatening infection. We report five cases of AK diagnosed from 2005 to 2009 in the Laboratory of Parasitology-Mycology at Habib Bourguiba Sfax Hospital, Tunisia. All were associated with improper care of contact lenses (rinsing of contact lenses with tap water and inappropriate cleaning) and lens storage. The patients displayed different clinical presentations: corneal inflammation, corneal ulceration, and corneal abscess. The diagnosis was made after direct examination, culture, and polymerase chain reaction amplification with specific primers. The genotype classification was based on the highly variable DF3 region in the 18S rRNA gene. This is the first study characterizing Acanthamoeba genotype in Tunisia and North Africa. All Acanthamoeba isolates were associated to the T4 genotype. Three different DF3 sequence types were related to AK infections T4/10, T4/15, and T4/16. PMID:23052779

Dendana, F; Sellami, H; Trabelsi, H; Neji, S; Cheikhrouhou, F; Makni, F; Ayadi, A

2013-01-01

203

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

NASA Astrophysics Data System (ADS)

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

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

2012-10-01

204

Characterization of tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa  

SciTech Connect

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

Kurochkina, Lidia P., E-mail: lpk@ibch.r [Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117997 (Russian Federation); Aksyuk, Anastasia A. [Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, IN 47907-2054 (United States); Sachkova, Maria Yu.; Sykilinda, Nina N.; Mesyanzhinov, Vadim V. [Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117997 (Russian Federation)

2009-12-20

205

Thrombocytopenia provoked by carrageenan in rabbits and the inhibitory effect of lysozyme  

Microsoft Academic Search

Hen egg white lysozyme (Fleming's lysozyme) prevented the thrombocytopenia provoked by carrageenan injected intravenously into rabbits but not that provoked by an anaphylactic shock. Lysozyme was given intravenously at 25, 50, 100 mg\\/kg 30 sec before carrageenan. Platelets were counted 15 and 60 min after carrageenan. Lysozyme was given intravenously to immunized animals at the dose of 100 mg\\/kg 5

Camillo Bianchi

1982-01-01

206

Lysozyme catalyzes the formation of antimicrobial silver nanoparticles.  

PubMed

Hen egg white lysozyme acted as the sole reducing agent and catalyzed the formation of silver nanoparticles in the presence of light. Stable silver colloids formed after mixing lysozyme and silver acetate in methanol and the resulting nanoparticles were concentrated and transferred to aqueous solution without any significant changes in physical properties. Activity and antimicrobial assays demonstrated lysozyme-silver nanoparticles retained the hydrolase function of the enzyme and were effective in inhibiting growth of Escherichia coli, Staphylococcus aureus, Bacillus anthracis, and Candida albicans. Remarkably, lysozyme-silver nanoparticles demonstrated a strong antimicrobial effect against silver-resistant Proteus mirabilis strains and a recombinant E. coli strain containing the multiple antibiotic- and silver-resistant plasmid, pMG101. Results of toxicological studies using human epidermal keratinocytes revealed that lysozyme-silver nanoparticles are nontoxic at concentrations sufficient to inhibit microbial growth. Overall, the ability of lysozyme to assemble silver nanoparticles in a one-step reaction offers a simple and environmentally friendly approach to form stable colloids of nontoxic silver nanoparticles that combine the antimicrobial properties of lysozyme and silver. The results expand the functionality of nanomaterials for biological systems and represent a novel antimicrobial composite for potential aseptics and therapeutic use in the future. PMID:19344124

Eby, D Matthew; Schaeublin, Nicole M; Farrington, Karen E; Hussain, Saber M; Johnson, Glenn R

2009-04-28

207

Nucleation and Growth According to Lysozyme  

NASA Technical Reports Server (NTRS)

How does one take a molecule, strongly asymmetric in both shape and charge distribution, and assemble it into a crystal? We propose a model for the nucleation and crystal growth process for tetragonal lysozyme that may be very germane to other monomeric proteins. The first species formed is postulated to be a dimer. Through repeating associations involving the same intermolecular interactions this becomes the 4(sub 3) helix, that in turn serves as the basic unit for nucleation and crystal growth. High salt attenuates surface charges while promoting hydrophobic interactions. Symmetry facilitates helix self-association. Assembly stability is enhanced when a four helix structure is obtained, with each bound to two neighbors. Only two unique interactions are required. The first are those for helix formation, where the dominant interaction is the intermolecular bridging anion. The second is the anti-parallel side-by-side helix-helix interaction, guided by alternating pairs of symmetry related salt bridges along each side. At this stage all eight unique positions of the P4(sub 3)2(sub 1)2(sub 1) unit cell are filled. From the above, the process is one of a) attenuating the most strongly interacting groups, such that b) the molecules begin to self-associate in defined patterns, so that c) symmetry is obtained, which d) propagates as a growing crystal. Simple and conceptually obvious in hindsight, this tells much about what we are empirically doing when we crystallize macromolecules. By adjusting the solution parameters we are empirically balancing the intermolecular interactions, preferentially attenuating the dominant strong (for lysozyme the charged groups) while strengthening the lesser strong (hydrophobic) interactions. Lysozyme is atypical in the breadth of its crystallization conditions; many proteins only crystallize under narrowly defined conditions, pointing to the criticality of the empirical balancing process. Lack of a singularly defined association pathway leads to formation of multiple species, i.e., amorphous precipitation. Weak interactions, such as hydrogen bonds, are promiscuous, serving to strengthen rather than define specific interactions. Participation in an interaction sequesters that surface from subsequent interactions, and we expect the strongest bonds to form first. When two molecules self associate the resulting species will have an axis of symmetry. Subsequent interactions between two associated species having equivalent interactions will also have symmetry. Only a few unique sets of interactions are required to give any of the commonly found space groups for monomeric proteins. This model and what it suggests will be discussed.

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

2002-01-01

208

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

SciTech Connect

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.

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

2014-01-01

209

My Life with Bacteriophage ?29  

PubMed Central

This article is a survey of my scientific work over 52 years. During my postdoctoral stay in Severo Ochoa's laboratory, I determined the direction of reading of the genetic message, and I discovered two proteins that I showed to be involved in the initiation of protein synthesis. The work I have done in Spain with bacteriophage ?29 for 45 years has been very rewarding. I can say that I was lucky because I did not expect that ?29 would give so many interesting results, but I worked hard, with a lot of dedication and enthusiasm, and I was there when the luck arrived. I would like to emphasize our work on the control of ?29 DNA transcription and, in particular, the finding for the first time of a protein covalently linked to the 5?-ends of ?29 DNA that we later showed to be the primer for the initiation of phage DNA replication. Very relevant was the discovery of the ?29 DNA polymerase, with its properties of extremely high processivity and strand displacement capacity, together with its high fidelity. The ?29 DNA polymerase has become an ideal enzyme for DNA amplification, both rolling-circle and whole-genome linear amplification. I am also very proud of the many brilliant students and collaborators with whom I have worked over the years and who have become excellent scientists. This Reflections article is not intended to be the end of my scientific career. I expect to work for many years to come. PMID:23124207

Salas, Margarita

2012-01-01

210

Bacteriophage-Based Pathogen Detection  

NASA Astrophysics Data System (ADS)

Considered the most abundant organism on Earth, at a population approaching 1031, bacteriophage, or phage for short, mediate interactions with myriad bacterial hosts that has for decades been exploited in phage typing schemes for signature identification of clinical, food-borne, and water-borne pathogens. With over 5,000 phage being morphologically characterized and grouped as to susceptible host, there exists an enormous cache of bacterial-specific sensors that has more recently been incorporated into novel bio-recognition assays with heightened sensitivity, specificity, and speed. These assays take many forms, ranging from straightforward visualization of labeled phage as they attach to their specific bacterial hosts to reporter phage that genetically deposit trackable signals within their bacterial hosts to the detection of progeny phage or other uniquely identifiable elements released from infected host cells. A comprehensive review of these and other phage-based detection assays, as directed towards the detection and monitoring of bacterial pathogens, will be provided in this chapter.

Ripp, Steven

211

The episodic evolution of fibritin: traces of ancient global environmental alterations may remain in the genomes of T4-like phages  

PubMed Central

The evolutionary adaptation of bacteriophages to their environment is achieved by alterations of their genomes involving a combination of both point mutations and lateral gene transfer. A phylogenetic analysis of a large set of collar fiber protein (fibritin) loci from diverse T4-like phages indicates that nearly all the modular swapping involving the C-terminal domain of this gene occurred in the distant past and has since ceased. In phage T4, this fibritin domain encodes the sequence that mediates both the attachment of the long tail fibers to the virion and also controls, in an environmentally sensitive way, the phage's ability to infect its host bacteria. Subsequent to its distant period of modular exchange, the evolution of fibritin has proceeded primarily by the slow vertical divergence mechanism. We suggest that ancient and sudden changes in the environment forced the T4-like phages to alter fibritin's mode of action or function. The genome's response to such episodes of rapid environmental change could presumably only be achieved quickly enough by employing the modular evolution mechanism. A phylogenetic analysis of the fibritin locus reveals the possible traces of such events within the T4 superfamily's genomes. PMID:24223296

Letarov, A V; Krisch, H M

2013-01-01

212

Complete Genome Sequence of Escherichia Phage Lw1, a New Member of the RB43 Group of Pseudo T-Even Bacteriophages  

PubMed Central

RB43-related bacteriophages have a specific genome type that clearly distinguishes them from other T4-like viruses. Here, we present the complete genome sequence of a new virulent phage, Lw1, isolated as an Escherichia coli BL21(DE3) contaminant. Lw1 shares an RB43-like genome organization, but it does not contain putative AP2-domain endonuclease genes. PMID:24336362

Tovkach, Fedor I.; Comeau, Andre M.; Kostetskii, Igor E.; Lisovski, Igor; Ostapchuk, Andriy M.; Voychuk, Sergiy I.; Gorb, Tetiana I.; Romaniuk, Liudmyla V.

2013-01-01

213

The DNA polymerase genes of several HMU-bacteriophages have similar group I introns with highly divergent open reading frames.  

PubMed Central

A previous report described the discovery of a group I, self-splicing intron in the DNA polymerase gene of the Bacillus subtilis bacteriophage SPO1 (1). In this study, the DNA polymerase genes of three close relatives of SPO1: SP82, 2C and phi e, were also found to be interrupted by an intron. All of these introns have group I secondary structures that are extremely similar to one another in primary sequence. Each is interrupted by an open reading frame (ORF) that, unlike the intron core or exon sequences, are highly diverged. Unlike the relatives of Escherichia coli bacteriophage T4, most of which do not have introns (2), this intron seems to be common among the relatives of SPO1. Images PMID:7937082

Goodrich-Blair, H; Shub, D A

1994-01-01

214

Intrinsic Mean Square Displacement in Lysozyme  

NASA Astrophysics Data System (ADS)

The internal dynamics of proteins is the essential interest of biophysics. The mean square displacement (MSD) of hydrogen in proteins and its associated hydration water is obtained by molecular dynamic (MD) simulation. The MSD as currently determined depends on the time of the MD simulation. A method is proposed in this paper to obtain the intrinsic MSD of hydrogen in the proteins. The intrinsic MSD is independent of the simulation time and defined as the infinite time value of calculated MSD that appears in the Debye-Waller factor. The method consists of fitting a model to the incoherent intermediate scattering function. The model contains the intrinsic MSD and a rate constant characterizing the motions of H in the protein. The method is illustrated by obtaining the intrinsic MSD of lysozyme in 100 ns and 1 ?s MD simulations.

Vural, Derya; Glyde, Henry R.; Hong, Liang

2013-03-01

215

Resistance of Streptococcus Strain Fa-1 to Lysozyme.  

National Technical Information Service (NTIS)

In the course of an investigation into the properties of bacterial membrane-associated adenosine triphosphatase activities, an attempt was made to obtain whole-membrane preparations from Streptococcus strain FA-1 by the lysozyme method. This organism prov...

D. J. Evans

1969-01-01

216

Preliminary crystallographic examination of a novel fungal lysozyme from Chalaropsis  

NASA Technical Reports Server (NTRS)

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.

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

1990-01-01

217

Production and Engineering of Human Lysozyme Using Recombinant DNA Techniques.  

National Technical Information Service (NTIS)

An artificial gene encoding human lysozyme was chemically synthesized and expressed both in E. coli and S. cerevisiae. The product in E. coli formed insoluble aggregates and had no enzymatic activity. Urea treatment of the aggregates regenerated the enzym...

M. Muraki, Y. Jigami, H. Tanaka

1989-01-01

218

Investigation of binding properties of umbelliferone (7hydroxycoumarin) to lysozyme.  

PubMed

The binding interaction of lysozyme and umbelliferone (7hydroxcoumarin, 7HC) was investigated by UV-vis absorption and fluorescence quenching. It was obtained from fluorescence spectra that the fluorescence quenching of lysozyme by 7HC was probably a result of the formation of lysozyme-7HC complex and binding parameters were determined according to the Stern-Volmer equation. The effects of various common metal ions on the binding were also studied. The thermodynamic parameters were calculated at different temperatures which indicated that hydrophobic interaction. The binding distance (r) between the donor (lysozyme) and the acceptor (7HC) was 3.81 nm based on the Förster theory of non-radioactive resonance energy transfer. PMID:23224618

Gök, Elmas

2013-03-01

219

Organic Cosolvents and Hen Egg White Lysozyme Folding  

E-print Network

of Chemistry, Peking University, BEIJING 100871, China Key words: lysozyme; organic cosolvent; circular, consequently we know little about the conformation and energetics during protein folding from a loose peptide

Luhua, Lai

220

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2010 CFR

...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...

2010-04-01

221

Bacteriophages with the Ability to Degrade Uropathogenic Escherichia Coli Biofilms  

PubMed Central

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

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

2012-01-01

222

T-4G Methodology: Undergraduate Pilot Training T-37 Phase.  

ERIC Educational Resources Information Center

The report's brief introduction describes the application of T-4G methodology to the T-37 instrument phase of undergraduate pilot training. The methodology is characterized by instruction in trainers, proficiency advancement, a highly structured syllabus, the training manager concept, early exposure to instrument training, and hands-on training.…

Woodruff, Robert R.; And Others

223

On the problem of phase transitions in lysozyme crystals  

E-print Network

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

Yu. Vasylkiv; Yu. Nastishin; R. Vlokh

2007-06-17

224

Penetration and fusion of phospholipid vesicles by lysozyme  

SciTech Connect

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.

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

1989-10-01

225

Study of ethanol-lysozyme interactions using neutron diffraction  

SciTech Connect

Single-crystal neutron diffraction has been used to observe the interactions between deuterated ethanol (CD3CD2OH) and lysozyme in triclinic crystals of hen egg white lysozyme soaked in 25% (v/v) ethanol solutions. A total of 6047 observed reflections to a resolution of 2 A were used, and 13 possible ethanol sites were identified. The three highest occupied sites are close to locations for bromoethanol found in an earlier study by Yonath et al. (Yonath, A., Podjarny, A., Honig, B., Traub, W., Sielecki, A., Herzberg, O., and Moult, J. (1978) Biophys. Struct. Mech. 4, 27-36). Structure refinements including a model for the flat solvent lead to a final crystallographic agreement factor of 0.097. Comparison with earlier neutron studies on triclinic lysozyme showed that neither the molecular structure nor the thermal motions were affected significantly by the ethanol. A detailed analysis of the ethanol-lysozyme contacts showed 61% of these to be with hydrophobic sites, in agreement with the dominant hydrophobic nature of ethanol. This, together with the fact that the molecular structure of lysozyme is not perturbed, suggests a model for denaturation of lysozyme by alcohol, which proceeds via a dehydration of the protein at high alcohol concentration.

Lehmann, M.S.; Mason, S.A.; McIntyre, G.J.

1985-10-08

226

Lysozyme detection on aptamer functionalized graphene-coated SPR interfaces.  

PubMed

The paper reports on a surface plasmon resonance (SPR)-based approach for the sensitive and selective detection of lysozyme. The SPR sensor consists of a 50 nm gold film coated with a thin film of reduced graphene oxide (rGO) functionalized with anti-lysozyme DNA aptamer. The SPR chip coating with rGO matrix was achieved through electrophoretic deposition of graphene oxide (GO) at 150 V. Electrophoretic deposition resulted in partial reduction of GO to rGO with a thickness depending on the deposition time. For very short time pulses of 20 s, the resulting rGO film had a thickness of several nanometers and was appropriate for SPR sensing. The utility of the graphene-based SPR sensor for the selective and sensitive detection of proteins was demonstrated using lysozyme as model protein. Functionalization of rGO matrix with anti-lysozyme DNA aptamer through ?-stacking interactions allowed selective SPR detection of lysozyme. The graphene-based SPR biosensor provides a means for the label-free, concentration-dependent and selective detection of lysozymes with a detection limit of 0.5 nM. PMID:23871871

Subramanian, Palaniappan; Lesniewski, Adam; Kaminska, Izabela; Vlandas, Alexis; Vasilescu, Alina; Niedziolka-Jonsson, Joanna; Pichonat, Emmanuelle; Happy, Henri; Boukherroub, Rabah; Szunerits, Sabine

2013-12-15

227

Cloning, characterization, and production of a novel lysozyme by different expression hosts.  

PubMed

Lysozyme is a protein found in egg white, tears, saliva, and other secretions. As a marketable natural alternative to preservatives, lysozyme can act as a natural antibiotic. In this study, we have isolated Bacillus licheniformis TIB320 from soil, which contains a lysozyme gene with various features. We have cloned and expressed the lysozyme in E. coli. The antimicrobial activity of the lysozyme showed that it had a broad antimicrobial spectrum against several standard strains. The lysozyme could maintain efficient activities in a pH range between 3 and 9 and from 20°C to 60°C, respectively. The lysozyme was resistant to pepsin and trypsin to some extent at 40°C. Production of the lysozyme was optimized by using various expression strategies in B. subtilis WB800. The lysozyme from B. licheniformis TIB320 will be promising as a food or feed additive. PMID:24986676

Zhang, Haifeng; Fu, Gang; Zhang, Dawei

2014-10-28

228

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

SciTech Connect

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

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

2006-01-01

229

Chemical Synthesis of Bacteriophage G4  

PubMed Central

Background Due to recent leaps forward in DNA synthesis and sequencing technology, DNA manipulation has been extended to the level of whole-genome synthesis. Bacteriophages occupy a special niche in the micro-organic ecosystem and have potential as a tool for therapeutic agent. The purpose of this study was to carry out chemical synthesis of the bacteriophage G4 and the study of its infectivity. Methodology/Principal Findings Full-sized genomes of bacteriophage G4 molecules were completed from short overlapping synthetic oligonucleotides by direct assembly polymerase chain reaction and ligase chain reaction followed by fusion polymerase chain reaction with flanking primers. Three novel restriction endonuclease sites were introduced to distinguish the synthetic G4 from the wild type. G4 particles were recovered after electroporation into Escherichia coli and were efficient enough to infect another strain. The phage was validated by electron microscope. Specific polymerase chain reaction assay and restriction analyses of the plaques verified the accuracy of the chemical synthetic genomes. Conclusions Our results showed that the bacteriophage G4 obtained is synthetic rather than a wild type. Our study demonstrated that a phage can be synthesized and manipulated genetically according to the sequences, and can be efficient enough to infect the Escherichia coli, showing the potential use of synthetic biology in medical application. PMID:22110602

Ye, Yiwang; Liu, Yuchen; Jiang, Zhimao; Gui, Yaoting; Cai, Zhiming

2011-01-01

230

Enteroviruses and Bacteriophages in Bathing Waters  

Microsoft Academic Search

A new procedure for detecting and counting enteroviruses based on the VIRADEN method applied to 10 liters of seawater was examined. It improved the efficiency of detection by taking into account both the number of positive isolations and numbers found with traditional methods. It was then used to quantify viruses in bathing waters. A number of bacterial indicators and bacteriophages

L. Moce-Llivina; Francisco Lucena; Juan Jofre

2005-01-01

231

Bacteriophage Ecology in Commercial Sauerkraut Fermentations  

Microsoft Academic Search

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

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

2003-01-01

232

ADSORPTION OF BACTERIOPHAGES ON CLAY MINERALS  

EPA Science Inventory

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

233

40 CFR 721.10337 - Copper, iodotris(triphenylphosphine)-, (T-4)-.  

Code of Federal Regulations, 2012 CFR

...Copper, iodotris(triphenylphosphine)-, (T-4)-. 721.10337 Section 721.10337...Copper, iodotris(triphenylphosphine)-, (T-4)-. (a) Chemical substance and...copper, iodotris(triphenylphosphine)-, (T-4)- (PMN P-04-6; CAS No....

2012-07-01

234

26 CFR 31.3402(t)-4 - Certain payments excepted from withholding.  

Code of Federal Regulations, 2012 CFR

...Certain payments excepted from withholding. 31.3402(t)-4 Section 31.3402(t)-4 Internal Revenue INTERNAL REVENUE SERVICE... Collection of Income Tax at Source § 31.3402(t)-4 Certain payments excepted from...

2012-04-01

235

40 CFR 721.10337 - Copper, iodotris(triphenylphosphine)-, (T-4)-.  

Code of Federal Regulations, 2013 CFR

...Copper, iodotris(triphenylphosphine)-, (T-4)-. 721.10337 Section 721.10337...Copper, iodotris(triphenylphosphine)-, (T-4)-. (a) Chemical substance and...copper, iodotris(triphenylphosphine)-, (T-4)- (PMN P-04-6; CAS No....

2013-07-01

236

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

Code of Federal Regulations, 2013 CFR

...vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261...vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption...vesicatoria and Pseudomonas syringae pv. tomato specific bacteriophages in or on...

2013-07-01

237

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

Code of Federal Regulations, 2012 CFR

...vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261...vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption...vesicatoria and Pseudomonas syringae pv. tomato specific bacteriophages in or on...

2012-07-01

238

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

Code of Federal Regulations, 2011 CFR

...vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261...vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption...vesicatoria and Pseudomonas syringae pv. tomato specific bacteriophages in or on...

2011-07-01

239

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

Code of Federal Regulations, 2010 CFR

...vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261...vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption...vesicatoria and Pseudomonas syringae pv. tomato specific bacteriophages in or on...

2010-07-01

240

Splint ligation of RNA with T4 DNA ligase  

PubMed Central

Splint ligation of RNA, whereby specific RNA molecules are ligated together, can be carried out using T4 DNA ligase and a bridging DNA oligonucleotide complementary to the RNAs. This method takes advantage of the property of T4 DNA ligase to join RNA molecules when they are in an RNA:DNA hybrid. Splint ligation is a useful tool for the introduction of modified nucleotides into RNA molecules, insertion of a radiolabel into a specific position within an RNA and for the assembly of smaller synthetic RNAs into longer RNA molecules. Such modifications enable a wide range of experiments to be carried out with the modified RNA including structural studies, co-immunoprecipitations, and the ability to map sites of RNA:RNA and RNA:protein interactions. PMID:23065567

Kershaw, Christopher J.; O’Keefe, Raymond T.

2014-01-01

241

Droplet hydrodynamics during lysozyme protein crystallization.  

PubMed

Various experimental studies in zero gravity have been reported in the literature for generation of superior quality crystals due to the importance of convective transport on protein crystal quality. However, limited experimental and numerical studies are available in the literature for a complete characterization of transport phenomena during the protein crystal growth process. The present investigation reports experimental results on convective motion inside the droplet during protein crystallization by the vapor diffusion method. Lysozyme crystals are grown using a sitting drop method and micro-particle image velocimetry is used for investigating the detailed hydrodynamics inside the droplet. Dynamic evolution of the flow field for the complete crystal growth process, i.e., during the prenucleation, nucleation, and postnucleation stage, is reported. Various flow transitions are observed during the complete cycle of the protein crystal growth process. Significant Marangoni convection is observed during the prenucleation period followed by buoyancy-driven convection during the postnucleation period. The Marangoni convection flow patterns observed during the prenucleation stage match those of evaporating droplets. The postnucleation convection patterns are similar to those of ethanol-water mixture evaporation with high ethanol concentration. PMID:23214788

Pradhan, T; Asfer, M; Panigrahi, P K

2012-11-01

242

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

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

2010-01-01

243

Killing of gram-negative bacteria by lactoferrin and lysozyme.  

PubMed Central

Although lactoferrin has antimicrobial activity, its mechanism of action is not full defined. Recently we have shown that the protein alters the Gram-negative outer membrane. As this membrane protects Gram-negative cells from lysozyme, we have studied whether lactoferrin's membrane effect could enhance the antibacterial activity of lysozyme. We have found that while each protein alone is bacteriostatic, together they can be bactericidal for strains of V. cholerae, S. typhimurium, and E. coli. The bactericidal effect is dose dependent, blocked by iron saturation of lactoferrin, and inhibited by high calcium levels, although lactoferrin does not chelate calcium. Using differing media, the effect of lactoferrin and lysozyme can be partially or completely inhibited; the degree of inhibition correlating with media osmolarity. Transmission electron microscopy shows that E. coli cells exposed to lactoferrin and lysozyme at 40 mOsm become enlarged and hypodense, suggesting killing through osmotic damage. Dialysis chamber studies indicate that bacterial killing requires direct contact with lactoferrin, and work with purified LPS suggests that this relates to direct LPS-binding by the protein. As lactoferrin and lysozyme are present together in high levels in mucosal secretions and neutrophil granules, it is probable that their interaction contributes to host defense. Images PMID:1918365

Ellison, R T; Giehl, T J

1991-01-01

244

Is Fleming's lysozyme an analgesic agent? Experiments on mice.  

PubMed

1. Antinociceptive activity of hen egg white lysozyme (Fleming's lysozyme) was determined against abdominal contractions provoked by irritants injected intraperitoneally into mice. Carrageenan (2 mg) (CA) injected with arachidonic acid (15 micrograms) (AA) or prostaglandins PGE1 or PGF2 alpha (0.04 ng), brewer's yeast (10 mg), caolin (10 mg), mepartricin (80 U) and phenylquinone (50 micrograms) were used as irritants. 2. Lysozyme was active at 400-800 mg/kg i.v. against CA + AA, CA + PG, brewer's yeast and caolin nociceptive stimulation. The compound was more effective against CA + AA than against CA + PG. Acetylsalicylic acid at 50-100-200 mg/kg p.o. was equally active against CA + AA and CA + PG. 3. Lysozyme was inactive in the tail pinch and hot plate tests that mainly detect central analgesics. 4. The results are discussed in relation to the claim advanced years ago that lysozyme is an effective analgesic agent in humans. The compound was found active against herpes zoster or cancer pain but did not find use despite the favourable reports presented. 5. The experimental results obtained on laboratory animals do not contradict the conclusions drawn after the clinical use of the compound. PMID:6220850

Bianchi, C

1983-01-01

245

Logarithmic Decay in Single-Particle Relaxation of Hydrated Lysozyme Powder Marco Lagi,1,2  

E-print Network

Logarithmic Decay in Single-Particle Relaxation of Hydrated Lysozyme Powder Marco Lagi,1,2 Piero-dynamics of protein amino acids of hydrated lysozyme powder around the physiological temperature by means of molecular

Chen, Sow-Hsin

246

Lysozyme Activity in the Serum, Saliva and Tears of Germfree and Conventional Rats and Mice.  

National Technical Information Service (NTIS)

Lysozyme levels in serum, saliva and tears of germfree, gnotobiotic, conventionalized as well as conventionally-reared rats and mice were studied. The results showed that lysozyme levels in serum and tears were quite similar in these groups. However, the ...

D. R. Makulu, M. Wagner

1967-01-01

247

Sensitization by Ethylenediaminetetraacetate of Clostridium perfringens Type A Spores to Germination by Lysozyme1  

PubMed Central

Clostridium perfringens spores (three strains) were normally resistant to germination by lysozyme. In the absence of disulfide bond-breaking reagents, tetrasodium ethylenediaminetetraacetate rapidly sensitized the spores to lysozyme. PMID:4355485

Adams, D. M.

1973-01-01

248

Bacteriophage therapy: a potential solution for the antibiotic resistance crisis.  

PubMed

The emergence of multiple drug-resistant bacteria has prompted interest in alternatives to conventional antimicrobials. One of the possible replacement options for antibiotics is the use of bacteriophages as antimicrobial agents. Phage therapy is an important alternative to antibiotics in the current era of drug-resistant pathogens. Bacteriophages have played an important role in the expansion of molecular biology and have been used as antibacterial agents since 1966. In this review, we describe a brief history of bacteriophages and clinical studies on their use in bacterial disease prophylaxis and therapy. We discuss the advantages and disadvantages of bacteriophages as therapeutic agents in this regard. PMID:24518621

Golkar, Zhabiz; Bagasra, Omar; Pace, Donald Gene

2014-02-01

249

[Lysozyme Reference Standard (Control 031) of National Institute of Health Sciences].  

PubMed

The "Lysozyme Reference Standard (Control 951031)" of the National Institute of Health Sciences was prepared. The lysozyme potency of the standard material was assayed against the Lysozyme Reference Standard (Control 951) by turbidimetric method two turbidimetric methods using the dried y-cells of Micrococcus luteus as a substrate. The potency of the standard material was in satisfactory agreement with that of Lysozyme Reference Standard (Control 951) and was defined as 1 mg [potency] per mg. PMID:14740415

Kaihara, Akiko; Murakami, Miho; Morita, Yukiko; Koide, Tatsuo; Murai, Toshimi; Saito, Hiroyuki; Tanimoto, Tsuyoshi

2003-01-01

250

Modeling Tetragonal Lysozyme Crystal Growth Rates  

NASA Technical Reports Server (NTRS)

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.

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

2003-01-01

251

T4 DNA condensation in water-alcohol media  

E-print Network

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.

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

2011-07-21

252

On symmetries of = (4, 4) sigma models on T 4  

NASA Astrophysics Data System (ADS)

Motivated by an analogous result for K3 models, we classify all groups of symmetries of non-linear sigma models on a torus T^4 that preserve the N=(4,4) superconformal algebra. The resulting symmetry groups are isomorphic to certain subgroups of the Weyl group of E8, that plays a role similar to the Conway group for the case of K3 models. Our analysis heavily relies on the triality automorphism of the T-duality group SO(4,4,Z). As a byproduct of our results, we discover new explicit descriptions of K3 models as asymmetric orbifolds of torus CFTs.

Volpato, Roberto

2014-08-01

253

??????????????????? ?????????????????????????????????????????????? Isolation of Bacteriophages Specific to Enteric Bacteria  

Microsoft Academic Search

Bacteria in family Enterobacteriaceae are pathogenic to human and animals. They often contaminate food and water. In this study four bacteriophages specific to Shigella dysenteriae DMS2137, Salmonella Typhi DMS5784, Escherichia coli ATCC25922 and Enterobacter aerogenes were isolated from sewage water in Ubon Ratchathani, Thailand, using double-layer plaque technique. All of them showed specificity only to their hosts. These results indicated

Kanjana Nukdee

254

Immune upregulation of novel antibacterial proteins from silkmoths (Lepidoptera) that resemble lysozymes but lack muramidase activity  

Microsoft Academic Search

Study on immune proteins in domesticated and wild silkmoths Bombyx mori and Antheraea mylitta, respectively, led to identification of a new class of antimicrobial proteins. We designated them as lysozyme-like proteins (LLPs) owing to their partial similarity with lysozymes. However, lack of characteristic catalytic amino acid residues essential for muramidase activity in LLPs puts them functionally apart from classical lysozymes.

Archana S. Gandhe; Gude Janardhan; Javaregowda Nagaraju

2007-01-01

255

Newly Discovered Antiterminator RNAs in Bacteriophage ?  

PubMed Central

Antiterminator RNA directly modifies the transcription elongation complex so that it terminates less efficiently at intrinsic and factor-dependent terminators. These unusual RNAs were first discovered in bacteriophage HK022, where the nascent transcripts of the phage put sites promote full expression of phage genes during lytic infection. The activity of antiterminator RNA depends on specific structural elements that form as the transcript exits RNA polymerase. To further our understanding of the critical sequence features that permit RNA to serve as a transcriptional antiterminator, we have identified eight antiterminator RNA sequences in bacteriophages or prophages. There is strong sequence conservation among most of the put sequences, but sequence divergence is tolerated if critical structural elements are preserved. The most diverged antiterminator RNA is found in bacteriophage HK639. The HK639 putL transcript is an efficient antiterminator, and it has a novel structural feature that is critical for its activity. HK639 also displays a unique pattern of sensitivity to amino acid substitutions in the ?? subunit zinc binding domain of RNA polymerase, adding to existing evidence that this domain interacts specifically with antiterminator RNA. PMID:21840976

King, Rodney A.; Wright, Alice; Miles, Courtney; Pendleton, Christopher S.; Ebelhar, Andrew; Lane, Stephanie; Parthasarathy, Prasanna Tamarapu

2011-01-01

256

Newly discovered antiterminator RNAs in bacteriophage.  

PubMed

Antiterminator RNA directly modifies the transcription elongation complex so that it terminates less efficiently at intrinsic and factor-dependent terminators. These unusual RNAs were first discovered in bacteriophage HK022, where the nascent transcripts of the phage put sites promote full expression of phage genes during lytic infection. The activity of antiterminator RNA depends on specific structural elements that form as the transcript exits RNA polymerase. To further our understanding of the critical sequence features that permit RNA to serve as a transcriptional antiterminator, we have identified eight antiterminator RNA sequences in bacteriophages or prophages. There is strong sequence conservation among most of the put sequences, but sequence divergence is tolerated if critical structural elements are preserved. The most diverged antiterminator RNA is found in bacteriophage HK639. The HK639 putL transcript is an efficient antiterminator, and it has a novel structural feature that is critical for its activity. HK639 also displays a unique pattern of sensitivity to amino acid substitutions in the ?' subunit zinc binding domain of RNA polymerase, adding to existing evidence that this domain interacts specifically with antiterminator RNA. PMID:21840976

King, Rodney A; Wright, Alice; Miles, Courtney; Pendleton, Christopher S; Ebelhar, Andrew; Lane, Stephanie; Parthasarathy, Prasanna Tamarapu

2011-10-01

257

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

PubMed Central

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

Dauden, Maria I.; Martin-Benito, Jaime; Sanchez-Ferrero, Juan C.; Pulido-Cid, Mar; Valpuesta, Jose M.; Carrascosa, Jose L.

2013-01-01

258

Spectrophotometric studies on the interaction between (-)-epigallocatechin gallate and lysozyme  

NASA Astrophysics Data System (ADS)

Various reported antibacterial activities of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea prompted us to study its binding with lysozyme. This has been investigated by fluorescence, circular dichroism (CD) and protein-ligand docking. The binding parameters were determined using a modified Stern-Volmer equation. The thermodynamic parameters are indicative of an initial hydrophobic association. The complex is, however, held together predominantly by van der Waals interactions and hydrogen bonding. CD studies do not indicate any significant changes in the secondary structure of lysozyme. Docking studies revealed that specific interactions are observed with residues Trp 62 and Trp 63.

Ghosh, Kalyan Sundar; Sahoo, Bijaya Ketan; Dasgupta, Swagata

2008-02-01

259

The solubility of hen egg-white lysozyme  

NASA Technical Reports Server (NTRS)

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.

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

1988-01-01

260

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

PubMed

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

Beyer, C

2013-09-01

261

Original article Effect of bacteriophage DC22 on Escherichia coli  

E-print Network

Original article Effect of bacteriophage DC22 on Escherichia coli O157:H7 in an artificial rumen -- The effect of a bacteriophage, DC22, on the survival of Escherichia coli O157:H7 in an artificial rumen of 105 PFU of DC22/CFU of E. coli O157:H7 (P Escherichia coli O157:H7 persisted in the control

Boyer, Edmond

262

Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy  

E-print Network

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

Collins, James J.

263

Targeting Antibacterial Agents by Using Drug-Carrying Filamentous Bacteriophages  

Microsoft Academic Search

Bacteriophages have been used for more than a century for (unconventional) therapy of bacterial infections, for half a century as tools in genetic research, for 2 decades as tools for discovery of specific target-binding proteins, and for nearly a decade as tools for vaccination or as gene delivery vehicles. Here we present a novel application of filamentous bacteriophages (phages) as

Iftach Yacoby; Marina Shamis; Hagit Bar; Doron Shabat; Itai Benhar

2006-01-01

264

Complete Genome Sequences of Five Paenibacillus larvae Bacteriophages  

PubMed Central

Paenibacillus larvae is a pathogen of honeybees that causes American foulbrood (AFB). We isolated bacteriophages from soil containing bee debris collected near beehives in Utah. We announce five high-quality complete genome sequences, which represent the first completed genome sequences submitted to GenBank for any P. larvae bacteriophage. PMID:24233582

Sheflo, Michael A.; Gardner, Adam V.; Merrill, Bryan D.; Fisher, Joshua N. B.; Lunt, Bryce L.; Breakwell, Donald P.; Grose, Julianne H.

2013-01-01

265

Complete Genome Sequence of a Mosaic Bacteriophage, Waukesha92  

PubMed Central

In this study, we determined the complete genome sequence of a mosaic bacteriophage, Waukesha92, which was isolated from soil using Bacillus thuringiensis as the host organism. This temperate Myoviridae bacteriophage has similarities to phages SpaA1 and BceA1 and the Bacillus thuringiensis plasmid pBMB165. PMID:25146131

Sauder, A. Brooke; Carter, Brandon; Langouet Astrie, Christophe

2014-01-01

266

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  

Microsoft Academic Search

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

Qinggang Xue; Michael E Hellberg; Kevin L Schey; Naoki Itoh; Ron I Eytan; Richard K Cooper; Jerome F La Peyre

2010-01-01

267

Some properties of a macromolecular conjugate of lysozyme prepared by modification with a monomethoxypolyethylene glycol derivative.  

PubMed

Hen egg-white lysozyme was modified with a succinyl ester derivative of monomethoxypolyethylene glycol (mPEG-COONSu), and some properties of the resulting conjugate (mPEG-lysozyme) were studied. The conjugate was prepared by modification of lysozyme with mPEG-COONSu and purified with use of columns of CM-Toyopearl 650M and Sephadex G-75. Analytical data indicated that in the conjugate, 1.05 moles of mPEG with an average molecular weight of 5,000 were covalently attached to the lysozyme molecule. Tryptic peptide analysis of the conjugate showed that Lys 33 in lysozyme is the residue mainly modified with mPEG-COONSu. Covalent attachment of the mPEG-derivative to amino groups greatly increased the thermostability of lysozyme without any conformational change of the protein molecule. mPEG-lysozyme retained full enzyme activity for glycol chitin, but lytic activity for Micrococcus luteus cells in neutral media was 75% of that of native lysozyme and its optimal pH was at pH 5.0. It was also found that the reactivity of lysozyme with anti-lysozyme antibody from BALB/c mice or human lymphocytes was decreased by modification with mPEG-COONSu. From these findings, it was suggested that mPEG-COONSu can be advantageously used for protein tailoring of lysozyme. PMID:10830491

Nodake, Y; Yamasaki, N

2000-04-01

268

Requirement for and Sensitivity to Lysozyme by Clostridium perfringens Spores Heated at Ultrahigh Temperatures1  

PubMed Central

The requirement of ultrahigh temperature (UHT)-treated Clostridium perfringens spores for lysozyme and the sensitivity of heated and unheated spores to lysozyme were studied. The UHT-treated spores requiring lysozyme for germination and colony formation originated from only a small portion of the non-UHT-treated spore population. This raised a question of whether the requirement for lysozyme was natural to the spores or was induced by the UHT treatments. However, these spores did not require lysozyme for germination before UHT treatment, which confirmed that the requirement for lysozyme had been induced by the UHT treatment. Only 1 to 2% of the spores were naturally sensitive to lysozyme; therefore, the mere addition of lysozyme to the plating medium did not permit the enumeration of all survivors. Treatment of UHT-treated spores with ethylenediaminetetraacetate (EDTA) sensitized the spores to lysozyme and increased by 10- to 100-fold the number of survivors that were detected on a medium containing lysozyme. Under the heating conditions used, spores that were naturally sensitive to lysozyme and spores that required EDTA treatment were equally heat resistant. PMID:4363559

Adams, D. M.

1974-01-01

269

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

NASA Astrophysics Data System (ADS)

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.

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

2005-03-01

270

The distribution of muramidase (lysozyme) in human tissues  

Microsoft Academic Search

The distribution of muramidase (lysozyme) in normal and pathological human tissues has been studied, using an immunohistological technique. The enzyme was demonstrated in a variety of healthy tissues, including serous salivary acinar cells, lactating mammary tissue, Paneth cells, renal tubular cells, myeloid cells (including eosinophils), and histiocytic cells. In pathological tissues the most striking positivity was encountered in reactive histiocytic

D Y Mason; C R Taylor

1975-01-01

271

Exons Encode Functional and Structural Units of Chicken Lysozyme  

Microsoft Academic Search

The nucleotide sequence was determined for the chicken egg white lysozyme mRNA and for the exons of the gene together with their flanking intron regions. The exon pattern is to some degree related to the structural subdivision of the final protein product. However, the relationship of exons to functional units of the enzyme is better established. Exon 2 codes for

Alexander Jung; Albrecht E. Sippel; Manuel Grez; Gunther Schutz

1980-01-01

272

The Paneth cell: a source of intestinal lysozyme  

Microsoft Academic Search

An antiserum prepared against lysozyme isolated from mucosal scrapings of mouse small intestine was used to stain sections of mouse small intestine with the indirect fluorescent antibody technique. Mucosal fluorescence was confined to the base of the crypts of Lieberkuhn, where Paneth cells are located. After the intravenous administration of 4 mg of pilocarpine fluorescence was no longer found in

T Peeters; G Vantrappen

1975-01-01

273

Effects of Acetate Buffer Concentration on Lysozyme Solubility.  

National Technical Information Service (NTIS)

The micro-solubility column technique was employed to systematically investigate the effects of buffer concentration on tetragonal lysozyme solubility. While keeping the NaCl concentrations constant at 2%, 3%, 4%, 5% and 7%, and the pH at 4.0, we have stu...

E. L. Forsythe, M. L. Pusey

1996-01-01

274

Science Study Aids 6: Lysozyme - The Cooperative Enzyme.  

ERIC Educational Resources Information Center

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…

Boeschen, John; Alderton, Gordon

275

HEMOLYSIN AND LYSOZYME PRODUCTION BY STAPHYLOCOCCI ISOLATED FROM  

E-print Network

out of i8 lysozyme-negative strains produced only beta lysin. Of the many extracellular substances in identifying potentially patho- genic strains. Closely associated with coagulase production as in vitro test of animal origin, especially in bovine mastitis, presence of beta hemo- lysin is considered to be very

Paris-Sud XI, Université de

276

Kinetic Roughening Transition and Energetics of Tetragonal Lysozyme Crystal Growth  

NASA Technical Reports Server (NTRS)

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.

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

2004-01-01

277

Synergistic effect of chitooligosaccharides and lysozyme for meat preservation  

Microsoft Academic Search

The objective of this study was to enhance the antibacterial spectrum of lysozyme with the use of chitooligosaccharides (COS) produced by radiation treatment of chitosan. Exposure of chitosan solution to ? radiation led to formation of oligosaccharides of different molecular weights. COS with molecular weight of 8.3kDa were found to exhibit highest antioxidant potential in free radical scavenging assay but

M. S. Rao; R. Chander; A. Sharma

2008-01-01

278

ANTIMICROBIAL EFFECTS ON STARCH-BASED FILMS INCORPORATED WITH LYSOZYMES  

Microsoft Academic Search

An antimicrobial (AM) Active Packaging can be made by incorporating and immobilizing suitable AM agents into food packages and applying a bio switch concept. A starch-based film was prepared and incorporated with antimicrobial agents, i.e. lysozyme and EDTA as chelating agent. This film was then inoculated with the bacteria Escherichia coli and Bacillus subtilis to carry out the microbial contamination

Nozieana Khairuddin; Ida Idayu Muhamad

279

Study of the Distribution of Pseudotuberculosis Bacteriophage in Nature. Communication I. Isolation of Bacteriophage from the Organs of Rodents.  

National Technical Information Service (NTIS)

During an investigation of the organs of 998 rodents, 36 strains of bacteriophage (3.6%) were isolated that lysed pseudotuberculosis cultures. The bacteriophage was detected in the organs of gray rats (3.8%), house mice, (2.6%), voles and common shrews. T...

N. M. Somova, N. A. Sergeeva

1965-01-01

280

M13 Bacteriophage Based Protein Sensors  

NASA Astrophysics Data System (ADS)

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

Lee, Ju Hun

281

Bacteriophage Diversity in the North Sea  

PubMed Central

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

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

1998-01-01

282

Exploiting gut bacteriophages for human health.  

PubMed

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

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

2014-07-01

283

Conductivity and Viscosity Measurements for Binary Lysozyme Chloride Aqueous Solution and Ternary Lysozyme-Salt-Water Solution  

E-print Network

We use the conductimetric method, adequate to electrolytes, to determine the lysozyme charge in lys-water and ternary lys-salt-water systems. We measured also the viscosities for the above binary and ternary systems in the same conditions at pH$=4.5$ and T$=298$ K, measurements that allow us to see any effect of viscosity on cations mobilities and implicitly on the lysozyme charge. The method is illustrated for the lysozyme chloride aqueous solution system at 25$^o$ C, using the data reported here for pH$=4.5$ at 0.15, 0.6, 0.8, 1., 1.5, 2., 2.5, 3., 3.5 mM (mg/mL) lysozyme chloride concentrations. The method was also applied to ternary lys-salt-water systems in the same conditions at pH$=4.5$ and T$=25^o$ C. Ternary conductivities are reported for a mean concentration 0.6 mM of lysozyme chloride in all systems and a mean concentration 0.01, 0.025, 0.05, 0.1, 0.175, 0.2, 0.5, 0.7, 0.9, 1.2, 1.3 and 1.4 M for NaCl; 0.005, 0.01, 0.05, 0.1, 0.175, 0.2, 0.5, 0.7, 0.9, 1.2, 1.3, 1.4 and 1.5 M for KCl; 0.005, 0.01,...

Buzatu, D; Buzatu, F D

2004-01-01

284

Structural transitions of monoolein bicontinuous cubic phase induced by inclusion of protein lysozyme solutions  

E-print Network

Inclusion of protein lysozyme molecules in lipidic monoolein cubic phase induces a transition from a $\\rm Pn\\bar{3}m$ structure to $\\rm Im\\bar{3}m$ one. Small-angle X-ray scattering (SAXS) method with high intensity synchrotron radiation enabled us to follow closely the transition depending on the conditions of lysozyme solutions. We showed that concentrated lysozyme solutions induced the appearance of the $\\rm Im\\bar{3}m$ structure coexisting with the $\\rm Pn\\bar{3}m$ structure. From the relation between the lattice parameters of these two structures it was shown that they were related by the Bonnet transformation of underlying triply periodic minimal surfaces. We found that the transition also occurred at lower lysozyme concentration when NaCl induced attraction between lysozyme molecules. The origin of the transition was considered as a frustration in the cubic phase where lysozyme molecules were highly confined. A simple estimation of the frustration was given, which took into account of the translational entropy of lysozyme molecules. At the highest concentration of lysozyme and NaCl the $\\rm Im\\bar{3}m$ structure was found to disappear and left only the $\\rm Pn\\bar{3}m$ structure. This was probably either due to the crystallization or phase separation of lysozyme solutions ongoing microscopically, which absorbed lysozyme molecules from channels of the cubic phase and thus removed the frustration.

S. Tanaka; S. Maki; M. Ataka

2006-05-25

285

Oxygen-17 and deuterium nuclear magnetic resonance studies of lysozyme hydration  

SciTech Connect

Oxygen-17 and deuterium NMR studies of lysozyme hydration are reported for a wide range of lysozyme concentrations, and the relationship between water activity and water mobility in the lysozyme-water system as determined by high-field NMR is examined. In a first approximation, the effect of lysozyme activity on hydration is considered to be small because of the relatively low charge on lysozyme at pH 7 and the absence of salt in the lysozyme solutions. Correlation times are determined for tightly bound water, weakly bound water, and multilayer or trapped water in lysozyme at 20 degrees C. Hydration numbers are also determined for these three different water populations interacting with lysozyme. Good agreement is found between the hydration numbers determined by 17O NMR and the calculations based on the D'Arcy and Watt analysis of water sorption isotherms for proteins that considered three major water populations in hydrated lysozyme. A molecular interpretation for the three components in the D'Arcy and Watt theory of sorption isotherms is also proposed on the basis of our NMR results. Previous proton NMR spin-echo results are shown to be consistent with our findings by 17O NMR and support the view that there are at least four regions of distinct hydration behavior of lysozyme which span the whole range from solutions to solid powders.

Lioutas, T.S.; Baianu, I.C.; Steinberg, M.P.

1986-05-15

286

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

Code of Federal Regulations, 2010 CFR

...acetato-.kappa.O)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880...acetato-.kappa.O)hydro-, sodium, (T-4)-. (a) Chemical substance and...acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

2010-07-01

287

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

Code of Federal Regulations, 2012 CFR

...acetato-.kappa.O)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880...acetato-.kappa.O)hydro-, sodium, (T-4)-. (a) Chemical substance and...acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

2012-07-01

288

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

Code of Federal Regulations, 2013 CFR

...acetato-.kappa.O)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880...acetato-.kappa.O)hydro-, sodium, (T-4)-. (a) Chemical substance and...acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

2013-07-01

289

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

Code of Federal Regulations, 2011 CFR

...acetato-.kappa.O)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880...acetato-.kappa.O)hydro-, sodium, (T-4)-. (a) Chemical substance and...acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

2011-07-01

290

[Friedrich Mauz: T4 assessor and military psychiatrist].  

PubMed

Friedrich Mauz is one of the medical perpetrators of the second tier whose biography is difficult to comprehend. Autobiographies from three different political systems exist - Weimar Republic, the Third Reich, and postwar Germany in which he constantly reinvented himself. While after 1933 he suddenly emphasized his participation in the civil war turmoil during the early period of the Weimar Republic and his patriotism, he then depicted himself after 1945 as an apolitical person characterized by Württemberg pietism who inwardly rejected the Nazi State but had found himself prepared to accept "all sorts of humiliating concessions." He claimed that he had always remained true to his scientific code of conduct and had distanced himself from psychiatric genetics. In point of fact, Mauz was among those exonerated in the denazification trial in 1946 and was able to pursue his career in the Federal Republic of Germany. However, if the sources are read against the grain, a different picture emerges. Mauz's career stalled in the 1930s, not because he had been politically offensive, but because his scientific work was flimsy and considered lacking originality, particularly since he had chosen constitution research and psychotherapy as his main fields of interest, which were overshadowed by research in genetic psychiatry in the 1930s. Mauz tendered his services to the Nazi policy of genetic health, served as a medical assessor in proceedings based on the "Law for the Prevention of Genetically Diseased Offspring," permitted himself to be recruited for the T4 program as a medical expert, even participated in the deliberations on a future "Law on Euthanasia," and as a consulting psychiatrist for the German Armed Forces contributed to military medicine. PMID:22399061

Silberzahn-Jandt, G; Schmuhl, H-W

2012-03-01

291

Protein crystal growth - Growth kinetics for tetragonal lysozyme crystals  

NASA Technical Reports Server (NTRS)

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

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

1986-01-01

292

Hydroxyl radicals do not crosslink a DNA-lysozyme complex  

SciTech Connect

The ionic complex between lysozyme and either Escherichia coli DNA or pBR322 DNA was not crosslinked by two systems capable of producing nanomolar amounts of hydroxyl radicals, the oxidation of xanthine by xanthine oxidase and the iron catalyzed oxidation of ascorbic acid. Nor did effective crosslinking occur with micromolar quantities of hydroxyl radicals raised by the addition of adenosine nucleotides to ferrous iron and hydrogen peroxide. In this case, radical content was estimated by colorimetric analysis of formaldehyde following hydroxyl radical oxidation of dimethyl sulfoxide. Similar amounts of radicals generated by pulse radiolysis in a nitrous oxide atmosphere failed also to induce crosslinking. These findings do not support a role for hydroxy radicals in the N-acetoxy-2-acetylaminofluorene induced crosslinking of DNA to lysozyme proposed earlier.

Werbin, H.; Cheng, C.J.

1985-12-01

293

Locations of Halide Ions in Tetragonal Lysozyme Crystals  

NASA Technical Reports Server (NTRS)

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

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

1998-01-01

294

Actin - Lysozyme Interactions in Model Cystic Fibrosis Sputum  

NASA Astrophysics Data System (ADS)

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.

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

2003-03-01

295

Tetragonal Chicken Egg White Lysozyme Solubility in Sodium Chloride Solutions  

NASA Technical Reports Server (NTRS)

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.

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

1998-01-01

296

Tunable supramolecular hydrogel for in situ encapsulation and sustained release of bioactive lysozyme.  

PubMed

To develop new matrices for the entrapment and sustained release of bioactive lysozyme, a series of supramolecular hydrogels based on ?-cyclodextrin (?-CD) and water-soluble poly(?-caprolactone)-poly(ethylene glycol) block copolymer (PCL-b-PEG) were prepared in the presence of chicken egg lysozyme. Different from commonly used polymeric microspheres and chemically crosslinked hydrogels for lysozyme encapsulation, such hydrogel matrices could be formed under mild conditions without high temperature and the use of chemical emulsifiers or crosslinkers. Their gelation rate, mechanical strength and shear viscosity as well as the release behavior for the encapsulated lysozyme could be tuned easily by the change of ?-CD or PCL-b-PEG amount. For the encapsulated lysozyme, its conformation and biological activity could be well maintained when compared to native lysozyme. For the resultant supramolecular hydrogels, they were also confirmed to have a good biocompatibility by MTT assay using mice skin fibroblast (L929). PMID:21536304

Ma, Dong; Zhang, Li-Ming; Xie, Xi; Liu, Tao; Xie, Min-Qiang

2011-07-15

297

Episodic evolution in the stomach lysozymes of ruminants  

Microsoft Academic Search

Summary  By sequencing lysozymesc from deer and pig stomachs and comparing them to the known amino acid sequences of other lysozymesc, it was possible to examine the rate of sequence change during and after the period in which this enzyme acquired a new function.\\u000a Evolutionary tree analysis suggests that the rate went up while lysozyme was being recruited to function as

Jacqueline Jollès; Pierre Jolles; Barbara H. Bowman; Ellen M. Prager; Caro-Beth Stewart; Allan C. Wilson

1989-01-01

298

Genetic engineering of bacteriophage and its applications for biomimetic materials  

E-print Network

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

Lee, Soo-Kwan

2006-01-01

299

Structural characterization of bacteriophage M13 solubilization by amphiphiles  

E-print Network

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

Hemminga, Marcus A.

300

The Tripartite Associations between Bacteriophage, Wolbachia, and Arthropods  

E-print Network

The Tripartite Associations between Bacteriophage, Wolbachia, and Arthropods Seth R. Bordenstein, United States of America By manipulating arthropod reproduction worldwide, the heritable endosymbiont densities and therefore reproductive parasitism. We conclude that phage, Wolbachia, and arthropods form

Bordenstein, Seth

301

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...

302

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

PubMed Central

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

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

2013-01-01

303

Relationship Between Equilibrium Forms of Lysozyme Crystals and Precipitant Anions  

NASA Technical Reports Server (NTRS)

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

Nadarajah, Arunan

1996-01-01

304

Shear-Induced Unfolding of Lysozyme Monitored In Situ  

PubMed Central

Conformational changes due to externally applied physiochemical parameters, including pH, temperature, solvent composition, and mechanical forces, have been extensively reported for numerous proteins. However, investigations on the effect of fluid shear flow on protein conformation remain inconclusive despite its importance not only in the research of protein dynamics but also for biotechnology applications where processes such as pumping, filtration, and mixing may expose protein solutions to changes in protein structure. By combining particle image velocimetry and Raman spectroscopy, we have successfully monitored reversible, shear-induced structural changes of lysozyme in well-characterized flows. Shearing of lysozyme in water altered the protein's backbone structure, whereas similar shear rates in glycerol solution affected the solvent exposure of side-chain residues located toward the exterior of the lysozyme ?-domain. The results demonstrate the importance of measuring conformational changes in situ and of quantifying fluid stresses by the three-dimensional shear tensor to establish reversible unfolding or misfolding transitions occurring due to flow exposure. PMID:19450493

Ashton, Lorna; Dusting, Jonathan; Imomoh, Eboshogwe; Balabani, Stavroula; Blanch, Ewan W.

2009-01-01

305

Overexpression and purification of recombinant lysozyme from Agrius convolvuli expressed as inclusion body in Escherichia coli  

Microsoft Academic Search

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase

Soon-Ik Park; Sung Moon Yoe

2012-01-01

306

Production of Lysozyme by Staphylococci and Its Correlation with Three Other Extracellular Substances1  

PubMed Central

Jay, James M. (Wayne State University, Detroit, Mich.). Production of lysozyme by staphylococci and its correlation with three other extracellular substances. J. Bacteriol. 91:1804–1810. 1966.—Lysozyme production was determined on plates containing 1 mg/ml of Lysozyme Substrate in Heart Infusion Agar with incubation at 37 C for 48 hr. Its production was compared with that of ?-hemolysin and sheep hemolysin and egg-yolk precipitation, by use of both coagulase-positive and coagulase-negative strains of staphylococci. Of 126 coagulase-positive strains tested, 120 or 95.2% produced lysozyme, 117 or 92.9% produced ?-hemolysin, 108 or 85.7% precipitated egg yolk, and 102 or 81% produced sheep hemolysin. Of the 49 coagulase-negative strains (which included 22 pathogens), only 4 or 8.1% produced lysozyme, 14 or 28.6% produced ?-hemolysin, 13 or 26.5% produced sheep hemolysins, and 5 or 10.2% precipitated egg yolk. Only two of the six coagulase-positive strains which failed to produce lysozyme showed any consistent patterns in relation to the four characteristics determined. The four coagulase-negative strains which produced lysozyme were inconsistent for the other characteristics measured. It is suggested that lysozyme production is more a property of coagulase-positive staphylococci, and therefore a better ancillary test of pathogenicity, than either production of ?-hemolysin or egg-yolk precipitation, because the incidence of lysozyme producers is higher among this group than among those producing the other substances and because fewer coagulase-negative staphylococci produced lysozyme than hemolysins or egg-yolk precipitation. Of 16 other species of bacteria and yeasts tested, all were found negative except Bacillus subtilis. Lysozyme production by staphylococci in heavily contaminated foods was not inhibited on plates containing sodium azide, whereas media containing 7.5% salt and sorbic acid were unsuitable. The possible relationship of lysozyme production to staphylococcal pathogenicity is discussed. PMID:5937238

Jay, James M.

1966-01-01

307

Effect of modified lysozyme on the microflora and sensory attributes of ground pork.  

PubMed

The effects of lysozyme monomer and thermochemically modified lysozyme on the microflora and sensory attributes of heated and unheated ground pork were investigated in this study. The dimer and trimer fractions of the modified lysozyme were 36.1 and 33.5%, respectively. The modified lysozyme exhibited higher hydrophobicity (40,600 U/mg of protein) and lower enzymatic activity (1,020 U/mg of protein) than the hydrophobicity (890 U/mg of protein) and activity (17,950 U/mg of protein) of the lysozyme monomer. Portions of ground pork (150 g) without lysozyme or supplemented with 5 mg/g lysozyme or modified lysozyme and either not heated or heated at 60°C for 10 min were stored at 4 ± 1°C and sampled at various times between 1 and 144 h. Meat color was not affected by either additive. After storage for 72 h, the mean odor score for meat supplemented with modified lysozyme and heated decreased from 5.0 at 1 h to 4.1, while the scores for all the other preparations were ?3.2. After 144 h, the counts of Pseudomonas and Enterobacteriaceae in meat that was supplemented with modified lysozyme and not heated were, respectively, 1.1 and 0.9 log less than in the controls, and the numbers in such meat that was heated were, respectively, 1.2 and 2.4 log less than the numbers in the controls. The counts in meat supplemented with lysozyme and the controls were comparable. Heat treatment increased the bacteriostatic effect of modified lysozyme on gram-negative bacteria. PMID:23433385

Cegielska-Radziejewska, Renata; Szablewski, Tomasz

2013-02-01

308

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

NASA Technical Reports Server (NTRS)

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.

Pusey, Marc L.

1991-01-01

309

Bacteriophage T7 DNA polymerase - sequenase  

PubMed Central

An ideal DNA polymerase for chain-terminating DNA sequencing should possess the following features: (1) incorporate dideoxy- and other modified nucleotides at an efficiency similar to that of the cognate deoxynucleotides; (2) high processivity; (3) high fidelity in the absence of proofreading/exonuclease activity; and (4) production of clear and uniform signals for detection. The DNA polymerase encoded by bacteriophage T7 is naturally endowed with or can be engineered to have all these characteristics. The chemically or genetically modified enzyme (Sequenase) expedited significantly the development of DNA sequencing technology. This article reviews the history of studies on T7 DNA polymerase with emphasis on the serial key steps leading to its use in DNA sequencing. Lessons from the study and development of T7 DNA polymerase have and will continue to enlighten the characterization of novel DNA polymerases from newly discovered microbes and their modification for use in biotechnology. PMID:24795710

Zhu, Bin

2014-01-01

310

Bacteriophages of leuconostoc, oenococcus, and weissella.  

PubMed

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

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

2014-01-01

311

Montmorillonite-induced Bacteriophage ?6 Disassembly  

NASA Astrophysics Data System (ADS)

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.

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

2012-12-01

312

Bacteriophage-based synthetic biology for the study of infectious diseases  

E-print Network

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 ...

Citorik, Robert James

313

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

E-print Network

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

Shi, Amy (Amy J.)

2007-01-01

314

3202 Biophysical Journal Volume 85 November 2003 32023213 Sound Velocity and Elasticity of Tetragonal Lysozyme Crystals by  

E-print Network

of Tetragonal Lysozyme Crystals by Brillouin Spectroscopy S. Speziale,* F. Jiang,*y C. L. Caylor,y S. Kriminski velocities and the second-order elastic moduli of tetragonal hen egg-white lysozyme crystals were determined

Duffy, Thomas S.

315

Measurement of spall strength of A12024-T4 and SS304 in plate impact experiments  

Microsoft Academic Search

Spall (dynamic fracture) strength of A12024-T4 and SS304 alloy have been determined at high strain rate. Gas gun experiments have been conducted on A12024-T4 and SS304 target plates in symmetric impact configuration. The spall in A12024-T4 and SS304 plates has been achieved by impacting them with parallel flying plates accelerated to velocities of 0.56 km\\/s and 0.6 km\\/s, respectively. The

K D Joshi; Amit S Rav; Satish C Gupta; S Banerjee

2010-01-01

316

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

PubMed

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

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

2014-09-11

317

Ancient origin of lactalbumin from lysozyme: Analysis of DNA and amino acid sequences  

Microsoft Academic Search

Summary Parsimony trees relating DNA sequences coding for lysozymesc and ?-lactalbumins suggest that the gene duplication that allowed lactalbumin to evolve from lysozyme preceded the divergence of mammals and birds. Comparisons of the amino acid sequences of additional lysozymes and lactalbumins are consistent with this view. When all base positions are considered, the probability that the duplication leading to the

Ellen M. Prager; Allan C. Wilson

1988-01-01

318

Engineered regulation of lysozyme by the SH3-CB1 binding interaction  

E-print Network

Engineered regulation of lysozyme by the SH3-CB1 binding interaction Elizabeth Pham1 and Kevin and diagnostic products. Using the protein structures of lambda lysozyme and the SH3 domain of human Crk, we active cleft through the binding of SH3 to its CB1 peptide-binding partner. First, several fusion protein

319

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

PubMed

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

Valachova, I; Takac, P; Majtan, J

2014-12-01

320

Isolation and complementation of mutants of Streptomyces coelicolor “Müller” DSM3030 deficient in lysozyme production  

Microsoft Academic Search

Streptomyces coelicolor “Müller” is known to excrete the lysozyme N-acetylmuramidase. Culture filtrates of this strain form a characteristic halo on agar plates containing freeze-dried Micrococcus luteus cells (lysoplate technique). The halo consists of a clear inner zone and a turbid outer ring. Simulation experiments showed that the turbid outer ring is most probably produced by lysozyme whereas the clear inner

Elli Birr; Wolfgang Wohlleben; Karin Aufderheide; Thomas Schneider; Alfred Piihler; Barbara Bräu; Rüdiger Marquardt; Gerhard Wöhner; Paul Präve; Merten Schlingmann

1989-01-01

321

Formation of amyloid fibrils from fully reduced hen egg white lysozyme  

E-print Network

Formation of amyloid fibrils from fully reduced hen egg white lysozyme AONENG CAO, DAOYING HU residues may play an important role. Keywords: Amyloid fibril formation; hen lysozyme; disulfide reduction Alzheimer's disease (the fourth most common cause of death in the Western world), Parkinson's disease, type

Luhua, Lai

322

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

Microsoft Academic Search

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

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

2009-01-01

323

Use of a Mixture of Bacteriophages for Biological Control of Salmonella enterica Strains in Compost?  

PubMed Central

Bacteriophages specific to Salmonella strains were isolated from sewage effluent and characterized. A five-strain bacteriophage mixture was applied to dairy manure compost inoculated with Salmonella enterica serotype Typhimurium. Bacteriophage treatment resulted in a greater than 2-log-unit reduction of Salmonella within 4 h at all moisture levels compared to the controls. PMID:20525859

Heringa, Spencer D.; Kim, JinKyung; Jiang, Xiuping; Doyle, M. P.; Erickson, M. C.

2010-01-01

324

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

EPA Science Inventory

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

325

The lysozyme locus in Drosophila melanogaster : an expanded gene family adapted for expression in the digestive tract  

Microsoft Academic Search

Lysozyme has been studied in insects as part of the system of inducible antibacterial defence in the haemolymph. We recently found two Drosophila lysozyme genes that are constitutively expressed in the digestive tract, and are probably involved in the digestion of bacteria in the food. To obtain an overview of the lysozyme genes in this species and their possible roles

Sirlei Daffre; Per Kylsten; Christos Samakovlis; Dan Hultmark

1994-01-01

326

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

Microsoft Academic Search

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

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

2000-01-01

327

Molecular cloning and characterization of a lysozyme cDNA from the mole cricket Gryllotalpa orientalis (Orthoptera: Gryllotalpidae).  

PubMed

A full-length lysozyme cDNA from Gryllotalpa orientalis was cloned and sequenced. The deduced amino acid sequence of the lysozyme protein was 143 amino acids in length, with a calculated molecular mass of 15.84 kDa and an isoelectric point of 4.74. Sequence motifs, together with alignment and phylogenetic results, confirmed that G. orientalis lysozyme belongs to the C (chicken)-type lysozyme family of proteins. The protein sequence of lysozyme from G. orientalis showed high identity to that of Drosophila melanogaster (51.7 %); however, in contrast to D. melanogaster lysozyme, G. orientalis lysozyme was immune inducible and expressed in a wide range of tissues. Expression of G. orientalis lysozyme mRNA was highest at 8 h post-infection and subsequently decreased with time after bacterial infection. We also expressed G. orientalis lysozyme protein in vitro using the pET expression system. Compared with the negative control, over-expressed G. orientalis lysozyme showed antimicrobial activity against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus subtilis by radial diffusion assay, with minimal inhibitory concentration values of 30.3 and 7.55 µM, respectively. These results indicate that G. orientalis lysozyme may have stronger antimicrobial activity than other lysozymes against a broad range of microorganisms. PMID:24929538

Kwon, Hyojung; Bang, Kyeongrin; Lee, Minsup; Cho, Saeyoull

2014-09-01

328

Colloids and Surfaces B: Biointerfaces 38 (2004) 6776 Calorimetric and structural investigation of the interaction of lysozyme  

E-print Network

of the interaction of lysozyme and bovine serum albumin with poly(ethylene oxide) and its copolymers Nara L. Almeidaa interactions between lysozyme or bovine serum albumin (BSA) and poly(ethylene oxide) and its copolymers: Protein denaturation; Calorimetry; Bovine serum albumin; Lysozyme; Poly(ethylene oxide) 1. Introduction

Loh, Watson

329

Determining the Molecular Growth Mechanisms of Tetragonal Lysozyme Crystals  

NASA Technical Reports Server (NTRS)

Studies of the growth of tetragonal lysozyme crystals employing atomic force microscopy (AFM) have shown the advantages of this technique in investigating the growth mechanisms of protein crystals [1]. The resolution of these studies was in the micron range, which revealed surface features such as the occurrence of dislocations and 2D nucleation islands, similar to those found in inorganic systems. They clearly showed that the crystals grew by these surface growth mechanisms. However, the studies also revealed some surprising features, such as bimolecular growth step heights and pronounced growth anisotropies on the (110) face, which could not be explained. In previous studies we employed Periodic Bond Chain (PBC) theory to tetragonal lysozyme crystal growth and found that the crystals were constructed by strongly bonded molecular chains forming helices about the 43 axes [2,3]. The helices were connected to each other with weaker bonds. The growth process was shown to proceed by the formation of these 43 helices, resulting in bimolecular growth steps on the (110) face. It was also shown to explain many other observations on tetragonal lysozyme crystal growth. Although PBC analysis is not a new technique [4], it has not been widely used as the mechanisms predicted from it could not be experimentally verified. In this study the growth process of these crystals was investigated, particularly for the (110) face, employing some newly developed high resolution AFM techniques. These techniques allowed individual lysozyme molecules on the crystal faces to be resolved and predictions from PBC analyses to be tested. The analyses had shown that of the two possible packing arrangements on (110) faces, only one would actually occur. Employing the first of the newly developed techniques, these faces were scanned by high resolution AFM. The resulting images were then compared with the theoretically constructed images for the two possible packing arrangements on the (110) face. The theoretical images were constructed by convolution of the crystal surface shape obtained from crystallographic data with the AFM tip shape. The comparison confirmed the prediction that the molecular packing arrangement of these faces corresponded to that for complete 43 helices. The second AFM technique that was developed was used to follow the growth process by measuring the dimensions of individual growth units on the (110) face. Linescans across a growth step, performed near the saturation limit of the crystals, allowed the growth unit dimensions to be measured. These measurements showed that growth on the (110) face proceeded by the formation of new 43 helices from the addition of at least tetramer units in the [110] direction. In the [001] direction growth proceeded by the addition of various aggregate units corresponding to the 4(sub 3) helices.

Li, Huayu; Nadarajah, Arunan; Konnert, John H.; Pusey, Marc L.

1998-01-01

330

Conformations and folding of lysozyme ions in vacuo.  

PubMed

Proton transfer reactivity of isolated charge states of the protein hen egg-white lysozyme shows that multiple distinct conformations of this protein are stable in the gas phase. The reactivities of the 9+ and 10+ charge state ions, formed by electrospray ionization of "native" (disulfide-intact) and "denatured" (disulfide-reduced) solutions, are consistent with values calculated for ions in their crystal structure and fully denatured conformations, respectively. Charge states below 8+ of both forms, formed by proton stripping, have similar or indistinguishable reactivities, indicating that the disulfide-reduced ions fold in the gas phase to a more compact conformation. PMID:8610183

Gross, D S; Schnier, P D; Rodriguez-Cruz, S E; Fagerquist, C K; Williams, E R

1996-04-01

331

Substrate stabilization of lysozyme to thermal and guanidine hydrochloride denaturation  

E-print Network

, is generally only 5 to 15 kcal/mole more stable than unfolded conformations which we designate D. Substrates and other small molecules which interact specif ical ly with enzymes generally bind tightly to N and weakly, if at all, to D. Consequently..., increasing the substrate concentration will shif t the N~~D equilibrium toward N and thereby stabilize the native conformation. In this thesis we have quantitatively investigated the stabi- lization of the enzyme lysozyme by a substrate, the p(1-a4...

McGrath, Timothy

2012-06-07

332

Antimicrobial and biosensing ultrasound-responsive lysozyme-shelled microbubbles.  

PubMed

Air-filled lysozyme microbubbles (LSMBs) were engineered as a support for the immobilization of gold nanoparticles and an enzyme, alkaline phosphatase, in order to develop micro-antimicrobial and biosensing devices. Gold nanoparticles immobilized on LSMBs significantly improved the antimicrobial efficacy of the microbubbles against M. lysodeikticus. The surface functionalization of the microbubbles with gold nanoparticles did not affect their echogenicity when exposed to an ultrasound imaging probe. Alkaline phosphatase was conjugated on the surface of microbubbles without compromising its enzymatic activity. The functionalized microbubbles were used for the detection of paraoxon in aqueous solutions. PMID:23265433

Cavalieri, Francesca; Micheli, Laura; Kaliappan, Subramanian; Teo, Boon M; Zhou, Meifang; Palleschi, Giuseppe; Ashokkumar, Muthupandian

2013-01-23

333

Renal Lysozyme Levels in Animals Developing "Sterile Pyelonephritis"  

PubMed Central

The induction of sterile unilateral pyelonephritis in rats with heat-killed Proteus mirabilis cells is described. The lesions were identical to those produced with viable bacteria. Lysozyme levels in both kidneys of rats developing unilateral sterile pyelonephritis underwent biphasic elevations similar to those produced with viable bacteria. In the injected kidney, the first elevation, associated with the trauma of injection, could be produced by injecting sterile saline. The second elevation was associated with the onset of chronicity in the injected kidney. The nonmanipulated, contralateral kidney showed a similar biphasic elevation, of equal duration but of greater magnitude. Images PMID:4949491

Eudy, W. W.; Burrous, S. E.; Sigler, F. W.

1971-01-01

334

Lysozyme net charge and ion binding in concentrated aqueous electrolyte solutions  

SciTech Connect

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.

Kuehner, D.E.; Engmann, J.; Fergg, F.; Wernick, M.; Blanch, H.W. [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering] [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering; Prausnitz, J.M. [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering] [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering; [Lawrence Berkeley National Lab., CA (United States). Chemical Sciences Div.

1999-02-25

335

Pulsed laser deposition of lysozyme: the dependence on shot numbers and the angular distribution  

NASA Astrophysics Data System (ADS)

The ejection of molecules from a pressed solid target of lysozyme induced by laser ablation in the UV-regime at a wavelength of 355 nm was investigated. The ablation studies were carried out in vacuum at a laser fluence of 2 J/cm2 for which a significant fraction of proteins remains intact. This was verified by matrix-assisted laser desorption ionization (MALDI) spectrometry of thin films deposited on silicon substrates. The deposition rate of lysozyme was found to decrease with the number of shots and was correlated with increasing thermal damage of the lysozyme. This was monitored by measurements of the optical reflectivity of dry lysozyme. The angular distribution of the mass deposition can be fitted well by Anisimov's hydrodynamic model. The total deposited yield over the entire hemisphere from direct laser ablation of lysozyme was estimated from this model and found to be three orders of magnitude less than the ablated mass.

Constantinescu, C.; Matei, A.; Schou, J.; Canulescu, S.; Dinescu, M.

2013-12-01

336

Bioconjugate of lysozyme and the antibacterial marine sesquiterpene quinone avarone and its derivatives  

PubMed Central

A conjugate of lysozyme with avarone, a bioactive sesquiterpene quinone of marine origin, and its three derivatives, was synthesized. MALDI TOF mass spectral analysis and tryptic digestion showed that the only residue in lysozyme that was modified by all derivatives was lysine 97. The identity of the residue was in full correlation with the prediction obtained by molecular modelling. All bioconjugates preserved most of the enzymatic activity of lysozyme. The melting point of the conjugates was slightly increased in comparison to lysozyme, indicating a slight stabilization of structure. The antibacterial activity of all the conjugates to both Gram positive and Gram negative bacteria was stronger than the activity of either lysozyme or the quinones, the MIC values being in low micromolar range for some conjugates. PMID:22148500

Novakovic, Irena; Annelkovic, Uros; Zlatovic, Mario; Gasic, Miroslav J.; Sladic, Dusan

2012-01-01

337

Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni  

PubMed Central

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

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

2014-01-01

338

AtlasT4SS: A curated database for type IV secretion systems  

PubMed Central

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

2012-01-01

339

Bacteriophage biocontrol in animals and meat products  

PubMed Central

Summary Since their discovery almost a century ago, bacterial viruses (bacteriophages or ‘phages’) have been used to prevent and treat a multitude of bacterial infections (phage therapy: PT). In addition, they have been the basis for many advances in genetics and biochemistry. Phage therapy was performed on human subjects in the United States, Europe and Asia in the few decades following their discovery. However, Western countries largely abandoned PT in favour of antibiotics in the 1940s. The relatively recent renaissance of PT in the West can be attributed partly to the increasing prevalence of antibiotic resistance in human and animal pathogens. However, the stringent controls on human trials now required in the United States and Europe have led to a greater number of domestic animal and agricultural applications as an alternative to PT in man. This trend is set to continue, at least in the short term, with recent approval from the Food and Drug Administration allowing commercial phage treatments to be used in human food in the USA. Nevertheless, despite these significant milestones and the growing number of successful PT trials, significant obstacles remain to their widespread use in animals, food and ultimately medicine in many parts of the world. This review will provide a brief overview of the history of PT in the West and will summarize some of the key findings of phage biocontrol studies in animals and meat products. PMID:21255295

Atterbury, R. J.

2009-01-01

340

Flap endonuclease of bacteriophage T7  

PubMed Central

Gene 6 protein of bacteriophage T7 has 5?-3?-exonuclease activity specific for duplex DNA. We have found that gene 6 protein also has flap endonuclease activity. The flap endonuclease activity is considerably weaker than the exonuclease activity. Unlike the human homolog of gene 6 protein, the flap endonuclease activity of gene 6 protein is dependent on the length of the 5?-flap. This dependency of activity on the length of the 5?-flap may result from the structured helical gateway region of gene 6 protein which differs from that of human flap endonuclease 1. The flap endonuclease activity provides a mechanism by which RNA-terminated Okazaki fragments, displaced by the lagging strand DNA polymerase, are processed. 3?-extensions generated during degradation of duplex DNA by the exonuclease activity of gene 6 protein are inhibitory to further degradation of the 5?-terminus by the exonuclease activity of gene 6 protein. The single-stranded DNA binding protein of T7 overcomes this inhibition. PMID:25105057

Mitsunobu, Hitoshi; Zhu, Bin; Lee, Seung-Joo; Tabor, Stanley; Richardson, Charles C

2014-01-01

341

Sampling Submicron T1 Bacteriophage Aerosols  

PubMed Central

Liquid impingers, filter papers, and fritted bubblers were partial viable collectors of radioactive submicron T1 bacteriophage aerosols at 30, 55, and 85% relative humidity. Sampler differences for viable collection were due to incomplete physical collection (slippage) and killing of phage by the samplers. Dynamic aerosols of a mass median diameter of 0.2 ? were produced with a Dautrebande generator from concentrated aqueous purified phage suspensions containing extracellular soluble radioactive phosphate as a physical tracer. There was considerable destruction of phage by the Dautrebande generator; phage titers of the Dautrebande suspension decreased exponentially, but there was a progressive (linear) increase in tracer titers. Liquid impingers recovered the most viable phage but allowed considerable (30 to 48%) slippage, which varies inversely with the aerosol relative humidity. Filter papers were virtually complete physical collectors of submicron particles but were the most destructive. Fritted bubbler slippage was more than 80%. With all samplers, phage kill was highest at 85% relative humidity and lowest at 55% relative humidity. An electrostatic precipitator was used to collect aerosol samples for particle sizing with an electron microscope. The particle size was slightly larger at 85% relative humidity than at 30 or 55% relative humidity. Images Fig. 1 Fig. 4 PMID:5866038

Harstad, J. Bruce

1965-01-01

342

Preliminary investigations into solutal flow about growing tetragonal lysozyme crystals  

NASA Technical Reports Server (NTRS)

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.

Pusey, Marc; Witherow, William; Naumann, Robert

1988-01-01

343

Amyloid Protofibrils of Lysozyme Nucleate and Grow Via Oligomer Fusion  

PubMed Central

The mechanisms linking deposits of insoluble amyloid fibrils to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings implicate transiently formed intermediates of mature amyloid fibrils as the principal toxic agent. Hence, determining which intermediate aggregates represent on-pathway precursors or off-pathway side branches is critical for understanding amyloid self-assembly, and for devising therapeutic approaches targeting relevant toxic species. We examined amyloid fibril self-assembly in acidic solutions, using the model protein hen egg-white lysozyme. Combining in situ dynamic light scattering with calibrated atomic-force microscopy, we monitored the nucleation and growth kinetics of multiple transient aggregate species, and characterized both their morphologies and physical dimensions. Upon incubation at elevated temperatures, uniformly sized oligomers formed at a constant rate. After a lag period of several hours, protofibrils spontaneously nucleated. The nucleation kinetics of protofibrils and the tight match of their widths and heights with those of oligomers imply that protofibrils both nucleated and grew via oligomer fusion. After reaching several hundred nanometers in length, protofibrils assembled into mature fibrils. Overall, the amyloid fibril assembly of lysozyme followed a strict hierarchical aggregation pathway, with amyloid monomers, oligomers, and protofibrils forming on-pathway intermediates for assembly into successively more complex structures. PMID:19413984

Hill, Shannon E.; Robinson, Joshua; Matthews, Garrett; Muschol, Martin

2009-01-01

344

The Effect of Protein Impurities on Lysozyme Crystal Growth  

NASA Technical Reports Server (NTRS)

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. In order 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 f {101} 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.

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

1998-01-01

345

Interaction of lactoferrin and lysozyme with casein micelles.  

PubMed

On addition of lactoferrin (LF) to skim milk, the turbidity decreases. The basic protein binds to the caseins in the casein micelles, which is then followed by a (partial) disintegration of the casein micelles. The amount of LF initially binding to casein micelles follows a Langmuir adsorption isotherm. The kinetics of the binding of LF could be described by first-order kinetics and similarly the disintegration kinetics. The disintegration was, however, about 10 times slower than the initial adsorption, which allowed investigating both phenomena. Kinetic data were also obtained from turbidity measurements, and all data could be described with one equation. The disintegration of the casein micelles was further characterized by an activation energy of 52 kJ/mol. The initial increase in hydrodynamic size of the casein micelles could be accounted for by assuming that it would go as the cube root of the mass using the adsorption and disintegration kinetics as determined from gel electrophoresis. The results show that LF binds to casein micelles and that subsequently the casein micelles partly disintegrate. All micelles behave in a similar manner as average particle size decreases. Lysozyme also bound to the casein micelles, and this binding followed a Langmuir adsorption isotherm. However, lysozyme did not cause the disintegration of the casein micelles. PMID:21932853

Anema, Skelte G; de Kruif, C G Kees

2011-11-14

346

Dynamics of lysozyme and its hydration water under electric field  

E-print Network

The effects of static electric field on the dynamics of lysozyme and its hydration water have been investigated by means of incoherent quasi-elastic neutron scattering (QENS). Measurements were performed on lysozyme samples, hydrated respectively with heavy water (D2O) to capture the protein dynamics, and with light water (H2O), to probe the dynamics of the hydration shell, in the temperature range from 210 $water per gram of dry protein. The field strengths investigated were respectively 0 kV/mm and 2 kV/mm (2 10$^6$ V/m) for the protein hydrated with D2O and 0 kV and 1 kV/mm for the H2O hydrated counterpart. While the overall internal protons dynamics of the protein appears to be unaffected by the application of electric field up to 2 kV/mm, likely due to the stronger intra-molecular interactions, there is also no appreciable quantitative enhancement of the diffusive dynamics of the hydration water, as would be anticipated based on our recent observations in water confined in silica pores under field values of 2.5 kV/mm. This may be due to the difference in surface interactions between water and the two adsorption hosts (silica and protein), or to the existence of a critical threshold field value Ec $\\sim$ 2-3 kV/mm for increased molecular diffusion, for which electrical breakdown is a limitation for our sample.

P. M. Favi; Q. Zhang; H. O'Neill; E. Mamontov; S. O. Diallo

2013-12-06

347

Lysozyme Protein Solution with an Intermediate Range Order Structure  

SciTech Connect

The formation of equilibrium clusters has been studied in both a prototypical colloidal system and protein solutions. The appearance of a low-Q correlation peak in small angle scattering patterns of lysozyme solution was attributed to the cluster-cluster correlation. Consequently, the presence of long-lived clusters has been established. By quantitatively analyzing both the SANS (small angle neutron scattering) and NSE (neutron spin echo) data of lysozyme solution using statistical mechanics models, we conclusively show in this paper that the appearance of a low-Q peak is not a signature of the formation of clusters. Rather, it is due to the formation of an intermediate range order structure governed by a short-range attraction and a long-range repulsion. We have further studied dynamic features of a sample with high enough concentration at which clusters are formed in solution. From the estimation of the mean square displacement by using short-time and long-time diffusion coefficient measured by NSE and NMR, we find that these clusters are not permanent but have a finite lifetime longer than the time required to diffuse over a distance of a monomer diameter.

Liu, Yun [National Institute of Standards and Technology (NIST); Porcar, L. [National Institute of Standards and Technology (NIST); Chen, Wei-Ren [ORNL; Chen, Jinhong [Memorial Sloan-Kettering Cancer Center; Falus, Peter [ORNL; Fratini, Emiliano [University of Florence; Faraone, Antonio [National Institute of Standards and Technology (NIST); Baglioni, P [University of Florence

2011-01-01

348

Bacteriophage Typing of Proteus mirabilis, Proteus vulgaris, and Proteus morganii  

PubMed Central

A bacteriphage typing scheme for differentiating Proteus isolated from clinical specimens was developed. Twenty-one distinct patterns of lysis were seen when 15 bacteriophages isolated on 8 Proteus mirabilis, 1 P. vulgaris, and 1 P. morganii were used to type 162 of 189 (85.7%) P. mirabilis and P. vulgaris isolates. Seven phages isolated on 3 P. morganii were used to type 13 of 19 (68.4%) P. morganii isolates. Overall, 84.1% of the 208 isolates were lysed by at least 1 phage at routine test dilution (RTD) or 1,000 × RTD. Fifty isolates, retyped several weeks after the initial testing, showed no changes in lytic patterns. The phages retained their titers after storage at 4 C for several months. A computer analysis of the data showed that there was no relationship between the source of the isolate and bacteriophage type. This bacteriophage typing system may provide epidemiological information on strains involved in human infections. PMID:4589141

Schmidt, William C.; Jeffries, Charles D.

1974-01-01

349

Different approaches for using bacteriophages against antibiotic-resistant bacteria  

PubMed Central

Bacterial resistance to antibiotics is an emerging threat requiring urgent solutions. Ever since their discovery, lytic bacteriophages have been suggested as therapeutic agents, but their application faces various obstacles: sequestration of the phage by the spleen and liver, antibodies against the phage, narrow host range, poor accessibility to the infected tissue, and bacterial resistance. Variations on bacteriophage use have been suggested, such as temperate phages as gene-delivery vehicles into pathogens. This approach, which is proposed to sensitize pathogens residing on hospital surfaces and medical personnel's skin, and its prospects are described in this addendum. Furthermore, phage-encoded products have been proposed as weapons against antibiotic resistance in bacteria. We describe a new phage protein which was identified during basic research into T7 bacteriophages. This protein may serendipitously prove useful for treating antibiotic-resistant pathogens. We believe that further basic research will lead to novel strategies in the fight against antibiotic-resistant bacteria. PMID:24653944

Yosef, Ido; Kiro, Ruth; Molshanski-Mor, Shahar; Edgar, Rotem; Qimron, Udi

2014-01-01

350

M13 bacteriophage production for large-scale applications.  

PubMed

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

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

2014-10-01

351

Replacing tryptophan-128 of T4 endonuclease V with a serine residue results in decreased enzymatic activity in vitro and in vivo.  

PubMed Central

Endonuclease V of bacteriophage T4 possesses two enzymatic activities, a DNA N-glycosylase specific for cyclobutane pyrimidine dimers (CBPD) and an associated apurinic/apyrimidinic (AP) lyase. Extensive structural and functional studies of endonuclease V have revealed that specific amino acids are associated with these two activities. Controversy still exists regarding the role of the aromatic amino acid stretch close to the carboxyl terminus, in particular the tryptophan at position 128. We have expressed wild-type and mutant W128S endonuclease V in Escherichia coli from an inducible tac promoter. Purified W128S endonuclease V demonstrated substantially decreased N-glycosylase (approximately 5-fold) and AP lyase (10- to 20-fold) activities in vitro compared to the wild-type enzyme when a UV-irradiated poly(dA)-poly(dT) substrate was used. However, a much smaller difference in AP lyase activity between the two forms was observed with a site-specific abasic oligonucleotide. The difference in enzymatic activity was qualitatively, but not quantitatively, reflected in the survival of UV-irradiated bacteria, that is the W128S cells were slightly less UV resistant than wild-type cells. No difference was observed in the complementation of UV repair using UV-damaged denV- T4 phage. A more pronounced difference between the wild-type and W128S proteins was observed in human xeroderma pigmentosum group A cells by host-cell reactivation of a UV-irradiated reporter gene. The relatively large discrepancy between the in vitro and in vivo results observed with bacteria may be because saturated levels of DNA repair are obtained in vivo with relatively low levels of endonuclease V. However, under limiting in vitro conditions and in human cells in vivo a considerable difference between the W128S mutant and wild-type endonuclease V activities can be detected. Our results demonstrate that tryptophan-128 is important for endonuclease V activity. Images PMID:7479008

Valerie, K

1995-01-01

352

Effects of sunlight on bacteriophage viability and structure.  

PubMed

Current estimates of viral abundance in natural waters rely on direct counts of virus-like particles (VLPs), using either transmission or epifluorescence microscopy. Direct counts of VLPs, while useful in studies of viral ecology, do not indicate whether the observed VLPs are capable of infection and/or replication. Rapid decay in bacteriophage viability under environmental conditions has been observed. However, it has not been firmly established whether there is a corresponding degradation of the virus particles. To address this question, viable and direct counts were carried out employing two Chesapeake Bay bacteriophages in experimental microcosms incubated for 56 h at two depths in the York River estuary. Viruses incubated in situ in microcosms at the surface yielded decay rates in full sunlight of 0.11 and 0.06 h-1 for CB 38 phi and CB 7 phi, respectively. The number of infective particles in microcosms in the dark and at a depth of 1 m was not significantly different from laboratory controls, with decay rates averaging 0.052 h-1 for CB 38 phi and 0.037 h-1 for CB 7 phi. Direct counts of bacteriophages decreased in teh estuarine microcosms, albeit only at a rate of 0.028 h-1, and were independent of treatment. Destruction of virus particles is concluded to be a process separate from loss of infectivity. It is also concluded that strong sunlight affects the viability of bacteriophages in surface waters, with the result that direct counts of VLPs overestimate the number of bacteriophage capable of both infection and replication. However, in deeper waters, where solar radiation is not a significant factor, direct counts should more accurately estimate numbers of viable bacteriophage. PMID:8919794

Wommack, K E; Hill, R T; Muller, T A; Colwell, R R

1996-04-01

353

Considerations for using bacteriophages for plant disease control  

PubMed Central

The use of bacteriophages as an effective phage therapy strategy faces significant challenges for controlling plant diseases in the phyllosphere. A number of factors must be taken into account when considering phage therapy for bacterial plant pathogens. Given that effective mitigation requires high populations of phage be present in close proximity to the pathogen at critical times in the disease cycle, the single biggest impediment that affects the efficacy of bacteriophages is their inability to persist on plant surfaces over time due to environmental factors. Inactivation by UV light is the biggest factor reducing bacteriophage persistence on plant surfaces. Therefore, designing strategies that minimize this effect are critical. For instance, application timing can be altered: instead of morning or afternoon application, phages can be applied late in the day to minimize the adverse effects of UV and extend the time high populations of phage persist on leaf surfaces. Protective formulations have been identified which prolong phage viability on the leaf surface; however, UV inactivation continues to be the major limiting factor in developing more effective bacteriophage treatments for bacterial plant pathogens. Other strategies, which have been developed to potentially increase persistence of phages on leaf surfaces, rely on establishing non-pathogenic or attenuated bacterial strains in the phyllosphere that are sensitive to the phage(s) specific to the target bacterium. We have also learned that selecting the correct phages for disease control is critical. This requires careful monitoring of bacterial strains in the field to minimize development of bacterial strains with resistance to the deployed bacteriophages. We also have data that indicate that selecting the phages based on in vivo assays may also be important when developing use for field application. Although bacteriophages have potential in biological control for plant disease control, there are major obstacles, which must be considered. PMID:23531902

Jones, Jeffrey B.; Vallad, Gary E.; Iriarte, Fanny B.; Obradovic, Aleksa; Wernsing, Mine H.; Jackson, Lee E.; Balogh, Botond; Hong, Jason C.; Momol, M.Timur

2012-01-01

354

Robert A. Weisberg (1937-2011): a Bacteriophage Pioneer?  

PubMed Central

With the untimely, sudden passing of Robert Weisberg on 1 September 2011, the bacteriophage community lost a shining light. Bob had a remarkable career and served his profession exceptionally well. He was an editor of the Journal of Virology (1983 to 1988) and the Journal of Bacteriology (1985 to 1995) and worked tirelessly to advance bacteriophage biology. He was my mentor when I was a Staff Fellow at the NIH in the mid-1970s. His long-time collaborator and colleague, Max Gottesman, has prepared a tribute to this stellar virologist.

2011-01-01

355

Engineered enzymatically active bacteriophages and methods of uses thereof  

DOEpatents

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.

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

356

Existence of lysogenic bacteriophages in Bacillus thuringiensis type strains.  

PubMed

We screened the existence of bacteriophages in 67 Bacillus thuringiensis type strains by phage DNA extraction and PCR using phage terminase small subunit (TerS)-specific primers to the supernatants and the precipitated pellets of Bt cultures, and by transmission electron microscopy. The various bacteriophages were observed from the supernatants of 22 type strains. Ten type strains showed the extracted phage DNAs and the amplified fragment by TerS PCR but 12 type strains showed only the phage DNAs. Their morphological characteristic suggests that they belong to Family Siphoviridae which had a long tail and symmetrical head. PMID:23632013

Roh, Jong Yul; Park, Jong Bin; Liu, Qin; Kim, Song Eun; Tao, Xueying; Choi, Tae Woong; Choi, Jae Young; Kim, Woo Jin; Jin, Byung Rae; Je, Yeon Ho

2013-07-01

357

Environmental augmentation with bacteriophage prevents colibacillosis in broiler chickens.  

PubMed

Bacteriophages are viruses that kill bacteria. They are plentiful in nature; are safe, having no known activity to human or animal cells; and are an attractive alternative to antibiotics. The objectives of this research were to establish an experimental model of colibacillosis induced by indirect exposure to Escherichia coli and to determine if bacteriophage could protect the birds from developing colibacillosis. In study 1 there were 6 treatments with 2 replicate pens of 25 birds. The treatments were control warm brooded; control cold stressed; litter inoculated with E. coli, warm brooded; litter inoculated with E. coli, cold stressed; seeder birds (5 per pen) challenged with E. coli, warm brooded; and seeder birds (5 per pen), cold stressed. The study concluded when the birds were 3 wk of age. Body weights at 1, 2, and 3 wk of age were significantly decreased (P ? 0.05) by cold stress, decreased at 1 and 2 wk of age by both the litter and seeder bird treatments compared with the control treatment and by the seeder bird treatment at 3 wk of age. Study 2 consisted of 8 treatments with 2 replicate pens of 20 birds per treatment. The treatments were control, warm brooded; control, cold stressed; litter inoculated with E. coli, cold stressed; and seeder birds (5/pen) challenged with E. coli, cold stressed with and without bacteriophage treatment. In the bacteriophage treatments the bacteriophages were sprayed on the litter. The study was concluded at 3 wk of age. Body weights at 1 wk of age were significantly (P ? 0.05) decreased from the control treatment by the seeder bird treatment and were significantly (P ? 0.05) higher in all the bacteriophage treatments compared with their matched untreated treatments, except in the control cold stressed treatment. Mortality was significantly (P ? 0.05) decreased by bacteriophage in the litter challenged treatment. These data suggest that augmentation of the environment with bacteriophage is a practical and efficacious way to prevent colibacillosis in broiler chickens. PMID:25214555

El-Gohary, F A; Huff, W E; Huff, G R; Rath, N C; Zhou, Z Y; Donoghue, A M

2014-11-01

358

CRISPR/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4  

E-print Network

Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain ...

Yaung, Stephanie J.

359

The complete amino acid sequence and enzymatic properties of an i-type lysozyme isolated from the common orient clam (Meretrix lusoria).  

PubMed

To determine the structure and functional relationships of invertebrate lysozymes, we isolated a new invertebrate (i)-type lysozyme from the common orient clam (Meretrix lusoria) and determined the complete amino acid sequence of two isozymes that differed by one amino acid. The determined sequence showed 65% similarity to a lysozyme from Venerupis philippinarum (Tapes japonica), and it was therefore classified as an i-type lysozyme. The lytic activities of this lysozyme were similar to those of previously reported bivalve i-type lysozymes, but unlike the V. philippinarum lysozyme, it did not exhibit an increase in activity in high ionic strength. Our data suggest that this lysozyme does not have a dimeric structure, due to the replacement of Lys108 which contributes to dimer formation in the V. philippinarum lysozyme. GlcNAc oligomer activities suggested an absence of transglycosylation activity and a higher number of subsites on this enzyme compared with hen egg lysozyme. PMID:24200802

Kuwano, Yuko; Yoneda, Kazunari; Kawaguchi, Yuya; Araki, Norie; Araki, Tomohiro

2013-01-01

360

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

SciTech Connect

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

Goven, A.J.; Chen, S.C.; Fitzpatrick, L.C. (Univ. of North Texas, Denton, TX (United States). Dept. of Biological Sciences); Venables, B.J. (Univ. of North Texas, Denton, TX (United States). Dept. of Biological Sciences TRAC Laboratories Inc., Denton, TX (United States))

1994-04-01

361

Affinity adsorption of lysozyme with Reactive Red 120 modified magnetic chitosan microspheres.  

PubMed

Reactive Red 120 was utilized as an affinity dye-ligand to modify the surface of magnetic chitosan microspheres to improve the adsorption capacity for lysozyme. The impact of pH, ionic strength, adsorption time and initial lysozyme concentration on the adsorption of lysozyme from aqueous solutions was investigated. An increased adsorption amount of 116.9 mg/g lysozyme on Reactive Red 120 modified microspheres was achieved in comparison to 24.6 mg/g on unmodified magnetic chitosan microspheres. The adsorption behaviour of lysozyme by the Reactive Red 120 modified magnetic chitosan microspheres fitted the pseudo second-order kinetic and the Langmuir thermodynamic model. The desorption ratio was 92.6% using 1M NaCl phosphate buffer (pH 6.0) as the desorption agent. The adsorption capacity of the Reactive Red 120 modified magnetic chitosan microspheres did not decrease significantly after four adsorption-desorption cycles (P>0.05). The as-prepared Reactive Red 120 modified magnetic chitosan microspheres were then used for the extraction of lysozyme from egg white solutions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the purity of the obtained lysozyme was ~80.7% and that the recovery yield was ~89.1%. PMID:24128540

Li, Zhonghong; Cao, Miao; Zhang, Wengang; Liu, Lizhi; Wang, Jianlong; Ge, Wupeng; Yuan, Yahong; Yue, Tianli; Li, Ronghua; Yu, William W

2014-02-15

362

Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications.  

PubMed

The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 ?g protein/cm(2) and 5.74 ?g protein/cm(2) for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH-lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29-lysozyme, 0.95 log for EVOH 44-lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging. PMID:23815412

Muriel-Galet, V; Talbert, J N; Hernandez-Munoz, P; Gavara, R; Goddard, J M

2013-07-10

363

Protein composition of different sized casein micelles in milk after the binding of lactoferrin or lysozyme.  

PubMed

Casein micelles with bound lactoferrin or lysozyme were fractionated into sizes ranging in radius from ?50 to 100 nm. The ?-casein content decreased markedly and the ?S-casein/?-casein content increased slightly as micelle size increased. For lactoferrin, higher levels were bound to smaller micelles. The lactoferrin/?-casein ratio was constant for all micelle sizes, whereas the lactoferrin/?S-casein and lactoferrin/?-casein ratio decreased with increasing micelle size. This indicates that the lactoferrin was binding to the surface of the casein micelles. For lysozyme, higher levels bound to larger casein micelles. The lysozyme/?S-casein and lysozyme/?-casein ratios were nearly constant, whereas the lysozyme/?-casein ratio increased with increasing micelle size, indicating that lysozyme bound to ?S-casein and ?-casein in the micelle core. Lactoferrin is a large protein that cannot enter the casein protein mesh; therefore, it binds to the micelle surface. The smaller lysozyme can enter the protein mesh and therefore binds to the more charged ?S-casein and ?-casein. PMID:23808832

Anema, Skelte G; de Kruif, C G Kees

2013-07-24

364

Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters.  

PubMed

Norovirus (NoV), the leading cause of adult non-bacterial gastroenteritis can be commonly detected in wastewater but the extent of NoV removal provided by wastewater treatment plants (WWTPs) is unclear. We monitored a newly commissioned WWTP with UV disinfection on a weekly basis over a six month period for NoV using RT-qPCR and for FRNA bacteriophage GA using both RT-qPCR (total concentration) and a plaque assay (infectious concentration). Mean concentrations of NoV GI and GII in influent wastewater were reduced by 0.25 and 0.41 log10 genome copies 100 ml(-1), respectively by the WWTP. The mean concentration of total FRNA bacteriophage GA was reduced by 0.35 log genome copies 100 ml(-1) compared to a reduction of infectious FRNA bacteriophage GA of 2.13 log PFU 100 ml(-1). A significant difference between concentrations of infectious and total FRNA bacteriophage GA was observed in treated, but not in untreated wastewaters. We conclude that RT-qPCR in isolation underestimates the reduction of infectious virus during wastewater treatment. We further compared the concentrations of infectious virus in combined sewer overflow (CSO) and UV treated effluents using FRNA bacteriophage GA. A greater percentage (98%) of infectious virus is released in CSO discharges than UV treated effluent (44%). Following a CSO discharge, concentrations of NoV GII and infectious FRNA bacteriophage GA in oysters from less than the limit of detection to 3150 genome copies 100 g(-1) and 1050 PFU 100 g(-1) respectively. PMID:23850211

Flannery, John; Keaveney, Sinéad; Rajko-Nenow, Paulina; O'Flaherty, Vincent; Doré, William

2013-09-15

365

Potential to use ultraviolet-treated bacteriophages to control foodborne pathogens.  

PubMed

The use of replication-deficient UV-treated bacteriophages, or phages, presents an alternative to viable phages for food biocontrol applications. Nontransducing UV-treated phages, if used correctly, are unlikely to produce viable progeny phages, which might otherwise mediate undesirable horizontal gene transfer events. Phage T4 and Escherichia coli were used as a model system to examine this possibility. UV-treated phages were able to cause a reduction in the optical density of outer membrane-free cell suspensions and they also killed host cells under conditions not permitting their multiplication, that is, 24 degrees C for 2 h and 37 degrees C for 15 min. Host cell reductions were also demonstrated in broth and on meat at 5 degrees C when high concentrations of phages of 2.3 x 10(9) PFU mL(-1) and 1.8 x 10(8) PFU cm(-2), respectively, were used. At 24 degrees C and 37 degrees C, "lysis from without" was likely to be the mechanism responsible for the reduction in host cell concentrations, but at 5 degrees C this may not have been the case. PMID:20113208

Hudson, John Andrew; Bigwood, Teresa; Premaratne, Aruni; Billington, Craig; Horn, Beverley; McIntyre, Lynn

2010-06-01

366

Novel Bacteriophages Containing a Genome of Another Bacteriophage within Their Genomes  

PubMed Central

A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 3? protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein-coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit) and B. cereus AH676 (additional regulatory and recombination genes), respectively. The third region represents an almost complete genome (except for the short terminal segments) of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a ‘fast track’ route of virus evolution and horizontal gene transfer. Another phage (BceA1) nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri. PMID:22815791

Makarova, Kira S.; Cock, Peter J.; Hopkins, David W.; Torrance, Lesley; Koonin, Eugene V.; Taliansky, Michael

2012-01-01

367

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

SciTech Connect

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

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

2008-07-11

368

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

SciTech Connect

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

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

2009-06-04

369

Molecular characterization, phylogeny, and expression pattern of c-type lysozyme in kelp grouper, Epinephelus bruneus.  

PubMed

Lysozymes are the key molecules in innate immune system and possess high bactericidal properties. In the present study, a full-length c-type lysozyme cDNA has been cloned and characterized by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques from kelp grouper, Epinephelus bruneus (Eb-CTL). The cDNA consists of 753 base pairs (bp) with a 432 bp open reading frame (ORF) that encodes 144 amino acids (aa) residues and polyadenylation signal sequence AATAAA. The deduced aa sequence polypeptide had a predicted molecular weight of 16 kDa and theoretical isoelectric point of 7.3. The deduced aa sequence have a long lysozyme domain which contains all catalytic sites and other conserved residues required for lysozyme activity. Pair-wise alignments showed a higher identity (76.4%) with Psetta maxima lysozyme and low identity (38.9%) with lysozymes of insect Bombyx mori. Interestingly, phylogenetic analysis revealed that the kelp grouper lysozyme was more closely related to other fish and vertebrate lysozymes. Quantitative real-time reverse transcriptase-PCR analysis showed that Eb-CTL transcripts are constitutively expressed in hematopoietic organs such as heart, spleen, and kidney after stimulation with LPS or infection with Vibrio anguillarum, indicating the involvement of Eb-CTL in the innate immunity of kelp grouper. This study is a first step on the genetics and functions of the c-type lysozyme in kelp grouper, and their role in anti-bacterial activity with reference to immunological properties which might have biotechnological applications. PMID:21752343

Harikrishnan, Ramasamy; Kim, Ju-Sang; Kim, Man-Chul; Balasundaram, Chellam; Heo, Moon-Soo

2011-10-01

370

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

PubMed

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?

Ercan, Duygu; Demirci, Ali

2013-07-01

371

Characterization of Helicobacter pylori Bacteriophage KHP30  

PubMed Central

Helicobacter pylori inhabits the stomach mucosa and is a causative agent of stomach ulcer and cancer. In general, bacteriophages (phages) are strongly associated with bacterial evolution, including the development of pathogenicity. Several tailed phages have so far been reported in H. pylori. We have isolated an H. pylori phage, KHP30, and reported its genomic sequence. In this study, we examined the biological characteristics of phage KHP30. Phage KHP30 was found to be a spherical lipid-containing phage with a diameter of ca. 69 nm. Interestingly, it was stable from pH 2.5 to pH 10, suggesting that it is adapted to the highly acidic environment of the human stomach. Phage KHP30 multiplied on 63.6% of clinical H. pylori isolates. The latent period was ca. 140 min, shorter than the doubling time of H. pylori (ca. 180 min). The burst size was ca. 13, which was smaller than the burst sizes of other known tailed or spherical phages. Phage KHP30 seemed to be maintained as an episome in H. pylori strain NY43 cells, despite a predicted integrase gene in the KHP30 genomic sequence. Seven possible virion proteins of phage KHP30 were analyzed using N-terminal protein sequencing and mass spectrometry, and their genes were found to be located on its genomic DNA. The genomic organization of phage KHP30 differed from the genomic organizations in the known spherical phage families Corticoviridae and Tectiviridae. This evidence suggests that phage KHP30 is a new type of spherical phage that cannot be classified in any existing virus category. PMID:23475617

Uchiyama, Jumpei; Takeuchi, Hiroaki; Kato, Shin-ichiro; Gamoh, Keiji; Takemura-Uchiyama, Iyo; Ujihara, Takako; Daibata, Masanori

2013-01-01

372

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

PubMed

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

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

2013-02-01

373

Structure and stability of self-assembled actin-lysozyme complexes in salty water  

E-print Network

Interactions between actin, an anionic polyelectrolyte, and lysozyme, a cationic globular protein, have been examined using a combination of synchrotron small-angle x-ray scattering and molecular dynamics simulations. Lysozyme initially bridges pairs of actin filaments, which relax into hexagonally-coordinated columnar complexes comprised of actin held together by incommensurate one-dimensional close-packed arrays of lysozyme macroions. These complexes are found to be stable even in the presence of significant concentrations of monovalent salt, which is quantitatively explained from a redistribution of salt between the condensed and the aqueous phases.

Lori K. Sanders; Camilo Guaqueta; Thomas E. Angelini; Jae-Wook Lee; Scott C. Slimmer; Erik Luijten; Gerard C. L. Wong

2005-07-25

374

Self-assembly of positively charged platinum nanoparticles in lysozyme crystal  

NASA Astrophysics Data System (ADS)

Assembling mechanism of platinum nanoparticles into a {1 0 1} sectors of a tetragonal lysozyme crystal was examined for two different types of the platinum nanoparticles which have either positive or negative ? potential at pH 3-7. The distribution coefficient of the positive ones inside the crystal is much higher than that of the negative ones in the whole pH range investigated. Dispersivity of the platinum nanoparticles before interaction with the lysozyme crystal surface, which is controlled by electrostatic interactions between nanoparticles, lysozyme and NaCl, causes a profound difference on the assembly of the nanoparticles into the crystal.

Takeda, Yoshihiro; Mafuné, Fumitaka

2014-06-01

375

Structure and Stability of Self-Assembled Actin-Lysozyme Complexes in Salty Water  

SciTech Connect

Interactions between actin, an anionic polyelectrolyte, and lysozyme, a cationic globular protein, have been examined using a combination of synchrotron small-angle x-ray scattering and molecular dynamics simulations. Lysozyme initially bridges pairs of actin filaments, which relax into hexagonally coordinated columnar complexes comprised of actin held together by incommensurate one-dimensional close-packed arrays of lysozyme macroions. These complexes are found to be stable even in the presence of significant concentrations of monovalent salt, which is quantitatively explained from a redistribution of salt between the condensed and the aqueous phases.

Sanders, Lori K.; Guaqueta, Camilo; Lee, Jae-Wook; Slimmer, Scott C.; Luijten, Erik [Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Angelini, Thomas E. [Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Wong, Gerard C.L. [Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States)

2005-09-02

376

Photochemical Protease: Site-Specific Photocleavage of Hen Egg Lysozyme and Bovine Serum Albumin  

Microsoft Academic Search

Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by N-(l-phenylalanine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 ± 0.3 × 105 M-1 and 6.5 ± 0.4 × 107 M-1, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, cobalt(III)

Challa V. Kumar; Apinya Buranaprapuk; Gregory J. Opiteck; Mary B. Moyer; Steffen Jockusch; Nicholas J. Turro

1998-01-01

377

[Mechanical denaturation of lysozyme in a crystal and film].  

PubMed

A method for taking stress-strain diagrams in microsamples prepared from glutaraldehyde-treated monocrystals and amorphous films of hen egg-white lysozyme has been developed. Analysis of the diagrams has shown that the deformation obeys Hooke's law within 0-2%. Upon further deformation of a crystalline sample (up to 6-10%) when a critical tension, sigma cr, is reached, the protein molecules in the sample denature and become greatly extended. Depending on crystal type and crystallographic direction the sample length increases 2 to 4 times. The sample deformation accompanying denaturation is reversible: when the sample is kept at high temperature it restores completely its initial length. The critical stress is essentially dependent on temperature, hydration level, urea concentration, the factors affecting intra- and intermolecular interactions. PMID:3390471

Gorelov, A V; Morozov, V N

1988-01-01

378

Effective interactions in molecular dynamics simulations of lysozyme solutions  

NASA Astrophysics Data System (ADS)

In this article we explore a problem of effective interactions between two rotationally restrained lysozyme molecules forming a crystal contact in aqueous solution. We perform non-equilibrium molecular dynamics simulations in order to estimate the interaction energy as a function of the distance between the two proteins obtained from direct application of the Jarzynski equality (JE), and compare it with that calculated by means of another non-equilibrium approach (Forward-Reverse method) and constrained force methods. The performance of the JE equality when applied to solvated protein interactions is discussed. All of the equilibrium and non-equilibrium methods show clear evidence that the potentials of mean force (PMF) are short-ranged, do not exceed few kTs, and that there is an accumulation of anions in the presence of hydrophobic surfaces.

Pellicane, Giuseppe; Sarkisov, Lev

2014-09-01

379

Therapeutic effects of bacteriophages against Salmonella gallinarum infection in chickens.  

PubMed

In this study the isolation and characterization of three bacteriophages (ST4, L13, and SG3) infecting Salmonella gallinarum were carried out. They were further tested for their in vivo efficacy in phage therapy. All three phages belong to the Siphoviridae family with isometric heads and non-contractile tails. They have a broad host range among serovars of Salmonella enterica. The burst sizes were observed to be 1670, 80, and 28 for ST4, L13, and SG3, respectively. The in vivo efficacy of the phages was tested in chickens. Layer chickens were challenged with S. gallinarum, whereas contact chickens were cohabited without direct challenge. Each bacteriophage was orally inoculated in the form of feed additives. Mortality was observed and S. gallinarum was periodically re-isolated from the livers, spleens, and cecums of the chickens. Bacterial re-isolation from the organs and mortality decreased significantly in both challenged and contact chickens treated with the bacteriophages compared with untreated chickens serving as the control. The three bacteriophages may be effective alternatives to antibiotics for the control of fowl typhoid disease in chickens. PMID:23801253

Hong, Sung Sik; Jeong, Jipseol; Lee, Jinju; Kim, Suk; Min, Wongi; Myung, Heejoon

2013-10-28

380

Engineering synthetic bacteriophage to combat antibiotic-resistant bacteria  

Microsoft Academic Search

Antibiotic resistance is a rapidly evolving problem that is not being adequately met by new antimicrobial drugs. Thus, there is a pressing need for effective antibacterial therapies that can be adapted against antibiotic-resistant bacteria. Here, we engineered synthetic bacteriophage to combat antibiotic-resistant bacteria by overexpressing proteins and attacking gene networks which are not directly targeted by antibiotics. By suppressing the

T. K. Lu; J. J. Collins

2009-01-01

381

The Tripartite Associations between Bacteriophage, Wolbachia, and Arthropods  

Microsoft Academic Search

By manipulating arthropod reproduction worldwide, the heritable endosymbiont Wolbachia has spread to pandemic levels. Little is known about the microbial basis of cytoplasmic incompatibility (CI) except that bacterial densities and percentages of infected sperm cysts associate with incompatibility strength. The recent discovery of a temperate bacteriophage (WO-B) of Wolbachia containing ankyrin-encoding genes and virulence factors has led to intensifying debate

Seth R. Bordenstein; Michelle L. Marshall; Adam J. Fry; Ulandt Kim; Jennifer J. Wernegreen

2006-01-01

382

Factors Affecting Survival of Bacteriophage on Tomato Leaf Surfaces  

Microsoft Academic Search

The ability of bacteriophage to persist in the phyllosphere for extended periods is limited by many factors, including sunlight irradiation, especially in the UV zone, temperature, desiccation, and exposure to copper bactericides. The effects of these factors on persistence of phage and formulated phage (phage mixed with skim milk) were evaluated. In field studies, copper caused significant phage reduction if

F. B. Iriarte; B. Balogh; M. T. Momol; L. M. Smith; M. Wilson; J. B. Jones

2007-01-01

383

In vitro translation of the three bacteriophage phi 6 RNAs.  

PubMed Central

In vitro translation of the three single-stranded RNAs transcribed in vitro by bacteriophage phi 6 RNA polymerase revealed that the large RNA codes for phage proteins P1, P2, P4, and P7, the medium RNA codes for P3, P6, and P10, and the smaller RNA for P5, P8, and P9. Images PMID:6997510

Cuppels, D A; Van Etten, J L; Burbank, D E; Lane, L C; Vidaver, A K

1980-01-01

384

Characterization of the Bacteriophage Lambda Holin and Its Membrane Lesion  

E-print Network

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...

Dewey, Jill Sayes

2011-10-21

385

Biochemical Characterization of Bacteriophage Lambda Genome Packaging in Vitro  

Microsoft Academic Search

Bacteriophage ? has been extensively studied, and the abundance of genetic and biochemical information available makes this an ideal model system to study virus DNA packaging at the molecular level. Limited in vitro packaging efficiency has hampered progress toward this end, however. It has been suggested that limited packaging efficiency is related to poor activity of purified procapsids. We describe

Qin Yang; Carlos Enrique Catalano

2003-01-01

386

First Complete Genome Sequence of Two Staphylococcus epidermidis Bacteriophages  

Microsoft Academic Search

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

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

2007-01-01

387

UPTAKE OF BACTERIOPHAGE F2 THROUGH PLANT ROOTS  

EPA Science Inventory

A model system was designed to measure viral uptake through the roots of plants and translocation to distal plant parts. For this study, uptake of bacteriophage f2 was measured in corn and bean plants growing in hydroponic solutions. Few phage were detected in plants with uncut r...

388

BACTERIOPHAGE TRANSPORT IN SANDY SOIL AND FRACTURED TUFF  

EPA Science Inventory

Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. In the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to ...

389

BACTERIOPHAGE TRANSPORT IN SANDY SOIL AND FRACTURED TUFF  

EPA Science Inventory

Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. n the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to 4...

390

Steel tanks T5 and T4 with overhead pipeline between. Redwood ...  

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Steel tanks T5 and T4 with overhead pipeline between. Redwood tanks seen in background - Hawaii Volcanoes National Park Water Collection System, Hawaii Volcanoes National Park, Volcano, Hawaii County, HI

391

_!'... ..t~\\ "4':=' .;;, .~.  

E-print Network

PASSAGE MEDIA CORP. #12;I~ I. I I i; Academic Ca~endar January 3 January 16 February 20 March 10 March 13-leave - Graduate Students........................................................10 Address Change TelephoneAssistance:...................................................................4 Calling STAR - Follow These Steps Before................................... 6 Registration Changes

Kaminsky, Werner

392

Protein-encapsulated gold cluster aggregates: the case of lysozyme  

NASA Astrophysics Data System (ADS)

We report the evolution and confinement of atomically precise and luminescent gold clusters in a small protein, lysozyme (Lyz) using detailed mass spectrometric (MS) and other spectroscopic investigations. A maximum of 12 Au0 species could be bound to a single Lyz molecule irrespective of the molar ratio of Lyz : Au3+ used for cluster growth. The cluster-encapsulated protein also forms aggregates similar to the parent protein. Time dependent studies reveal the emergence of free protein and the redistribution of detached Au atoms, at specific Lyz to Au3+ molar ratios, as a function of incubation time, proposing inter-protein metal ion transfer. The results are in agreement with the studies of inter-protein metal transfer during cluster growth in similar systems. We believe that this study provides new insights into the growth of clusters in smaller proteins.We report the evolution and confinement of atomically precise and luminescent gold clusters in a small protein, lysozyme (Lyz) using detailed mass spectrometric (MS) and other spectroscopic investigations. A maximum of 12 Au0 species could be bound to a single Lyz molecule irrespective of the molar ratio of Lyz : Au3+ used for cluster growth. The cluster-encapsulated protein also forms aggregates similar to the parent protein. Time dependent studies reveal the emergence of free protein and the redistribution of detached Au atoms, at specific Lyz to Au3+ molar ratios, as a function of incubation time, proposing inter-protein metal ion transfer. The results are in agreement with the studies of inter-protein metal transfer during cluster growth in similar systems. We believe that this study provides new insights into the growth of clusters in smaller proteins. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33180b

Baksi, Ananya; Xavier, Paulrajpillai Lourdu; Chaudhari, Kamalesh; Goswami, N.; Pal, S. K.; Pradeep, T.

2013-02-01

393

Multilayer adsorption of lysozyme on a hydrophobic substrate.  

PubMed Central

Macromolecular adsorption is known to occur as a complex process, often in a series of steps. Several models are discussed in the literature which describe the microscopic structure of the adsorbate. In the present study we investigated the adsorption of hen egg white lysozyme on alkylated silicon oxide surfaces. A combination of fluorescence excitation in the evanescent field and fluorescence recovery after photobleaching allowed us to measure the amount of adsorbed fluorescent lysozyme and the equilibrium exchange kinetics with molecules in solution. We found that a model with at least three classes of adsorbed molecules is necessary to describe the experimental results. A first layer is formed by the molecules which adsorb within a short time after the beginning of the incubation. These molecules make up approximately 65% of the final coverage. They are quasi-irreversibly adsorbed and do not measurably exchange with bulk molecules within one day even at temperatures up to 55 degrees C. A second layer, which reaches equilibrium only after several hours of incubation, shows a pronounced exchange with bulk molecules. The on-off kinetics show a distinct temperature dependence from which an activation barrier of delta E approximately 22 kcal/mol is derived. A third layer of molecules that exchange rapidly with the bulk can be seen to comprise approximately 10% of the total coverage. The exchange rate is on the order of fractions of a second. The binding of the latter two classes of adsorbed molecules is exothermic. From the temperature dependence of the coverage, the binding enthalpy of the slowly exchanging layer was estimated to be delta Hads approximately 3.8 kcal/mol. The second and third class of molecules remain enzymatically active as a muramidase, which was tested by the lysis of the cell walls of Micrococcus lysodeiktikus. The molecules in the first layer, on the other hand, showed no enzymatic activity. Images FIGURE 1 FIGURE 2 FIGURE 10 PMID:2306502

Schmidt, C F; Zimmermann, R M; Gaub, H E

1990-01-01

394

Crystallization of Chicken Egg White Lysozyme from Assorted Sulfate Salts  

NASA Technical Reports Server (NTRS)

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.

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

1999-01-01

395

Crystallization of Chicken Egg White Lysozyme from Sulfate Salts  

NASA Technical Reports Server (NTRS)

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.

Forsythe, Elizabeth; Pusey, Marc

1998-01-01

396

Multilayer adsorption of lysozyme on a hydrophobic substrate.  

PubMed

Macromolecular adsorption is known to occur as a complex process, often in a series of steps. Several models are discussed in the literature which describe the microscopic structure of the adsorbate. In the present study we investigated the adsorption of hen egg white lysozyme on alkylated silicon oxide surfaces. A combination of fluorescence excitation in the evanescent field and fluorescence recovery after photobleaching allowed us to measure the amount of adsorbed fluorescent lysozyme and the equilibrium exchange kinetics with molecules in solution. We found that a model with at least three classes of adsorbed molecules is necessary to describe the experimental results. A first layer is formed by the molecules which adsorb within a short time after the beginning of the incubation. These molecules make up approximately 65% of the final coverage. They are quasi-irreversibly adsorbed and do not measurably exchange with bulk molecules within one day even at temperatures up to 55 degrees C. A second layer, which reaches equilibrium only after several hours of incubation, shows a pronounced exchange with bulk molecules. The on-off kinetics show a distinct temperature dependence from which an activation barrier of delta E approximately 22 kcal/mol is derived. A third layer of molecules that exchange rapidly with the bulk can be seen to comprise approximately 10% of the total coverage. The exchange rate is on the order of fractions of a second. The binding of the latter two classes of adsorbed molecules is exothermic. From the temperature dependence of the coverage, the binding enthalpy of the slowly exchanging layer was estimated to be delta Hads approximately 3.8 kcal/mol. The second and third class of molecules remain enzymatically active as a muramidase, which was tested by the lysis of the cell walls of Micrococcus lysodeiktikus. The molecules in the first layer, on the other hand, showed no enzymatic activity. PMID:2306502

Schmidt, C F; Zimmermann, R M; Gaub, H E

1990-03-01

397

Antitumor activity of endogenous mFlt4 displayed on a T4 phage nanoparticle surface  

PubMed Central

Aim: Flt4 plays a key role in promoting tumor metastasis by stimulating solid tumor lymphangiogenesis. In this study, mouse Flt4 (mFlt4) was displayed on T4 phage in order to explore the feasibility of breaking immune tolerance to “self-antigens” and to evaluate the phage's antitumor activity. Methods: A T4 phage nanometer particle expressing mFlt4 on the surface was constructed for evaluation as a recombinant vaccine. The presence of the mFlt4 gene in the T4-mFlt4 recombinant vaccine was verified by PCR and Western blot analysis. The immunotherapeutic potential of T4-mFlt4 was tested in mice injected with Lewis lung carcinoma (LLC) cells. Anti-Flt4 antibody producing B cells were detected by ELISPOT. The effects of T4-mFlt4 on lymphatic metastasis and lymphangiogenesis were investigated in a mouse antimetastasis assay and by Flt4 and CD105 immunohistochemistry. Results: The T4-mFlt4 recombinant vaccine demonstrated antitumor activity and elicited autoantibodies against mFlt4. Mice carrying LLC-derived tumors exhibited prolonged survival when given the vaccine compared with control-treated animals. The vaccine also inhibited lymphangiogenesis and tumor metastasis in the mouse models. However, T4-mFlt4 was not observed to inhibit tumor growth. Conclusion: The T4-mFlt4 recombinant vaccine induced protective antitumor immunity and antimetastasis against LLC. Induction of an autoimmune response directed against tumor progression merits further study as a new strategy for immunotherapy in cancer. PMID:19417736

Ren, Shun-xiang; Ren, Zhao-jun; Zhao, Min-yi; Wang, Xiao-bin; Zuo, Shu-guang; Yu, Feng

2009-01-01

398

Biochemical Analysis of Human T Lymphocyte Differentiation Antigens T4 and T5  

Microsoft Academic Search

Two major functionally distinct T cell subsets in man have been defined with heteroantiserums and monoclonal antibodies directed against stable cell surface antigens that appear during thymic ontogeny. A monoclonal antibody to T4 antigen (anti-T4) is reactive with the peripheral inducer T cell population while a monoclonal antibody to T5 antigen (anti-T5) is reactive with the cytotoxic and suppressor population.

Cox Terhorst; Andre van Agthoven; Ellis Reinherz; Stuart Schlossman

1980-01-01

399

DNA sequences of the tail fiber genes of bacteriophage P2: evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages.  

PubMed Central

We have determined the DNA sequence of the bacteriophage P2 tail genes G and H, which code for polypeptides of 175 and 669 residues, respectively. Gene H probably codes for the distal part of the P2 tail fiber, since the deduced sequence of its product contains regions similar to tail fiber proteins from phages Mu, P1, lambda, K3, and T2. The similarities of the carboxy-terminal portions of the P2, Mu, ann P1 tail fiber proteins may explain the observation that these phages in general have the same host range. The P2 H gene product is similar to the products of both lambda open reading frame (ORF) 401 (stf, side tail fiber) and its downstream ORF, ORF 314. If 1 bp is inserted near the end of ORF 401, this reading frame becomes fused with ORF 314, creating an ORF that may represent the complete stf gene that encodes a 774-amino-acid-long side tail fiber protein. Thus, a frameshift mutation seems to be present in the common laboratory strain of lambda. Gene G of P2 probably codes for a protein required for assembly of the tail fibers of the virion. The entire G gene product is very similar to the products of genes U and U' of phage Mu; a region of these proteins is also found in the tail fiber assembly proteins of phages TuIa, TuIb, T4, and lambda. The similarities in the tail fiber genes of phages of different families provide evidence that illegitimate recombination occurs at previously unappreciated levels and that phages are taking advantage of the gene pool available to them to alter their host ranges under selective pressures. PMID:1531648

Haggård-Ljungquist, E; Halling, C; Calendar, R

1992-01-01

400

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

PubMed

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

Burke, Thomas P; Loukitcheva, Anastasia; Zemansky, Jason; Wheeler, Richard; Boneca, Ivo G; Portnoy, Daniel A

2014-11-01

401

Renaturation of reduced hen egg white lysozyme containing two blocked sulfhydryl groups  

SciTech Connect

Formation of native lysozyme from the reduced form involves many pathways in two processes: incorrect pairing of half-cystine residues by oxidation and rearrangement of disulfide (SS) bonds. The energy barrier against suflhydryl (SH)-disulfide interchange of the native or nativelike species thus formed causes accumulation of these species. For example, the enzymatically active isomers containing three presumably native SS bonds and one open SS bond may be thermodynamically favorable over the nonnative isomers and can be formed from reduced lysozyme or lysozyme containing scrambled SS bonds by nonobligatory and flexible pathways. As an extension of these observations formation of nativelike species from reduced lysozyme containing the average of two carboxymethyl (CM)-cysteine was investigated.

Acharya, A.S.; Taniuchi, H.

1980-10-01

402

Logarithmic Decay in Single-Particle Relaxation of Hydrated Lysozyme Powder  

E-print Network

We present the self-dynamics of protein amino acids of hydrated lysozyme powder around the physiological temperature by means of molecular dynamics simulations. The self-intermediate scattering functions of the amino acid ...

Baglioni, Piero

403

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

NASA Astrophysics Data System (ADS)

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.

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

2014-11-01

404

Stability of Lysozyme in Aqueous Extremolyte Solutions during Heat Shock and Accelerated Thermal Conditions  

PubMed Central

The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin) under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectroscopy and a bioactivity assay. During heat shock (10 min at 70°C), betaine, trehalose, ectoin and firoin protected lysozyme against inactivation while hydroxyectoine, did not have a significant effect. During accelerated thermal conditions (4 weeks at 55°C), firoin also acted as a stabilizer. In contrast, betaine, hydroxyectoine, trehalose and ectoine destabilized lysozyme under this condition. These findings surprisingly indicate that some extremolytes can stabilize a protein under certain stress conditions but destabilize the same protein under other stress conditions. Therefore it is suggested that for the screening extremolytes to be used for protein stabilization, an appropriate storage conditions should also be taken into account. PMID:24465983

van Streun, Erwin L. P.; Frijlink, Henderik W.; Hinrichs, Wouter L. J.

2014-01-01

405

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

NASA Astrophysics Data System (ADS)

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.

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

2008-04-01

406

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

NASA Astrophysics Data System (ADS)

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.

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

2010-03-01

407

Structural Studies of Bacteriophage ?3 Assembly  

PubMed Central

Bacteriophage ?3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include ?X174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108 S empty procapsid that requires the external D and internal B scaffolding proteins for its formation. The ?3 “open” procapsid structural intermediate was determined to 15Å resolution by cryo-electron microscopy (cryo-EM). Unlike the ?X174 “closed” procapsid and the infectious virion, the ?3 open procapsid has 30Å wide pores at the 3-fold vertices and 20Å wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid. Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30Å wide pores. The structure of the ?3 mature virion was solved to 3.5Å resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the ?3 and ?X174 virion structures are in the spike and the DNA-binding proteins. The ?3 pentameric spikes have a rotation of 3.5° compared to those of ?X174. The ?3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the ?X174 J protein, retains its carboxy-terminal-binding site on the internal surface of the capsid protein. The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in ?3 compared to ?X174, allowing the building of about 10% of the ribose-phosphate backbone. PMID:12473449

Bernal, Ricardo A.; Hafenstein, Susan; Olson, Norman H.; Bowman, Valorie D.; Chipman, Paul R.; Baker, Timothy S.; Fane, Bentley A.; Rossmann, Michael G.

2014-01-01

408

Engineering bacteriophage P22 as a nanomaterial  

NASA Astrophysics Data System (ADS)

The precise architectures of viruses and virus-like particles are highly advantageous in synthetic materials applications. These nano-size compartments are perfectly suited to act as containers of designed cargo. Not only can these nano-containers be harnessed as active materials, but they can be exploited for examining the effects of in vivo "cell-like" crowding and confinement on the properties of the encapsulated cargo. The high concentration of many different types of mutually volume excluding macromolecules in the cell causes it to be a crowded and confining environment in which to carry out reactions. Herein, the molecular design of the bacteriophage P22 encapsulation system is described and utilized for the synthesis of active nanomaterials and to explore the effect of encapsulation on the entrapped proteins' properties. In the designed system, any gene can be inserted and results in the fusion of the insert to a truncated form of the P22 scaffold protein. This scaffold protein fusion templates the spontaneous in vivo assembly of P22 capsids and also acts as an encapsulation signal. Once encapsulated, we can examine how crowding and confinement affect inter-molecular communication and activity of the cargo molecules. The P22 system is unique in that the capsid morphology can be altered, without losing the encapsulated cargo, resulting in a doubling of the capsid volume. Thus, the encapsulated fusions can be examined at two different internal concentrations. The packaged capsids contain up to 300 copies of fusion and fill more than 24% of the internal volume with the internal concentration of the fusions in the millimolar range. Not only are these fusions densely and efficiently packaged, but they retain their activity. Described herein is the packaging of fluorescent proteins, enzymes, and active peptides. In all cases, it is shown that the enforced proximity via encapsulation greatly affects the fusions activity compared to the fusion free in solution. To expand the utility of the P22 capsid as a nanomaterial, the inherent asymmetry implored by the portal complex has also been exploited. The P22 encapsulation system has proved to be an effective and versatile vehicle for nanomaterials design.

O'Neil, Alison Linsley

409

Non-equilibrium behavior of lysozyme solutions: beads, clusters and gels  

E-print Network

Observation of salt-induced aggregation of lysozyme at pH = 4.5, 22$^{\\circ}$C by optical microscopy revealed four regimes: bicontinuous texture, `beads', large aggregates, and transient gelation. The interaction of a metastable liquid-liquid binodal and an ergodic to non-ergodic transition boundary inside the equilibrium crystallization region can explain our findings. Lysozyme data at -2$^{\\circ}$C, as well as many literature results, are consistent with the relative movement of these boundaries.

H. Sedgwick; K. Kroy; A. Salonen; M. B. Robertson; S. U. Egelhaaf; W. C. K. Poon

2003-09-26

410

Evaluation of alternatives for human lysozyme purification from transgenic rice: impact of phytic acid and buffer.  

PubMed

Producing economically competitive recombinant human lysozyme from transgenic rice demands an inexpensive purification process for nonpharmaceutical applications. Human lysozyme is a basic protein, and thus, cation exchange chromatography was the selected method for lysozyme purification. Similar to other protein production systems, the identification of critical impurities in the rice extract was important for the development of an efficient purification process. Previous adsorption data indicated that phytic acid was probably responsible for an unacceptably low cation exchange adsorption capacity. In this study, we confirm that reducing phytic acid concentration improves lysozyme binding capacity and investigate alternative process conditions that reduce phytic acid interference. Compared with the previous best process, the adsorption capacity of human lysozyme was increased from 8.6 to 19.7 mg/mL when rice extract was treated with phytase to degrade phytic acid. Using tris buffer to adjust pH 4.5 extract to pH 6 before adsorption reduced phytic acid interference by minimizing phytic acid-lysozyme interactions, eliminated the need for phytase treatment, and increased the binding capacity to 25 mg/mL. Another method of reducing phytic acid concentration was to extract human lysozyme from rice flour at pH 10 with 50 mM NaCl in 50 mM sodium carbonate buffer. A similar binding capacity (25.5 mg/mL) was achieved from pH 10 extract that was clarified by acidic precipitation and adjusted to pH 6 for adsorption. Lysozyme purities ranged from 95 to 98% for all three processing methods. The tris-mediated purification was the most efficient of the alternatives considered. PMID:20945487

Wilken, Lisa R; Nikolov, Zivko L

2010-01-01

411

Calcium-Independent Phospholipase A2 Is Required for Lysozyme Secretion in U937 Promonocytes1  

Microsoft Academic Search

As a part of their surveillance functions in the immune system, monocytes\\/macrophages secrete large amounts of the bactericidal enzyme lysozyme to the extracellular medium. We report here that lysozyme secretion in activated U937 promonocytes depends on a functional calcium-independent phospholipase A2 (iPLA2). Inhibition of the enzyme by bromoenol lactone or by treatment with a specific antisense oligonucleotide results in a

Maria A. Balboa; Yolanda Saez; Jesus Balsinde

412

Lysozyme transgenic goats’ milk positively impacts intestinal cytokine expression and morphology  

Microsoft Academic Search

In addition to its well-recognized antimicrobial properties, lysozyme can also modulate the inflammatory response. This ability\\u000a may be particularly important in the gastrointestinal tract where inappropriate inflammatory reactions can damage the intestinal\\u000a epithelium, leading to significant health problems. The consumption of milk from transgenic goats producing human lysozyme\\u000a (hLZ) in their milk therefore has the potential to positively impact intestinal

Caitlin A. Cooper; Dottie R. Brundige; Wade A. Reh; Elizabeth A. Maga; James D. Murray

413

Cloning and characterization of an invertebrate type lysozyme from Venerupis philippinarum  

Microsoft Academic Search

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections and providing nutrition as digestion enzymes. In the present study, an invertebrate type lysozyme (denoted as VpLYZ) was identified from Venerupis philippinarum haemocytes by cDNA library and RACE approaches. The full-length cDNA of VpLYZ consisted of 805 nucleotides with a canonical polyadenylation signal sequence AATAAA and

Jianmin Zhao; Lihua Qiu; Xuanxuan Ning; Aiqin Chen; Huifeng Wu; Chenghua Li

2010-01-01

414

YY1 binds to regulatory element of chicken lysozyme and ovalbumin promoters  

Microsoft Academic Search

Chicken lysozyme is highly expressed in the oviduct. The 5? regulatory region of this gene contains a negative element that\\u000a represses transcription. To assess the molecular basis underlying the regulation of lysozyme gene expression, we investigated\\u000a the binding protein to this region. Sequence motif analysis suggested the existence of putative YY1 binding sites in this\\u000a regulatory region. Electrophoretic mobility shift

Mahboob Morshed; Munetoshi Ando; Junko Yamamoto; Akitsu Hotta; Hidenori Kaneoka; Jun Kojima; Ken-ichi Nishijima; Masamichi Kamihira; Shinji Iijima

2006-01-01

415

Purification and translation of ovalbumin, conalbumin, ovomucoid and lysozyme messenger RNA  

Microsoft Academic Search

Messenger RNAs for 4 egg white proteins (ovalbumin, conalbumin, ovomucoid and lysozyme) were assayed in a cell-free, protein-synthesizing system derived from rabbit reticulocytes. Each of these messengers was purified about 33-fold from hen oviduct polysomal RNA using oligo(dT) cellulose. The apparent minimal translational efficiencies varied from 22 translations for each ovalbumin mRNA to less than 1 for lysozyme mRNA. These

Richard D. Palmiter; Lynne T. Smith

1973-01-01

416

Lysozyme enhances the inhibitory effects of the peroxidase system on glucose metabolism of Streptococcus mutans.  

PubMed

The combined effect of the salivary peroxidase system and lysozyme on the glucose uptake of Streptococcus mutans NCTC 10449 was investigated. The bacteria were grown to late-exponential phase, washed, re-suspended in buffer at pH6, and incubated with (1) 50 micrograms/mL lysozyme from human milk for 60 min; (2) 7-15 mumol/L hypothiocyanous acid/hypothiocyanite for 10 min; and (3) lysozyme for 60 min prior to addition of and incubation with hypothiocyanous acid/hypothiocyanite for 10 min. Glucose uptake was initiated by adding the bacterial suspensions to 10 mL of pre-warmed 50 mumol/L glucose containing 0.98 mumol/L D-(U-14C-)-glucose, and the mixture was incubated in a shaking water-bath at 37 degrees C. Samples were withdrawn at various time intervals, rapidly filtered through 0.45-microns membranes, washed with ice-chilled buffer, and the incorporated radioactivity determined. Lysozyme stimulated S. mutans glucose uptake slightly, but significantly inhibited S. rattus glucose metabolism. A 20-30% inhibition of radiolabeled glucose incorporation was observed with hypothiocyanous acid/hypothiocyanite alone. Incubation of the bacteria with lysozyme prior to addition of hypothiocyanous acid/hypothiocyanite containing peroxidase resulted in a total inhibition of the glucose uptake. In contrast, lysozyme in combination with hypothiocyanous acid/hypothiocyanite without peroxidase gave only a 30-50% inhibition. The addition of 5 mmol/L dithiothreitol after incubation with lysozyme and hypothiocyanous acid/hypothiocyanite eliminated the inhibition of the bacterial glucose uptake. The viability of S. mutans was not affected by treatment with any of the components used. Our results indicate that physiological concentrations of lysozyme and the salivary peroxidase system components have a synergistic effect which results in a significant inhibition of glucose metabolism by S. mutans. PMID:1573081

Lenander-Lumikari, M; Månsson-Rahemtulla, B; Rahemtulla, F

1992-03-01

417

The Lysozyme from Insect (Manduca sexta) is a Cold-Adapted Enzyme  

SciTech Connect

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of {alpha}-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 {sup o}C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.

Sotelo-Mundo,R.; Lopez-Zavala, A.; Garcia-Orozco, K.; Arvizu-Flores, A.; Velazquez-Contreras, E.; Valenzuela-Soto, E.; Rojo-Dominguez, A.; Kanost, M.

2007-01-01

418

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

PubMed Central

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

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

2007-01-01

419

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

PubMed

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

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

2007-07-01

420

Two-stage, self-cycling process for the production of bacteriophages  

PubMed Central

Background A two-stage, self-cycling process for the production of bacteriophages was developed. The first stage, containing only the uninfected host bacterium, was operated under self-cycling fermentation (SCF) conditions. This automated method, using the derivative of the carbon dioxide evolution rate (CER) as the control parameter, led to the synchronization of the host bacterium. The second stage, containing both the host and the phage, was operated using self-cycling infection (SCI) with CER and CER-derived data as the control parameters. When each infection cycle was terminated, phages were harvested and a new infection cycle was initiated by adding host cells from the SCF (first stage). This was augmented with fresh medium and the small amount of phages left from the previous cycle initiated the next infection cycle. Both stages were operated independently, except for this short period of time when the SCF harvest was added to the SCI to initiate the next cycle. Results It was demonstrated that this mode of operation resulted in stable infection cycles if the growth of the host cells in the SCF was synchronized. The final phage titers obtained were reproducible among cycles and were as good as those obtained in batch productions performed under the same conditions (medium, temperature, initial multiplicity of infection, etc.). Moreover, phages obtained in different cycles showed no important difference in infectivity. Finally, it was shown that cell synchronization of the host cells in the first stage (SCF) not only maintained the volumetric productivity (phages per volume) but also led to higher specific productivity (phage per cell per hour) in the second stage (SCI). Conclusions Production of bacteriophage T4 in the semi-continuous, automated SCF/SCI system was efficient and reproducible from cycle to cycle. Synchronization of the host in the first stage prior to infection led to improvements in the specific productivity of phages in the second stage while maintaining the volumetric productivity. These results demonstrate the significant potential of this approach for both upstream and downstream process optimization. PMID:21040541

2010-01-01