These are representative sample records from Science.gov related to your search topic.
For comprehensive and current results, perform a real-time search at Science.gov.
1

What is the Average Conformation of Bacteriophage T4 Lysozyme in Solution? A Domain Orientation  

E-print Network

What is the Average Conformation of Bacteriophage T4 Lysozyme in Solution? A Domain Orientation & Department of Chemistry University of Oregon, Eugene OR 97403, USA Lysozyme from T4 bacteriophage couplings; NMR*Corresponding author Introduction Bacteriophage T4 lysozyme is an endomurami- dase utilized

Skrynnikov, Nikolai

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

Bacteriophage T4 Genome†  

PubMed Central

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

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

2003-01-01

4

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

5

Structure of the bacteriophage T4 DNA adenine methyltransferase  

Microsoft Academic Search

DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains

Zhe Yang; John R Horton; Lan Zhou; Xu Jia Zhang; Aiping Dong; Xing Zhang; Samuel L Schlagman; Valeri Kossykh; Xiaodong Cheng

2003-01-01

6

Structure and assembly of bacteriophage T4 head  

Microsoft Academic Search

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

Venigalla B Rao; Lindsay W Black

2010-01-01

7

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

8

Effect of alpha particles on bacteriophage T4Br(+)  

SciTech Connect

The effects of heavy particle radiation, which is believed to be responsible for the high relative biological effectiveness (RBE) of space hadrons, on bacteriophages are investigated. Dry film cultures of bacteriophage T4 were irradiated with 5.3 MeV Po-210 alpha particles to doses from 5 to 60 Gray, and compared with cultures irradiated by Co-60 gamma radiation. Examination of the exponential dose-response curves for bacteriophage survival indicates an RBE of 4.68 for the alpha particles. The r-mutation frequency per 10,000 surviving phages is found to peak at 7.1 at doses between 65 and 85 Gray for gamma radiation, however it declines steadily from a level of 10.2 per 10,000 survivors with increasing dose of alpha radiation. Comparison of the mutation frequencies at the same levels of lethality and the spectra of mutations produced by the two types of radiation indicates alpha and gamma radiation to differ as well in the mechanisms of mutation production. It is concluded that the observed high RBE of space hadrons cannot be explained by the presence of high-energy particles in the secondary radiation. 13 references.

Leonteva, G.A.; Akoev, I.G.; Grigorev, A.E.

1983-01-01

9

RAPID COMMUNICATION The Structure of Isometric Capsids of Bacteriophage T4  

E-print Network

RAPID COMMUNICATION The Structure of Isometric Capsids of Bacteriophage T4 Norman H. Olson,* Mari-dimensional structure of DNA-filled, bacteriophage T4 isometric capsids has been determined by means of cryoelectron of the main capsid pro- tein, gp23; and (4) isometric and intermediate-length phage produced by "petite

Baker, Timothy S.

10

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

E-print Network

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

Müftü, Sinan

11

Investigation of bacteriophage T4 by atomic force microscopy  

PubMed Central

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

Kuznetsov, Yuri G; Chang, Sheng-Chieh

2011-01-01

12

A Promiscuous DNA Packaging Machine from Bacteriophage T4  

PubMed Central

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

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

2011-01-01

13

Genomes of the T4-related bacteriophages as windows on microbial genome evolution  

Microsoft Academic Search

The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes.

Vasiliy M Petrov; Swarnamala Ratnayaka; James M Nolan; Eric S Miller; Jim D Karam

2010-01-01

14

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

15

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

NASA Astrophysics Data System (ADS)

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

Sims, Patrick Craig

16

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

17

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

E-print Network

ON THE THERMODYNAMICS AND KINETICS OF THE COOPERATIVE BINDING OF BACTERIOPHAGE T4- CODED GENE 32 of thermodynamic, and preliminary kinetic, studies on the molecular details and specificity of interaction of phage into the molecular origins of binding cooperativity is obtained by determining these thermodynamic parameters also

Kowalczykowski, Stephen C.

18

Restriction endonuclease inhibitor IPI* of bacteriophage T4  

PubMed Central

SUMMARY Phage T4 protects its DNA from the two gene encoded gmrS/gmrD (glucose modified hydroxymethylcytosine (gHMC) restriction endonuclease) (CT), of pathogenic E. coli CT596, by injecting several hundred copies of the 76 amino acid residue nuclease inhibitor, IPI*, into the infected host. Here, the three-dimensional solution structure of mature IPI* is reported as determined by nuclear magnetic resonance (NMR) techniques using 1290 experimental NOE and dipolar coupling constraints (?17 constraints/residue). Close examination of this oblate-shaped protein structure reveals a novel fold consisting of two small ?-sheets (?1: B1, B2; ?2: B3-B5), flanked at the N- and C-termini by alpha helices (H1 & H2). Such a fold is very compact in shape, and allows ejection of IPI* through the narrow 30Å portal and tail tube apertures of the virion without unfolding. Structural and dynamic measurements identify an exposed hydrophobic knob that is a putative gmrS/gmrD binding site. A single gene from the uropathogenic E. coli UT189, which codes for a gmrS/gmrD-like fusion protein (?90% identity to the heterodimeric CT enzyme) has evolved IPI* inhibitor immunity. Analysis of the gmrS/gmrD restriction endonuclease enzyme family and its IPI* family phage antagonists reveals an evolutionary pathway that has elaborated a surprisingly diverse and specifically fitted set of co-evolving attack and defense structures. PMID:18037438

Rifat, Dalin; Wright, Nathan T.; Varney, Kristen M.; Weber, David J.; Black, Lindsay W.

2008-01-01

19

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

20

Expression of the bacteriophage T4 denV structural gene in Escherichia coli  

SciTech Connect

The expression of the T4 denV gene, which previously had been cloned in plasmid constructs downstream of the bacteriophage lambda hybrid promoter-operator oLpR, was analyzed under a variety of growth parameters. Expression of the denV gene product, endonuclease V, was confirmed in DNA repair-deficient Escherichia coli (uvrA recA) by Western blot analyses and by enhancements of resistance to UV irradiation.

Recinos, A. III; Augustine, M.L.; Higgins, K.M.; Lloyd, R.S.

1986-11-01

21

The dynamics of lysozyme from bacteriophage lambda in solution probed by NMR and MD simulations.  

PubMed

(15) N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and H(N) -H(?) coupling constants, and molecular dynamics (MD) simulations have been used to characterise the behaviour of lysozyme from bacteriophage lambda (? lysozyme) in solution. The lower and upper lip regions in ? lysozyme (residues 51-60 and 128-141, respectively) show reduced (1) H-(15) N order parameters indicating mobility on a picosecond timescale. In addition, residues in the lower and upper lips also show exchange contributions to T2 indicative of slower timescale motions. The chemical shift, RDC, coupling constant and NOE data for ? lysozyme indicate that two fluctuating ?-strands (?3 and ?4) are populated in the lower lip region while the N terminus of helix ?6 (residues 136-139) forms dynamic helical turns in the upper lip region. This behaviour is confirmed by MD simulations that show hydrogen bonds, indicative of the ?-sheet and helical secondary structure in the lip regions, with populations of 40-60?%. Thus in solution ? lysozyme adopts a conformational ensemble that will contain both the open and closed forms observed in the crystal structures of the protein. PMID:23801644

Smith, Lorna J; Bowen, Alice M; Di Paolo, Alexandre; Matagne, André; Redfield, Christina

2013-09-23

22

Plasmid models for bacteriophage T4 DNA replication: requirements for fork proteins.  

PubMed Central

Bacteriophage T4 DNA replication initiates from origins at early times of infection and from recombinational intermediates as the infection progresses. Plasmids containing cloned T4 origins replicate during T4 infection, providing a model system for studying origin-dependent replication. In addition, recombination-dependent replication can be analyzed by using cloned nonorigin fragments of T4 DNA, which direct plasmid replication that requires phage-encoded recombination proteins. We have tested in vivo requirements for both plasmid replication model systems by infecting plasmid-containing cells with mutant phage. Replication of origin and nonorigin plasmids strictly required components of the T4 DNA polymerase holoenzyme complex. Recombination-dependent plasmid replication also strictly required the T4 single-stranded DNA-binding protein (gene product 32 [gp32]), and replication of origin-containing plasmids was greatly reduced by 32 amber mutations. gp32 is therefore important in both modes of replication. An amber mutation in gene 41, which encodes the replicative helicase of T4, reduced but did not eliminate both recombination- and origin-dependent plasmid replication. Therefore, gp41 may normally be utilized for replication of both plasmids but is apparently not required for either. An amber mutation in gene 61, which encodes the T4 RNA primase, did not eliminate either recombination- or origin-dependent plasmid replication. However, plasmid replication was severely delayed by the 61 amber mutation, suggesting that the protein may normally play an important, though nonessential, role in replication. We deleted gene 61 from the T4 genome to test whether the observed replication was due to residual gp61 in the amber mutant infection. The replication phenotype of the deletion mutant was identical to that of the amber mutant. Therefore, gp61 is not required for in vivo T4 replication. Furthermore, the deletion mutant is viable, demonstrating that the gp61 primase is not an essential T4 protein. Images PMID:1433501

Benson, K H; Kreuzer, K N

1992-01-01

23

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

PubMed

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

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

2014-09-23

24

Mass distribution of a probable tail-length-determining protein in bacteriophage T4  

SciTech Connect

Analysis of dark-field scanning transmission electron micrographs of unstained freeze-dried specimens established that the interior of the intact bacteriophage T4 tail tube contains extra density that is missing in tubes artificially emptied by treatment with 3 M guanidine hydrochloride. The mass of the tail tube is 3.1 x 10W daltons, and the central channel is 3.2 nm in diameter. Quantitative analysis of the density data is consistent with the presence of up to six strands of a protein molecule in the central channel that could serve as the template or ruler structure that determines the length of the bacteriophage tail and that could be injected into the cell with the phage DNA.

Duda, R.L.; Wall, J.S.; Hainfeld, J.F.; Sweet, R.M.; Eiserling, F.A.

1985-08-01

25

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

E-print Network

Electrostatic Contributions to T4 Lysozyme Stability: Solvent-Exposed Charges versus Semi mutations on five other proteins, we conclude that 1) the popular view that electrostatic interactions Electrostatic interactions play important roles in the stability of proteins, as illustrated by protein

Weston, Ken

26

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

PubMed

The Bacteriophage T4 Lysozyme (T4L) is a prototype modular protein comprised of an N-terminal and a C-domain domain, which was extensively studied to understand the folding/unfolding mechanism of modular proteins. To offer detailed structural and dynamic insights to the folded-state stability and the mechanical unfolding behaviors of T4L, we have performed extensive equilibrium and steered molecular dynamics simulations of both the wild-type (WT) and a circular permutation (CP) variant of T4L using all-atom and coarse-grained force fields. Our all-atom and coarse-grained simulations of the folded state have consistently found greater stability of the C-domain than the N-domain in isolation, which is in agreement with past thermostatic studies of T4L. While the all-atom simulation cannot fully explain the mechanical unfolding behaviors of the WT and the CP variant observed in an optical tweezers study, the coarse-grained simulations based on the Go model or a modified elastic network model (mENM) are in qualitative agreement with the experimental finding of greater unfolding cooperativity in the WT than the CP variant. Interestingly, the two coarse-grained models predict different structural mechanisms for the observed change in cooperativity between the WT and the CP variant-while the Go model predicts minor modification of the unfolding pathways by circular permutation (i.e., preserving the general order that the N-domain unfolds before the C-domain), the mENM predicts a dramatic change in unfolding pathways (e.g., different order of N/C-domain unfolding in the WT and the CP variant). Based on our simulations, we have analyzed the limitations of and the key differences between these models and offered testable predictions for future experiments to resolve the structural mechanism for cooperative folding/unfolding of T4L. PMID:25612731

Zheng, Wenjun; Glenn, Paul

2015-01-21

27

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

NASA Astrophysics Data System (ADS)

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

Zheng, Wenjun; Glenn, Paul

2015-01-01

28

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

PubMed Central

Tailed bacteriophages are the most abundant biological entities in marine environments. However, most of these marine phages are uncharacterized because few of their hosts have been cultivated. To learn more about such phages, we designed a set of degenerate PCR primers for phage T4 g23, which encodes the major capsid protein in all of the T4-type phages, an important family of the tailed phage. These primers were used to amplify g23-related sequences from diverse marine environments (fjords and bays of British Columbia, the eastern Gulf of Mexico, and the western Arctic Ocean) revealing a remarkable level of molecular diversity, which in some cases was correlated with morphological variation of the virions. Phylogenetic analysis showed that although some of these sequences were closely related to well studied subgroups of the T4-type phage, such as the T-evens, the majority of them belong to five previously uncharacterized subgroups. These data indicate that the host range of T4-type phages is much broader than previously imagined and that the laboratory isolate T4 belongs to a phage family that is extraordinarily widespread and diverse in the biosphere. PMID:16116082

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

2005-01-01

29

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

30

Translational repression in vitro by the bacteriophage T4 regA protein.  

PubMed

The bacteriophage T4 translational repressor regA protein has been purified from an overproducing strain, and its activity has been studied in simple in vitro protein synthesis reactions. RegA protein was found to inhibit the translation of T4 genes 44, 45, and ORF45-1 in a concentration-dependent fashion. Expression of two other T4 genes which are insensitive to regA protein in vivo, genes 32 and 43, was unaffected by the presence of regA protein. Specific inhibition of synthesis of genes 44, 45, and ORF 45-1 proteins was achieved with 5-20 microM concentrations of regA protein, without the addition of any other T4 encoded proteins or cofactors. When in vitro protein synthesis was performed in two steps, uncoupling translation from transcription, regA protein had an inhibitory effect regardless of whether it was added at the initiation of transcription or only at the translation step. This indicates that regA protein functions during the translation step of protein synthesis in vitro in agreement with previous in vivo studies of regA protein. PMID:3502424

Adari, H Y; Spicer, E K

1986-10-01

31

Focused genetic recombination of bacteriophage t4 initiated by double-strand breaks.  

PubMed Central

A model system for studying double-strand-break (DSB)-induced genetic recombination in vivo based on the ets1 segCDelta strain of bacteriophage T4 was developed. The ets1, a 66-bp DNA fragment of phage T2L containing the cleavage site for the T4 SegC site-specific endonuclease, was inserted into the proximal part of the T4 rIIB gene. Under segC(+) conditions, the ets1 behaves as a recombination hotspot. Crosses of the ets1 against rII markers located to the left and to the right of ets1 gave similar results, thus demonstrating the equal and symmetrical initiation of recombination by either part of the broken chromosome. Frequency/distance relationships were studied in a series of two- and three-factor crosses with other rIIB and rIIA mutants (all segC(+)) separated from ets1 by 12-2100 bp. The observed relationships were readily interpretable in terms of the modified splice/patch coupling model. The advantages of this localized or focused recombination over that distributed along the chromosome, as a model for studying the recombination-replication pathway in T4 in vivo, are discussed. PMID:12399370

Shcherbakov, Victor; Granovsky, Igor; Plugina, Lidiya; Shcherbakova, Tamara; Sizova, Svetlana; Pyatkov, Konstantin; Shlyapnikov, Michael; Shubina, Olga

2002-01-01

32

Late effect of bacteriophage T4D on the permeability barrier of Escherichia coli.  

PubMed Central

Cold centrifugation of lysis-inhibited Escherichia coli B infected with wild-type T4D results in extensive lysis beginning around 20 min after infection at 37 degrees C. Infection with an e mutant, which fails to make lysozyme, prevents lysis, but does not prevent a marked loss of K+ and Mg3+. The t gene product, thought to disrupt the cytoplasmic membrane in natural lysis, is not required for this handling-induced cation loss or lysis. Three lines of evidence argue that late protein synthesis is required to develop this potential for cation loss; the potential does not develop in infections by: (i) mutants defective in DNA synthesis, (ii) mutants defective in gene 55, and (iii) wild-type T4 when chloramphenicol is added at 6 min after infection. All late mutants examined, which are blocked in the major pathways of morphogenesis, do not prevent development of the potential. The evidence argues for a new, late effect of T4 infection on the cytoplasmic membrane. PMID:342725

Thompson, S; Wiberg, J S

1978-01-01

33

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

PubMed Central

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

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

2009-01-01

34

A Late Exclusion of Bacteriophage T4 Can Be Suppressed by Escherichia Coli Groel or Rho  

PubMed Central

A litCon mutation in Escherichia coli TU6 results in exclusion of bacteriophage T4 during the late, morphogenetic stage of its development at low temperatures. DNA was synthesized continuously in the infected cells, but less than 10% of the DNA made by 90 min after infection was packaged into DNAase-resistant particles, few viable phage were formed, and the cells lysed poorly. The exclusion could be relieved by conditions leading to elevated levels, determined immunologically, of the E. coli Rho protein (believed to be involved in regulation of T4 transcription), or chromosomally encoded E. coli GroEL (a chaperone known to be involved in phage assembly), or by supplying GroEL in trans from a plasmid. The two suppressing proteins appeared to act independently of each other. GroEL-suppression restored packaging to normal levels, perhaps by preventing GP23 from activating the host Lit protein; in addition DNA synthesis was delayed and reduced and cell lysis enhanced, demonstrating involvement of GroEL in both these processes. Rho suppression was less efficient. Since both transcription-termination-proficient and transcription-termination-deficient Rho suppressed, the results raise the possibility that Rho has a role during T4 development not directly involving transcription regulation. PMID:7916307

Linder, C. H.; Carlson, K.; Albertioni, F.; Soderstrom, J.; Pahlson, C.

1994-01-01

35

Occurrence of Lysophosphatides in Bacteriophage T4rII-Infected Escherichia coli S/6  

PubMed Central

Hydrolysis of phospholipids was observed to start about 15 min after Escherichia coli S/6 cells were infected with T4rII bacteriophage mutants. Hydrolysis continued through the latent period and well past the time when cell lysis occurs. The hydrolytic products that accumulated were free fatty acids, 2-acyl lysophosphatidylethanolamine, and various lysocardiolipins. These products indicated the action of phospholipase A1. From 15 to 22 min after infection, there were equivalent amounts of fatty acids and lysophosphatides in extracts of cellular lipids. Thereafter, free fatty acids were produced in excess. This suggests that lysophospholipase was active at the later time. We also observed a stoichiometric relation between loss of phosphatidylglycerol and increase of cardiolipin plus lysocardiolipins. This continued well past the normal lysis time (25 min). The appearance of lipase activities during the latent period seems to be specific to infection with rII mutants. Neither the wild-type bacteriophage nor rI mutants produced similar activities by 22 min after infection. PMID:5002011

Bradley, W. E. C.; Astrachan, L.

1971-01-01

36

Evidence that bacteriophage T4 ephl is a missense hoc mutation  

SciTech Connect

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

Childs, J.D.; Pilon, R.

1983-05-01

37

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

38

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

PubMed Central

How viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here, we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force, and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free energy profile of motor conformational states with that of the ATP hydrolysis cycle. PMID:24937091

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

2014-01-01

39

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

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

40

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

PubMed

How viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free-energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle. PMID:24937091

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

2014-01-01

41

A Mutant of E. COLI That Restricts Growth of Bacteriophage T4 at Elevated Temperatures  

PubMed Central

After nitrosoguanidine mutagenesis, a Phage Host Defective (phd) mutant of E. coli HfrH was isolated that supported the growth of T4D wild-type bacteriophage at 30°, but not at 40° or higher. Eleven independent spontaneous mutants of T4 (go mutants) were isolated that overcame the growth restriction at high temperature. All of these mutants were located within three percent recombination of a gene 39 amber mutation in the clockwise direction on the standard map. In mixed infections, the representative go mutant chosen for further study seems to be recessive to its wild-type allele. Temperature-shift experiments suggested that the mutated host function involved in phage growth is a "late" function, beginning in mid-eclipse.—Electrophoresis of phage proteins labelled early and late in infection showed that under restrictive conditions early protein synthesis was normal, but that certain late proteins were absent. However, measurements of DNA synthesis showed that under restrictive conditions the amount of phage DNA synthesized, and especially the amount of DNA sedimenting as high molecular weight replicative intermediate, was reduced. Pulse-chase experiments showed that the phage DNA made under restrictive conditions was not rapidly degraded. PMID:6993283

Jensen, Jean L.; Susman, Millard

1980-01-01

42

The activation domain of the MotA transcription factor from bacteriophage T4.  

PubMed Central

Bacteriophage T4 encodes a transcription factor, MotA, that binds to the -30 region of middle-mode promoters and activates transcription by host RNA polymerase. We have solved the structure of the MotA activation domain to 2.2 A by X-ray crystallography, and have also determined its secondary structure by NMR. An area on the surface of the protein has a distinctive patch that is populated with acidic and hydrophobic residues. Mutations within this patch cause a defective T4 growth phenotype, arguing that the patch is important for MotA function. One of the mutant MotA activation domains was purified and analyzed by NMR, and the spectra clearly show that the domain is properly folded. The mutant full-length protein appears to bind DNA normally but is deficient in transcriptional activation. We conclude that the acidic/hydrophobic surface patch is specifically involved in transcriptional activation, which is reminiscent of eukaryotic acidic activation domains. PMID:9155025

Finnin, M S; Cicero, M P; Davies, C; Porter, S J; White, S W; Kreuzer, K N

1997-01-01

43

Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase.  

PubMed Central

The seven-protein bacteriophage T4 DNA replication complex can be manipulated in vitro to study mechanistic aspects of the elongation phase of DNA replication. Under physiological conditions, the processivity of DNA synthesis catalyzed by the T4 polymerase (gp43) is greatly increased by the interaction of this enzyme with its accessory proteins (gp44/62 and gp45) and the T4 single-stranded DNA binding protein (gp32). The assembly of this T4 holoenzyme requires hydrolysis of ATP by the gp44/62 complex. We demonstrate here that processive T4 holoenzyme-like DNA synthesis can be obtained without hydrolysis of ATP by simply adding gp45 to the T4 DNA polymerase at extremely high concentrations, effectively bypassing the ATPase subunits (gp44/62) of the accessory protein complex. The amount of gp45 required for the gp43-gp45 heteroassociation event is reduced by addition of the macromolecular crowding agent polyethylene glycol (PEG) as well as gp32. A chromatographic strategy involving PEG has been used to demonstrate the gp43-gp45 interaction. These results suggest that gp45 is ultimately responsible for increasing the processivity of DNA synthesis via a direct and functionally significant interaction with the T4 DNA polymerase. A corollary to this notion is that the specific role of the gp44/62 complex is to catalytically link gp45 to gp43. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8475061

Reddy, M K; Weitzel, S E; von Hippel, P H

1993-01-01

44

DNA nick processing by exonuclease and polymerase activities of bacteriophage T4 DNA polymerase accounts for acridine-induced mutation specificities in T4.  

PubMed Central

Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase. In the absence of a functional T4 topoisomerase, in vivo acridine-induced mutagenesis is reduced to background levels. Further, the in vivo sites of acridine-induced deletions and duplications correlate precisely with in vitro sites of acridine-induced T4 topoisomerase cleavage. These correlations suggest that acridine-induced discontinuities introduced by topoisomerase could be processed into frameshift mutations. The induced mutations at these sites have a specific arrangement about the cleavage site. Deletions occur adjacent to the 3' end and duplications occur adjacent to the 5' end of the cleaved bond. It was proposed that at the nick, deletions could be produced by the 3'-->5' removal of bases by DNA polymerase-associated exonuclease and duplications could be produced by the 5'-->3' templated addition of bases. We have tested in vivo for T4 DNA polymerase involvement in nick processing, using T4 phage having DNA polymerases with altered ratios of exonuclease to polymerase activities. We predicted that the ratios of the deletion to duplication mutations induced by acridines in these polymerase mutant strains would reflect the altered exonuclease/polymerase ratios of the mutant T4 DNA polymerases. The results support this prediction, confirming that the two activities of the T4 DNA polymerase contribute to mutagenesis. The experiments show that the influence of T4 DNA polymerase in acridine-induced mutation specificities is due to its processing of acridine-induced 3'-hydroxyl ends to generate deletions and duplications by a mechanism that does not involve DNA slippage. Images Fig. 1 Fig. 3 Fig. 5 PMID:7892253

Kaiser, V L; Ripley, L S

1995-01-01

45

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

46

Impact of the cell life-cycle on bacteriophage T4 infection.  

PubMed

Synchronized Escherichia coli cultures were infected with bacteriophage T4 at discrete points in the cell growth cycle. The cell cycle had a significant impact on the outcome of infection. Cell burst size was smallest for newly formed cells and increased dramatically as these progressed in the cell cycle. The largest burst sizes were achieved when infecting cells immediately prior to cell division. When cells were infected during cell division, the burst size was reduced back to its initial value. Interestingly, lysis time was longest for young cells, reached a minimum at the same point that burst size reached its maximum value, and then increased at the commencement of cell division. Consequently, phage productivity in cells about to undergo cell division was almost three times greater than the productivity of young, newly formed cells. The availability of intracellular resources is believed to be the major driving force behind phage productivity during infection. Indeed, intracellular RNA contents at the time of infection were found to correlate strongly with phage productivity. There was no significant relationship between cell DNA levels and phage productivity. Finally, burst size experiments suggested that the cell cycle also influenced the likelihood of a phage to undergo productive infection. PMID:24822278

Storms, Zachary J; Brown, Tobin; Cooper, David G; Sauvageau, Dominic; Leask, Richard L

2014-04-01

47

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

48

High-molecular-weight DNA and the sedimentation coefficient: a new perspective based on DNA from T7 bacteriophage and two novel forms of T4 bacteriophage.  

PubMed Central

The DNA molecules from T7 bacteriophage and a recently obtained mutant form of T4D were studied. The DNA of this T4 mutant contains cytosine in place of all of the glucosylated hydroxymethylcytosines normally present in T4. Molecular weights were measured with an electron microscope technique, and sedimentation coefficients were determined in isokinetic sucrose gradients. T7 DNA was found to have an Mr of 26.5 x 10(6). The T4 mutant, which we have termed T4c, produces two distinct phage head and DNA size clases. DNA from the standard heads (T4c DNA) has an Mr of 114.9 x 10(6), and DNA from the petite heads (T4cp DNA) has an Mr of 82.9 x 10(6). This enabled the derivation of an equation of sedimentation coefficient at zero concentration corrected to water at 20 degrees C versus Mr for the molecular weight range of 25 x 10(6) to 115 x 10(6) that is based solely on cytosine-containing DNA standards, thereby avoiding possible anomalies introduced by the glucosylation and hydroxymethylation of cytosine. The theory of Gray et al. provided the best description of the sedimentation coefficient versus Mr relationship, based on the sedimentation coefficients and the molecular weights of the three DNA standards and other evidence. Images PMID:7365870

Clark, R W; Wever, G H; Wiberg, J S

1980-01-01

49

In vivo cleavage of cytosine-containing bacteriophage T4 DNA to genetically distinct, discretely sized fragments  

SciTech Connect

Mutants of bacteriophage T4D that are defective in genes 42 (dCMP hydroxymethylase), 46 (DNA exonuclease), and 56 (dCTPase) produce limited amounts of phage DNA in Escherichia coli B. In this DNA, glucosylated 5-hydroxymethylcytosine is completely replaced by cytosine. It is found that this DNA rapidly becomes fragmented in vivo to at least 16 discrete bands as visualized on agarose gels subjected to electrophoresis. The sizes of the fragments ranged from more than 20 to less than 2 kilobase pairs. When DNAs from two of these bands were radioactively labeled in vitro by nick translation and hybridized to XbaI restiction fragments of cytosine-containing T4 DNA, evidence was obtained that the two bands are genetically distinct, i.e., they contain DNA from different parts of the T4 genome. Mutational inactivation of T4 endonuclease II (gene denA) prevented the fragmentation. Three different mutations in T4 endonuclease IV (gene denB) caused the same minor changes in the pattern of fragments. It is concluded that T4 endonuclease II is required, and endonuclease IV is involved to a minor extent, in the in vivo production of these cytosine-containing T4 DNA fragments. These DNA fragments are viewed as ''restriction fragments'' since they represent degradation products of DNA ''foreign'' to T4, they are of discrete size, and they are genetically distinct.

Carlson, K.; Wiberg, J.S.

1983-10-01

50

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

PubMed Central

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

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

2013-01-01

51

Complete Genome Sequence of the Broad-Host-Range Vibriophage KVP40: Comparative Genomics of a T4-Related Bacteriophage  

PubMed Central

The complete genome sequence of the T4-like, broad-host-range vibriophage KVP40 has been determined. The genome sequence is 244,835 bp, with an overall G+C content of 42.6%. It encodes 386 putative protein-encoding open reading frames (CDSs), 30 tRNAs, 33 T4-like late promoters, and 57 potential rho-independent terminators. Overall, 92.1% of the KVP40 genome is coding, with an average CDS size of 587 bp. While 65% of the CDSs were unique to KVP40 and had no known function, the genome sequence and organization show specific regions of extensive conservation with phage T4. At least 99 KVP40 CDSs have homologs in the T4 genome (Blast alignments of 45 to 68% amino acid similarity). The shared CDSs represent 36% of all T4 CDSs but only 26% of those from KVP40. There is extensive representation of the DNA replication, recombination, and repair enzymes as well as the viral capsid and tail structural genes. KVP40 lacks several T4 enzymes involved in host DNA degradation, appears not to synthesize the modified cytosine (hydroxymethyl glucose) present in T-even phages, and lacks group I introns. KVP40 likely utilizes the T4-type sigma-55 late transcription apparatus, but features of early- or middle-mode transcription were not identified. There are 26 CDSs that have no viral homolog, and many did not necessarily originate from Vibrio spp., suggesting an even broader host range for KVP40. From these latter CDSs, an NAD salvage pathway was inferred that appears to be unique among bacteriophages. Features of the KVP40 genome that distinguish it from T4 are presented, as well as those, such as the replication and virion gene clusters, that are substantially conserved. PMID:12923095

Miller, Eric S.; Heidelberg, John F.; Eisen, Jonathan A.; Nelson, William C.; Durkin, A. Scott; Ciecko, Ann; Feldblyum, Tamara V.; White, Owen; Paulsen, Ian T.; Nierman, William C.; Lee, Jong; Szczypinski, Bridget; Fraser, Claire M.

2003-01-01

52

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

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

Slow switchover from host RNA synthesis to bacteriophage RNA synthesis after infection of Escherichia coli with a T4 mutant defective in the bacteriophage T4-induced unfolding of the host nucleoid.  

PubMed Central

Most, if not all, host RNA synthesis was shut off after infection of Escherichia coli strain B/5 with a bacteriophage T4 multiple mutant defective in the abilities to induce (i) unfolding of the host nucleoid (unf-), (ii) nuclear disruption (ndd-), and (iii) host DNA degradation (denA-, denB-). The shutoff of host RNA synthesis and turn-on of phage RNA synthesis were slower after infection of E. coli with unf- phage than after infection with unf+ phage. This delay in the switchover from host RNA synthesis to phage RNA synthesis in unf- infections did not result in a measurable delay in the onset of nuclear disruption, deoxyribonucleoside monophosphate kinase synthesis, or DNA synthesis. unf39 did not complement alc (allows late transcription on cytosine-containing DNA) mutants, supporting the proposal of Sirotkin et al. [Nature (London) 265:28-32, 1977] that alc and unf are possibly the same gene. PMID:201776

Tigges, M A; Bursch, C J; Snustad, D P

1977-01-01

55

Inhibition of Host Protein Synthesis During Infection of Escherichia coli by Bacteriophage T4  

PubMed Central

Two gene clusters on the Escherichia coli chromosome were induced at early times after T4 infection when >99% of the cells were infected: the lactose (lac) operon and prophage ?. Their messenger ribonucleic acid (mRNA) was detected by hybridization to ?80 dlac deoxyribonucleic acid (DNA) and ?DNA, respectively. Synthesis of host mRNA could be initiated during the first few minutes after T4 infection, although no ?-galactosidase activity could be detected. Hybridization analyses of selected fractions from sucrose gradients revealed that most of this lac mRNA induced at very early times of T4 infection was not associated with ribosomes. In contrast, virtually all lac mRNA in uninfected bacteria was associated with polysomes. This exclusion affected all host mRNA; about 70% of E. coli3H-mRNA, labeled from 2 to 3 min after T4 infection, was excluded from polysomes. Infection even reduced the yield of ?-galactosidase from lac mRNA induced before infection. Gradients from rifampicin-inhibited cells showed the normal growth of lac mRNA polysomes; in contrast, T4 infection prevented growth of the preinduced lac polysomes. It is concluded that T4 infection interferes within seconds with the reassociation of ribosomes to host mRNA. PMID:4925774

Kennell, David

1970-01-01

56

In vitro and in vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine  

PubMed Central

The bacteriophage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56-µm-long, 170-kb viral genome into 120 nm × 86 nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, highly antigenic outer capsid protein (Hoc) and small outer capsid protein (Soc). T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein in the T4 head elicited robust antibody and cellular immune responses. This “progene delivery” approach might lead to new types of vaccines and genetic therapies. PMID:23530211

Tao, Pan; Mahalingam, Marthandan; Marasa, Bernard S.; Zhang, Zhihong; Chopra, Ashok K.; Rao, Venigalla B.

2013-01-01

57

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

58

Some properties of HU are modified after the infection of Escherichia coli by bacteriophage T4.  

PubMed Central

Escherichia coli HU, an abundant, nucleoid-associated, DNA-binding protein, plays a role in several biological processes including DNA replication. Many other bacteria have well-conserved HU homologs, and there are several more-distantly related members of the family, including TF1, encoded by Bacillus subtilis phage SPO1. We have asked whether coliphage T4, like SPO1, encodes an HU homolog or whether it alters the properties of host HU. We have been unable to detect a T4-specified HU homolog, but we have shown that E. coli HU extracted from phage-infected cells differs in some properties from that extracted from uninfected cells. First, HU from uninfected cells inhibits a reconstituted T4 DNA replication system, whereas HU from infected cells does not. Second, HU from infected cells appears to bind a T4-encoded polypeptide, as shown by coimmunoprecipitation. We propose that such binding alters HU function in T4-infected cells. Images PMID:8132451

Bensaid, A; Uzan, M; Jacq, A; Hibner, U; Brody, E; Rouvière-Yaniv, J

1994-01-01

59

Some properties of HU are modified after the infection of Escherichia coli by bacteriophage T4.  

PubMed

Escherichia coli HU, an abundant, nucleoid-associated, DNA-binding protein, plays a role in several biological processes including DNA replication. Many other bacteria have well-conserved HU homologs, and there are several more-distantly related members of the family, including TF1, encoded by Bacillus subtilis phage SPO1. We have asked whether coliphage T4, like SPO1, encodes an HU homolog or whether it alters the properties of host HU. We have been unable to detect a T4-specified HU homolog, but we have shown that E. coli HU extracted from phage-infected cells differs in some properties from that extracted from uninfected cells. First, HU from uninfected cells inhibits a reconstituted T4 DNA replication system, whereas HU from infected cells does not. Second, HU from infected cells appears to bind a T4-encoded polypeptide, as shown by coimmunoprecipitation. We propose that such binding alters HU function in T4-infected cells. PMID:8132451

Bensaid, A; Uzan, M; Jacq, A; Hibner, U; Brody, E; Rouvière-Yaniv, J

1994-03-01

60

Regulation of the bacteriophage T4 Dda helicase by Gp32 single-stranded DNA-binding protein.  

PubMed

Dda, one of three helicases encoded by bacteriophage T4, has been well-characterized biochemically but its biological role remains unclear. It is thought to be involved in origin dependent DNA replication, recombination-dependent replication, anti-recombination, and recombination repair. The Gp32 protein of bacteriophage T4 plays critical roles in DNA replication, recombination, and repair by coordinating protein components of the replication fork and by stabilizing ssDNA. Previous work demonstrated that stimulation of DNA synthesis by Dda helicase appears to require direct Gp32-Dda protein-protein interactions and that Gp32 and Dda form a tight complex in the absence of ssDNA. Here we characterize the effects of Gp32-Dda physical and functional interactions through changes in the duplex DNA unwinding and ATPase activities of Dda helicase in the presence of different variants of Gp32 and different DNA repair and replication intermediate structures. Results show that Gp32-Dda interactions can be enhancing or inhibitory, depending on the Gp32 domain seen by Dda. Protein-protein interactions with Gp32 stimulate the unwinding activity of Dda, an effect associated with increased turnover of ATP, suggesting a higher rate of ATPase-driven translocation. Dda-Gp32 interactions also promote the unwinding of DNA substrates at higher salt concentrations and in the presence of substrate-bound DNA polymerase. Conversely, the formation of Gp32 clusters on ssDNA can inhibit unwinding, suggesting that Gp32-ssDNA formation sterically regulates which portions of replication and recombination intermediates are accessible for processing by Dda helicase. The data suggest a mechanism of replication fork restart in which Gp32 promotes Dda activity in template switching while preventing premature fork progression. PMID:25481875

Jordan, Christian S; Morrical, Scott W

2015-01-01

61

Photodynamic inactivation and mutagenesis by angelicin (isopsoralen) or thiopyronin (methylene red) in wild-type and repair-deficient strains of bacteriophage T4  

SciTech Connect

Photodynamic inactivation of bacteriophage T4 particles, mediated by either angelicin or thiopyronin, is enhanced by defects in the T4 uvsW-uvsX-uvsY postreplication repair system but not by a defect in the denV pyrimidine-dimer-excision system. There was no evidence for functional interactions between the two repair systems. As observed previously with 8-methoxypsoralen, photodynamic mutagenesis with angelicin is abolished by defects in the uvsW-uvsX-uvsY system.

Drake, J.W.

1985-06-01

62

Use of experimental crystallographic phases to examine the hydration of polar and nonpolar cavities in T4 lysozyme  

PubMed Central

There is conflicting evidence as to whether cavities in proteins that are nonpolar and large enough to accommodate solvent are empty or are occupied by disordered water molecules. Here, we use multiple-wavelength x-ray data collected from crystals of the selenomethionine-substituted L99A/M102L mutant of T4 lysozyme to obtain a high-resolution electron density map free of bias that is unavoidably associated with conventional model-based structure determination and refinement. The mutant, L99A/M102L, has four cavities, two being polar in character and the other two nonpolar. Cavity 1 (polar, volume 45.2 ?3) was expected to contain two well ordered water molecules, and this is confirmed in the experimental electron density map. Likewise, cavity 2 (polar, 16.9 ?3) is confirmed to contain a single water molecule. Cavity 3 (nonpolar, 21.4 ?3) was seen to be empty in conventional x-ray refinement, and this is confirmed in the experimental map. Unexpectedly, however, cavity 4 (nonpolar, volume 133.5 ?3) was seen to contain diffuse electron density equivalent to ?1.5 water molecules. Although cavity 4 is largely nonpolar, it does have some polar character, and this apparently contributes to the presence of solvent. The cavity is large enough to accommodate four to five water molecules, and it appears that a hydrogen-bonded chain of three or more solvent molecules could occupy the cavity at a given time. The results are consistent with theoretical predictions that cavities in proteins that are strictly nonpolar will not contain solvent until the volume is large enough to permit mutually satisfying water–water hydrogen bonds. PMID:18780783

Liu, Lijun; Quillin, Michael L.; Matthews, Brian W.

2008-01-01

63

Replication of bacteriophage T4 DNA in vitro. I. Basic properties of the system.  

PubMed Central

A new in vitro system for T4 DNA replication was developed by concentrating cell lysates on cellophane disks. The time course of [3H]dTTP incorporation into DNA by the system was separated into two phases: one was a very rapid incorporation which was terminated within 2 min (phase I reaction), and the other was a slow but continuous incorporation thereafter (phase II reaction). More than half of the phase I reaction product was Escherichia coli DNA, but the phase II reaction was mostly T4 DNA. Phase II reaction required four deoxyribonucleoside triphosphates, ATP, Mg2+, and KCl. 5-Hydroxymethyldeoxycytidine triphosphate was essential for the reaction and not substitutable by dCTP. The presence of KCN or NaN3 in the reaction mixture did not interfere with [3H]dTTP incorporation, but the addition of deoxyribonuclease completely degraded the system. Alkaline sucrose sedimentation analysis of phage II reaction product revealed that phase II reaction proceeded by the discontinuous mode of DNA replication as in vivo. After T4 infection, the activity for phase II reaction appeared in parallel with the activity of T4 phage DNA replication in vivo. PMID:785023

Imae, Y; Okazaki, R

1976-01-01

64

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

65

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

E-print Network

to LIN ..................................... 104 4.1 Amino acid sequence of T, its derivatives and possible topologies of T .................................................................................................. 109 4.2 Lysis...). Locations of known viral proteins are shown. 17 the exterior surface of the outer membrane (41)). Reversible binding of the long tail fibers allows T4 to ?walk? on the surface of the host and eventually bring the baseplate closer to the cell surface...

Tran, Tram Anh Thi

2009-05-15

66

Genetic Control of Capsid Length in Bacteriophage T4 I. Isolation and Preliminary Description of Four New Mutants  

PubMed Central

Four new mutants are described whose phenotypic expression affects the length of the head of bacteriophage T4D. All mutants produce some phenotypically normal phage particles. Mutant pt21-34 also produces at least two size classes of phage particle which have heads that are shorter than normal. The other three mutants, ptg19-2, ptg19-80, and ptg191, produce, in addition to phages with normal and with shorter-than-normal heads, giant phages with heads from 1.5 to at least 10 times the normal length. All mutations are clustered near gene 23. Giant phage particles have the following properties: they are infectious and contain and inject multiple genomes as a single continuous bihelical DNA molecule of greater-than-unit length. Their frequency, relative to the total plaque-former population, increases late in the infectious cycle. They have a normal diameter, variable length, and a buoyant density range in CsCl from equal to slightly greater than that of normal phage. The arrangement of capsomers is visible in the capsids, which are composed of cleaved gene 23 protein. Images PMID:4127051

Doermann, A. H.; Eiserling, F. A.; Boehner, Linde

1973-01-01

67

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

68

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

69

Suppression of gene 49 mutations of bacteriophage T4 by a second mutation in gene X: structure of pseudorevertant DNA.  

PubMed Central

Mutations in gene 49 of bacteriophage T4 were suppressed by a second mutation in gene X. Mapping studies located gene X between genes 41 and 42. Complementation results indicated that mutations in FdsA gene (a suppressor of gene 49 mutants) were in gene X. The intracellular pseudorevertant DNA was examined for unusual properties which could explain its successful encapsidation. After the in vivo inactivation of a temperature-sensitive gene 32 (DNA unwinding) protein, the intracellular pseudorevertant DNA was converted into DNA pieces of approximately genome size. A similar conversion was observed after in vitro digestion of pseudorevertant DNA with single-strand-specific S1 endonuclease. Appreciable quantities of oligomeric intermediates were not produced during this conversion process. These data indicate that pseudorevertant DNA contains sizable single-stranded gaps and has a conformation similar to that of wild-type DNA. The results further suggest that the suppression of gene 49 mutant abnormal DNA phenotype and the encapsidation defect by a second mutation in gene X is associated with the formation of sizable single-stranded gaps. These studies raise the possibility that single-stranded gaps may be involved directly in the DNA encapsidation process, or may act indirectly as a consequence of their effect on the organization of intracellular DNA. PMID:592466

Shah, D B; DeLorenzo, L

1977-01-01

70

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions.  

PubMed

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a dimer in protein crystals. Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA. Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer. However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex. Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution. However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers. Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA. Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer. PMID:8932389

Phillips, C A; Gordon, J; Spicer, E K

1996-11-01

71

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions.  

PubMed Central

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a dimer in protein crystals. Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA. Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer. However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex. Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution. However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers. Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA. Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer. PMID:8932389

Phillips, C A; Gordon, J; Spicer, E K

1996-01-01

72

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.

Gene Mayer

73

Functions of replication factor C and proliferating-cell nuclear antigen: Functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4  

SciTech Connect

The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase {delta} on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4.

Tsurimoto, Toshiki; Stillman, B. (Cold Spring Harbor Laboratory, NY (USA))

1990-02-01

74

Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4.  

PubMed Central

The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase delta on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4. Images PMID:1967833

Tsurimoto, T; Stillman, B

1990-01-01

75

The Genome of S-PM2, a “Photosynthetic” T4-Type Bacteriophage That Infects Marine Synechococcus Strains  

PubMed Central

Bacteriophage S-PM2 infects several strains of the abundant and ecologically important marine cyanobacterium Synechococcus. A large lytic phage with an isometric icosahedral head, S-PM2 has a contractile tail and by this criterion is classified as a myovirus (1). The linear, circularly permuted, 196,280-bp double-stranded DNA genome of S-PM2 contains 37.8% G+C residues. It encodes 239 open reading frames (ORFs) and 25 tRNAs. Of these ORFs, 19 appear to encode proteins associated with the cell envelope, including a putative S-layer-associated protein. Twenty additional S-PM2 ORFs have homologues in the genomes of their cyanobacterial hosts. There is a group I self-splicing intron within the gene encoding the D1 protein. A total of 40 ORFs, organized into discrete clusters, encode homologues of T4 proteins involved in virion morphogenesis, nucleotide metabolism, gene regulation, and DNA replication and repair. The S-PM2 genome encodes a few surprisingly large (e.g., 3,779 amino acids) ORFs of unknown function. Our analysis of the S-PM2 genome suggests that many of the unknown S-PM2 functions may be involved in the adaptation of the metabolism of the host cell to the requirements of phage infection. This hypothesis originates from the identification of multiple phage-mediated modifications of the host's photosynthetic apparatus that appear to be essential for maintaining energy production during the lytic cycle. PMID:15838046

Mann, Nicholas H.; Clokie, Martha R. J.; Millard, Andrew; Cook, Annabel; Wilson, William H.; Wheatley, Peter J.; Letarov, Andrey; Krisch, H. M.

2005-01-01

76

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

77

Fidelity of DNA replication catalysed in vitro on a natural DNA template by the T4 bacteriophage multi-enzyme complex.  

PubMed

More than 50 copies of a phi X174 DNA template can be made in 60 min in an in vitro DNA replication system consisting of seven purfied replication proteins isolated from T4 bacteriophage-infected cells. By transfecting with the DNA products and assaying for the reversion of specific amber mutants, the high degree of base-pairing fidelity in this system is revealed; the in vitro system is also shown to respond to the mutagenic effect of Mn2+ and to display strong base-pair context effects on fidelity, as expected from in vivo studies. PMID:6246450

Hibner, U; Alberts, B M

1980-05-29

78

Mechanisms of assembly of the enzyme-ssDNA complexes required for recombination-dependent DNA synthesis and repair in bacteriophage T4  

SciTech Connect

During late stages of bacteriophage T4 infection in E. coli, the initiation of phage DNA replication is dependent on the homologous recombination activity of the T4 uvsX protein. In vitro, uvsX protein initiates DNA synthesis on a duplex template by inserting the 3{prime} end of a homologous ssDNA molecule into the duplex. The resulting D-loop structure serves as a primer-template junction for the assembly of the T4 replication fork. Two key steps in this initiation process are (A) the assembly of uvsX-ssDNA complexes necessary for recombination activity and for the priming of lead-strand DNA synthesis, and (B) the assembly of the T4 primosome (gp41 helicase/gp61 primase complex) onto the single-stranded template for lagging-strand synthesis. Our laboratory is focusing on the mechanisms of these two different but related enzyme-ssDNA assembly processes. In this extended abstract, we describe recent efforts in our laboratory to elucidate the mechanism by which the gp41 helicase enzyme is assembled onto gp32-covered ssDNA, a process requiring the activity of a special helicase assembly factor, the T4 gp59 protein.

Morrical, S.; Hempstead, K.; Morrical, M. [Univ. of Vermont College of Medicine, Burlington, VT (United States)

1994-12-31

79

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

PubMed Central

T4-like bacteriophages have been explored for phage therapy and are model organisms for phage genomics and evolution. Here, we describe the sequencing of 11 T4-like phages. We found a high nucleotide similarity among the T4, RB55, and RB59; RB32 and RB33; and RB3, RB5, RB6, RB7, RB9, and RB10 phages. PMID:25555735

Esvelt, Kevin M.; Church, George M.

2015-01-01

80

Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns  

PubMed Central

All genetic markers from phage T2 are partially excluded from the progeny of mixed infections with the related phage T4 (general, or phage exclusion). Several loci, including gene 56 of T2, are more dramatically excluded, being present in only ?1% of the progeny. This phenomenon is referred to as localized marker exclusion. Gene 69 is adjacent to gene 56 of T4 but is absent in T2, being replaced by completely nonhomologous DNA. We describe SegF, a novel site-specific DNA endonuclease encoded by gene 69, which is similar to GIY–YIG homing endonucleases of group I introns. Interestingly, SegF preferentially cleaves gene 56 of T2, both in vitro and in vivo, compared with that of phage T4. Repair of the double-strand break (DSB) results in the predominance of T4 genes 56 and segF in the progeny, with exclusion of the corresponding T2 sequences. Localized exclusion of T2 gene 56 is dependent on full-length SegF and is likely analogous to group I intron homing, in which repair of a DSB results in coconversion of markers in the flanking DNA. Phage T4 has many optional homing endonuclease genes similar to segF, whereas similar endonuclease genes are relatively rare in other members of the T-even family of bacteriophages. We propose that the general advantage enjoyed by T4 phage, over almost all of its relatives, is a cumulative effect of many of these localized events. PMID:11825876

Belle, Archana; Landthaler, Markus; Shub, David A.

2002-01-01

81

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

82

gpwac of the T4-Type Bacteriophages: Structure, Function, and Evolution of a Segmented Coiled-Coil Protein That Controls Viral Infectivity  

PubMed Central

The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the ?50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins. PMID:15659683

Letarov, A.; Manival, X.; Desplats, C.; Krisch, H. M.

2005-01-01

83

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

PubMed

Although the structure, function, conformational dynamics, and controlled thermodynamics of proteins are manifested by their corresponding amino acid sequences, the natural rules for molecular design and their corresponding interplay remain obscure. In this study, we focused on the role of internal cavities of proteins in conformational dynamics. We investigated the pressure-induced responses from the cavity-enlarged L99A mutant of T4 lysozyme, using high-pressure NMR spectroscopy. The signal intensities of the methyl groups in the (1)H/(13)C heteronuclear single quantum correlation spectra, particularly those around the enlarged cavity, decreased with the increasing pressure, and disappeared at 200 MPa, without the appearance of new resonances, thus indicating the presence of heterogeneous conformations around the cavity within the ground state ensemble. Above 200 MPa, the signal intensities of >20 methyl groups gradually decreased with the increasing pressure, without the appearance of new resonances. Interestingly, these residues closely matched those sensing a large conformational change between the ground- and high-energy states, at atmospheric pressure. (13)C and (1)H NMR line-shape simulations showed that the pressure-induced loss in the peak intensity could be explained by the increase in the high-energy state population. In this high-energy state, the aromatic side chain of F114 gets flipped into the enlarged cavity. The accommodation of the phenylalanine ring into the efficiently packed cavity may decrease the partial molar volume of the high-energy state, relative to the ground state. We suggest that the enlarged cavity is involved in the conformational transition to high-energy states and in the volume fluctuation of the ground state. PMID:25564860

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

2015-01-01

84

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

PubMed Central

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (?100 ?s) samples to those from commonly shock-frozen (slow freeze, 1s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions. PMID:22341208

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

2012-01-01

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

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

NASA Astrophysics Data System (ADS)

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

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

2015-01-01

87

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

SciTech Connect

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

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

2002-01-01

88

Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase.  

PubMed Central

The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or single-stranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent. PMID:8668550

Belanger, K G; Mirzayan, C; Kreuzer, H E; Alberts, B M; Kreuzer, K N

1996-01-01

89

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

90

Expression of cloned bacteriophage T4 uvsW and uvsY genes in rec+ and rec- Escherichia coli  

SciTech Connect

Chimeric plasmids containing the uvsY uvsW region of the T4 genome were examined for the expression of these genes. Certain of these plasmids were shown to express the uvsY or the uvsW gene products by their ability to complement the UV sensitivity of infecting uvsW or uvsY mutant phage. Also, a chimeric plasmid containing both the uvsW and uvsY genes increases the survival of UV-irradiated, methyl methane sulfonate- or ethyl methane sulfonate-treated recA hosts.

DeVries, J.K.; Wallace, S.S.

1983-09-01

91

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

PubMed

TiO(2)-coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with thin films of TiO(2), CuO and hybrid CuO/TiO(2) prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO(2) prepared by a sol-gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of viruses. The chemical oxidising activity was also determined by measuring stearic acid oxidation. The results showed that the rate of inactivation of bacteriophage T4 increased with increasing chemical oxidising activity with the maximum rate obtained on highly active sol-gel preparations. However, these were delicate and easily damaged unlike the Ap-CVD coatings. Inactivation rates were highest on CuO and CuO/TiO(2) which had the lowest chemical oxidising activities. The inactivation of T4 was higher than that of Escherichia coli on low activity surfaces. The combination of photocatalysis and toxicity of copper acted synergistically to inactivate bacteriophage T4 and retained some self-cleaning activity. The presence of phosphate ions slowed inactivation but NaCl had no effect. The results show that TiO(2)/CuO coated surfaces are highly antiviral and may have applications in the food and healthcare industries. PMID:18317747

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

2008-05-01

92

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

PubMed Central

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

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

2013-01-01

93

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

PubMed

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

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

2014-11-01

94

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

95

Acta Cryst. (1999). D55, 10611063 Sohn et al. T4 deoxycytidylate hydroxymethylase 1061 crystallization papers  

E-print Network

and preliminary X-ray crystallographic analysis of deoxycytidylate hydroxymethylase from bacteriophage T4 Se Hui bacteriophage T4 is a homodimeric enzyme in which each polypeptide chain consists of 246 amino-acid residues September 1998 Accepted 4 February 1999 1. Introduction Bacteriophage T4 modi®es deoxycytidine monophosphate

Suh, Se Won

96

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme  

E-print Network

), the sliding clamp (gp45), and the clamp loader (gp44 62) to form the bacteriophage T4 DNA polymerase of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymer- and one for lagging-strand synthesis, proceeds stepwise with many intermediates. The bacteriophage T4 DNA

Ahmad, Sajjad

97

Expression of bacteriophage phiEa1h lysozyme in Escherichia coli and its activity in growth inhibition of Erwinia amylovora.  

PubMed

A 3.3 kb fragment from Erwinia amylovora phage Ea1h in plasmid pJH94 was previously characterized and found to contain an exopolysaccharide depolymerase (dpo) gene and two additional ORFs encoding 178 and 119 amino acids. ORF178 (lyz) and ORF119 (hol) were found to overlap by 19 bp and they resembled genes encoding lysozymes and holins. In nucleotide sequence alignments, lyz had structurally conserved regions with residues important for lysozyme function. The lyz gene was cloned into an expression vector and expressed in Escherichia coli. Active lysozyme was detected only when E. coli cells with the lyz gene and a kanamycin-resistance cassette were grown in the presence of kanamycin. Growth of Erw. amylovora was inhibited after addition of enzyme exceeding a threshold for lysozyme to target cells. When immature pears were soaked in lysates of induced cells, symptoms such as ooze formation and necrosis were retarded or inhibited after inoculation with Erw. amylovora. PMID:15289567

Kim, Won-Sik; Salm, Heike; Geider, Klaus

2004-08-01

98

Immobilization of biotinylated bacteriophages on biosensor surfaces  

Microsoft Academic Search

Bacteriophages are viruses that recognize specific receptors on the bacterium surface to which they bind and inject genetic material. The specificity of this recognition opens remarkable possibilities for biosensor development. The chemical attachment of T4 bacteriophages onto gold surfaces is being reported. This attachment leverages the genetic biotinylation of the capsid heads of bacteriophages, and the natural affinity of the

L. Gervais; M. Gel; B. Allain; M. Tolba; L. Brovko; M. Zourob; R. Mandeville; M. Griffiths; S. Evoy

2007-01-01

99

On the thermodynamics and kinetics of the cooperative binding of bacteriophage T4-coded gene 32 (helix destabilizing) protein to nucleic acid lattices.  

PubMed Central

In this paper we summarize a series of thermodynamic, and preliminary kinetic, studies on the molecular details and specificity of interaction of phage T4-coded gene 32-protein (GP32) with nucleic acid lattices. It is shown that the binding of GP32 to short (l = 2--8 residues) oligonucleotides is essentially independent of base composition and sugar-type, as well as of salt concentration. In contrast, cooperative (continuous) or isolated binding of GP32 to single-stranded polynucleotides is base and sugar composition-dependent (binding is tighter to DNA than to RNA) and highly dependent on salt concentrations. Binding constants (K), cooperativity parameters (w), and binding site sizes (n) are determined for binding to various nucleic acid lattices under a variety of environmental conditions. These results are used to show that GP32 can bind to nucleic acid lattices in two different conformations, and to characterize the molecular details of these binding species. Further insight into the molecular origins of binding cooperativity is obtained by determining these thermodynamic parameters also for the specifically proteolytically degraded GP32 fragments GP32 I (C-terminal peptide removed) and GP32 III (C- and N-terminal peptides removed). It is also shown that these GP32-nucleic acid binding measurements can be used to provide a quantitative molecular interpretation of the sequential (competitive) binding equilibria involved in the autogenous translational regulation of GP32 synthesis (Lemaire et al., 1978, J. Mol. Biol. 126:73, 1978), and to illustrate some general principles of the development of interactional specificity in cooperatively binding protein-nucleic acid complexes. Preliminary experiments have also been carried out on the kinetics of GP32 association to, and dissociation from, single-stranded nucleic acid lattices. In particular, fluorescence stopped-flow measurements of the dissociation of GP32 from such lattices as a function of lattice saturation (and protein cluster size) can be interpreted to suggest that the protein may translocate ("slide") on the lattice before dissociation, These studies permit an approach to possible rates and mechanisms of such translocation events. PMID:6264988

Kowalczykowski, S C; Lonberg, N; Newport, J W; Paul, L S; von Hippel, P H

1980-01-01

100

Isolation of Lactococcus lactis Mutants Simultaneously Resistant to the Cell Wall-Active Bacteriocin Lcn972, Lysozyme, Nisin, and Bacteriophage c2  

PubMed Central

Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits cell wall biosynthesis by binding to lipid II. In this work, two mutants resistant to Lcn972, Lactococcus lactis D1 and D1-20, with high (>320 arbitrary units [AU]/ml) and low (80 AU/ml) susceptibilities, respectively, have been isolated. Resistance to Lcn972 did not impose a burden to growth under laboratory conditions, nor did it substantially alter the physicochemical properties of the cell surface. However, the peptidoglycan of the mutants featured a higher content of muropeptides with tripeptide side chains than the wild-type strain, linking for the first time peptidoglycan remodelling to bacteriocin resistance. Moreover, L. lactis lacking a functional d,d-carboxypeptidase DacA (i.e., with a high content of pentapeptide side chain muropeptides) was shown to be more susceptible to Lcn972. Cross-resistance to lysozyme and nisin and enhanced susceptibility to penicillin G and bacitracin was also observed. Intriguingly, the Lcn972-resistant mutants were not infected by the lytic phage c2 and less efficiently infected by phage sk1. Lack of c2 infectivity was linked to a 22.6-kbp chromosomal deletion encompassing the phage receptor protein gene pip. The deletion also included maltose metabolic genes and the two-component system (TCS) F. However, a clear correlation between these genes and resistance to Lcn972 could not be clearly established, pointing to the presence of as-yet-unidentified mutations that account for Lcn972 resistance. PMID:22504807

Roces, Clara; Courtin, Pascal; Kulakauskas, Saulius; Rodríguez, Ana; Chapot-Chartier, Marie-Pierre

2012-01-01

101

Cloning and expression of T4 DNA polymerase.  

PubMed Central

The structural gene coding for bacteriophage T4 DNA polymerase (gene 43) has been cloned into inducible plasmid vectors, which provide a source for obtaining large amounts of this enzyme after induction. The T4 DNA polymerase produced in this fashion was purified by an innovative three-step procedure and was fully active. Images PMID:3478676

Lin, T C; Rush, J; Spicer, E K; Konigsberg, W H

1987-01-01

102

Electrochemical Detection of Bacteria Using Bacteriophage  

Microsoft Academic Search

Immobilization of bacteriophage T4 onto the SPEs was achieved by formation of amide bonds between the protein coating of the phage and the electrochemically generated carboxylic groups at the carbon surface. The immobilized T4 were then used to specifically detect target bacteria (E.coli). Impedance measurements (Nyquist plots) show significant shifts in both the real (Zr) and imaginary (Zi) impedances due

Arghavan Shabani; Mohammed Zourob; Beatrice Allain; Marcus Lawrence; Rosemonde Mandeville

2007-01-01

103

Molecular Structure of Lysozyme  

NSDL National Science Digital Library

Alexander Fleming discovered the antibiotic activity of lysozyme in 1922 when he dropped mucus into a culture of bacteria and noticed that the bacteria were killed. In humans, lysozyme is in the blood, mucus, tears and saliva. The mechanism by which lysozyme kills bacteria is by hydrolyzing the glycosidic bond of the bacterial cell wall, the protective outer coating of the organism. This causes cell lysis, hence the name lysozyme (zyme is for enzyme).

2003-01-23

104

StructureFunction Analysis of the DNA Translocating Portal of the Bacteriophage  

E-print Network

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

Kihara, Daisuke

105

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

106

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

107

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display  

PubMed Central

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; D?browska, Krystyna

2013-01-01

108

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

E-print Network

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

Sparks, Donald L.

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

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

111

Crystallization of Hevamine, an Enzyme with Lysozyme\\/Chitinase Activity from Hevea brasiliensis Latex  

Microsoft Academic Search

Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain “pathogenesis-related” proteins from plants, but not to hen egg-white or phage T4 lysozyme. To investigate the atomic details of the substrate specificity and the cause for hevamine’s low pH optimum (pH 4.0),

Bauke W. Dijkstra; Jaap J. Beintema; Asmini Budiani; Henriëtte J. Rozeboom

1990-01-01

112

Lysozymes in the animal kingdom  

Microsoft Academic Search

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

Lien Callewaert; Chris W. Michiels

2010-01-01

113

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

114

Microneedle-mediated transdermal bacteriophage delivery.  

PubMed

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 × 10(6)PFU/ml (plaque forming units per ml) was detected in the receiver compartment when delivered across dermatomed skin and 4.0 × 10(3)PFU/ml was detected in the receiver compartment when delivered across full thickness skin. An in vivo study resulted in 4.13 × 10(3)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 24h, 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

2012-09-29

115

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

116

The Tape Measure Protein of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA35 Has an Active Muramidase Domain  

PubMed Central

Tailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein from Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibits in vitro lytic activity against live S. aureus cells, an activity that could eventually find use in the treatment of infections. PMID:22729533

Rodríguez-Rubio, Lorena; Gutiérrez, Dolores; Martínez, Beatriz; Rodríguez, Ana; Götz, Friedrich

2012-01-01

117

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

118

Antiviral effects on bacteriophages and rotavirus by cranberry juice.  

PubMed

Studies were undertaken to investigate the antiviral effects of comestible juices, especially cranberry juice, on non-related viral species. After exposure of bacteriophage T2 to a commercially available cranberry (Vaccinium macrocarpon) juice cocktail (CJ), virus infectivity titer was no longer detectible. After a 60-min exposure to orange (OJ) and grapefruit juices (GJ), phage infectivity was reduced to 25-35% of control, respectively. Similar data were observed for the bacteriophage T4. CJ inactivation of phage T4 was rapid, dose-dependent, and occurred at either 4 or 23 degrees C. Neither pH nor differences in sugar/carbohydrate levels among the juices may be ascribed to the recognized antiviral effects. Further studies were performed to identify the occurrence of antiviral activity by CJ to a mammalian enteric virus. The treatment of the simian rotavirus SA-11 with a 20% CJ suspension was sufficient to inhibit hemagglutination. Under scanning and transmission electron microscopy, CJ was observed to inhibit the adsorption of phage T4 to its bacterial host cells and prevented the replication of rotavirus in its monkey kidney (MA-104) host cells, respectively. The data suggest, for the first time, a non-specific antiviral effect towards unrelated viral species (viz., bacteriophages T2 and T4 and the simian rotavirus SA-11) by a commercially available cranberry fruit juice drink. PMID:17140784

Lipson, S M; Sethi, L; Cohen, P; Gordon, R E; Tan, I P; Burdowski, A; Stotzky, G

2007-01-01

119

Optical Activity of Human Lysozyme  

Microsoft Academic Search

The ultraviolet circular dichroism spectra of human lysozyme are presented. Effects of pH and added inhibitor (N-acetyl-D-glucosamine) were examined and the results were compared with similar measurements of hen egg-white lysozyme. The near-ultraviolet CD spectral bands are substantially different in the human and hen egg-white enzymes. In addition to marked dissimilarities in the spectral interval 260-300 nm, an unusual CD

J. P. Halper; N. Latovitzki; H. Bernstein; S. Beychok

1971-01-01

120

Optical activity of human lysozyme.  

PubMed

The ultraviolet circular dichroism spectra of human lysozyme are presented. Effects of pH and added inhibitor (N-acetyl-D-glucosamine) were examined and the results were compared with similar measurements of hen egg-white lysozyme. The near-ultraviolet CD spectral bands are substantially different in the human and hen egg-white enzymes. In addition to marked dissimilarities in the spectral interval 260-300 nm, an unusual CD band occurs at an anomalous wavelength (313 nm) in human lysozyme. The pH dependence of the latter suggests a possible interaction, absent in hen egg-white lysozyme, between a tryptophan and a tyrosine residue. Analysis of the spectra furthermore suggests lesser net rotational strengths of tryptophan bands in hen egg-white lysozyme than in human lysozyme, although the latter has one less tryptophan residue. The relationship between the CD spectra and the sequence differences of the proteins is discussed, as well as the CD spectra (published by others) of a closely related protein, bovine alpha-lactalbumin. Contributions of cystine residues to the spectra are examined in the light of possible differences in chirality of one of the four disulfide bridges.The far-ultraviolet CD spectra of human and egg-white lysozyme are quite similar, though not identical. In view of the pronounced differences in side-chain optical activity, and of the effect of pH variation on the far-ultraviolet CD spectrum of human lysozyme, it is likely that at least part of the observed difference in spectra is due to nonpeptide optical activity, and that the proteins have a secondary structure in common. PMID:5276753

Halper, J P; Latovitzki, N; Bernstein, H; Beychok, S

1971-03-01

121

Biophysical aspects of lysozyme adduct with monocrotophos.  

PubMed

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

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

2014-09-01

122

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

SciTech Connect

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

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

2010-07-22

123

Bacteriophage therapy against Enterobacteriaceae.  

PubMed

The Enterobacteriaceae are a class of gram-negative facultative anaerobic rods, which can cause a variety of diseases, such as bacteremia, septic arthritis, endocarditis, osteomyelitis, lower respiratory tract infections, skin and soft-tissue infections, urinary tract infections, intra-abdominal infections and ophthalmic infections, in humans, poultry, animals and fish. Disease caused by Enterobacteriaceae cause the deaths of millions of people every year, resulting in enormous economic loss. Drug treatment is a useful and efficient way to control Enterobacteriaceae infections. However, with the abuse of antibiotics, drug resistance has been found in growing number of Enterobacteriaceae infections and, as such, there is an urgent need to find new methods of control. Bacteriophage therapy is an efficient alternative to antibiotics as it employs a different antibacterial mechanism. This paper summarizes the history of bacteriophage therapy, its bacterial lytic mechanisms, and the studies that have focused on Enterobacteriaceae and bacteriophage therapy. PMID:25662887

Xu, Youqiang; Liu, Yong; Liu, Yang; Pei, Jiangsen; Yao, Su; Cheng, Chi

2015-02-01

124

T4 Phage and Its Head Surface Proteins Do Not Stimulate Inflammatory Mediator Production  

PubMed Central

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1?, IL-1?, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-?, TNF-?, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages. PMID:23976975

Miernikiewicz, Paulina; D?browska, Krystyna; Piotrowicz, Agnieszka; Owczarek, Barbara; Wojas-Turek, Justyna; Kicieli?ska, Jagoda; Rossowska, Joanna; Pajtasz-Piasecka, El?bieta; Hodyra, Katarzyna; Macegoniuk, Katarzyna; Rzewucka, Kamila; Kopciuch, Agnieszka; Majka, Tomasz; Letarov, Andrey; Kulikov, Eugene; Maciejewski, Henryk; Górski, Andrzej

2013-01-01

125

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.

American Society for Microbiology. Division M.

1998-01-01

126

BACTERIOPHAGE THERAPY AND CAMPYLOBACTER  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

127

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.

128

Coevolutionary arms races between bacteria and bacteriophage  

E-print Network

Coevolutionary arms races between bacteria and bacteriophage J. S. Weitz* , H. Hartman , and S. A coevolutionary dynamics of bacteriophage and bac- teria in their ecological context. Bacteriophage enter host

Weitz, Joshua S.

129

FASP Kit Protocol -ORNL Developed for Bacteriophage  

E-print Network

FASP Kit Protocol - ORNL Developed for Bacteriophage This method was optimized for bacteriophage by Kristen Corrier undergraduate research assistant at Oak. This method is designed to prepare small quantities (~1-10ug) of isolate bacteriophage

Sullivan, Matthew B.

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

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.

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

1998-06-16

132

Single-molecule dynamics of lysozyme processing distinguishes linear and cross-linked peptidoglycan substrates.  

PubMed

The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single-molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field-effect transistors through pyrene linker molecules. The substrate-driven hinge-bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibited processive low turnover rates of 20-50 s(-1) and rapid (200-400 s(-1)) nonproductive motions. The latter nonproductive binding events occupied 43% of the enzyme's time in the presence of the cross-linked peptidoglycan but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate but appeared to sidestep the peptide cross-links to zigzag through the wild-type substrate. PMID:22239748

Choi, Yongki; Moody, Issa S; Sims, Patrick C; Hunt, Steven R; Corso, Brad L; Seitz, David E; Blaszczak, Larry C; Blaszcazk, Larry C; Collins, Philip G; Weiss, Gregory A

2012-02-01

133

Enhanced Human Lysozyme Production by Kluyveromyces lactis  

Microsoft Academic Search

An attempt to enhance recombinant human lysozyme production by Kluyveromyces lactis K7 was performed in this study. In this study, the production of recombinant human lysozyme was investigated using shake\\u000a flasks and bioreactor under different cultivation conditions. It was demonstrated that 25 °C could enhance human lysozyme\\u000a production when compared with other temperatures tested. This study also demonstrated that higher biomass

Eric Lu Huang; Ali Demirci

2009-01-01

134

THE MORTALITY OF BACTERIOPHAGE CONTAINING ASSIMILATED RADIOACTIVE PHOSPHORUS  

PubMed Central

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

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

1951-01-01

135

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

DOEpatents

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

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

1998-10-20

136

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

DOEpatents

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

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

1998-11-03

137

Detection of lysozyme and alpha 2-macroglobulin--lysozyme complexes by immunoblotting.  

PubMed

An immunoblotting technique was developed to detect human lysozyme and lysozyme complexes in body fluids. The unoccupied binding capacity of proteins was demonstrated by addition of surplus lysozyme. The sensitivity of immunoblotting to the free enzyme in human albumin solution was less than 5 ng. In serum and pleural fluid, part of exogenous lysozyme was bound to alpha 2-macroglobulin (alpha 2-M). At high concentrations of lysozyme in leukemic sera, part of the enzyme formed an endogenous alpha 2-M complex. On the other hand, the formation of alpha 2-M complexes with exogenous lysozyme was especially striking in sera from nephrotic patients with elevated alpha 2-M. The findings corroborate with previous reports on lysozyme binding to purified alpha 2-M in vitro and suggest that the binding is concentration-dependent with respect to both reaction partners. In vivo the mechanism may provide a pathway for extrarenal lysozyme catabolism medicated by reticuloendothelial cells. No other binding proteins were seen in the present study: lysozyme did not bind to serum immunoglobulins in 35 samples with an immunoglobulin paraprotein, three samples with polyclonally elevated gamma-globulins, 20 other patient sera and 10 normal sera. Neither did lysozyme bind to urinary proteins in five samples from patients with myeloic leukemias nor in 10 samples from myeloma patients with urinary excretion of a monoclonal immunoglobulin light chain. PMID:2467766

Mörsky, P

1988-12-30

138

Inhibition of lysozyme by taurine dibromamine.  

PubMed

Hypobromous acid (HOBr) is a powerful oxidant produced by stimulated neutrophils and eosinophils. Taurine, a non-protein amino acid present in high amounts in the leukocytes, reacts instantaneously with HOBr leading to their haloamine derivative taurine dibromamine (Tau-NBr2). Lysozyme is a bactericidal enzyme also present in leukocytes and in secretory fluids. The inhibition of lysozyme is a pathway for bacterial proliferation in inflammatory sites. Here, we investigated the inhibition of the enzymatic activity of lysozyme when it was submitted to oxidation by Tau-NBr2. We found that the oxidation of lysozyme by Tau-NBr2 decreased its enzymatic activity in 80%, which was significant higher compared to the effect of its precursor HOBr (30%). The study and comparison of Tau-NBr2 and HOBr regarding the alterations provoked in the intrinsic fluorescence, synchronous fluorescence, resonance light scattering and near and far-UV circular dichroism spectra of lysozyme and oxidized lysozyme revealed that tryptophan residues in the active site of the protein were the main target for Tau-NBr2 and could explain its efficacy as inhibitor of lysozyme enzymatic activity. This property of Tau-NBr2 may have pathological significance, since it can be easily produced in the inflammatory sites. PMID:23590281

Petrônio, M S; Ximenes, V F

2013-11-01

139

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

140

Microbiology (1997), 143, 179185 Printed in Great Britain Bacteriophage T4 development depends on the  

E-print Network

by pretreatment with low penicillin (Pn) concentrations; it was directly proportional to total cellular surface). ................................................................................................................................................. Abbreviations: -MG, methyl -D-glucoside; Pn, penicillin; PSS, protein- synthesizing system. Phage

Zaritsky, Arieh

141

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

Microsoft Academic Search

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

Craig Tuerk; Larry Gold

1990-01-01

142

Purification and concentration of bacteriophage T4 using monolithic chromatographic supports.  

PubMed

Phages are gaining importance due to their wide usage. In this work strong anion exchange monolithic chromatographic column was used for single step phage purification. Most of the proteins and DNA were removed and recovery of approximately 70% of infective virus was reproducibly achieved. 30 ml of phage sample was purified in around 10 min. PMID:17588505

Smrekar, F; Ciringer, M; Peterka, M; Podgornik, A; Strancar, A

2008-01-15

143

Inactivation of Bacteriophages via Photosensitization of Fullerol  

E-print Network

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

Alvarez, Pedro J.

144

Dispersing biofilms with engineered enzymatic bacteriophage  

E-print Network

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

Collins, James J.

145

Widespread genetic exchange among terrestrial bacteriophages  

E-print Network

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

Hartl, Daniel L.

146

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

Microsoft Academic Search

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

A. D. Hershey; MARTHA CHASE

1952-01-01

147

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

148

Acetylated Lysozyme as Impurity in Lysozyme Crystals: Constant Distribution Coefficient  

NASA Technical Reports Server (NTRS)

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

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

2000-01-01

149

Kinetics of Competitive Adsorption between Lysozyme and Lactoferrin on Silicone Hydrogel Contact Lenses and the Effect on Lysozyme Activity.  

PubMed

Abstract Purpose: To determine the effect of competitive adsorption between lysozyme and lactoferrin on silicone hydrogel contact lenses and the effect on lysozyme activity. Methods: Three commercially available silicone hydrogel contact lens materials (senofilcon A, lotrafilcon B and balafilcon A) were examined, for time points ranging from 10?s to 2?h. Total protein deposition was determined by I(125) radiolabeling of lysozyme and lactoferrin, while the activity of lysozyme was determined by a micrococcal activity assay. Results: Senofilcon A and balafilcon A did not show any relevant competitive adsorption between lysozyme and lactoferrin. Lotrafilcon B showed reduced protein deposition due to competitive adsorption for lactoferrin at all time points and lysozyme after 7.5?min. Co-adsorption of lactoferrin and lysozyme decreased the activity of lysozyme in solution for senofilcon A and lotrafilcon B, but co-adsorption had no effect on the surface activity of lysozyme for all lens types investigated. Conclusions: Competition between lysozyme and lactoferrin is material specific. Co-adsorption of lysozyme and lactoferrin does not affect the activity of surface-bound lysozyme but can reduce the activity of subsequently desorbed lysozyme. PMID:25251834

Hall, Brad; Jones, Lyndon; Forrest, James A

2014-09-24

150

Amino acid sequences of stomach and nonstomach lysozymes of ruminants.  

PubMed

Complete amino acid sequences are presented for lysozymes c from camel and goat stomachs and compared to sequences of other lysozymes c. 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 probably took place before the divergence of cow, goat, and deer about 25 million years ago. Partial sequences of three lysozymes from goat tears indicated that (a) the goat tear family of lysozymes may have diverged from the stomach lysozyme family by an ancient duplication and (b) later duplications are probably responsible for the multiple forms of tear and milk lysozymes in ruminants. PMID:2111849

Jollès, J; Prager, E M; Alnemri, E S; Jollès, P; Ibrahimi, I M; Wilson, A C

1990-04-01

151

Novel Bacteriophage lambda Cloning Vector  

Microsoft Academic Search

A simple method for generating phage collections representing eukaryotic genomes has been developed by using a novel bacteriophage lambda vector, lambda 1059. The phage is a BamHI substitution vector that accommodates DNA fragments 6-24 kilobases long. Production of recombinants in lambda 1059 requires deletion of the lambda red and gamma genes. The recombinants are therefore spi- and may be separated

Jonathan Karn; Sydney Brenner; Leslie Barnett; Gianni Cesareni

1980-01-01

152

ISOLATION OF LYTIC SALMONELLA BACTERIOPHAGES  

Technology Transfer Automated Retrieval System (TEKTRAN)

This research was based on the hypothesis that Salmonella bacteriophages (phages) occur naturally in manure and can be isolated for future characterization and potential use as typing reagents, indicators and biocontrol agents. The purpose of this research was to test a protocol for isolation of ly...

153

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

154

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

155

Structure-Sweetness Relationship in Egg White Lysozyme: Role of Lysine and Arginine Residues on the Elicitation of Lysozyme Sweetness  

Microsoft Academic Search

Lysozyme is one of the sweet-tasting proteins. To clarify the structure-sweetness relationship and the basicity-sweetness relationship in lysozyme, we have generated lysozyme mutants with Pichia systems. Alanine substitution of lysine residues demonstrated that two out of six lysine residues, Lys13 and Lys96, are required for lysozyme sweetness, while the remaining four lysine residues do not play a significant role in

Tetsuya Masuda; Nobuyuki Ide; Naofumi Kitabatake

2005-01-01

156

LYSOZYME IN EPIPHYSEAL CARTILAGE: II. The Effect of Egg White Lysozyme on Mouse Embryonic Femurs in Organ Cultures  

Microsoft Academic Search

Embryonic mouse femoral cartilage, like the epiphyseal cartilage of the calf scapula, contains large amounts of lysozyme . The addition of egg white lysozyme to organ cultures of embryonic mouse femurs induces unique alterations in the gross and microscopic mor- phology of the femurs . The sites of these alterations are precisely related to the natural distribution of lysozyme in

K. E. Kuettner; L. W. SOBLE; R. D. RAY; R. L. CROXEN; M. PASSOVOY; R. EISENSTEIN

1970-01-01

157

Productive and unproductive lysozyme-chitosaccharide complexes. Kinetic investigations.  

PubMed

Flow and relaxation methods were used to study the kinetics of oligosaccharides binding to lysozyme and the pre-steady-state kinetics of the lysozyme-catalyzed, hydrolysis of chitohexose. The minimal mechanism demonstrated, and the kinetics parameters pertaining to the elementary steps, allow interpretations of previous equilibrium and steady-state kinetic measurements which had yielded only complex constants, reflecting both productive and unproductive lysozyme-substrate complexes. In contrast to previous assumptions, the data presented in this paper provide evidence for "stable" productive lysozyme-substrate complexes. Our proposed mechanism utilizes structural information and accounts for the difference in efficiency of lysozyme-catalyzed hydrolysis of chitopentose and chitohexose. PMID:1138867

Holler, E; Rupley, J A; Hess, G P

1975-06-01

158

Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49  

PubMed Central

RB49 is a virulent bacteriophage that infects Escherichia coli. Its virion morphology is indistinguishable from the well-known T-even phage T4, but DNA hybridization indicated that it was phylogenetically distant from T4 and thus it was classified as a pseudo-T-even phage. To further characterize RB49, we randomly sequenced small fragments corresponding to about 20% of the ?170-kb genome. Most of these nucleotide sequences lacked sufficient homology to T4 to be detected in an NCBI BlastN analysis. However, when translated, about 70% of them encoded proteins with homology to T4 proteins. Among these sequences were the numerous components of the virion and the phage DNA replication apparatus. Mapping the RB49 genes revealed that many of them had the same relative order found in the T4 genome. The complete nucleotide sequence was determined for the two regions of RB49 genome that contain most of the genes involved in DNA replication. This sequencing revealed that RB49 has homologues of all the essential T4 replication genes, but, as expected, their sequences diverged considerably from their T4 homologues. Many of the nonessential T4 genes are absent from RB49 and have been replaced by unknown sequences. The intergenic sequences of RB49 are less conserved than the coding sequences, and in at least some cases, RB49 has evolved alternative regulatory strategies. For example, an analysis of transcription in RB49 revealed a simpler pattern of regulation than in T4, with only two, rather than three, classes of temporally controlled promoters. These results indicate that RB49 and T4 have diverged substantially from their last common ancestor. The different T4-type phages appear to contain a set of common genes that can be exploited differently, by means of plasticity in the regulatory sequences and the precise choice of a large group of facultative genes. PMID:11976309

Desplats, Carine; Dez, Christophe; Tétart, Françoise; Eleaume, Heïdy; Krisch, H. M.

2002-01-01

159

Blocking the T4 lysis inhibition phenotype.  

PubMed

Nonlysogenic Escherichia coli K cells exhibit a delay in lysis when infected by T4rII phage termed lysis inhibition (LIN). E. coli K cells expressing lambda rexB from either a prophage defective for rexA, or a multicopy plasmid supported T4rII infection, but prevented the establishment of LIN. In addition, E. coli null mutations in either the periplasmic "tail-specific protease" tsp, or the 10Sa RNA ssrA, completely blocked the establishment of LIN following T4 infections. The expression of rexB in the absence of rexA resulted in several cellular phenotypes, including aberrant cell surface morphology, the partial to near complete suppression of mutations of lambda S and T4t holin genes, and lysis by cells aging on plates or growing with high rexB expression at elevated temperatures. These activities of RexB were impeded in the presence of RexA. PMID:14637004

Slavcev, Roderick A; Hayes, Sidney

2003-12-01

160

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

161

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

162

Proton glass freezing in hydrated lysozyme powders  

Microsoft Academic Search

At room temperature, the dielectric relaxation of hydrated powder of the protein lysozyme is known to be due to protons migrating between ionized side chains. A recent study of this relaxation at lower temperatures suggested a behavior typical of proton glasses. An analysis of the complex dielectric susceptibility by a temperature-frequency plot presented here has revealed that ergodicity is broken

Adrijan Levstik; Cene Filipic; Zdravko Kutnjak; Giorgio Careri; Giuseppe Consolini; Fabio Bruni

1999-01-01

163

Recombinant bacteriophage lysins as antibacterials  

PubMed Central

With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential. PMID:21327123

Fenton, Mark; Ross, Paul; McAuliffe, Olivia; O'Mahony, Jim

2010-01-01

164

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

165

Stationary phase-like properties of the bacteriophage lambda Rex exclusion phenotype.  

PubMed

The rex genes of bacteriophage lambda were found to protect lysogenic Escherichia coliK host cells against killing by phage T4 rII, when compared in parallel to isogenic Rex(-) lysogens and nonlysogens. This protective effect was abrogated upon mutation of the host stationary-phase sigma factor RpoS. Rex(+) lysogens infected by T4 rII contracted, formed aggregates and shed flagella, thus resembling cells entering stationary phase. These phenotypes were accentuated in nonlysogenic cells carrying multicopy plasmids expressing rexA-rexB: cells were about two-fold contracted in length, expressed membrane-bound and detached flagella, were insensitive to infection by a variety of phages and clumped extensively; in addition, cultures of these cells were odorous. Our observations support the hypothesis that the Rex system can cause a stationary-phase-like response that protects the host against infection by T4 rII. PMID:12715152

Slavcev, R A; Hayes, S

2003-04-01

166

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. PMID:25258475

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

2014-01-01

167

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

168

Lysozyme depolymerization of photo-activated chitosan adhesive films.  

PubMed

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

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

2015-05-01

169

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

2005-12-14

170

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

171

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

172

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

E-print Network

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

Gruner, Sol M.

173

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

174

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

175

The amino acid sequence of reeves' pheasant (Syrmaticus reevesii) lysozyme.  

PubMed

The amino acid sequence of reeves' pheasant lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and resulting peptides were analyzed using the DABITC/PITC double coupling manual Edman method. The established amino acid sequence had seven substitutions, Tyr3, Leu15, His41, His77, Ser79, Arg102, and Asn121, compared with hen egg-white lysozyme. Ser79 was the first found in a bird lysozyme. A substitution in the active site was found in position 102 which has been considered to participate in the substrate binding at subsites A-C. PMID:1368713

Araki, T; Kuramoto, M; Torikata, T

1991-07-01

176

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2013 CFR

...CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1490 Lysozyme... (b) Classification. Class I (general controls). The device is exempt...

2013-04-01

177

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2012 CFR

...CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1490 Lysozyme... (b) Classification. Class I (general controls). The device is exempt...

2012-04-01

178

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2014 CFR

...CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1490 Lysozyme... (b) Classification. Class I (general controls). The device is exempt...

2014-04-01

179

Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved  

Microsoft Academic Search

There are common themes among bacteriophage-encoded virulence factors, which include the well-characterized bacterial toxins and proteins that alter antigenicity as well as several new classes of bacteriophage-encoded proteins such as superantigens, effectors translocated by a type III secretion system, and proteins required for intracellular survival and host cell attachment. These virulence factors are encoded by a diversity of bacteriophages, members

E. Fidelma Boyd; Harald Brüssow

2002-01-01

180

Lysozyme pattern formation in evaporating droplets  

NASA Astrophysics Data System (ADS)

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

Gorr, Heather Meloy

181

Genetic Dissection of T4 Lysis  

PubMed Central

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

Moussa, Samir H.; Lawler, Jessica L.

2014-01-01

182

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

PubMed

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

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

1999-01-01

183

Impurity effects of lysozyme molecules specifically labeled with a fluorescent reagent on the crystallization of tetragonal and monoclinic lysozyme crystals  

NASA Astrophysics Data System (ADS)

To study the effects of impure proteins, i.e. proteins with heterogeneous features that are minor constituents within "purified" protein solutions, on the crystallization of target proteins, and in particular the effects of such impurities on intermolecular contacts, we modified only the ?-amino group of the N-terminal lysine of hen egg-white lysozyme with a fluorescent reagent, tetramethylrhodamine-5-isothiocyanate (5-TRITC), which has a high detection sensitivity. We then investigated the effects of the fluorescent-labeled lysozyme (F-lysozyme) on the nucleation and growth of tetragonal and monoclinic lysozyme crystals. In the tetragonal and monoclinic crystals, the fluorescent labels were located at molecular surfaces of lysozyme inside and outside of the intermolecular contact areas, respectively. We found that addition of a low concentration (0.02 mg/ml) of F-lysozyme significantly suppressed nucleation of the tetragonal crystals but had no effect on that of the monoclinic crystals. In contrast, addition of a higher concentration (0.05 mg/ml) of F-lysozyme significantly promoted the heterogeneous nucleation of both polymorphs. In addition, the decrease in the growth rate of the tetragonal crystal by F-lysozyme was much more significant than that of the monoclinic one, although the effective distribution coefficients of F-lysozyme for the tetragonal and monoclinic crystals were similar (2.9 and 3.2, respectively). These results clearly indicate that inhibiting the formation of specific intermolecular contacts plays a crucial role in the effects of impure proteins.

Matsui, Takuro; Sazaki, Gen; Hondoh, Hironori; Matsuura, Yoshiki; Nakada, Toshitaka; Nakajima, Kazuo

2006-08-01

184

Novel bacteriophage lambda cloning vector.  

PubMed

A simple method for generating phage collections representing eukaryotic genomes has been developed by using a novel bacteriophage lambda vector, lambda 1059. The phage is a BamHI substitution vector that accommodates DNA fragments 6-24 kilobases long. Production of recombinants in lambda 1059 requires deletion of the lambda red and gamma genes. The recombinants are therefore spi- and may be separated from the spi+ vector phages by plating on strains lysogenic for bacteriophage P2. Random fragments suitable for insertion into lambda 1059 are obtained by partial digestion of high molecular weight eukaryotic DNA with Sau3a. This restriction enzyme cleaves at the sequence G-A-T-C and leaves a 5'-tetranucleotide "sticky end." Because G-A-T-C extensions are also produced by BamHI cleavage, these fragments may be annealed directly to BamHI-cleaved lambda 1059. By using these methods, a set of clones covering the entire Caenorhabditis elegans genome was constructed. DNA segments which include the unc-54 myosin heavy chain gene have been isolated from this collection. PMID:6254062

Karn, J; Brenner, S; Barnett, L; Cesareni, G

1980-09-01

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

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

188

Original article Selective extraction of lysozyme from a mixture  

E-print Network

Original article Selective extraction of lysozyme from a mixture with lactoferrin mixture with lactoferrin (77 000 g.mol ­1, in monomer form) by ultrafiltration (UF). The system protein- brane partly fouled by the charged protein. This approach was successfully used to achieve the lysozyme/lactoferrin

Paris-Sud XI, Université de

189

Lysozyme diffusion adjacent to the (1 1 0) crystal surface  

Microsoft Academic Search

Measurements of lysozyme diffusion within the “depletion zone” of the (110) face of lysozyme crystals are reported. The measurements were performed using the technique of microscope light scattering , capable of measuring small sampling areas (?2?m2). Measurements within such small areas allowed for the determination of intensity correlation functions at precise locations (at ?10?m intervals) adjacent to the (110) crystal

Sridhar Gorti; William M. Zuk; John Konnert; Keith Ward; Toyoichi Tanaka; Hua Yang

2001-01-01

190

Purification, characterization, and biosynthesis of bovine cartilage lysozyme isoforms.  

PubMed

The cationic protein, lysozyme, has an extracellular distribution in cartilage; however, its biological role in this tissue still remains unclear. This study describes a simple and high yielding procedure for the purification of four novel isoforms of lysozyme from the functionally different articular (metacarpalphalangeal joint) and nonarticular (nasal septum) bovine cartilages. Chromatography of the cartilage extracts on S-Sepharose revealed the presence of four major lysozyme active peaks each of which was further purified to homogeneity by gel filtration and reversed-phase chromatography. Each peak yielded a different molecular mass when analyzed by ion spray mass spectrometry, and material isolated from either cartilage source displayed an identical molecular mass for each lysozyme preparation. N-terminal amino acid sequence and amino acid composition analyses confirmed the presence of four novel lysozyme isoforms in both bovine articular and nonarticular cartilages. The lytic activity of each lysozyme isoform toward Micrococcus lysodeikticus was dependent on both the ionic strength and pH of the buffer, where an increase in activity accompanied an increase in ionic strength. The lysozymes were shown to be synthesized by chondrocytes in vitro, which in addition to the relatively high chemical amounts of lysozyme present in cartilage, would suggest that this small cationic protein has some as yet undetermined biological role within the cartilage extracellular matrix. PMID:9056247

Moss, J M; Van Damme, M P; Murphy, W H; Stanton, P G; Thomas, P; Preston, B N

1997-03-01

191

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

192

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; Górski, Andrzej; De Vos, Daniel

2014-01-01

193

Evidence That the Heterogeneity of a T4 Population Is the Result of Heritable Traits  

PubMed Central

Many bacteriophage populations display heterogeneity in their adsorption characteristics; a portion of the phage population remains free in solution throughout adsorption experiments (residual fraction). This residual fraction generally constitutes a minority of phages that exhibit significantly slower adsorption kinetics than the main phage stock (main fraction). While this phenomenon is likely the result of evolutionary driving forces, the present study demonstrates that the residual fraction is not always the result of phenotypic variations within a single genotype, as is generally thought. Experiments with phage T4 showed that two subgroups with distinct adsorption traits that were passed on to their progeny could be isolated from the original phage stock. Sequencing of genes involved in adsorption revealed two point mutations in gene 37 of residual fraction isolates, which resulted in modifications to the long tail-fiber, the organelle of attachment and host cell recognition. Adsorption studies consistently showed that T4 phage stocks amplified from residual fraction isolates had significantly lower adsorption efficiencies than those amplified from main fractions. The conducted experiments provide convincing evidence that the observed heterogeneity in T4 adsorption behavior is the result of conserved mutations to the phage genome and is not exclusively the result of phenotypic variations within the population. While it is believed high mutation rates exist to hasten phage adaptation, this study shows that this bet hedging strategy can also, in the short term, inadvertently handicap the phage's adsorption capabilities to a given host under normal infection conditions, resulting in the residual fraction observed in adsorption experiments. PMID:25551763

Storms, Zachary J.; Sauvageau, Dominic

2014-01-01

194

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

195

Tradeoffs in bacteriophage life histories  

PubMed Central

Viruses are the most abundant biological entities on the planet, yet most classical principles of evolutionary biology and ecology were not developed with viruses in mind. Here, the concept of biological tradeoffs, a fundamental tenet of life history theory, is examined in the context of bacteriophage biology. Specifically, several important parameters of phage life histories—replication, persistence, host range, and adsorption—are evaluated for tradeoffs. Available data indicate that replication rate is strongly negatively correlated with both persistence and host range, suggesting that the well-documented tradeoff in macroorganisms between offspring production and offspring quality also applies to phages. The biological tradeoffs that appear to characterize viruses’ life histories have potential importance for viral evolution, ecology, and pathogenesis. PMID:24616839

Keen, Eric C

2014-01-01

196

Visible Light Sensitized Inactivation of MS-2 Bacteriophage by a  

E-print Network

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

Alvarez, Pedro J.

197

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.

198

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

E-print Network

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

Harrison, Stephen C.

199

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, we engineered bacteriophage to overexpress proteins and attack gene networks that are not directly bacteriophage en- hances killing by quinolones by several orders of magnitude in vitro and significantly

Collins, James J.

200

BACTERIOPHAGE: A SAFE AND NATURAL ALTERNATIVE TO ANTIMICROBIAL GROWTH PROMOTERS  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

201

Modeling and analysis of a marine bacteriophage infection  

E-print Network

Modeling and analysis of a marine bacteriophage infection Edoardo Beretta a,1 , Yang Kuang b,*,2 Abstract A mathematical model for the marine bacteriophage infection is proposed and its es- sential mathematical features are analyzed. Since bacteriophage infection induces bacte- rial lysis which releases

Kuang, Yang

202

Real time observations of single bacteriophage DNA ejections in vitro.  

E-print Network

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

Phillips, Rob

203

Local Adaptation of Bacteriophages to Their Bacterial Hosts in Soil  

E-print Network

Local Adaptation of Bacteriophages to Their Bacterial Hosts in Soil Michiel Vos,1 * Philip J and their parasitic viruses (bacteriophages, phages). Lytic phages bind to the bacterial cell surface, inject and bacteriophage population structure is of particular importance because microbial diver- sity ultimately

Nuismer, Scott L.

204

Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida).  

PubMed

In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes - four times, in comparison to the control. In three cases: of the coelomic fluid, the homogenates from cocoons and the extract from coelomocytes, the antibody against HEWL (hen egg white lysozyme) recognized only one protein with a molecular mass of about 14.4 kDa. In the coelomic fluid, apart from the protein with molecular mass of 14.4 kDa the antibody directed against human lysozyme recognized an additional protein of 22 kDa. Using the bioautography technique after electrophoretic resolution of native proteins in acidic polyacrylamide gels, two lytic zones of M. luteus were observed in the case of the coelomic fluid and three after the analysis of the extract of coelomocytes and the egg homogenates. The results indicated the existence of several forms of lysozyme with a different electric charge in the analyzed D. veneta samples. The highest lysozyme activity in the intestine of D. veneta was observed in the midgut. The antibody directed against human lysozyme indicated a strong positive signal in epidermal and midgut cells of earthworm. PMID:22019387

Fio?ka, Marta J; Zagaja, Miros?aw P; Hu?as-Stasiak, Monika; Wielbo, Jerzy

2012-01-01

205

The Effects of Acetate Buffer Concentration on Lysozyme Solubility  

NASA Technical Reports Server (NTRS)

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

Forsythe, Elizabeth L.; Pusey, Marc L.

1996-01-01

206

Mesoscopic coarse-grained simulations of lysozyme adsorption.  

PubMed

Coarse-grained simulations are adopted to study the adsorption behavior of lysozyme on different (hydrophobic, neutral hydrophilic, zwitterionic, negatively charged, and positively charged) surfaces at the mesoscopic microsecond time scale (1.2 ?s). Simulation results indicate the following: (i) the conformation change of lysozyme on the hydrophobic surface is bigger than any other studied surfaces; (ii) the active sites of lysozyme are faced to the hydrophobic surface with a "top end-on" orientation, while they are exposed to the liquid phase on the hydrophilic surface with a "back-on" orientation; (iii) the neutral hydrophilic surface can induce the adsorption of lysozyme, while the nonspecific protein adsorption can be resisted by the zwitterionic surface; (iv) when the solution ionic strength is low, lysozyme can anchor on the negatively charged surface easily but cannot adsorb on the positively charged surface; (v) when the solution ionic strength is high, the positively charged lysozyme can also adsorb on the like-charged surface; (vi) the major positive potential center of lysozyme, especially the residue ARG128, plays a vital role in leading the adsorption of lysozyme on charged surfaces; (vii) when the ionic strength is high, a counterion layer is formed above the positively charged surface, which is the key factor why lysozyme can adsorb on a like-charged surface. The coarse-grained method based on the MARTINI force field for proteins and the BMW water model could provide an efficient way to understand protein interfacial adsorption behavior at a greater length scale and time scale. PMID:24785197

Yu, Gaobo; Liu, Jie; Zhou, Jian

2014-05-01

207

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

Microsoft Academic Search

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,

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

2004-01-01

208

Arrangement of coding and intervening sequences of chicken lysozyme gene.  

PubMed Central

Hybrid phages that contain chicken lysozyme gene sequences have been isolated from a chicken DNA library. Two overlapping clones covering a region of 22 kilobase pairs around this gene have been studied by restriction mapping. Southern hybridization, and electron microscopic analysis of hybrids between lysozyme mRNA and the cloned cellular DNA. Three intervening sequences interrupt the lysozyme structural gene. The cellular gene is at least 3.9 kilobases long, about 6 times the length of the structural gene. Images PMID:293713

Lindenmaier, W; Nguyen-Huu, M C; Lurz, R; Stratmann, M; Blin, N; Wurtz, T; Hauser, H J; Sippel, A E; Schütz, G

1979-01-01

209

Cations reduce antimicrobial efficacy of lysozyme-chelator combinations.  

PubMed

Reduction of the antimicrobial efficacy of lysozyme-chelator combinations against two Escherichia coli O157:H7 strains on addition of mineral salts was studied. The objective of the study was to determine the effect of type and concentration of mono-, di-, and trivalent mineral salts on the antimicrobial effectiveness of lysozyme and various chelators against E. coli O157:H7. Seven salts (Al3+, Ca2+, Fe2+, Fe3+, K+, Mg2+, and Na+) at 1 to 10 mM were added to aqueous solutions of lysozyme and disodium ethylenediamine tetraacetic acid (EDTA), disodium pyrophosphate (DSPP), or pentasodium tripolyphosphate (PSTPP) at pH 6, 7, or 8 and applied to cultures of E. coli O157:H7 strains 932 and H1730. Inhibitory activity of lysozyme chelator combinations against both strains was completely lost after addition of > or = 1 mM Ca2+ and Mg2+ at pH 7 and 8. At pH 6, antimicrobial activity of lysozyme-EDTA against both strains was retained in the presence of calcium or magnesium cations. DSPP-lysozyme inhibited strain H1730 at pH 6 despite the presence of Mg2+. Concentrations above 4 mM Fe2+ neutralized activity of all lysozyme-chelator combinations. Reversal of inhibition by lysozyme-chelator complexes by the monovalent Na+ and K+ ions depended on E. coli O157:H7 strain type. Neither monovalent cation reversed inhibition of strain 932. However, Na+ and K+ reversed lysozyme-chelator inhibition of strain H1730. The addition of > or = 1 mM Fe3+ or Al3+ was effective in reversing inhibition of both strains by lysozyme and EDTA at pH 6, 7, and 8. Isothermal titration calorimetry was used to determine the amount of ion-specific competitive binding of free cations by EDTA-lysozyme combinations. A mechanistic model for the antimicrobial functionality of chelator-lysozyme combinations is proposed. PMID:14968960

Boland, J S; Davidson, P M; Bruce, B; Weiss, J

2004-02-01

210

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

NASA Astrophysics Data System (ADS)

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

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

2014-05-01

211

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

PubMed

Viral fibers play a central role in many virus infection mechanisms since they recognize the corresponding host and establish a mechanical link to its surface. Specifically, bacteriophages have to anchor to bacteria through the fibers surrounding the tail before starting the viral DNA translocation into the host. The protein gene product (gp) 37 from bacteriophage T4 long tail fibers forms a fibrous parallel homotrimer located at the distal end of the long tail fibers. Biochemical data indicate that, at least, three of these fibers are required for initial host cell interaction but do not reveal why three and no other numbers are required. By using atomic force microscopy, we obtained high-resolution images of gp37 fibers adsorbed on a mica substrate in buffer conditions and probed their local mechanical properties. Our experiments of radial indentation at the nanometer scale provided a radial stiffness of ? 0.08 N/m and a breaking force of ? 120 pN. In addition, we performed finite element analysis and determined a Young's modulus of ? 20 MPa. From these mechanical parameters, we hypothesize that three viral fibers provide enough mechanical strength to prevent a T4 virus from being detached from the bacteria by the viral particle Brownian motion, delivering a biophysical justification for the previous biochemical data. PMID:25353832

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

2014-05-01

212

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

213

Proton glass freezing in hydrated lysozyme powders.  

PubMed

At room temperature, the dielectric relaxation of hydrated powder of the protein lysozyme is known to be due to protons migrating between ionized side chains. A recent study of this relaxation at lower temperatures suggested a behavior typical of proton glasses. An analysis of the complex dielectric susceptibility by a temperature-frequency plot presented here has revealed that ergodicity is broken due to the divergence of the longest relaxation time at 266 K, indicating specifically that this hydrated protein is a proton glass. A change in the temperature behavior of the static dielectric constant and the average relaxation frequency at 273 K indicates a further transition occurring at this temperature, whose nature remains to be investigated. PMID:11970721

Levstik, A; Filipic, C; Kutnjak, Z; Careri, G; Consolini, G; Bruni, F

1999-12-01

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

Photodynamic Inactivation of Mammalian Viruses and Bacteriophages  

PubMed Central

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

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

2012-01-01

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

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2011 CFR

...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...

2011-04-01

218

[Temperate Legionella bacteriophage: discovery and characteristics].  

PubMed

For the first time, temperate Legionella bacteriophage was isolated from organs of guinea pig infected with Philadelphia 1 strain of Legionella pneumophila. Negative colonies of bactriophage from 1.5 to 2.5 mm in diameter were detected. Central part of them was transparent and surrounded by peripheral zone of partial lysis. Electron microscopy showed that corpuscles of the phage consist from multifaceted elongated head of stretched hexagonal form and short tail. The bacteriophage lyzed bacteria, which cause Legionnaires' disease, and also had certain lytic activity against causative agent of tularemia. PMID:18819413

Grigor'ev, A A; Bondarev, V P; Borisevich, I V; Darmov, I V; Mironin, A V; Zolotarev, A G; Pogorel'ski?, I P; Ianov, D S

2008-01-01

219

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

2014-10-01

220

Strong and selective adsorption of lysozyme on graphene oxide.  

PubMed

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

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

2014-04-23

221

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

PubMed Central

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides –9 to –3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides –8 to –3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an ?7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix–loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix–loop groove, which may also play a role in RNA binding. PMID:11222767

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

2001-01-01

222

The associative nature of adenylyl transfer catalyzed by T4 DNA ligase.  

PubMed

DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD(+) and assistance of a divalent metal cofactor Mg(2+). Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover (31)P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase-nucleotide complex is triggered in situ by the photo release of caged Mg(2+), and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4-5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations. PMID:18562298

Cherepanov, Alexey V; Doroshenko, Elena V; Matysik, Jörg; de Vries, Simon; de Groot, Huub J M

2008-06-24

223

Bacteriophage lysis: mechanism and regulation.  

PubMed Central

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

Young, R

1992-01-01

224

Selective Antibacterial Properties of Lysozyme for Oral Microorganisms  

PubMed Central

The antibacterial properties of lysozyme were investigated with oral microorganisms representing the seven serotypes (a through g) of Streptococcus mutans, Veillonella alcalescens, and the virulent (V) and avirulent (AV) strains of Actinomyces viscosus T14. Growth of bacteria in defined medium was monitored spectrophotometrically after the addition of various amounts (25 ?g to 5 mg/ml) of enzyme. No growth inhibition of V. alcalescens was observed. Inhibition of A. viscosus T14(V) and A. viscosus T14(AV) occurred with 160 ?g of lysozyme per ml. Of the S. mutans cultures tested, the serotype a and b strains were inhibited with as little as 25 ?g of enzyme per ml, whereas e and f strains were most resistant to the bacteriostatic activity of lysozyme. The presence of dl-threonine or sucrose in growth medium did not significantly affect the results. A lysoplate assay was developed to rapidly survey the bacterial cultures for their susceptibility to the lytic ability of the enzyme. Lysis, as a measure of a zone of clearing in agarose plates, occurred for all microorganisms in the presence of lysozyme after the subsequent addition of NaCl or detergent. The bactericidal activity of lysozyme was determined on S. mutans BHT and S. mutans LM-7 by the pour plate technique. Preincubation of S. mutans LM-7 with as much as 1 mg of enzyme for 90 min did not affect viability or growth, whereas preincubation of S. mutans BHT with 1 mg of lysozyme resulted in no recoverable colony-forming units. An antigen containing extract of S. mutans LM-7 blocked the growth inhibitory property of lysozyme. Human lysozyme was a more effective antibacterial factor than hen egg white lysozyme. Total growth inhibition of S. mutans BHT was effected with 40 ?g of human enzyme, and as little as 10 ?g of human enzyme inhibited growth for greater than 20 h. The data presented indicate that different mechanisms may be responsible for the bacteriostatic, lytic, and bactericidal properties of the enzyme and that lysozyme is a selective but effective antibacterial factor for oral microorganisms. ImagesFig. 3 PMID:7216430

Iacono, Vincent J.; MacKay, Bruce J.; DiRienzo, Sharon; Pollock, Jerry J.

1980-01-01

225

Bacteriophage ecology in commercial sauerkraut fermentations  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

226

Bacteriophage Host Range and Bacterial Resistance  

Microsoft Academic Search

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

Paul Hyman; Stephen T. Abedon

2010-01-01

227

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...

228

GENETIC HETEROZYGOSITY IN UNREPLICATED BACTERIOPHAGE h RECOMBINANTS  

Microsoft Academic Search

Bacteriophage crosses using density-labeled parents have been carried out under conditions restricting DNA synthesis. The parental material and genetic contributions to progeny manifesting recombination within a genetic interval sufficiently short to exhibit high negative interference have been examined. The unreplicated products of recombination isolated as phage particles appear to contain long continuous heteroduplex regions which are heterozygous for the closely

RAYMOND L. WHITE; MAURICE S. FOX

229

Long-Circulating Bacteriophage as Antibacterial Agents  

Microsoft Academic Search

The increased prevalence of multidrugresistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the

Carl R. Merril; Biswajit Biswas; Richard Carlton; Nicole C. Jensen; G. Joseph Creed; Steve Zullo; Sankar Adhya

1996-01-01

230

Comparative electrochemical inactivation of bacteria and bacteriophage  

Microsoft Academic Search

Electric fields and currents have been shown to be capable of disinfecting drinking water and reducing the numbers of bacteria and yeast in food. However, little research has been conducted regarding the effectiveness of electric fields and currents in the inactivation of viruses. The objective of this study was to compare the ability of bacteria and bacteriophage to survive exposure

Kevin P. Drees; Morteza Abbaszadegan; Raina M. Maier

2003-01-01

231

Effectiveness of bacteriophages in the sputum of cystic fibrosis patients.  

PubMed

Bacteriophages have been shown to be effective for treating acute infections of the respiratory tract caused by antibiotic-resistant bacteria in animal models, but no evidence has yet been presented of their activity against pathogens in complex biological samples from chronically infected patients. We assessed the efficacy of a cocktail of ten bacteriophages infecting Pseudomonas aeruginosa following its addition to 58 sputum samples from cystic fibrosis (CF) patients collected at three different hospitals. Ten samples that did not contain P. aeruginosa were not analysed further. In the remaining 48 samples, the addition of bacteriophages led to a significant decrease in the levels of P. aeruginosa strains, as shown by comparison with controls, taking two variables (time and bacteriophages) into account (p = 0.024). In 45.8% of these samples, this decrease was accompanied by an increase in the number of bacteriophages. We also tested each of the ten bacteriophages individually against 20 colonies from each of these 48 samples and detected bacteriophage-susceptible bacteria in 64.6% of the samples. An analysis of the clinical data revealed no correlation between patient age, sex, duration of P. aeruginosa colonization, antibiotic treatment, FEV1 (forced expiratory volume in the first second) and the efficacy of bacteriophages. The demonstration that bacteriophages infect their bacterial hosts in the sputum environment, regardless of the clinical characteristics of the patients, represents a major step towards the development of bacteriophage therapy to treat chronic lung infections. PMID:24920209

Saussereau, E; Vachier, I; Chiron, R; Godbert, B; Sermet, I; Dufour, N; Pirnay, J-P; De Vos, D; Carrié, F; Molinari, N; Debarbieux, L

2014-12-01

232

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

Code of Federal Regulations, 2013 CFR

...2013-07-01 2013-07-01 false Bacteriophage of Clavibacter michiganensis subspecies...Exemptions From Tolerances § 180.1307 Bacteriophage of Clavibacter michiganensis subspecies...is established for residues of lytic bacteriophage of Clavibacter michiganensis...

2013-07-01

233

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

Code of Federal Regulations, 2014 CFR

...2014-07-01 2014-07-01 false Bacteriophage of Clavibacter michiganensis subspecies...Exemptions From Tolerances § 180.1307 Bacteriophage of Clavibacter michiganensis subspecies...is established for residues of lytic bacteriophage of Clavibacter michiganensis...

2014-07-01

234

Novel control of the sheep scab mite, Psoroptes ovis, through the application of bacteriophage therapy   

E-print Network

trails were used to isolate bacteriophage from environmental samples. Sixteen bacteriophage were successfully isolated, which were infective against three mite faecal bacteria. Isolated bacteriophage were characterised by a number of methods including...

Hall, Sarah Alice

2011-11-24

235

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

236

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

Code of Federal Regulations, 2012 CFR

...2012-07-01 2012-07-01 false Bacteriophage of Clavibacter michiganensis subspecies...Exemptions From Tolerances § 180.1307 Bacteriophage of Clavibacter michiganensis subspecies...is established for residues of lytic bacteriophage of Clavibacter michiganensis...

2012-07-01

237

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

Code of Federal Regulations, 2014 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...

2014-07-01

238

STUDIES ON THE PURIFICATION OF BACTERIOPHAGE  

PubMed Central

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

Kalmanson, G.; Bronfenbrenner, J.

1939-01-01

239

Lytic sensitivity of Actinobacillus actinomycetemcomitans Y4 to lysozyme.  

PubMed Central

The ability of both human and hen egg white lysozymes to lyse Actinobacillus actinomycetemcomitans Y4 was investigated. Lysis was followed optically at 540 nm by measuring the percent reduction in turbidity of freshly harvested log-phase cells suspended in Tris-maleate buffers within a wide range of pH (5.2 to 8.5) and molarity (0.01 to 0.2 M) and containing various amounts of enzyme and EDTA. In several instances, treated microorganisms were subsequently examined in thin sections by electron microscopy. Reductions in turbidity and clearing of suspensions occurred with small amounts of lysozyme (less than 1 microgram) under relatively alkaline conditions and at low ionic strength and in the presence of small amounts of EDTA (greater than 0.01 mM). Under the most alkaline conditions, EDTA alone effected turbidity reductions similar to those observed in the presence of lysozyme, which suggested that EDTA not only increased outer membrane permeability but also caused cell lysis. Ultrastructural analysis did not always correspond to turbidimetric observations. Cell lysis was virtually complete in suspensions containing both lysozyme and EDTA. However, in contrast to turbidimetric findings, a significant percentage of cells (greater than 25%) was lysed in the presence of lysozyme alone. Furthermore, significant damage occurred in the presence of EDTA alone. Spheroplast-like cell ghosts were present which surrounded condensed cytoplasm or relatively clear spaces. These findings further support the concept of the requirement for electron microscopy to assess lytic damage in addition to turbidimetric and biochemical methods. Our results are the first to demonstrate the remarkable sensitivity of A. actinomycetemcomitans Y4 to lysozyme and to show that EDTA not only affects outer membrane permeability but effects cell lysis, possibly through activation of autolytic enzymes at the cytoplasmic membrane. The exquisite sensitivity of A. actinomycetemcomitans Y4 to lysis could be an important mechanism by which lysozyme participates in the regulation of this suspected periodontal pathogen. Images PMID:6404833

Iacono, V J; Boldt, P R; MacKay, B J; Cho, M I; Pollock, J J

1983-01-01

240

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

241

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

242

Effects of Purification on the Crystallization of Lysozyme  

NASA Technical Reports Server (NTRS)

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

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

1996-01-01

243

Regulatory and Structural Genes for Lysozymes of Mice  

PubMed Central

The molecular and genetic basis of large differences in the concentration of P lysozyme in the small intestine has been investigated by crossing inbred strains of two species of house mouse (genus Mus). The concentration of P in domesticus is about 130-fold higher than in castaneus . An autosomal genetic element determining the concentration of P has been identified and named the P lysozyme regulator, Lzp-r . The level of P in interspecific hybrids (domesticus x castaneus) as well as in certain classes of backcross progeny is intermediate relative to parental levels, which shows that the two alleles of Lzp-r are inherited additively. There are two forms of P lysozyme in the intestine of the interspecific hybrid—one having the heat stability of domesticus P, the other being more stable and presumably the product of the castaneus P locus. These two forms occur in equal amounts, and it appears that Lzp-r acts in trans. The linkage of Lzp-r to three structural genes (Lzp-s, Lzm-s1, and Lzm-s2), one specifying P lysozyme and two specifying M lysozymes, was shown by electrophoretic analysis of backcrosses involving domesticus and castaneus and also domesticus and spretus . The role of regulatory mutations in evolution is discussed in light of these results. PMID:3569879

Hammer, Michael F.; Wilson, Allan C.

1987-01-01

244

Absence of equilibrium cluster phase in concentrated lysozyme solutions  

PubMed Central

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

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

2008-01-01

245

Surfactant Copolymers Prevent Aggregation of Heat Denatured Lysozyme  

PubMed Central

We investigated the ability of certain triblock copolymer surfactant poloxamers of the form polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO), to prevent formation of stable aggregates of heat denatured hen egg lysozyme. Differential scanning calorimetry (DSC) and synchrotron small angle x-ray scattering (SAXS) experiments were performed to study the thermodynamics and solution structures of lysozyme at temperatures between 20 and 90°C in the presence and absence of poloxamers with various molecular weights (8.4–14.3 kDa), but similar hydrophile/hydrophobe (PEO:PPO) ratio of 80%. Poloxmer 188 was found to be very effective in preventing aggregation of heat denatured lysozyme and those functioned as a synthetic surfactant, thus enabling them to refold when the conditions become optimal. For comparison, we measured the ability of 8 kDa polyethylene glycol (PEG) to prevent lysozyme aggregation under same conditions. The results of these studies suggest that poloxamers are more efficient than PEG in preventing aggregation of heat denaturated lysozyme, To achieve equivalence, more than an order of magnitude higher concentration of PEG concentration was needed. Apparently, the presence of a hydrophobic segment in the poloxamers increases their ability to target the hydrophobic region of the unfolded proteins and protect them from self association. Given their biocompatibility and the low concentrations at which they effectively facilitate refolding of denatured proteins, they may be useful in the treatment of burns and other conditions resulting in the denaturation of proteins. PMID:16786393

Lee, Raphael C.; Despa, Florin; Guo, L.; Betala, Pravin; Kuo, Anne; Thiyagarajan, P.

2007-01-01

246

The EFTTRA-T4 experiment on americium transmutation  

Microsoft Academic Search

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

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

2000-01-01

247

Magnetic Cation Exchange Isolation of Lysozyme from Native Hen Egg White  

Microsoft Academic Search

Summary Two magnetic macroporous cellulose cation exchangers (Iontosorb MG CM 100 and Iontosorb MG SHP 100) were used for one-step isolation of lysozyme from native, undilu- ted hen egg white. Highly purified lysozyme (purity >96 %) with specific activity similar to that of commercial lysozyme preparations was obtained in both cases. Carboxymethyl- -based cation exchanger exhibited substantially higher capacity for

Ivo Safarik; Zdenka Sabatkova; Oldrich Tokar; Mirka Safarikova

2007-01-01

248

Sonochemical synthesis of liquid-encapsulated lysozyme microspheres.  

PubMed

Liquid-encapsulated lysozyme microspheres were successfully synthesized using a sonochemical method. The encapsulation of four different liquids, namely, sunflower oil, tetradecane, dodecane and perfluorohexane on the formation, stability and morphology of the lysozyme microspheres was studied. Among the four different liquids used for encapsulation, perfluorohexane-filled microspheres were found to be most stable in the dried state with a narrow size distribution. In order to explore the possibility of encapsulating biofunctional molecules (e.g., drugs) within these microspheres, liquids containing a fluorescent dye (Nile red) were encapsulated and the ultrasound-induced release of these dye-loaded liquids was studied. The fluorescence data for the liquid-filled lysozyme microspheres demonstrated the potential use of the sonochemical technique for synthesizing these "vehicles" for the encapsulation and the controlled delivery of dyes, flavours, fragrances or drugs. PMID:19850505

Zhou, Meifang; Leong, Thomas Seak Hou; Melino, Sonia; Cavalieri, Francesca; Kentish, Sandra; Ashokkumar, Muthupandian

2010-02-01

249

Analysis of Monomer Aggregation and Crystal Growth Rates of Lysozyme  

NASA Technical Reports Server (NTRS)

This project was originally conceived to analyze the extensive data of tetragonal lysozyme crystal growth rates collected at NASA/MSFC by Dr. Marc L. Pusey's research group. At that time the lack of analysis of the growth rates was hindering progress in understanding the growth mechanism of tetragonal lysozyme and other protein crystals. After the project was initiated our initial analysis revealed unexpected complexities in the growth rate behavior. This resulted in an expansion in the scope of the project to include a comprehensive investigation of the growth mechanisms of tetragonal lysozyme crystals. A discussion of this research is included as well a list of presentations and publications resulting from the research. This project contributed significantly toward the education of several students and fostered extensive collaborations between investigators.

Nadarajah, Arunan

1996-01-01

250

Binding of lysozyme to brush border membranes of rat kidney.  

PubMed

The binding of 125I-labelled egg-white lysozyme to isolated brush border membranes of rat kidney cortex was investigated. The lysozyme binding was reversible and saturable. The Scatchard plot revealed a one-component binding type with a dissociation constant of 7.8 microM and 15.6 nmol/mg membrane protein for the number of binding sites. The binding of the basic lysozyme could be reduced by basic amino acids such as L-lysine, L-ornithine or L-arginine, while neutral amino acids such as L-citrulline or L-alanine had no effect. The inhibitory effect of lysine was competitive. PMID:6871205

Beyer, G; Bode, F; Baumann, K

1983-07-27

251

Detection of bacteria with bioluminescent reporter bacteriophage.  

PubMed

Bacteriophages are viruses that exclusively infect bacteria. They are ideally suited for the development of highly specific diagnostic assay systems. Bioluminescent reporter bacteriophages are designed and constructed by integration of a luciferase gene in the virus genome. Relying on the host specificity of the phage, the system enables rapid, sensitive, and specific detection of bacterial pathogens. A bioluminescent reporter phage assay is superior to any other molecular detection method, because gene expression and light emission are dependent on an active metabolism of the bacterial cell, and only viable cells will yield a signal. In this chapter we introduce the concept of creating reporter phages, discuss their advantages and disadvantages, and illustrate the advances made in developing such systems for different Gram-negative and Gram-positive pathogens. The application of bioluminescent reporter phages for the detection of foodborne pathogens is emphasized. PMID:25084997

Klumpp, Jochen; Loessner, Martin J

2014-01-01

252

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

253

Surface Display of Proteins on Bacteriophage ? Heads  

Microsoft Academic Search

We have developed plasmid and phage vectors for the display of foreign proteins on the surface of bacteriophage ? capsid by modifying theDgene which encodes the major head protein gpD. The vectors have multiple cloning sites, and permit colour selection and conditional chain termination for recombinants. Displayed proteins can be fused to either the N or C terminus of gpD

Y. Gi Mikawa; Ichi N. Maruyama; Sydney Brenner

1996-01-01

254

Single M13 bacteriophage tethering and stretching  

PubMed Central

The ability to present biomolecules on the highly organized structure of M13 filamentous bacteriophage is a unique advantage. Where previously this viral template was shown to direct the orientation and nucleation of nanocrystals and materials, here we apply it in the context of single-molecule (SM) biophysics. Genetically engineered constructs were used to display different reactive species at each of the filament ends and along the major capsid, and the resulting hetero-functional particles were shown to consistently tether microscopic beads in solution. With this system, we report the development of a SM assay based on M13 bacteriophage. We also report the quantitative characterization of the biopolymer's elasticity by using an optical trap with nanometer-scale position resolution. Expanding the fluctuating rod limit of the wormlike chain to incorporate enthalpic polymer stretching yielded a model capable of accurately capturing the full range of extensions. Fits of the force-extension measurements gave a mean persistence length of ?1,265 nm, lending SM support for a shorter filamentous bacteriophage persistence length than previously thought. Furthermore, a predicted stretching modulus roughly two times that of dsDNA, coupled with the system's linkage versatility and load-bearing capability, makes the M13 template an attractive candidate for use in tethered bead architectures. PMID:17360403

Khalil, Ahmad S.; Ferrer, Jorge M.; Brau, Ricardo R.; Kottmann, Stephen T.; Noren, Christopher J.; Lang, Matthew J.; Belcher, Angela M.

2007-01-01

255

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

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

2013-01-01

256

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

257

[Role of thymine dimers in the UV mutagenesis of bacteriophage sd].  

PubMed

The sensitized irradiation (lambda greater than 310 nm) with AcphiM induces plaque mutants of extracellular phage Sd. Thus, TT in phage Sd DNA are both lethal and mutagenic. The yield of plaque mutants is about 3-fold lower as compared with the yield after UV-irradiation (lambda = 254 nm). The theoretical analysis of UV-induced mutagenesis curves of phage Sd was carried out. It is concluded that the following photoproducts make a contribution in the mutagenic effect of near-UV (lambda = 254 nm): 1) lethal pyrimidine dimers; 2) other lethal photolesions; and 3) non-lethal mutagenic photoproducts of cytosine. The cytosine photohydrate contribution nearly twice exceeds the total contribution of lethal photoproducts. It is assumed that bacteriophages T4 and Sd have their own system of repair, like SOS-repair in bacteria. PMID:612486

Esipova, V V; Zavil'gel'ski?, G B; Kriviski?, A S

1977-01-01

258

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

259

Sequences of two highly divergent canine type c lysozymes: implications for the evolutionary origins of the lysozyme/alpha-lactalbumin superfamily.  

PubMed

The amino acid sequences of two canine lysozymes, from milk and spleen, have been elucidated by direct sequence analyses of the purified proteins and fragments generated from them. The two enzymes are highly divergent, differing from each other by 45% in sequence, but each is closely similar to lysozymes previously obtained from other mammalian species. The milk lysozyme is similar in sequence to equine and donkey milk lysozymes (83% identity) and, like these enzymes, contains a bound Ca2+ ion while the spleen enzyme is most similar in sequence to the majority of previously studied mammalian and avian lysozymes (80 to 83% identity) and, based on its sequence, does not contain a Ca(2+)-binding site. This demonstrates that Ca(2+)-binding lysozymes are expressed in at least two mammalian orders, the carnivores and perissodactyls, as well as confirming that the genes for the Ca(2+)-binding and conventional lysozymes are paralogous. The latter point was further confirmed by the isolation and partial sequence analysis of a conventional lysozyme from equine spleen. The relationships of these new lysozyme sequences to those of other lysozymes and their homologues, the alpha-lactalbumins, were analyzed using different molecular phylogeny algorithms, producing a new model for the evolutionary origins of the superfamily. The most significant conclusion to be drawn from this model is that Ca(2+)-binding activity was an ancient feature of this protein superfamily which was lost during the evolutionary development of the conventional lysozymes. It also supports a previous suggestion that the alpha-lactalbumins and lysozymes diverged at a time earlier than the divergence of the fishes and tetrapods. PMID:8080284

Grobler, J A; Rao, K R; Pervaiz, S; Brew, K

1994-09-01

260

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

E-print Network

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

Priscu, John C.

261

2d-LC-MS/MS Method-ORNL Developed for Bacteriophage  

E-print Network

2d-LC-MS/MS Method- ORNL Developed for Bacteriophage This multidimensional chromatography tandem mass spectrometry (2d-LC-MS/MS) method was optimized for bacteriophage by Kristen Corrier undergraduate-10ug) of isolate bacteriophage or mixed bacterial/bacteriophage communities prepared via the FASP

Sullivan, Matthew B.

262

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

263

RESULTS OF IRRADIATING STAPHYLOCOCCUS AUREUS BACTERIOPHAGE WITH MONOCHROMATIC ULTRAVIOLET LIGHT  

Microsoft Academic Search

Ever since d'Herelle's announcement of the discovery of lyric prin- ciples for bacteria now collectively called bacteriophage, interest and debate have centered on the nature of the active agent. The two main points of view and bases of contention relate bacteriophages re- spectively to living organisms (ultramicroscopic viruses parasitic on living bacteria), and to agents of unknown but simpler structure,

FREDERICK L. GATES

264

Complete Genome Sequences of Five Paenibacillus larvae Bacteriophages.  

PubMed

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; Burnett, Sandra H

2013-01-01

265

Potential of Bacteriophage to Prevent and Treat Poultry Diseases  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

266

iBioSeminar: Bacteriophages: Genes and Genomes  

NSDL National Science Digital Library

Bacteriophage, viruses that specifically infect bacteria, are, by far, the majority of all biological entities in the biosphere. The viral population, including bacteriophage, is very diverse yet relatively few viral genomes have been sequenced. In this series of lectures, Hatfull argues that viral genomes provide a great source of new genes, potentially with new functions and structures.

Graham Hatfull (University of Pittsburgh and Howard Hughes Medical Institute; )

2011-06-06

267

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

268

Activation of a Naive T4 Lymphocyte by an Antigen-Presenting Cell and Its Subsequent Proliferation and Differentiation into Effector T4 Lymphocytes  

NSDL National Science Digital Library

Following the activation of a naive T4 lymphocyte by an antigen-presenting cell, that T4 cell subsequently proliferates into a large clone of identical T4 cells and most of these then differentiate into effector T4 lymphocytes.

American Society For Microbiology

2005-07-14

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

Quantitative tear lysozyme assay: a new technique for transporting specimens.  

PubMed Central

We have developed a method for assaying the concentration of tear lysozyme using eluates of tear fluid collected on filter paper discs. Specimens can be stored and transported to remote laboratories for assay. We have shown that the 'indirect' or eluate method gives statistically comparable results to the 'direct' method using fresh, neat tear fluid. Images PMID:7426595

Seal, D V; Mackie, I A; Coakes, R L; Farooqi, B

1980-01-01

271

The effects of bacteriophage and nanoparticles on microbial processes  

NASA Astrophysics Data System (ADS)

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

Moody, Austin L.

272

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

273

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

274

Study of CCD eyepiece on T-4 theodolite  

Microsoft Academic Search

This document describes the effort of the University of Maryland to develop a Charge Coupled Device (CCD) Camera System, with the necessary support hardware and analysis software, to act as an impersonal electronic eyepiece on the T-4 theodolite for astronomical longitude and latitude determinations. This report will describe the concept, the implementation, and the current status of this project. Analysis

D. G. Currie

1982-01-01

275

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

276

Chemical mutations of the catalytic carboxyl groups in lysozyme to the corresponding amides.  

PubMed

In a two-step process, esterification and ammonolysis, Glu-35 and Asp-52 in lysozyme were amidated to glutamine and asparagine residues. Since the side chains of glutamine and asparagine are almost equal in size to those of glutamic acid and aspartic acid, these conversions would provide appropriate derivatives to elucidate the catalytic participations of these residues. The enzymatic activities of the resulting [Gln35]lysozyme and [Asn52]lysozyme were found to be less than 4% of that of native lysozyme in a pH range of 3.4-8.0. As these derivatives were inactive, we could determine the dissociation constants (Ks values) for the binding of beta-1,4-linked n-mer, a hexasaccharide of N-acetyl-D-glucosamine, to [Gln35]lysozyme and [Asn52] lysozyme. The values of Ks at pH 5.5 and 40 degrees C were 1.6 X 10(-5) M for [Gln35]lysozyme and 2.7 X 10(-5) M for [Asn52]lysozyme. These values are similar to that for native lysozyme. The results are direct proof for the involvements of Glu35 and Asp52 in the catalytic action of lysozyme. A method for ammonolysis of ester groups in proteins in liquid ammonia is described and will be useful for amidation of carboxyl groups of proteins. PMID:3759981

Kuroki, R; Yamada, H; Moriyama, T; Imoto, T

1986-10-15

277

Bacteriophage lambda display systems: developments and applications.  

PubMed

Bacteriophage (phage) Lambda (?) has played a key historic role in driving our understanding of molecular genetics. The lytic nature of ? and the conformation of its major capsid protein gpD in capsid assembly offer several advantages as a phage display candidate. The unique formation of the ? capsid and the potential to exploit gpD in the design of controlled phage decoration will benefit future applications of ? display where steric hindrance and avidity are of great concern. Here, we review the recent developments in phage display technologies with phage ? and explore some key applications of this technology including vaccine delivery, gene transfer, bio-detection, and bio-control. PMID:24442507

Nicastro, Jessica; Sheldon, Katlyn; Slavcev, Roderick A

2014-04-01

278

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

279

Non-covalent conjugation of CdTe QDs with lysozyme binding DNA for fluorescent sensing of lysozyme in complex biological sample.  

PubMed

Water-soluble cysteamine (CA) capped CdTe quantum dots (QDs) conjugated with lysozyme binding DNA (LBD) was constructed for luminescent sensing of lysozyme by forming a ternary self-assembly complex. Addition of negatively charged lysozyme binding DNA to the positively charged CA capped CdTe QDs buffer solution (Tris-HCl pH 7.4) could lead to the formation of QDs-LBD complex through electrostatic interactions. Once lysozyme was introduced into the CdTe QDs-LBD system, it could bind specifically with the QDs-LBD complex, resulting in fluorescence emission enhancement of the QDs due to the surface inert of QDs. At a given amount of LBD and CdTe QDs (LBD: QDs=2: 1), the fluorescence intensity enhancement of QDs was linear with lysozyme concentration over the range of 8.9-71.2 nM, with a detection limit of 4.3 nM. Due to the specific binding of LBD with lysozyme, this approach displayed high selectivity for lysozyme recognition. The sensing mechanism was confirmed by DLS and zeta potential measurement, and agarose gel electrophoresis experiment. Furthermore, the proposed CA-capped CdTe QDs-LBD sensor was applied to lysozyme detection in mouse serum and human morning urine samples, which showed high sensitivity and selectivity in the complex biological sample. PMID:25127568

Li, Shujia; Gao, Zhidan; Shao, Na

2014-11-01

280

T4 genes in the marine ecosystem: studies of the T4-like cyanophages and their role in marine ecology  

Microsoft Academic Search

From genomic sequencing it has become apparent that the marine cyanomyoviruses capable of infecting strains of unicellular cyanobacteria assigned to the genera Synechococcus and Prochlorococcus are not only morphologically similar to T4, but are also genetically related, typically sharing some 40-48 genes. The large majority of these common genes are the same in all marine cyanomyoviruses so far characterized. Given

Martha RJ Clokie; Andrew D Millard; Nicholas H Mann

2010-01-01

281

Bacteriophage recombination systems and biotechnical applications.  

PubMed

Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, ?, and N15, the integrase from the Streptomyces phage, ?C31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

Nafissi, Nafiseh; Slavcev, Roderick

2014-04-01

282

Making temporal maps using bacterial luciferase: Bacteriophage  

NASA Astrophysics Data System (ADS)

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

Kuhn, Jonathan; Broza, Rachel; Verkin, Ekaterina

2004-06-01

283

Understanding Bacteriophage Specificity in Natural Microbial Communities  

PubMed Central

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

Koskella, Britt; Meaden, Sean

2013-01-01

284

Recognition of bacterial lipopolysaccharide using bacteriophage-adhesin-coated long-period gratings.  

PubMed

In this paper we present a new type of highly sensitive label-free sensor based on long-period gratings (LPG) coated with T4 bacteriophage (phage) adhesin. The adhesin (gp37) binds Escherichia coli B (E. coli B) by recognizing its bacterial lipopolysaccharide (LPS). The LPG biofunctionalization methodology is based on coating LPG surface with nickel ions capable of gp37 histidine tag reversible binding. For the first time recombinant adhesive phage protein has been used as a receptor molecule in biosensing scheme. The specificity of LPS binding by adhesin has been tested with LPG-based device and confirmed using Western blot, Enzyme-Linked Immunosorbent Assay (ELISA) and BIACORE methods. The LPG-based sensor can measure bacterial contamination in real time and with a high accuracy. We show that T4 phage adhesin binds E. coli B LPS in its native or denatured form. The binding is highly specific and irreversible. The applied procedure allows for obtaining reusable biosensors. PMID:25067838

Brzozowska, Ewa; ?mietana, Mateusz; Koba, Marcin; Górska, Sabina; Pawlik, Krzysztof; Gamian, Andrzej; Bock, Wojtek J

2015-05-15

285

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

PubMed

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

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

2008-01-01

286

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

287

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

288

Mouse lysozyme M gene: isolation, characterization, and expression studies.  

PubMed Central

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 tissue specificities of expression. The M gene is expressed weakly in myeloblasts, moderately in immature macrophages, and strongly in both mature macrophages and macrophage-rich tissues, while high levels of P transcripts are present only in small intestine. Sites of protein accumulation, rather than gene expression, have been identified by comparative quantitation of mRNA and enzyme levels. Images PMID:3413093

Cross, M; Mangelsdorf, I; Wedel, A; Renkawitz, R

1988-01-01

289

Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles  

Microsoft Academic Search

Functionalized Fe3O4 nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl

Jun Sun; Yujie Su; Shengqi Rao; Yanjun Yang

2011-01-01

290

Substrate stabilization of lysozyme to thermal and guanidine hydrochloride denaturation  

E-print Network

SUBSTRATE STABILIZATION OF I YSOZYNE TO THERNAL AND GUANIDINE HYDROCHLORIDE DENATURATION A Thesis Timothy NcGrath Submitted to the Graduate College of Texas ABH University in partial fulf illment of the requirement for the degree of NASTER... OF SCIENCE August %977 Na jor Sub j ect: Biochemistry SUBSTRATE STABILE IZATION OF LYSOZYME TO THERMAL AND GUAM IDINE HYDROCHLORIDE DENATURATION A Thesis Timothy NcGrath Approved as to style and content by: Chairman of Commi t tee (Head of De artment...

McGrath, Timothy

1977-01-01

291

An integrated process for purification of lysozyme, ovalbumin, and ovomucoid from hen egg white  

Microsoft Academic Search

This article describes an integrated process for simultaneous purification of lysozyme, ovalbumin, and ovomucoid from hen\\u000a egg white. The crude egg white extract was passed through a cation exchanger Streamline™ SP and the bound lysozyme was eluted with 5% ammonium carbonate, pH 9.0, containing 1 M NaCl after elution of avidin. This partially purified lysozyme was further purified 639-fold on

Ipsita Roy; M. V. S. Rao; Munishwar N. Gupta

2003-01-01

292

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

PubMed Central

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

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

2013-01-01

293

Aggregation of silica nanoparticles directed by adsorption of lysozyme.  

PubMed

The interaction of the globular protein lysozyme with silica nanoparticles of diameter 20 nm was studied in a pH range between the isoelectric points (IEPs) of silica and the protein (pH 3-11). The adsorption affinity and capacity of lysozyme on the silica particles is increasing progressively with pH, and the adsorbed protein induces bridging aggregation of the silica particles. Structural properties of the aggregates were studied as a function of pH at a fixed protein-to-silica concentration ratio which corresponds to a surface concentration of protein well below a complete monolayer in the complete-binding regime at pH > 6. Sedimentation studies indicate the presence of compact aggregates at pH 4-6 and a loose flocculated network at pH 7-9, followed by a sharp decrease of aggregate size near the IEP of lysozyme. The structure of the bridged silica aggregates was studied by cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering. The structure factor S(q) derived from the scattering profiles displays characteristic features of particles interacting by a short-range attractive potential and can be represented by the square-well Percus-Yevick potential model, with a potential depth not exceeding 3k(B)T. PMID:21728288

Bharti, Bhuvnesh; Meissner, Jens; Findenegg, Gerhard H

2011-08-16

294

Design and structural analysis of an engineered thermostable chicken lysozyme.  

PubMed Central

A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (delta Tm approximately +10.5 degrees C) and chemical (delta Cm for guanidine hydrochloride denaturation = +1.3 M) stabilities relative to wild-type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 degrees C) to 4.0 (differential scanning calorimetry, 74 degrees C) kcal mol-1 greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came from diverse sources: (1) The I55L/S91T core construct with delta Tm = 3.3 degrees C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062). (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 31 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The D101S variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive. PMID:8535242

Shih, P.; Kirsch, J. F.

1995-01-01

295

Modeling the electrophoresis of lysozyme. II. Inclusion of ion relaxation.  

PubMed Central

In this work, boundary element methods are used to model the electrophoretic mobility of lysozyme over the pH range 2-6. The model treats the protein as a rigid body of arbitrary shape and charge distribution derived from the crystal structure. Extending earlier studies, the present work treats the equilibrium electrostatic potential at the level of the full Poisson-Boltzmann (PB) equation and accounts for ion relaxation. This is achieved by solving simultaneously the Poisson, ion transport, and Navier-Stokes equations by an iterative boundary element procedure. Treating the equilibrium electrostatics at the level of the full rather than the linear PB equation, but leaving relaxation out, does improve agreement between experimental and simulated mobilities, including ion relaxation improves it even more. The effects of nonlinear electrostatics and ion relaxation are greatest at low pH, where the net charge on lysozyme is greatest. In the absence of relaxation, a linear dependence of mobility and average polyion surface potential, (lambda zero)s, is observed, and the mobility is well described by the equation [formula: see text] where epsilon 0 is the dielectric constant of the solvent, and eta is the solvent viscosity. This breaks down, however, when ion relaxation is included and the mobility is less than predicted by the above equation. Whether or not ion relaxation is included, the mobility is found to be fairly insensitive to the charge distribution within the lysozyme model or the internal dielectric constant. PMID:9199778

Allison, S A; Potter, M; McCammon, J A

1997-01-01

296

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

297

Genetic Evolution of Bacteriophage, I. Hybrids between Unrelated Bacteriophages P22 and Fels 2  

Microsoft Academic Search

A new bacteriophage species, designated F22, was isolated from phage P22 stocks grown on Salmonella typhimurium Q1 lysogenic for Fels 2 at a frequency of less than 10-11. P22 has a very short tail with a hexagonal base plate and six spikes. Phage Fels 2 is morphologically similar to E. coli T-even phages, having a long tail with a contractile

Nobuto Yamamoto

1969-01-01

298

[Highly active fractions of the medicinal leech recombinant destabilase-lysozyme].  

PubMed

From the highly purified but lowly active recombinant protein Destabilas-Lysozyme (Dest-Lys) by use cation-exchange column TSK CM 3-SW chromatography, it was separated non-active fraction IV, contained 90% of protein. Fractions I, II and III, represented proteins with lysozyme and isopeptidase activities. Their lysozyme activity correlates with the activity of natural Des-Lys. The ratio of the activities in fractions I - III is such, that maximal lysozyme activity is concentrated in fraction III, isopeptidase - in fraction I. It is discussed the possibility of Dest-Lys different functions regulation is depended on the formation of protein complex forms. PMID:25019395

Fadeeva, Iu I; Antipova, N V; Baskova, I P; Zavalova, L L

2014-01-01

299

Engineering Escherichia coli for Soluble Expression and Single Step Purification of Active Human Lysozyme  

PubMed Central

Genetically engineered variants of human lysozyme represent promising leads in the battle against drug-resistant bacterial pathogens, but early stage development and testing of novel lysozyme variants is constrained by the lack of a robust, scalable and facile expression system. While wild type human lysozyme is reportedly produced at 50 – 80 kg per hectare of land in recombinant rice, this plant-based system is not readily scaled down to bench top production, and it is therefore not suitable for development and characterization of novel lysozyme variants. Here, we describe a novel and efficient expression system capable of producing folded, soluble and functional human lysozyme in E. coli cells. To achieve this goal, we simultaneously co-express multiple protein folding chaperones as well as harness the lysozyme inhibitory protein, Ivy. Our strategy exploits E. coli’s ease of culture, short doubling time, and facile genetics to yield upwards of 30 mg/L of soluble lysozyme in a bioreactor system, a 3000-fold improvement over prior efforts in E. coli. Additionally, molecular interactions between lysozyme and a his-tagged Ivy allows for one-step purification by IMAC chromatography, yielding as much as 21 mg/L of purified enzyme. We anticipate that our expression and purification platform will facilitate further development of engineered lysozymes having utility in disease treatment and other practical applications. PMID:23220215

Lamppa, John W.; Tanyos, Sam A.; Griswold, Karl E.

2012-01-01

300

Organization and dynamics of the bacteriophage Mu transpososome  

E-print Network

Transposition of a linear mobile genetic element, such as the bacteriophage Mu, requires a set of spatially and temporally coordinated DNA phosphoryl transfer reactions to move the element from an existing DNA location to ...

Williams, Tanya L. (Tanya Lynn), 1970-

2002-01-01

301

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.

302

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

SciTech Connect

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

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

2006-01-01

303

Lytic bacteriophages reduce Escherichia coli O157  

PubMed Central

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

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

2013-01-01

304

Triclosan-lysozyme complex as novel antimicrobial macromolecule: a new potential of lysozyme as phenolic drug-targeting molecule.  

PubMed

A novel anti-infection strategy to alleviate antibiotic-resistance problem and non-specific toxicity associated with chemotherapy is explored in this study. It is based on utilizing a bacteriolytic enzyme (lysozyme) as a carrier to allow specific targeting of a potential phenolic antimicrobial drug (triclosan) to microbial cells. Lysozyme (LZ) was complexed, via electrostatic and hydrophobic condensation at alkaline pH, to various degrees with triclosan (TCS), a negatively charged phenolic antimicrobial that inhibits bacterial fatty acid synthesis. Fluorescence and absorbance spectra analysis revealed non-covalent association of TCS with the aromatic residues at the interior of LZ molecule. The conjugation greatly promoted the lytic activity of LZ as the degree of TCS derivatization increased. The complexation with LZ turned TCS into completely soluble in aqueous solution. TCS-LZ complexes showed significantly enhanced bactericidal activity against several strains of Gram-positive and Gram-negative bacteria compared to the activity of TCS or LZ alone when tested at the same molar basis. Strikingly, TCS-LZ complex, but not LZ or TCS alone, exhibited unique specificity to scavenge superoxide radicals, generated by the natural xanthine/xanthine oxidase coupling system, without affecting the catalytic function of oxidase. This finding is the first to describe that the membrane disrupting function of lysozyme can be utilized to specifically target antimicrobial drug(s) to pathogen cells and heralding a fascinating opportunity for the potential candidacy of TCS-LZ as novel antimicrobial strategy for human therapy. PMID:18439671

Hoq, Md Imranul; Mitsuno, Koji; Tsujino, Yoshio; Aoki, Takayoshi; Ibrahim, Hisham R

2008-06-01

305

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage.  

PubMed

Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene. PMID:14651626

Pullinger, Gillian D; Bevir, Thomas; Lax, Alistair J

2004-01-01

306

Characterization of streptococcal bacteriophage c6A.  

PubMed

Bacteriophage c6A is a lytic phage that infects strains of Streptococcus lactis. Infection of S. lactis C6 under standard conditions yielded 124 +/- 8 p.f.u. per infected cell after a latent period of 25 min at 30 degrees C. The virion of c6A was shown to contain at least 12 polypeptides and a 21.9 kilobase double-stranded, linear DNA genome with complementary 5'-protruding single-stranded termini. The (G + C) content of this DNA was estimated to be 36.7%. A restriction map was constructed which indicates that a number of restriction endonucleases did not digest the DNA and that others cleaved with a much lower frequency than expected. PMID:2999317

Powell, I B; Davidson, B E

1985-12-01

307

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

308

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

309

Bacteriophages of Leuconostoc, Oenococcus, and Weissella  

PubMed Central

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

310

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 = (4 , 4) superconformal algebra. The resulting symmetry groups are isomorphic to certain subgroups of the Weyl group of E 8, 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 , ?). 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

311

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

312

FRAP-T4 best estimate sensitivity study. [PWR; BWR  

SciTech Connect

Sensitivity studies of five postulated reactor transient and accident events were performed with the FRAP-T4 transient fuel behavior code. The purpose of the study was to determine the influence of variation in selected input parameters on fuel performance characteristics. The range of the parameters perturbed during the study were supplied by the major US reactor vendors. The five different events examined in the analysis were: (1) locked rotor, (2) control element ejection, (3) steam line break, (4) loss of flow, and (5) turbine trip without bypass.

Hansen, B.L.

1980-02-01

313

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

E-print Network

Temperature Affects the Tripartite Interactions between Bacteriophage WO, Wolbachia that temperatures at the extreme edges of an insect's habitable range alter bacteriophage WO inducibility cases where Wolbachia densities decline due to temperature changes, phage WO densities increase

Bordenstein, Seth

314

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

Federal Register 2010, 2011, 2012, 2013, 2014

...Refs. 1 through 8). Bacteriophage are obligate parasites of bacteria, which means they attach to, infect, and reproduce in bacteria, and are host-specific for bacteria, with specific bacteriophage attacking only one bacterial...

2011-10-26

315

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

E-print Network

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

Levitt, Michael

316

ASSESSING THE MICROBIAL COMMUNITY DYNAMICS AND THE ROLE OF BACTERIOPHAGES IN BACTERIAL  

E-print Network

ASSESSING THE MICROBIAL COMMUNITY DYNAMICS AND THE ROLE OF BACTERIOPHAGES IN BACTERIAL MORTALITY IN LAKE GENEVA Dynamique des communautés microbiennes et rôle fonctionnel des virus bacteriophages du lac

Jacquet, Stéphan

317

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

318

Effect of combined lysozyme and lipase treatment on the survival of Listeria monocytogenes  

Microsoft Academic Search

The effectiveness of polyphosphates or lipases to increase the lytic activity of lysozyme was evaluated both on Listeria monocytogenes suspended in buffer and on growing cultures incubated at different temperatures. At 5 °C and 37 °C polyphosphates combined with lysozyme did not result in the decrease of the number of non-growing L. monocytogenes cells. At the same incubation conditions, the

Rosella Liberti; Giovanna Franciosa; Monica Gianfranceschi; Paolo Aureli

1996-01-01

319

Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin  

Microsoft Academic Search

A microtiter plate assay was employed to systematically assess the interaction between ethylenediaminetetraacetic acid (EDTA) or lactoferrin and nisin, lysozyme, or monolaurin against strains of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Pseudomonas fluorescens. Low levels of EDTA acted synergistically with nisin and lysozyme against L. monocytogenes but EDTA and monolaurin interacted additively against this microorganism. EDTA synergistically enhanced the

Jill K. Branen; P. Michael Davidson

2004-01-01

320

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

ERIC Educational Resources Information Center

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

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

2010-01-01

321

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

322

Three in one: Identification, expression and enzymatic activity of lysozymes in amphioxus.  

PubMed

The lysozymes identified so far in animals belong to the g-type, c-type, and i-type. Vertebrate animals possess only the former two types, i.e., g- and c-types, while all the three types have been reported in invertebrates. Here we demonstrate that (1) three cDNAs that encode g-, c-, and i-type lysozymes, respectively, were identified in a single species of the amphioxus Branchiostoma japonicum; (2) all the 3-type genes displayed distinct tissue-specific expression pattern; (3) recombinant g-, c-, and i-type lysozymes all exhibited enzymatic activities; and (4) native g-, c-, and i-type lysozymes were identified in the different tissues of amphioxus. Collectively, these results suggest the presence of all the 3-type lysozymes in a single animal species, first such data ever reported. The presence of biologically active i-type lysozyme in amphioxus also suggests that i-type lysozyme gene is retained at least in Protochordata, contrasting to the previous proposal that i-type lysozyme gene has been lost in a common ancestor of all chordates. PMID:24968076

Xu, Na; Pan, Junli; Liu, Shousheng; Xue, Qinggang; Zhang, Shicui

2014-10-01

323

[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

324

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

325

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

326

Bacteriophage cocktail for biocontrol of salmonella in dried pet food.  

PubMed

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

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

2015-01-01

327

Structural Basis of Protein Oxidation Resistance: A Lysozyme Study  

PubMed Central

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

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

2014-01-01

328

Identification and cloning of an invertebrate-type lysozyme from Eisenia andrei.  

PubMed

Lysozyme is a widely distributed antimicrobial protein having specificity for cleaving the beta-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (GlcNAc) of peptidoglycan of the bacterial cell walls and thus efficiently contributes to protection against infections caused mainly by Gram-positive bacteria. In the present study, we assembled a full-length cDNA of a novel invertebrate-type lysozyme from Eisenia andrei earthworm (EALys) by RT-PCR and RACE system. The primary structure of EALys shares high homology with other invertebrate lysozymes; however the highest, 72% identity, was shown for the destabilase I isolated from medicinal leech. Recombinant EALys expressed in Escherichia coli exhibited the lysozyme and isopeptidase activity. Moreover, real-time PCR revealed increased levels of lysozyme mRNA in coelomocytes of E. andrei after the challenge with both Gram-positive and Gram-negative bacteria. PMID:19454335

Josková, Radka; Silerová, Marcela; Procházková, Petra; Bilej, Martin

2009-08-01

329

Genetic analysis of a switch in cell specificity of P lysozyme expression in molossinus mice.  

PubMed

An electrophoretic survey of concentrations of lysozymes M and P was carried out with seven species in the house mouse group (spretus, hortulanus, abbotti, musculus, castaneus, domesticus and molossinus). In most species M is the predominant lysozyme in all tissues tested, except the small intestine, where P predominates if present. In inbred strains of molossinus mice P is more abundant than M in all tissues tested. The phenotypes of high expression of P lysozyme and low expression of M lysozyme in peritoneal and alveolar macrophages were examined genetically. Results of interspecific crosses and backcrosses to domesticus mice support the model that the phenotypes are caused by mutation(s) tightly linked to the lysozyme locus. Alleles at the regulatory loci show additive inheritance. PMID:1765259

Cortopassi, G A; Wilson, A C

1991-10-01

330

Study on the interaction mechanism of lysozyme and bromophenol blue by fluorescence spectroscopy.  

PubMed

The interaction of lysozyme with bromophenol blue (BPB) in acetate buffer (pH 6.0) was studied by fluorescence quenching method for the first time. It was found that BPB could conspicuously quench the fluorescence of lysozyme by the static quenching process, possibly due to the binding on the active site near Trp62. The binding parameters including the binding constant and the number of binding site were calculated. The thermodynamic parameters DeltaH degrees, DeltaS degrees and DeltaG degrees at different temperatures were obtained. The formation of lysozyme-BPB complex depended on the cooperation of the hydrophobic and electrostatic forces. And the binding average distance between lysozyme and BPB was determined. The effect of common metal ions on the binding constant of lysozyme-BPB was also examined. PMID:17682927

Yue, Qiaoli; Niu, Lichuan; Li, Xin; Shao, Xiaodong; Xie, Xiaofeng; Song, Zhenghua

2008-01-01

331

Three distinct epitopes within the loop region of hen egg lysozyme defined with monoclonal antibodies.  

PubMed Central

Five monoclonal antibodies specific for the loop region of hen egg lysozyme were prepared by immunisation with a synthetic conjugate of a proteolytic fragment of lysozyme coupled to bovine serum albumin. Their fine specificities were investigated using a panel of variant lysozymes and peptide fragments of lysozyme in a quantitative radio-immunoassay procedure. Knowledge of the structure of hen lysozyme to high resolution and the use of computer graphics enables the localisation of the epitopes recognised by the antibodies with some precision. The antibodies were shown to define three distinct, overlapping epitopes within what was previously considered to be a single antigenic site. These results are discussed in relation to current ideas of the antigenic nature of proteins and other recent studies in which anti-protein antibodies have been elicited by immunisation with small peptides. Images Fig. 6. PMID:2410255

Darsley, M J; Rees, A R

1985-01-01

332

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

333

Effect of Ultraviolet Irradiation on Bacteriophage Active against Streptococcus Lactis1  

Microsoft Academic Search

Various methods have been advocated for decreasing the incidence of slow acid production due to bacteriophage action during the manufacture of cheese. The control measures have included protection of the mother culture, bulk culture and cheese milk from bacteriophage, and also meth- ods for the destruction of bacteriophage within the cheese plant. Chlori- nation and irradiation with ultraviolet light commonly

G. I. GREENEu; F. J. Babel

1948-01-01

334

Evaluating Bacteriophage P22 as a Tracer in a Complex Surface Water  

E-print Network

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

335

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

E-print Network

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

Bohannan, Brendan

336

A STOCHASTIC MODEL FOR BACTERIOPHAGE THERAPIES X. BARDINA, D. BASCOMPTE, C. ROVIRA, AND S. TINDEL  

E-print Network

A STOCHASTIC MODEL FOR BACTERIOPHAGE THERAPIES X. BARDINA, D. BASCOMPTE, C. ROVIRA, AND S. TINDEL Abstract. In this article, we analyze a system modeling bacteriophage treatments for infections in a noisy. Introduction In the last years Bacteriophage therapies are attracting the attention of several sci- entific

Tindel, Samy - Institut de Mathématiques �lie Cartan, Université Henri Poincaré

337

MODELING AND ANALYSIS OF A MARINE BACTERIOPHAGE INFECTION WITH LATENCY PERIOD  

E-print Network

MODELING AND ANALYSIS OF A MARINE BACTERIOPHAGE INFECTION WITH LATENCY PERIOD EDOARDO BERETTA-965-8119 Abstract. A mathematicalmodel forthe marine bacteriophage infection with explicit latency period: August 4, 2000. Key words and phrases. marine bacteriophage infection, time delay, Liapunov functional

Kuang, Yang

338

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

E-print Network

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

Dobson, Stephen L.

339

Two-site kinetic modeling of bacteriophages transport through columns of saturated dune sand  

E-print Network

Two-site kinetic modeling of bacteriophages transport through columns of saturated dune sand Jack F in the simulation of breakthrough curves from a recent field study on the removal of bacteriophages MS2 and PRD1 of this issue are presented. Breakthrough curves of salt and bacteriophages MS2, PRD1, and fX174 in 1D column

Hassanizadeh, S. Majid

340

Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus  

E-print Network

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

Ely, Bert

341

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

E-print Network

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

Harrison, Stephen C.

342

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

E-print Network

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

Phillips, Rob

343

Modeling removal of bacteriophages MS2 and PRD1 by dune recharge at Castricum, Netherlands  

E-print Network

Modeling removal of bacteriophages MS2 and PRD1 by dune recharge at Castricum, Netherlands Jack F was dosed with bacteriophages MS2 and PRD1 for 11 days at a constant concentration in a 10- by 15-m attachment as well as first-order inactivation was employed to simulate the bacteriophage breakthrough curves

Hassanizadeh, S. Majid

344

Water Research 37 (2003) 21862194 Bacteriophages and clostridium spores as indicator organisms  

E-print Network

Water Research 37 (2003) 2186­2194 Bacteriophages and clostridium spores as indicator organisms production, bacteriophage MS2 was shown to be removed 8 log10 by passage through the dune sand. The question of Cryptosporidium. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bacteriophage; MS2; Poliovirus

Hassanizadeh, S. Majid

345

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

E-print Network

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

Ryan, Joe

346

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

E-print Network

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

Hillis, David

347

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

E-print Network

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

348

Column experiments to study nonlinear removal of bacteriophages by passage through  

E-print Network

Column experiments to study nonlinear removal of bacteriophages by passage through saturated dune 2002; accepted 12 April 2002 Abstract In a recent field study on dune recharge, bacteriophages MS2. Bacteriophage fX174 was included for comparison. The high initial removal in the field was found not to be due

Hassanizadeh, S. Majid

349

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

E-print Network

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

350

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

E-print Network

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

Turner, Paul

351

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

E-print Network

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

Augustine, Mathew P.

352

A DELAY REACTION-DIFFUSION MODEL OF THE SPREAD OF BACTERIOPHAGE INFECTION  

E-print Network

A DELAY REACTION-DIFFUSION MODEL OF THE SPREAD OF BACTERIOPHAGE INFECTION STEPHEN A. GOURLEY modeling, the dynamics of marine bacteriophage infections. Previous authors have proposed systems0036139903436613 1. Introduction. It is known that bacteriophage infection can be a signifi- cant mechanism

Kuang, Yang

353

Filter-Based Assay for Escherichia coli in Aqueous Samples Using Bacteriophage-Based Amplification  

E-print Network

Filter-Based Assay for Escherichia coli in Aqueous Samples Using Bacteriophage-Based Amplification on a syringe filter, and the infection of targeted bacterial species with a bacteriophage (phage). The use. This method could potentially be extended to detect many different bacteria with bacteriophages that deliver

Weitz, David

354

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...

355

A comparative assessment of bacteriophages as tracers and models for virus removal waste stabilisation ponds  

Microsoft Academic Search

The suitability of five strains of bacteriophage as hydraulic tracers and as models of pathogenic virus removal in waste stabilisation ponds was determined in vitro. The bacteriophages investigated included Serratia marcescens, Pseudomonas syringae, Erwinia amanas, and two phages of Erwinia amylovora. The study modeled the survival of these bacteriophages under the physico-chemical conditions typically encountered in WSP systems. This demonstrated

C. A. Vorkas; B. J. Lloyd

356

Towards lysozyme nanotube and 3D hybrid self-assembly.  

PubMed

We report lysozyme self-assembly into nanotubes, under the effect of hydrolysis at pH 2 and 90 °C. We resolve the final steps of the fibrillation pathway, entailing the closure of multi-stranded helical ribbons into nanotubes, and we provide evidence of ?-sheet arrangement within the nanotubes, demonstrating amyloid-like aggregation. Addition of chloroauric acid to the self-assembled structures can lead to generation of either gold single crystal nanoplatelets or gold nanoparticles (when a reducing agent is added) decorating the nanotube and ribbon surfaces. The crystal-based organic-inorganic hybrids further assemble into 3D "sandwiched" structures. PMID:23824259

Lara, Cecile; Handschin, Stephan; Mezzenga, Raffaele

2013-08-21

357

Control of solvent evaporation in hen egg white lysozyme crystallization  

NASA Technical Reports Server (NTRS)

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

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

1992-01-01

358

Control of solvent evaporation in hen egg white lysozyme crystallization  

NASA Astrophysics Data System (ADS)

An investigation of the role of solvent evaporation in tetragonal lysozyme crystallization was preformed with a device that employs N 2(g) to control the evaporation of solvent from a micro-volume crystallization hanging drop. The number of crystals was found to vary with the rate at which the final supersaturation level was achieved. It was found that the more rapid the approach to supersaturation the larger the number of crystals. Accordingly, the crystals reached a smaller terminal size. Elongation of the (110) face parallel to the four-fold axis was observed with the slower evaporation rates.

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

1992-02-01

359

Polar solvation dynamics of lysozyme from molecular dynamics studies  

NASA Astrophysics Data System (ADS)

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

Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy

2012-05-01

360

The effect of mineral substrates on the crystallization of lysozyme  

NASA Astrophysics Data System (ADS)

The effects of exogenous mineral substrates on the induction time for nucleation, and on the number, morphology and purity of protein crystals were investigated in a series of experiments using chicken egg-white lysozyme (CEWL) as a model protein. CEWL was crystallized using the vapor-diffusion technique in the absence of substrates (control) and in the presence of mineral substrates exhibiting various degrees of crystalline lattice match to CEWL (experiments). Results indicate that mineral substrates with a close lattice match to CEWL had a greater influence on the induction time for nucleation and crystal properties.

Kimble, W. L.; Paxton, T. E.; Rousseau, R. W.; Sambanis, A.

1998-05-01

361

The Latex of Hevea brasiliensis Contains High Levels of Both Chitinases and Chitinases/Lysozymes 1  

PubMed Central

The latex of the commercial rubber tree, Hevea brasiliensis, was fractionated by ultracentrifugation as described by G. F. J. Moir ([1959] Nature 184: 1626-1628) into a top layer of rubber particles, a cleared cytoplasm, and a pellet that contains primarily specialized vacuoles known as lutoids. The proteins in each fraction were resolved by two-dimensional gel electrophoresis. Both the pellet fraction and cleared cytoplasm contained large amounts of relatively few proteins, suggesting that laticifers serve a very specialized function in the plant. More than 75% of the total soluble protein in latex was found in the pellet fraction. Twenty-five percent of the protein in the pellet was identified as chitinases/lysozymes, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of bacterial cell walls. Both the chitinase and lysozyme activities were localized exclusively in the pellet or lutoid fraction. The chitinases/lysozymes were resolved into acidic and basic classes of proteins and further purified. An acidic protein (molecular mass 25.5 kD) represented 20% of the chitinase activity in latex; this protein lacked the low level of lysozyme activity that is associated with many plant chitinases. Six basic proteins, having both chitinase and lysozyme activities in various ratios and molecular mass of 27.5 or 26 kD, were resolved. Two of the basic proteins had very high lysozyme specific activities which were comparable to the specific activities reported for animal lysozymes. Like animal lysozymes, but unlike previously characterized plant chitinases/lysozymes, these basic chitinases/lysozymes were also capable of completely lysing or clearing suspensions of bacterial cell walls. These results suggest that laticifers may serve a defensive role in the plant. Images Figure 2 Figure 5 PMID:16668007

Martin, Melinda N.

1991-01-01

362

A three-disulphide derivative of hen lysozyme. Structure, dynamics and stability.  

PubMed Central

A three-disulphide derivative of hen egg-white lysozyme was made by selective reduction and carboxymethylation of one of the four original disulphide bridges. N-Terminal sequencing and two-dimensional 1H-n.m.r. spectroscopy revealed that the disulphide bridge linking cysteine residues 6 and 127 had been modified and that the three remaining disulphide bonds were native-like in nature. Analysis of COSY and NOESY spectra indicated that the three-disulphide lysozyme (CM6.127-lysozyme retains the same secondary and tertiary structure as its four-disulphide counterpart; its stability to pH and temperature is, however, dramatically decreased. N.m.r. spectroscopy was used to characterize the thermal folding and unfolding transition of CM6.127-lysozyme. Not only is the transition still a highly co-operative event, but the enthalpy change associated with folding and unfolding resembles that of intact lysozyme when their differences in thermal stability are taken into consideration. The significance of these results in terms of the folding process of lysozyme is discussed. By contrast with authentic lysozyme, CM6.127-lysozyme was found to exist in an unfolded state at pH 2 at room temperature. N.m.r. spectroscopy and c.d. were used to characterize this state. Unlike their homologous relative, alpha-lactalbumin, which exists in a partially folded molten globule state under these conditions, only residual non-native-like structure persists in the acid-unfolded state of CM6.127-lysozyme. These results indicate that the difference in folding behaviour of lysozyme and alpha-lactalbumin cannot be accounted for simply by their differences in thermal stability. Images Fig. 1. PMID:1989584

Radford, S E; Woolfson, D N; Martin, S R; Lowe, G; Dobson, C M

1991-01-01

363

Kinetics of lysozyme activity recovered from conventional and silicone hydrogel contact lens materials.  

PubMed

We determined the activity of lysozyme recovered from various conventional and silicone hydrogel (SH) contact lens materials as a function of time, using an in vitro model. Polymacon, omafilcon, etafilcon, vifilcon, lotrafilcon A, lotrafilcon B, balafilcon A, galyfilcon A and senofilcon A contact lenses (n = 5) were incubated in lysozyme solution for time periods ranging from 1 h to 28 days. Following the specified incubation period, the lysozyme deposited on the lenses was extracted and the sample extracts were assessed for lysozyme activity and total lysozyme. We found no significant difference (NSD) between omafilcon and polymacon lens materials for the initial 3 days (P > 0.05); however, there was a significant difference between the two lenses from 5 to 28 days (P < 0.05). There was NSD (P > 0.05) between etafilcon and vifilcon lens materials at all time-points and significant differences were seen between various SH lens materials at different time points. After 28 days, lysozyme deposited on etafilcon (90 +/- 3%) and vifilcon (91.4 +/- 3%) exhibited the greatest activity. Lysozyme deposited on polymacon (17.8 +/- 4%), lotrafilcon A (23.4 +/- 4%) and lotrafilcon B (24 +/- 5%) exhibited the lowest activity. Lysozyme deposited on omafilcon, galyfilcon, senofilcon and balafilcon exhibited 38 +/- 3%, 62.3 +/- 8%, 47 +/- 6% and 61 +/- 7% of activity, respectively. The reduction in activity of lysozyme deposited on contact lens materials is time-dependent and the rate of reduction varies between lens materials. This variation in activity recovered from lenses could be due to the differences in surface/bulk material properties or the location of lysozyme on these lenses. PMID:20178690

Subbaraman, Lakshman N; Jones, Lyndon

2010-01-01

364

Redesign of the substrate-binding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes.  

PubMed

On the basis of the molecular evolution of hen egg white, human, and turkey lysozymes, three replacements (Trp62 with Tyr, Asn37 with Gly, and Asp101 with Gly) were introduced into the active-site cleft of hen egg white lysozyme by site-directed mutagenesis. The replacement of Trp62 with Tyr led to enhanced bacteriolytic activity at pH 6.2 and a lower binding constant for chitotriose. The fluorescence spectral properties of this mutant hen egg white lysozyme were found to be similar to those of human lysozyme, which contains Tyr at position 62. The replacement of Asn37 with Gly had little effect on the enzymatic activity and binding constant for chitotriose. However, the combination of Asn37----Gly (N37G) replacement with Asp101----Gly (D101G) and Trp62----Tyr (W62Y) conversions enhanced bacteriolytic activity much more than each single mutation and restored hydrolytic activity toward glycol chitin. Consequently, the mutant lysozyme containing triple replacements (N37G, W62Y, and D101G) showed about 3-fold higher bacteriolytic activity than the wild-type hen lysozyme at pH 6.2, which is close to the optimum pH of the wild-type enzyme. PMID:1537843

Kumagai, I; Sunada, F; Takeda, S; Miura, K

1992-03-01

365

Predicting Tensile Stretchability of Trimmed AA6111-T4 Sheets  

SciTech Connect

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

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

2014-02-15

366

Study of CCD eyepiece on T-4 theodolite  

NASA Astrophysics Data System (ADS)

This document describes the effort of the University of Maryland to develop a Charge Coupled Device (CCD) Camera System, with the necessary support hardware and analysis software, to act as an impersonal electronic eyepiece on the T-4 theodolite for astronomical longitude and latitude determinations. This report will describe the concept, the implementation, and the current status of this project. Analysis of the field test data shows that the developed system has a performance level significantly higher than the original design goals, as well as being far above the best human skill level. The system is now being operated by personnel of the Geodetic Survey Squadron. The results of the overall operation will be addressed with respect to the current performance and future potential of this system.

Currie, D. G.

1982-11-01

367

Lysozyme Aggregation and Fibrillation Monitored by Dynamic Light Scattering  

NASA Astrophysics Data System (ADS)

The aggregation of amyloidogenic proteins provides a rich phase space with significant biomedical implications, including a link with several age-related diseases. We employed dynamic light scattering to monitor the aggregation of lysozyme, a model protein, from a monomeric state until the formation of micron-sized fibrils. For an aqueous lysozyme solution buffered at pH 2, the auto-correlation function of the scattered light intensity was found to be well-fit by a single exponential function with decay time ? = 1/(2Dq^2) = 0.25 ms, which corresponds to a mean hydrodynamic radius (RH) of 2.2 nm, very likely generated by monomers. Ethanol (4% v/v final concentration) induced a partial unfolding, to RH = 4.6 nm. The subsequent addition of 70 mM KCl was found to shrink the size back to RH = 2.5 nm, as expected when a denatured protein refolds due to partial screening of the intramolecular repulsion. However, further aggregation was not observed. At pH 4, using a low-salt acetate buffer, more ethanol (10% v/v) was required to initiate unfolding, but once it occurred, larger aggregates formed. These results are consistent with the model that partial unfolding, which exposes beta-motif secondary structure, is a prerequisite for aggregation and fibrillation, but the aggregation fate depends on the protein charge state (pH) and screening (salt concentration).

Nemzer, Louis; Flanders, Bret; Schmit, Jeremy; Sorensen, Christopher

2012-02-01

368

Retention of anomeric form in lysozyme-catalyzed reaction.  

PubMed

A lysozyme-catalyzed reaction is initiated by a cleavage of the beta-1, 4-glucosaminide linkage, followed by hydration and transglycosylation. Since all glycosides produced by transglycosylation have beta-glycosidic linkages between the sugar and the acceptor moieties, the lysozyme-catalyzed reaction has been classified as an anomer-retention reaction. However, there is no experimental evidence on the anomer retention of the new reducing residue produced by the hydrolysis of the substrate. In the present study, an attempt was made to determine the anomeric form of the GlcNAc residue at the reducing end in nascent hydrolytic products. The anomeric forms of the enzymatic products were separated and quantitatively analyzed by high-performance liquid chromatography. The amounts of alpha- and beta-anomers in the product were plotted against the reaction time. Computer analysis of the experimental data indicated that the nascent hydrolytic product takes only the beta-anomeric form and that the alpha-anomer is formed from beta-anomer by mutarotation. PMID:3813561

Yanase, Y; Fukamizo, T; Hayashi, K; Goto, S

1987-02-15

369

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

370

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

371

Towards lysozyme nanotube and 3D hybrid self-assembly  

NASA Astrophysics Data System (ADS)

We report lysozyme self-assembly into nanotubes, under the effect of hydrolysis at pH 2 and 90 °C. We resolve the final steps of the fibrillation pathway, entailing the closure of multi-stranded helical ribbons into nanotubes, and we provide evidence of ?-sheet arrangement within the nanotubes, demonstrating amyloid-like aggregation. Addition of chloroauric acid to the self-assembled structures can lead to generation of either gold single crystal nanoplatelets or gold nanoparticles (when a reducing agent is added) decorating the nanotube and ribbon surfaces. The crystal-based organic-inorganic hybrids further assemble into 3D ``sandwiched'' structures.We report lysozyme self-assembly into nanotubes, under the effect of hydrolysis at pH 2 and 90 °C. We resolve the final steps of the fibrillation pathway, entailing the closure of multi-stranded helical ribbons into nanotubes, and we provide evidence of ?-sheet arrangement within the nanotubes, demonstrating amyloid-like aggregation. Addition of chloroauric acid to the self-assembled structures can lead to generation of either gold single crystal nanoplatelets or gold nanoparticles (when a reducing agent is added) decorating the nanotube and ribbon surfaces. The crystal-based organic-inorganic hybrids further assemble into 3D ``sandwiched'' structures. Electronic supplementary information (ESI) available: Materials and methods, further images and FTIR data. See DOI: 10.1039/c3nr02194g

Lara, Cecile; Handschin, Stephan; Mezzenga, Raffaele

2013-07-01

372

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

373

Stopped-flow fluorescence studies on saccharide binding to lysozyme.  

PubMed Central

The binding of the beta-1-4-linked trimer of N-acetyl-D-glucosamine to hen egg-white lysozyme was studied by rapid-reaction-kinetic methods with tryptophyl fluorescence observation of the transients. It was found that discrete segments of the fluorescence-difference spectrum from this reaction were perturbed at different time-points during the binding process. The results were interpretated as the formation of the initial complex, the fast phase of the reaction, perturbing the environment of tryptophan-62 and a subsequent and slower rearrangement of the initial complex perturbing the environment of tryptophan-108. At pH 4.4, the release of protons from aspartate-101 occurred during the rearrangement step of the binding reaction. A model for the reaction is presented (E, enzyme; L, ligand): (see article) The association of this ligand with lysozyme may be visualized in three-dimensional terms as initial complex-formation across the top of the active-site cleft followed by a diving motion of the ligand into the cleft. Images Fig. 1. Fig. 3. Fig. 5. Fig. 8. PMID:1180904

Halford, S E

1975-01-01

374

Bacteriophage host range and bacterial resistance.  

PubMed

Host range describes the breadth of organisms a parasite is capable of infecting, with limits on host range stemming from parasite, host, or environmental characteristics. Parasites can adapt to overcome host or environmental limitations, while hosts can adapt to control the negative impact of parasites. We consider these adaptations as they occur among bacteriophages (phages) and their bacterial hosts, since they are significant to phage use as antibacterials (phage therapy) or to protection of industrial ferments from phage attack. Initially, we address how phage host range can (and should) be defined plus summarize claims of host ranges spanning multiple bacterial genera. Subsequently, we review bacterial mechanisms of phage resistance. These include adsorption resistance, which results in reduced interaction between phage and bacterium; what we describe as "restriction," where bacteria live but phages die; and abortive infections, where both phage and bacterium die. Adsorption resistance includes loss of phage receptor molecules on hosts as well as physical barriers hiding receptor molecules (e.g., capsules). Restriction mechanisms include phage-genome uptake blocks, superinfection immunity, restriction modification, and CRISPR, all of which function postphage adsorption but prior to terminal phage takeover of host metabolism. Standard laboratory selection methods, involving exposure of planktonic bacteria to high phage densities, tend to directly select for these prehost-takeover resistance mechanisms. Alternatively, resistance mechanisms that do not prevent bacterium death are less readily artificially selected. Contrasting especially bacteria mutation to adsorption resistance, these latter mechanisms likely are an underappreciated avenue of bacterial resistance to phage attack. PMID:20359459

Hyman, Paul; Abedon, Stephen T

2010-01-01

375

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

376

STUDIES ON BACTERIOPHAGES OF HEMOLYTIC STREPTOCOCCI  

PubMed Central

The host ranges of bacteriophages for group A, types 1, 6, 12, and 25 and group C streptococci have been determined. The findings indicate that the susceptibility to these phages is primarily a group-specific phenomenon, although it is modified by several factors such as the hyaluronic acid capsule, lysogeny, and possibly the presence of surface proteins. Phage antibody studies indicate that while the group A phages are antigenically related, they are distinct from the group C phage. This is in agreement with the observation that group A phages are not specific for their homologous streptococcal types. The purified group C carbohydrate inactivates group C phage but not the group A phages, thus suggesting that the carbohydrate, a component of the cell wall, may serve as the phage receptor site. It has not been possible to inactivate the group A phages with group A carbohydrate. Phage lysis of groups A and C streptococci is accompanied by fragmentation of the cell wall since the C carbohydrate has been identified serologically and chemically in the supernate of centrifuged lysates. The immediate lysis of groups A and C hemolytic streptococci and their isolated cell walls by an accesory heat-labile lytic factor in fresh group C lysates is also described. PMID:13463248

Krause, Richard M.

1957-01-01

377

Hurdles in bacteriophage therapy: deconstructing the parameters.  

PubMed

Bacterial infections in animals impact our food production, leading to economic losses due to food rejection and the need for preventive and curative measures. Since the onset of the antibiotic era, the rise of antibiotic-resistant pathogens is causing scares in health care and food producing facilities worldwide. In the search of new therapeutics, re-evaluation of bacteriophage (phage) therapy, using naturally occurring bacterial viruses to tackle infections, is gaining interest. Many studies report about phage therapy success, showing the value and power of these natural viruses. Although phages carry some interesting traits and their basic biology is now well understood, this review argues that phage therapy has not revealed all of its secrets and many parameters remain understudied, making the outcome of phage therapy highly variable depending on the disease incidence. These difficulties include poorly understood mechanisms of phage penetration and distribution throughout the body, the variable expression and accessibility of phage receptors on the bacterial host in in vivo conditions and the unusual (non-linear) phage pharmacokinetics. These parameters are not easily measured in realistic in vivo settings, but are nevertheless important hurdles to overcome the high variability of phage therapy trials. This critical approach is in accordance with Goethe's statement; "Difficulties increase the nearer we get to the goal". However, since the importance of the goal itself also rises, both difficulties and goal justify the need for additional in depth research in this domain. PMID:24315040

Tsonos, Jessica; Vandenheuvel, Dieter; Briers, Yves; De Greve, Henri; Hernalsteens, Jean-Pierre; Lavigne, Rob

2014-07-16

378

Characterisation of methacrylate monoliths for bacteriophage purification.  

PubMed

Binding of three different bacteriophages (phages), namely T7, lambda and M13 on methacrylate monoliths was investigated. Phage M13 exhibited the highest dynamic binding capacity of 4.5×10(13) pfu/mL while T7 and lambda showed capacity of 1×10(13) pfu/mL, all corresponding to values of around 1mg/mL. Interestingly, capacity for lambda phage was increased 5-fold by increasing NaCl concentration in a loaded sample from 0 to 0.2M while there was a constant capacity decrease for T7 and M13 phages. Under optimal conditions, recovery for all three phages approached 100%. Measurement of a pressure drop increase during loading enabled estimation of adsorbed phage layer thickness. At a maximal capacity it was calculated to be around 50 nm for T7 phage and 60 nm for lambda phage matching closely capside size thus indicating monolayer adsorption while 80 nm layer thickness was estimated for M13 phage showing its orientation along the pore. PMID:21238969

Smrekar, Franc; Ciringer, Mateja; Strancar, Aleš; Podgornik, Aleš

2011-04-29

379

Inactivation of clay-associated bacteriophage MS-2 by chlorine.  

PubMed

The model system consisted of bacteriophage MS-2, bentonite clay, and hypochlorous acid (HOC1). Factors that influenced association of the bacterial virus with bentonite were the titer of unadsorbed viruses, clay concentration, cation concentration, temperature, stirring rate, and the presence of soluble organics. Variation of the kinetic adsorption rate constant with stirring speed indicates that phage attachment is a diffusion-limited process; the attachment reaction has an apparent activation energy of 1 kcal/mol. About 18% of clay-associated bacteriophages was recovered by mixing the suspension with an organic eluent. Inactivation data were obtained from batch reactors operated under those conditions in which loss of HOC1 was minimal during the reaction. Bacteriophages attached to clay were more resistant to HOC1 than were freely suspended phages; for equivalent HOC1 concentrations, clay-associated phages required about twice the time that freely suspended phages required for loss of 99% of the initial virus titer. PMID:192148

Stagg, C H; Wallis, C; Ward, C H

1977-02-01

380

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

381

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

382

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

383

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

384

Phenotypic and genotypic variations within a single bacteriophage species  

Microsoft Academic Search

Background  Although horizontal gene transfer plays a pivotal role in bacteriophage evolution, many lytic phage genomes are clearly shaped\\u000a by vertical evolution. We investigated the influence of minor genomic deletions and insertions on various phage-related phenotypic\\u000a and serological properties.\\u000a \\u000a \\u000a \\u000a \\u000a Findings  We collected ten different isolates of Pseudomonas aeruginosa bacteriophage ?KMV. All sequenced genomes (42-43 kb, long direct terminal repeats) are nearly identical,

Pieter-Jan Ceyssens; Thea Glonti; ndrew M Kropinski; Rob Lavigne; Nina Chanishvili; Leonid Kulakov; Nino Lashkhi; Marina Tediashvili; Maya Merabishvili

2011-01-01

385

Structure of the Ribonucleic Acid Bacteriophage R17  

PubMed Central

Vasquez, Cesar (Institut de Recherches sur le Cancer, Villejuif, Seine, France), Nicole Granboulan, and Richard M. Franklin. Structure of the ribonucleic acid bacteriophage R17. J. Bacteriol. 92:1779–1786. 1966.—The morphology of bacteriophage R17 was studied by electron microscopy of negatively stained virions. The hexagonal shape, the presence of a maximum of 10 units at the periphery, and especially the observation of central fivefold points of symmetry with neighboring five and six coordinated units indicated icosahedral symmetry with 32 morphological units. Although the exact shape of the polyhedron could not be specified, the number of morphological units agreed with the chemically estimated number of structural units. Images PMID:5958109

Vasquez, Cesar; Granboulan, Nicole; Franklin, Richard M.

1966-01-01

386

Engineered enzymatically active bacteriophages and methods of uses thereof  

SciTech Connect

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

387

Genetically engineered acidophilic heterotrophic bacteria by bacteriophage transduction  

SciTech Connect

A bacteriophage capable of infecting acidophilic heterotrophic bacteria and processes for genetically engineering acidophilic bacteria for biomining or sulfur removal from coal are disclosed. The bacteriophage is capable of growth in cells existing at pH at or below 3.0. Lytic forms of the phage introduced into areas experiencing acid drainage kill the bacteria causing such drainage. Lysogenic forms of the phage having genes for selective removal of metallic or nonmetallic elements can be introduced into acidophilic bacteria to effect removal of the desired element from ore or coal. 1 fig., 1 tab.

Ward, T.E.; Bruhn, D.F.; Bulmer, D.F.

1989-05-10

388

The lysis genes of bacteriophage 21: structure and function  

E-print Network

THE LYSIS GENES OF BACTERIOPHAGE 21: STRUCTURE AND FUNCTION A Thesis by MARIA TERESA BONOVICH Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE... December 1991 Major Subject: Biology THE LYSIS GENES OP BACTERIOPHAGE 2l: STRUCTURE AND PUNCTION A Thesis by MARIA TERESA BONOVICH Approved as to style and content by: Ryland Young (Co-Chair of Co 'ttee) Ni enedik (Co-Chair of Committee) Susan...

Bonovich, Maria Teresa

1991-01-01

389

The antibacterial protein lysozyme identified as the termite egg recognition pheromone.  

PubMed

Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus 'termite-ball' and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic microbes on the evolution of social behaviour in termites. PMID:17726543

Matsuura, Kenji; Tamura, Takashi; Kobayashi, Norimasa; Yashiro, Toshihisa; Tatsumi, Shingo

2007-01-01

390

Studies on the role of insect hemolymph polypeptides: Galleria mellonella anionic peptide 2 and lysozyme.  

PubMed

The lysozymes are well known antimicrobial polypeptides exhibiting antibacterial and antifungal activities. Their antibacterial potential is related to muramidase activity and non-enzymatic activity resembling the mode of action of cationic defense peptides. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozyme are still not clear. In the present study, the anti-Candida albicans activity of Galleria mellonella lysozyme and anionic peptide 2 (AP2), defense factors constitutively present in the hemolymph, was examined. The lysozyme inhibited C. albicans growth in a dose-dependent manner. The decrease in the C. albicans survival rate caused by the lysozyme was accompanied by a considerable reduction of the fungus metabolic activity, as revealed by LIVE/DEAD staining. In contrast, although AP2 reduced C. albicans metabolic activity, it did not influence its survival rate. Our results suggest fungicidal action of G. mellonella lysozyme and fungistatic activity of AP2 toward C. albicans cells. In the presence of AP2, the anti-C. albicans activity of G. mellonella lysozyme increased. Moreover, when the fungus was incubated with both defense factors, true hyphae were observed besides pseudohyphae and yeast-like C. albicans cells. Atomic force microscopy analysis of the cells exposed to the lysozyme and/or AP2 revealed alterations in the cell surface topography and properties in comparison with the control cells. The results indicate synergistic action of G. mellonella AP2 and lysozyme toward C. albicans. The presence of both factors in the hemolymph of naive larvae suggests their important role in the early stages of immune response against fungi in G. mellonella. PMID:24472857

Sowa-Jasi?ek, Aneta; Zdybicka-Barabas, Agnieszka; St?czek, Sylwia; Wydrych, Jerzy; Mak, Pawe?; Jakubowicz, Teresa; Cytry?ska, Ma?gorzata

2014-03-01

391

High-level potentiation of lysostaphin anti-staphylococcal activity by lysozyme.  

PubMed Central

The purpose of this study was to determine whether lysostaphin would enhance its anti-staphylococcal efficacy in combination with lysozyme. Minimum inhibitory concentrations (MICs) of lysostaphin and lysozyme were separately determined for 41 strains belonging to 10 different species of human staphylococci. Lysozyme was virtually inactive and showed MICs of 15 mg/ml. On the contrary, all strains were susceptible to lysostaphin and showed MICs ranging from 2.5 to 60 micrograms/ml for the different Staphylococcus species. When the MIC of lysostaphin was determined in media containing submultiples of the MIC of lysozyme, the values obtained were much lower. The reduction of the lysostaphin MIC ranged from 16- to 200-fold in the different species tested. In Staphylococcus aureus, in particular, the combination of lysostaphin with 1.5 mg of lysozyme per ml reduced the MIC of lysostaphin by 25-fold. The activities of two combinations of the two enzymes were evaluated: one combination was expected to be active on S. aureus only, and the other combination was expected to inhibit all Staphylococcus strains. The first combination (0.5 micrograms of lysostaphin plus 0.5 mg of lysozyme per ml) was inhibitory to all of the 84 S. aureus strains tested, whereas 137 of 151 strains of other Staphylococcus species were unaffected. On the contrary, all of the 235 Staphylococcus strains tested were inhibited by the second combination (4 micrograms of lysostaphin plus 5 mg of lysozyme per ml). The possible mechanisms of lysostaphin potentiation by lysozyme are considered, and the potential use of a lysostaphin-lysozyme combination for topical therapy of staphylococcal infections resistant to other antibiotics is discussed. Images PMID:7081976

Cisani, G; Varaldo, P E; Grazi, G; Soro, O

1982-01-01

392

Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme.  

PubMed Central

To assess the respective roles of local and long-range interactions during protein folding, the influence of the native disulfide bonds on the early formation of secondary structure was investigated using continuous-flow circular dichroism. Within the first 4 ms of folding, lysozyme with intact disulfide bonds already had a far-UV CD spectrum reflecting large amounts of secondary structure. Conversely, reduced lysozyme remained essentially unfolded at this early folding time. Thus, native disulfide bonds not only stabilize the cfinal conformation of lysozyme but also provide, in early folding intermediates, the necessary stabilization that favors the formation of secondary structure. PMID:8069219

Goldberg, M. E.; Guillou, Y.

1994-01-01

393

In situ study of nanotemplate-induced growth of lysozyme microcrystals by submicrometer GISAXS.  

PubMed

Ultrasmall lysozyme microcrystals are grown by classical hanging-drop vapor diffusion and by its modification using a homologous protein thin-film template displaying long-range order. The nucleation and growth mechanisms of lysozyme microcrystals are studied at the thin lysozyme film surface using a new in situ µGISAXS (microbeam grazing-incidence small-angle X-ray scattering) technique recently developed at the microfocus beamline of the ESRF in Grenoble, France. New insight on the nucleation and crystallization processes appear to emerge. PMID:21335918

Pechkova, Eugenia; Nicolini, Claudio

2011-03-01

394

The use of lysozyme modified with fluorescein for the detection of Gram-positive bacteria.  

PubMed

Lysozyme (1,4-?-N-acetylmuramidase) is commonly applied in the food, medical, and pharmaceutical industries. In this study, we tested a novel application of fluorescein-modified lysozyme (using carboxyfluorescein with a triazine-based coupling reagent) as a new tool for the detection of Gram-positive soil bacteria. The results, obtained by cultivation methods, fluorescence analysis, and laser interferometry, showed that, after optimization, fluorescein-modified lysozyme could be used to evaluate the prevalence of Gram-positive bacteria essential in bioremediation of soils with low pH, such as those degraded by sulfur. PMID:24916601

Arabski, Micha?; Konieczna, Iwona; Tusi?ska, Ewa; W?sik, S?awomir; Relich, Inga; Zaj?c, Krzysztof; Kami?ski, Zbigniew J; Kaca, Wies?aw

2015-01-01

395

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

396

Partial characterization of Acanthamoeba castellanii (T4 genotype) DNase activity.  

PubMed

The deoxyribonuclease (DNase) activities of Acanthamoeba castellanii belonging to the T4 genotype were investigated. Using zymographic assays, the DNase activities had approximate molecular masses of 25 and 35 kDa. A. castellanii DNases exhibited activity at wide-ranging temperature of up to 60 °C and at pH ranging from 4 to 9. The DNases activities were unaffected by proteinase-K treatment, divalent cations such as Ca(++), Cu(++), Mg(++), and Zn(++), or divalent cation chelating agent ethylenediaminetetraacetic acid (EDTA) or sodium dodecyl sulfate (SDS). The non-reliance on divalent cations and homology data suggests that A. castellanii DNases belong to the class of eukaryotic lysosomal DNase II but exhibit robust properties. The DNases activity in A. castellanii interfered with the genomic DNA extraction. Extraction methods involving EDTA, SDS, and proteinase-K resulted in low yield of genomic DNA. On the other hand, these methods resulted in high yield of genomic DNA from human cells suggesting the robust nature of A. castellanii DNases that are unaffected by reagents normally used in blocking eukaryotic DNases. In contrast, the use of chaotropic agent such as guanidine thiocyanate improved the yield of genomic DNA from A. castellanii cells significantly. Further purification and characterization of Acanthamoeba DNases is needed to study their non-classic distinct properties and to determine their role in the biology, cellular differentiation, cell cycle progression, and arrest of Acanthamoeba. PMID:25358239

Iqbal, Junaid; Panjwani, Shamvil; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

2014-10-31

397

Bacteriophages and their enzymes in biofilm control.  

PubMed

Although free-swimming planktonic bacteria historically have been the typical focus of microbiological studies, the natural state of many or most bacteria is one where they instead are associated with surfaces and/or each other. For many pathogenic as well as nuisance bacteria, including biofouling bacteria, it consequently is within the context of this biofilm state that antibacterial strategies must be implemented. For reasons that are not fully understood, however, biofilm-associated bacteria tend to be less susceptible to treatments with standard chemical antibacterial agents than are planktonic bacteria, and this appears to be especially an issue with the use of less-harsh agents such as antibiotics. Within a variety of contexts the development of less- or selectively toxic antibacterial agents capable of clearing biofilms therefore would be welcome. In this review we consider the use of three categories of such agents as anti-biofilm antibacterials. These are lytic viruses of bacteria, that is, bacteriophages, effecting phage-mediated biocontrol of bacteria (a.k.a., phage therapy); purified phage-encoded enzymes that digest bacterial cell-wall material (endolysins or simply lysins); and a second category of phage-encoded enzymes that digest the extracellular polymeric substance (EPS) that are particularly notable components of bacterial biofilms (EPS depolymerases). These agents have been shown to reduce the bacterial density of a diversity of biofilms and, in many cases, tend to be lacking in inherent toxicity against the tissues of animals. Here we consider these phage-based anti-biofilm strategies with emphasis on ecological aspects of their action and with particular consideration of EPS depolymerases. PMID:25189866

Chan, Benjamin K; Abedon, Stephen T

2015-01-01

398

Effects of bacteriophage traits on plaque formation  

PubMed Central

Background The appearance of plaques on a bacterial lawn is one of the enduring imageries in modern day biology. The seeming simplicity of a plaque has invited many hypotheses and models in trying to describe and explain the details of its formation. However, until now, there has been no systematic experimental exploration on how different bacteriophage (phage) traits may influence the formation of a plaque. In this study, we constructed a series of isogenic ? phages that differ in their adsorption rate, lysis timing, or morphology so that we can determine the effects if these changes on three plaque properties: size, progeny productivity, and phage concentration within plaques. Results We found that the adsorption rate has a diminishing, but negative impact on all three plaque measurements. Interestingly, there exists a concave relationship between the lysis time and plaque size, resulting in an apparent optimal lysis time that maximizes the plaque size. Although suggestive in appearance, we did not detect a significant effect of lysis time on plaque productivity. Nonetheless, the combined effects of plaque size and productivity resulted in an apparent convex relationship between the lysis time and phage concentration within plaques. Lastly, we found that virion morphology also affected plaque size. We compared our results to the available models on plaque size and productivity. For the models in their current forms, a few of them can capture the qualitative aspects of our results, but not consistently in both plaque properties. Conclusions By using a collection of isogenic phage strains, we were able to investigate the effects of individual phage traits on plaque size, plaque productivity, and average phage concentration in a plaque while holding all other traits constant. The controlled nature of our study allowed us to test several model predictions on plaque size and plaque productivity. It seems that a more realistic theoretical approach to plaque formation is needed in order to capture the complex interaction between phage and its bacterium host in a spatially restricted environment. PMID:21827665

2011-01-01

399

The Bacteriophage T4 Transcription Activator MotA Interacts with the Far-C-Terminal Region of the  70 Subunit of Escherichia coli RNA Polymerase  

Microsoft Academic Search

portion of the 70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at 30, and also interacts with 70. We show here that the N-terminal half of MotA (MotANTD), which is thought to include the activation domain, interacts with the C-terminal region of 70 in an E. coli two-hybrid assay. Replacement of

Suchira Pande; Anna Makela; Simon L. Dove; Bryce E. Nickels; Ann Hochschild; Deborah M. Hinton

2002-01-01

400

Sequence analysis of bacteriophage T4 DNA packaging\\/terminase genes 16 and 17 reveals a common ATPase center in the large subunit of viral terminases  

Microsoft Academic Search

Phage DNA packaging is believed to be driven by a rotary device coupled to an ATPase 'motor'. Recent evidence suggests that the phage DNA packaging motor is one of the strongest force-generating molecular motors reported to date. However, the ATPase center that is responsible for generating this force is unknown. In order to identify the DNA translocating ATPase, the sequences

Michael S. Mitchell; Shigenobu Matsuzaki; Shosuke Imai; Venigalla B. Rao

2002-01-01

401

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

402

Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis  

NASA Technical Reports Server (NTRS)

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

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

1999-01-01

403

Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis  

NASA Technical Reports Server (NTRS)

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

Crawford, Lisa; Karr, Laurel; Pusey, Marc

1998-01-01

404

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

405

Crystallization of chicken egg white lysozyme from assorted sulfate salts  

NASA Astrophysics Data System (ADS)

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 32 12. Crystallization at 20°C typically resulted in the formation of orthorhombic crystals, space group P2 12 12 1. The tetragonal ? 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 12 1, a= b=87.4, c=73.7, ?=120°, which diffracted to 2.8 Å. 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, ?=119.2°. A crystal of ˜0.2×0.2×0.5 mm grown from bulk solution diffracted to ˜3.5 Å.

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

1999-01-01

406

Crystal structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product.  

PubMed

The crystal structure of a mutant hen egg white lysozyme, in which the key catalytic residue aspartic acid 52 has been changed to a serine residue (D52S HEWL), has been determined and refined to a crystallographic R value of 0.173 for all data F > 0 between 8 and 1.9 A resolution. The D52S HEWL structure is very similar to the native HEWL structure (r.m.s. deviation of main-chain atoms 0.20 A). Small shifts that result from the change in hydrogen bonding pattern on substitution of Asp by Ser were observed in the loop between beta-strands in the region of residues 46 to 49. D52S HEWL exhibits less than 1% activity against the bacterial cell wall substrate. Cocrystallisation experiments with the hexasaccharide substrate beta(1-4) polymer of N-acetyl-D-glucosamine (GlcNAc6) resulted in crystals between 5 days and 14 days after the initial mixing of enzyme and substrate. Analysis by laser absorption mass spectrometry of the oligosaccharides present after incubation with native and D52S HEWL under conditions similar to those used for crystal growth showed that after 14 days with native HEWL complete catalysis to GlcNAc3. GlcNAc2 and GlcNac had occurred but with D52S HEWL only partial catalysis to the major products GlcNAc4 and GlcNAc2 had occurred and at least 50% of the GlcNAc6 remained intact. X-ray analysis of the D52S-oligosaccharide complex crystals showed that they contained the product GlcNAc4. The structure of the D52S HEWL-GlcNAc4 complex has been determined and refined to an R value of 0.160 for data between 8 and 2 A resolution. GlcNAc4 occupies sites A to D in the active site cleft. Careful refinement and examination of 2Fo-Fc electron density maps showed that the sugar in site D has the sofa conformation, a conformation previously observed with the HEWL complex with tetra-N-acetylglucosamine lactone transition state analogue, the HEWL complex with the cell wall trisaccharide and the phage T4 lysozyme complex with a cell wall product. The semi-axial C(5)-C(6) geometry of the sofa is stabilised by hydrogen bonds from the O-6 hydroxyl group to the main-chain N of Val109 and main-chain O of Ala107. The sugar in site D adopts the alpha configuration, seemingly in conflict with the observation that the hydrolysis of beta (1-4) glycosidie linkage by HEWL proceeds with 99.9% retention of beta-configuration.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7966306

Hadfield, A T; Harvey, D J; Archer, D B; MacKenzie, D A; Jeenes, D J; Radford, S E; Lowe, G; Dobson, C M; Johnson, L N

1994-11-11

407

Lysozyme is an inducible marker of macrophage activation in murine tissues as demonstrated by in situ hybridization.  

PubMed

This study demonstrates the induction of lysozyme mRNA expression in situ in tissue macrophages (M phi) of mice following in vivo stimulation. The resting resident tissue M phi of most tissues do not contain enough lysozyme mRNA to be detected by in situ hybridization using 35S-labeled RNA probes. Following Bacille Calmette Guerin or Plasmodium yoelli infection, however, M phi recruited to liver and spleen hybridize strongly to the lysozyme probe. Within 24 h of infection, cells found in the marginal zone of the spleen begin to produce lysozyme mRNA. This response is also evoked by a noninfectious agent (intravenously injected sheep erythrocytes), and is possibly the result of an early phagocytic interaction. Later in the infection, other cells in the red and white pulp of the spleen, and cells in granulomas in the liver, become lysozyme-positive. Kupffer cells are rarely lysozyme-positive. Lysozyme mRNA levels in liver granulomas remain relatively constant during the infection, and lysozyme is produced by most granuloma cells. This contrasts with tumor necrosis factor alpha (TNF alpha) mRNA, which is produced by fewer cells in the granuloma, and which can be massively induced by lipopolysaccharide administration. The production of lysozyme, previously considered a constitutive function of M phi, is therefore an indicator of M phi activation in vivo, where immunologically specific and nonspecific stimuli both stimulate lysozyme production at high levels in subpopulations of cells occupying discrete anatomical locations. PMID:1940787

Keshav, S; Chung, P; Milon, G; Gordon, S

1991-11-01

408

Genome Landscapes and Bacteriophage Codon Usage Julius B. Lucks1  

E-print Network

Genome Landscapes and Bacteriophage Codon Usage Julius B. Lucks1 , David R. Nelson1,2 , Grzegorz R exhibit unequal usage of synonymous codons. Although alternative theories abound, translational selection has been accepted as an important mechanism that shapes the patterns of codon usage in prokaryotes

Plotkin, Joshua B.

409

Genetic consequences of transfection with heteroduplex bacteriophage ? DNA  

Microsoft Academic Search

The role of rectification of heteroduplex heterozygotes in the formation of recombinant genotypes involving closely liked markers has been examined. Heteroduplex molecules of bacteriophage ? DNA, heterozygous at several alleles, have been constructed and the genetic composition of phage present in infective centers derived by transfection with such molecules has been determined. Allele loss and concomitant recombinant formation is frequent,

Raymond L. White; Maurice S. Fox

1975-01-01

410

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...

411

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 ...

412

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...

413

Symmetry mismatch and DNA packaging in large bacteriophages.  

PubMed Central

A model is presented for the mechanism of packaging double-stranded DNA into phage heads. The model is based on, and rationalizes, the mismatch in symmetry between the heads and tails of large bacteriophages. DNA movement is postulated to be mediated by a rotating protein structure at the tail-proximal vertex of the head. PMID:283391

Hendrix, R W

1978-01-01

414

[Photoinactivation of bacteriophage PM2 by cyanine dyes].  

PubMed

Photoinactivation of the lipid-containing bacteriophage PM2 by visible light and cyanine dyes (carbo- and dicarbocyanines), aluminum phtalocyanine tetrasulfonate and methylene blue was studied. It was concluded that cyanine dye aggregates adsorbed on phage particles and oxygen are essential for phage photoinactivation. PMID:11449549

Gonikberg, E M; Kuznetsova, N V

2001-01-01

415

Factors Affecting Survival of Bacteriophage on Tomato Leaf Surfaces  

Technology Transfer Automated Retrieval System (TEKTRAN)

The ability of bacteriophage to persist in the phyllosphere for extended periods is limited by many factors, including sunlight irradiation, especially in the UV zone, temperature, desiccation, and exposure to copper bactericides. The effects of these factors on persistence of phage and formulated p...

416

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

417

Shape evolution and thermal stability of lysozyme crystals: effect of pH and temperature.  

PubMed

The properties of crystalline protein materials are closely linked to crystal shape. However, the effective strategies for the shape control of protein crystals are lacking. The conventional sitting-drop vapor-diffusion method was employed to investigate the influence of pH and temperature on the crystal nucleation behavior of hen egg white lysozyme. Moreover, the size distributions of protein crystals grown at different conditions were analyzed. Differential scanning calorimetry was employed to evaluate the thermal stability of lysozyme crystals. The results indicated that pH and temperature will affect the supersaturation and electrostatic interactions among protein molecules in the nucleation process. In particular, the crystals with different aspect ratios can be selectively nucleated, depending upon the choice of pH and temperature. Therefore, this study provided a simple method for obtaining shape-controlled lysozyme crystals and supplied some information on thermal behaviors of lysozyme crystals grown at different pH values. PMID:22729674

Liang, Miao; Jin, Fengmin; Liu, Rui; Yu, Yanjun; Su, Rongxin; Wang, Libing; Qi, Wei; He, Zhimin

2013-01-01

418

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

419

Salivary immunoglobulins, lysozyme, pH, and microbial counts in children receiving anti-neoplastic therapy.  

PubMed

Salivary immunoglobulins, lysozyme, pH, and microbial counts were determined in 55 children with cancer diseases (37 cured subjects and 18 acute ones) and 103 healthy subjects. 5-10 ml unstimulated whole saliva was collected and pH, immunoglobulins and lysozyme were measured. Chairside dip-slide cultivations were used for microbiologic cultures. Reduced salivary pH and an increased amount of lysozyme were found in the saliva of those children who had been cured of their cancer diseases, but ongoing cancer disease or the treatment provided for it reduced pH and increased the amounts of lysozyme, lactobacillus, Streptococcus mutans and some immunoglobulins. These findings suggest that children with childhood cancer may be more susceptible to dental diseases than healthy ones. PMID:2704978

Pajari, U; Poikonen, K; Larmas, M; Lanning, M

1989-04-01

420

Lactoferrin and lysozyme in milk during acute mastitis and their inhibitory effect in Delvotest P.  

PubMed

Microbiological methods for detection of antibiotic residues in milk give no explanations regarding the identity of the inhibitory substance(s). Natural antibacterial substances, present at higher concentrations in mastitic milk and in colostrum, occasionally cause false positive results in antibiotic assays. In an earlier investigation, lysozyme and lactoferrin were shown to inhibit the growth of Bacillus stearothermophilus var. calidolactis spores, used as test organism in Delvotest P. To study the effect of high lysozyme and lactoferrin concentrations in milk on the Delvotest P, cows were subjected to acute experimental mastitis by infusion of Salmonella typhimurium SH 4809 endotoxin. Milk samples were collected up to 11 h postinfusion. Concentrations of lactoferrin and lysozyme, somatic cell count, and effect on Delvotest P were determined. A positive reaction in the Delvotest correlated well with an increase in lactoferrin and lysozyme concentrations. The nature of the inhibitory effect is briefly discussed. PMID:2628440

Carlsson, A; Björck, L; Persson, K

1989-12-01

421

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

NASA Astrophysics Data System (ADS)

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

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

1995-01-01

422

A study on photochemical behavior of lysozyme-bromophenol blue complex and its analytical application  

NASA Astrophysics Data System (ADS)

It was found that macromolecular complexes were formed between lysozyme and bromophenol blue (BPB) with the electrostatic attraction in acetate medium (pH 6.5). The binding constant and the number of binding site for lysozyme-BPB complex were obtained, and the thermodynamic parameters were given. In addition, a remarkable enhancement of resonance light scattering (RLS) intensity for the macromolecular complex was observed with a scattering peak at 336 nm. And the increment of RLS intensity was proportional to the concentration of lysozyme in the range of 5 ng ml -1 to 10.0 ?g ml -1. The influence of experimental conditions including pH, BPB concentration, and ionic strength on RLS system were tested, especially the effect of temperature was examined in detail. The proposed method was successfully applied to determine lysozyme in human saliva and tear samples without any special pretreatment. Compared with other methods the proposed method is of higher sensitivity and wider linear range.

Yue, Qiaoli; Song, Zhenghua; Dong, Faxin

2008-04-01

423

Binding of Lysozyme to Phospholipid Bilayers: Evidence for Protein Aggregation upon Membrane Association  

PubMed Central

Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5?-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated protein species is likely to be the mechanism responsible for the cytotoxicity of lysozyme. PMID:17434939

Gorbenko, Galyna P.; Ioffe, Valeriya M.; Kinnunen, Paavo K. J.

2007-01-01

424

Aggregation of aqueous lysozyme solutions followed by dynamic light scattering and 1H NMR spectroscopy  

Microsoft Academic Search

In the presented work, we followed our previous calorimetric investigation [J. Pozna?ski, M. Wszelaka-Rylik, W. Zielenkiewicz, Termochim. Acta 409 (2004) 25–32], analyzing the kinetics of NaCl-induced lysozyme aggregation. The aggregates' size distribution was monitored independently by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR). Experiments were conducted for 2.1 mM lysozyme solution in acetate buffer, pH=4.25, in the NaCl

Jaros?aw Pozna?ski; J?drzej Szyma?ski; Teresa Basi?ska; Stanis?aw S?omkowski; Wojciech Zielenkiewicz

2005-01-01

425

Identification and cloning of an invertebrate-type lysozyme from Eisenia andrei  

Microsoft Academic Search

Lysozyme is a widely distributed antimicrobial protein having specificity for cleaving the ?-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (GlcNAc) of peptidoglycan of the bacterial cell walls and thus efficiently contributes to protection against infections caused mainly by Gram-positive bacteria.In the present study, we assembled a full-length cDNA of a novel invertebrate-type lysozyme from Eisenia andrei earthworm (EALys) by

Radka Josková; Marcela Šilerová; Petra Procházková; Martin Bilej

2009-01-01

426

Production of recombinant human lysozyme in the milk of transgenic pigs  

Microsoft Academic Search

In the swine industry pathogenic infections have a significant negative impact on neonatal survival. Piglets fed with human\\u000a lysozyme, a natural antibiotic, might be more resistant to gastrointestinal infections. Here we describe the generation of\\u000a transgenic swine expressing recombinant human lysozyme by somatic cell nuclear transfer. Three cloned female pigs were born,\\u000a one of which expressed rhLZ at 0.32 ± 0.01 ?g\\/ml in

Jia Tong; HengXi Wei; XiaoFang Liu; WenPing Hu; MingJun Bi; YuanYuan Wang; QiuYan Li; Ning Li

2011-01-01

427

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

Microsoft Academic Search

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.

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

2007-01-01

428

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

PubMed Central

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

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

1985-01-01

429

A study on photochemical behavior of lysozyme–bromophenol blue complex and its analytical application  

Microsoft Academic Search

It was found that macromolecular complexes were formed between lysozyme and bromophenol blue (BPB) with the electrostatic attraction in acetate medium (pH 6.5). The binding constant and the number of binding site for lysozyme–BPB complex were obtained, and the thermodynamic parameters were given. In addition, a remarkable enhancement of resonance light scattering (RLS) intensity for the macromolecular complex was observed

Qiaoli Yue; Zhenghua Song; Faxin Dong

2008-01-01

430

Study on the Interaction Mechanism of Lysozyme and Bromophenol Blue by Fluorescence Spectroscopy  

Microsoft Academic Search

The interaction of lysozyme with bromophenol blue (BPB) in acetate buffer (pH 6.0) was studied by fluorescence quenching method\\u000a for the first time. It was found that BPB could conspicuously quench the fluorescence of lysozyme by the static quenching\\u000a process, possibly due to the binding on the active site near Trp62. The binding parameters including the binding constant\\u000a and the number

Qiaoli Yue; Lichuan Niu; Xin Li; Xiaodong Shao; Xiaofeng Xie; Zhenghua Song

2008-01-01

431

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

PubMed Central

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

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

2011-01-01

432

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

433

Effect of Fe3O4 magnetic nanoparticles on lysozyme amyloid aggregation  

NASA Astrophysics Data System (ADS)

Peptide amyloid aggregation is a hallmark of several human pathologies termed amyloid diseases. We have investigated the effect of electrostatically stabilized magnetic nanoparticles of Fe3O4 on the amyloid aggregation of lysozyme, as a prototypical amyloidogenic protein. Thioflavin T fluorescence assay and atomic force microscopy were used for monitoring the inhibiting and disassembly activity of magnetic nanoparticles of Fe3O4. We have found that magnetic Fe3O4 nanoparticles are able to interact with lysozyme amyloids in vitro leading to a reduction of the amyloid aggregates, thus promoting depolymerization; the studied nanoparticles also inhibit lysozyme amyloid aggregation. The ability to inhibit lysozyme amyloid formation and promote lysozyme amyloid disassembly exhibit concentration-dependent characteristics with IC50 = 0.65 mg ml-1 and DC50 = 0.16 mg ml-1 indicating that nanoparticles interfere with lysozyme aggregation already at stoichiometric concentrations. These features make Fe3O4 nanoparticles of potential interest as therapeutic agents against amyloid diseases and their non-risk exploitation in nanomedicine and nanodiagnostics.

Bellova, Andrea; Bystrenova, Eva; Koneracka, Martina; Kopcansky, Peter; Valle, Francesco; Tomasovicova, Natalia; Timko, Milan; Bagelova, Jaroslava; Biscarini, Fabio; Gazova, Zuzana

2010-02-01

434

Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris.  

PubMed

Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia pastoris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of ?14 kD, as shown by SDS-PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni-NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M.lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture. PMID:21241808

Wang, Tingting; Xu, Yongping; Liu, Wenjia; Sun, Yongxin; Jin, Liji

2011-05-01

435

Liquid-liquid extraction of lysozyme using a dye-modified ionic liquid.  

PubMed

An affinity-dye, Cibacron Blue 3GA (CB), derivatized organic salt [BMIM]3[CB] was synthesized for lysozyme extraction. This compound was formed by mixing an ionic liquid (IL) [BMIM][Cl] and the silver salt of CB. Liquid-liquid extractions of lysozyme from the aqueous and [BMIM]3[CB] in [BMIM][PF6] solutions were examined in this study. The transfer of lysozyme from the aqueous phase to the IL phase decreased while the pH of the aqueous phase increased. An extraction higher than 90% was observed at pH 4. Under a high ionic strength, the lysozyme would transform back from the IL phase into the aqueous phase. Lysozyme molecules were almost quantitatively recovered from the IL phase to the aqueous solutions of 1M KCl under pH 9-11. It appeared that the extraction was specific for lysozyme in contrast to cytochrome c, ovalbumin, and bovine serum albumin. The extraction efficiency of the IL phase remained essentially the same after eight cycles of extraction. PMID:18462742

Tzeng, Yu-Ping; Shen, Ching-Wei; Yu, Tiing

2008-06-01

436

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

Microsoft Academic Search

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

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

1991-01-01

437

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

438

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

439

Development of an enzyme-linked immunosorbent assay for determination of lysozyme in human parotid and submandibular-sublingual salivas.  

PubMed Central

The specificity of lysozyme determinations in human parotid and submandibular-sublingual salivas of two subjects was assessed by comparison of lysozyme concentrations in native acidified salivas with purified enzyme obtained by immunoadsorbent fractionation of the salivas. Lysozyme concentrations were measured by the turbidimetric catalytic method and by a newly developed enzyme-linked immunosorbent assay (ELISA). The validity of the assays was established by comparing assay results with enzyme concentration values determined from optical density-extinction coefficient calculations of the purified lysozyme peak. Values for purified enzyme were found to be similar, irrespective of the assay used to determine lysozyme concentrations, and were in agreement with extinction coefficient calculations. Based on the ELISA technique, recoveries of lysozyme from both parotid and submandibular-sublingual salivas were greater than 75 and 90%, respectively. Similar recoveries were noted for parotid saliva when determinations were based on the turbidimetric assay. However, the ELISA and turbidimetric assays differed with respect to lysozyme levels in submandibular-sublingual saliva because of the apparent presence of an enhancement factor which gave rise to higher lysozyme values in the catalytic assay and therefore resulted in low recoveries of purified enzyme. This catalytic enhancement factor was present in the nonadsorbed fraction of both subjects, as higher lysozyme activities were noted when nonadsorbed fractions were added to affinity-purified lysozymes. Lysozyme levels were also determined in the parotid and submandibular-sublingual salivas of caries-resistant and -susceptible adults. In general, levels of lysozyme in parotid saliva were lower in comparison to submandibular -sublingual saliva; however, significant differences in enzyme concentration were not evident between the