Sample records for bacteriophage t4 lysozyme

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

    E-print Network

    Skrynnikov, Nikolai

    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

  2. Mutations in an upstream regulatory sequence that increase expression of the bacteriophage T4 lysozyme gene.

    PubMed Central

    Knight, J A; Hardy, L W; Rennell, D; Herrick, D; Poteete, A R

    1987-01-01

    A P22 hybrid phage bearing the bacteriophage T4 lysozyme gene (e), as well as T4 sequences upstream from the lysozyme gene, was constructed. Amber mutations were introduced into gene e in the hybrid phage, and the resulting mutant phages were tested for the ability to form plaques on amber suppressor strains. Revertant phages that were able to form plaques on amber suppressors that did not suppress the parent amber mutant phages were isolated following UV mutagenesis. Secondary site pseudorevertants were identified among the revertants by a genetic test. Four of the suppressing secondary site mutations were mapped and sequenced. They were found to consist of small sequence alterations immediately upstream from gene e, all of which would tend to destabilize potential base-pairing interactions in the transcript. The mutations were shown to increase lysozyme expression when introduced into an otherwise wild-type hybrid phage, but were found to have little effect on transcription of the lysozyme gene. Images PMID:3654580

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

    SciTech Connect

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

    2010-07-19

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

  4. Mutagenic effects on the fluorescence of tryptophan residues in bacteriophage T4 lysozyme: correlation with dynamics

    NASA Astrophysics Data System (ADS)

    Hudson, Bruce S.; Harris, Dan

    1992-04-01

    Site-directed mutagenesis of specific residues in bacteriophage T4 lysozyme is shown to result in changes in the emission spectra of the tryptophan residues of this protein. In some cases a significant red-shift is observed. This is interpreted in terms of enhanced dielectric relaxation due to fluctuations that expose a buried residue to the aqueous solvent. For substitutions at position 146, the spectral shift is strongly correlated with the rate of a specific proteolytic digestion of the T4 lysozyme by trypsin as determined by Signor, Dalzoppo, and Schellman. In cases where a spectral shift is observed there is also an enhancement of internal mobility of a tryptophan residue as indicated by the amplitude of a short correlation time component of the anisotropy decay. All of these spectral and enzymatic susceptibility effects are reversed by introduction of a disulfide linkage spanning the two lobes of the protein. The interpretation of these results in terms of molecular dynamics is discussed. The effect of mutational changes on the average fluorescence lifetime and quantum yield of tryptophan fluorescence is also discussed in terms of collisional quenching of tryptophan residues by neighboring groups.

  5. Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage

    Microsoft Academic Search

    Yasunori Tanji; Chiaki Furukawa; Suk-Hyun Na; Tomonori Hijikata; Kazuhiko Miyanaga; Hajime Unno

    2004-01-01

    Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E. coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid.

  6. The bacteriophage T4 DNA injection machine Michael G Rossmann1,

    E-print Network

    Rossmann, Michael G.

    The bacteriophage T4 DNA injection machine Michael G Rossmann1,Ã , Vadim V Mesyanzhinov2 , Fumio Arisaka3 and Petr G Leiman1 The tail of bacteriophage T4 consists of a contractile sheath surrounding bacteriophage T4L phage T4 lysozyme Introduction Bacterial viruses, or bacteriophages, have developed var- ious

  7. Molecular dynamics study of an insertion/duplication mutant of bacteriophage T4 lysozyme reveals the nature of ??? transition in full protein context.

    PubMed

    Kaur, Harpreet; Sasidhar, Yellamraju U

    2013-05-28

    An ??? transition underlies the first step of disease causing amyloidogenesis in many proteins. In view of this, many studies have been carried out using peptide models to characterize these secondary structural transitions. In this paper we show that an insertion/duplication mutant 'L20' of bacteriophage T4 lysozyme (M. Sagermann, W. A. Baase and B. W. Matthews, Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 6078) displays an ??? transition. We performed molecular dynamics (MD) simulation of L20, using the GROMACS package of programs and united atom GROMOS 53a6 force field for a time period of 600 ns at 300 K, in explicit water. Our MD simulation demonstrated that the transition occurs in a duplicated ?-helical region inserted tandemly at the N-terminus of the 'parent' helix. We show that a C-terminal ?-sheet anchors the parent helix while the loosely held N-terminal loop in the duplicate region is vulnerable to solvent attack and thus undergoes an ??? transition. Main chain-solvent interactions were seen to stabilize the observed ?-structure. Thus L20 serves as a good protein model for characterization of ??? transition in a full length protein. PMID:23598905

  8. Bacteriophage T4 Genome†

    PubMed Central

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

    2003-01-01

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

  9. Environmental polarity induces conformational transitions in a helical peptide sequence from bacteriophage T4 lysozyme and its tandem duplicate: a molecular dynamics simulation study.

    PubMed

    Kaur, Harpreet; Sasidhar, Yellamraju U

    2015-04-01

    Our recent molecular dynamics (MD) simulation of an insertion/duplication mutant 'L20' of bacteriophage T4 lysozyme demonstrated a solvent induced ??? transition in a loosely held duplicate helical region, while ?-helical conformation in the parent region was relatively stabilized by its tertiary interactions with the neighboring residues. The solution NMR of the parent helical sequence, sans its protein context, showed no inherent tendency to adopt a particular secondary structure in pure water but showed ?-helical propensity in TFE/water and SDS micelles. In this study we investigate the conformational preference of the 'parent' and 'duplicate' sequences, sans the protein context, in pure water and an apolar TFE/water solution. Apolar TFE/water solution is a model for non-polar protein context. We performed MD simulations of the two peptides, in explicit water and 80 % (v/v) TFE/water, using GROMOS 53a6 force field, at 300 K and 1 bar (under NPT conditions). We show that in TFE/water mixture, salt bridges are stabilized by apolar TFE molecules and main chain-main chain hydrogen bonds promote the ?-helical conformation, particularly in the duplicate peptide. Solvent exposure, in pure water, resulted in an ??? transition to form a triple stranded ?-sheet structure in the 'duplicate' sequence, with a rare psi-loop topology, while a mixture of turn/bend conformations were adopted by the 'parent' sequence. Thus the differences in conformational preference of the parent and duplicate sequence sans protein context, in pure water and TFE/water, implicate the importance of the environment polarity in dictating the peptide conformation. Mechanism of folding of the observed psi-loop in the duplicate sequence gives insights into folding of this rare ?-sheet topology. PMID:25773700

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Stabilization of phage T4 lysozyme by engineered disulfide bonds.

    PubMed Central

    Matsumura, M; Becktel, W J; Levitt, M; Matthews, B W

    1989-01-01

    Four different disulfide bridges (linking positions 9-164, 21-142, 90-122, and 127-154) were introduced into a cysteine-free phage T4 lysozyme at sites suggested by theoretical calculations and computer modeling. The new cysteines spontaneously formed disulfide bonds on exposure to air in vitro. In all cases the oxidized (crosslinked) lysozyme was more stable than the corresponding reduced (noncrosslinked) enzyme toward thermal denaturation. Relative to wild-type lysozyme, the melting temperatures of the 9-164 and 21-142 disulfide mutants were increased by 6.4 degrees C and 11.0 degrees C, whereas the other two mutants were either less stable or equally stable. Measurement of the equilibrium constants for the reduction of the engineered disulfide bonds by dithiothreitol indicates that the less thermostable mutants tend to have a less favorable crosslink in the native structure. The two disulfide bridges that are most effective in increasing the stability of T4 lysozyme have, in common, a large loop size and a location that includes a flexible part of the molecule. The results suggest that stabilization due to the effect of the crosslink on the entropy of the unfolded polypeptide is offset by the strain energy associated with formation of the disulfide bond in the folded protein. The design of disulfide bridges is discussed in terms of protein flexibility. PMID:2671995

  12. Molecular architecture of the prolate head of bacteriophage T4

    E-print Network

    Rossmann, Michael G.

    Molecular architecture of the prolate head of bacteriophage T4 Andrei Fokine , Paul R. Chipman and Molecular Biology, Catholic University of America, Washington, DC 20064 Edited by David S. Eisenberg long tail fibers. The architecture and the molecular composition of the T4 head, tail, and fibers have

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

    E-print Network

    Baker, Timothy S.

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

  14. Ribonucleotides Covalently Linked to Deoxyribonucleic Acid in T4 Bacteriophage

    PubMed Central

    Speyer, J. F.; Chao, J.; Chao, L.

    1972-01-01

    Bacteriophage T4 was grown in the presence of labeled uridine. The deoxyribonucleic acid (DNA) of the phage was shown to contain covalently attached ribonucleotides. The label appears not to be internal in the DNA strands. Presumably, it is at the ends of the DNA strands and this may be related to DNA initiation. PMID:4564585

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

    PubMed

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

    1983-01-01

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

  16. Structure and assembly of bacteriophage T4 head

    PubMed Central

    2010-01-01

    The bacteriophage T4 capsid is an elongated icosahedron, 120 nm long and 86 nm wide, and is built with three essential proteins; gp23*, which forms the hexagonal capsid lattice, gp24*, which forms pentamers at eleven of the twelve vertices, and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. The past twenty years of research has greatly elevated the understanding of phage T4 head assembly and DNA packaging. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as that found in phage HK97 and several other icosahedral bacteriophages. Folding of gp23 requires the assistance of two chaperones, the E. coli chaperone GroEL and the phage coded gp23-specific chaperone, gp31. The capsid also contains two non-essential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. The structure of Soc shows two capsid binding sites which, through binding to adjacent gp23 subunits, reinforce the capsid structure. Hoc and Soc have been extensively used in bipartite peptide display libraries and to display pathogen antigens including those from HIV, Neisseria meningitides, Bacillus anthracis, and FMDV. The structure of Ip1*, one of the components of the core, has been determined, which provided insights on how IPs protect T4 genome against the E. coli nucleases that degrade hydroxymethylated and glycosylated T4 DNA. Extensive mutagenesis combined with the atomic structures of the DNA packaging/terminase proteins gp16 and gp17 elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. Cryo-EM structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at a rate of up to 2000 bp/sec, the fastest reported to date of any packaging motor. FRET-FCS studies indicate that the DNA gets compressed during the translocation process. The current evidence suggests a mechanism in which electrostatic forces generated by ATP hydrolysis drive the DNA translocation by alternating the motor between tensed and relaxed states. PMID:21129201

  17. Bi-functional activities of chimeric lysozymes constructed by domain swapping between bacteriophage T7 and K11 lysozymes.

    PubMed

    Alcantara, Ethel H; Kim, Dong Hee; Do, Su-Il; Lee, Sang Soo

    2007-07-31

    The lysozymes encoded by bacteriophage T7 and K11 are both bifunctional enzymes sharing an extensive sequence homology (75%). The constructions of chimeric lysozymes were carried out by swapping the N-terminal and C-terminal domains between phage T7 and K11 lysozymes. This technique generated two chimeras, T7K11-lysozyme (N-terminal T7 domain and C-terminal K11 domain) and K11T7-lysozyme (N-terminal K11 domain and C-terminal T7 domain), which are both enzymatically active. The amidase activity of T7K11-lysozyme is comparable with the parental enzymes while K11T7-lysozyme exhibits an activity that is approximately 45% greater than the wild-type lysozymes. Moreover, these chimeric constructs have optimum pH of 7.2-7.4 similar to the parental lysozymes but exhibit greater thermal stabilities. On the other hand, the chimeras inhibit transcription comparable with the parental lysozymes depending on the source of their N-terminals. Taken together, our results indicated that domain swapping technique localizes the N-terminal region as the domain responsible for the transcription inhibition specificity of the wild type T7 and K11 lysozymes. Furthermore, we were able to develop a simple and rapid purification scheme in purifying both the wild-type and chimeric lysozymes. PMID:17669270

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

    E-print Network

    Müftü, Sinan

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

  19. Investigation of bacteriophage T4 by atomic force microscopy

    PubMed Central

    Kuznetsov, Yuri G; Chang, Sheng-Chieh

    2011-01-01

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

  20. Increased Killing of Bacillus subtilis on the Hair Roots of Transgenic T4 Lysozyme-Producing Potatoes

    Microsoft Academic Search

    INGRID AHRENHOLTZ; KLAUS HARMS; JOHANN DE VRIES; WILFRIED WACKERNAGEL

    2000-01-01

    Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium Erwinia carotovora subsp. carotovora have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to

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

    PubMed

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

    2010-08-01

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

  2. Structure of Bacteriophage T4 Gene Product 11, the Interface Between the Baseplate and Short Tail Fibers

    E-print Network

    Rossmann, Michael G.

    Structure of Bacteriophage T4 Gene Product 11, the Interface Between the Baseplate and Short Tail/10 Miklukho- Maklaya Street, 117871 Moscow, Russia Bacteriophage T4, like all other viruses, is required Academic Press Keywords: bacteriophage T; baseplate; gp11; ®ber attachment protein; X-ray crystallography

  3. Modified T4 Lysozyme Fusion Proteins Facilitate G Protein-Coupled Receptor Crystallogenesis.

    PubMed

    Thorsen, Thor Seneca; Matt, Rachel; Weis, William I; Kobilka, Brian K

    2014-10-23

    G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. Most GPCR crystal structures have been obtained using a fusion protein strategy where the flexible third intracellular loop is replaced by T4 lysozyme (T4L). However, wild-type T4L may not be ideally suited for all GPCRs because of its size and the inherent flexibility between the N- and C-terminal subdomains. Here we report two modified T4L variants, designed to address flexibility and size, that can be used to optimize crystal quality or promote alternative packing interactions. These variants were tested on the M3 muscarinic receptor (M3). The original M3-T4L fusion protein produced twinned crystals that yielded a 3.4 Å structure from a 70 crystal data set. We replaced T4L with the modified T4L variants. Both T4L variants yielded M3 muscarinic receptor crystals with alternate lattices that were not twinned, including one that was solved at 2.8 Å resolution. PMID:25450769

  4. The Functional Domains of Bacteriophage T4 Terminase* Received for publication, April 1, 2004, and in revised form, June 24, 2004

    E-print Network

    Rossmann, Michael G.

    The Functional Domains of Bacteriophage T4 Terminase* Received for publication, April 1, 2004 into some viral and all bacteriophage capsids is driven by powerful molecular motors. In bacteriophage T4 by progressively more force to encapsidate the genome. Bacteriophage T4 requires a 56- m-long double-stranded DNA

  5. Initiation of bacteriophage T4 DNA replication and replication fork dynamics: a review in the Virology Journal series on bacteriophage T4 and its relatives

    Microsoft Academic Search

    Kenneth N Kreuzer; J Rodney Brister

    2010-01-01

    Bacteriophage T4 initiates DNA replication from specialized structures that form in its genome. Immediately after infection, RNA-DNA hybrids (R-loops) occur on (at least some) replication origins, with the annealed RNA serving as a primer for leading-strand synthesis in one direction. As the infection progresses, replication initiation becomes dependent on recombination proteins in a process called recombination-dependent replication (RDR). RDR occurs

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

    SciTech Connect

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

    2003-07-16

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

  7. Rapid Isolation and Identification of Bacteriophage T4-Encoded Modifications of Escherichia coli RNA Polymerase: A Generic

    E-print Network

    Chait, Brian T.

    Rapid Isolation and Identification of Bacteriophage T4-Encoded Modifications of Escherichia coli RNA Polymerase: A Generic Method to Study Bacteriophage/Host Interactions Lars F. Westblade, Leonid of Wisconsin-Madison, Madison, Wisconsin 53706 Received July 19, 2007 Bacteriophages are bacterial viruses

  8. An Introduction to the Bacteriophage T4 Virus.

    NSDL National Science Digital Library

    dFORM: Projects

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

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

    Microsoft Academic Search

    J DUBOCHET

    1975-01-01

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

  10. Characterization of New Regulatory Mutants of Bacteriophage T4

    PubMed Central

    Johnson, James R.; Hall, Dwight H.

    1974-01-01

    Plating techniques which eliminate T4 plaque formation on Escherichia coli by folate analogue inhibition of dihydrofolate (FH2) reductase (EC 1.5.1.3) allowed the isolation of folate analogue-resistant (far) mutants of T4. One class of far mutants overproduces the phage-induced FH2 reductase. Deoxycytidylate deaminase (EC 3.5.4.12), thymidine kinase (EC 2.7.1.21), and deoxycytidine triphosphatase (EC 3.6.1.12) are also overproduced by 20 min after infection at 37 C. The overproduction of FH2 reductase by these far mutants is not affected by the absence of DNA synthesis. Other types of mutations that affect the synthesis of early enzymes cause overproduction in the absence of DNA synthesis of some of the above enzymes but not of FH2 reductase. Therefore, overproducing far mutants apparently have mutations in previously undescribed genes controlling the expression of the T4 genome. Three of four mutants under study map near gene 56, and one maps near gene 52. All of these mutants show delays in DNA synthesis, phage production, and lysis and appear to show decreased levels of RNA synthesis based on the cumulative incorporation of uridine. PMID:4362869

  11. Focused Genetic Recombination of Bacteriophage T4 Initiated by Double-Strand Breaks

    Microsoft Academic Search

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

    A model system for studying double-strand-break (DSB)-induced genetic recombination in vivo based on the ets1 segC 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

  12. Cloning and Physical Mapping of an Early Region of the Bacteriophage T4 Genome

    PubMed Central

    Macdonald, Paul M.; Mosig, Gisela

    1984-01-01

    We have cloned DNA restriction fragments from the largely nonessential region of bacteriophage T4 located between genes 39 and 56. The cloned DNA fragments were used to construct a precise map of the sites in this region recognized by eight restriction endonucleases. This restriction map allowed us to compare the cytosine-containing T4 DNA used for cloning with the hydroxymethylcytosine-containing DNA of wild-type T4; there were no detectable rearrangements in the region tested. We were also able to determine the physical locations of several deletion end points and of several genes. PMID:6319228

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

    PubMed Central

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

    2014-01-01

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

  14. Restriction endonuclease inhibitor IPI* of bacteriophage T4

    PubMed Central

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

    2008-01-01

    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

  15. Structure of the bacteriophage T4 long tail fiber receptor-binding tip

    PubMed Central

    Bartual, Sergio G.; Otero, José M.; Garcia-Doval, Carmela; Llamas-Saiz, Antonio L.; Kahn, Richard; Fox, Gavin C.; van Raaij, Mark J.

    2010-01-01

    Bacteriophages are the most numerous organisms in the biosphere. In spite of their biological significance and the spectrum of potential applications, little high-resolution structural detail is available on their receptor-binding fibers. Here we present the crystal structure of the receptor-binding tip of the bacteriophage T4 long tail fiber, which is highly homologous to the tip of the bacteriophage lambda side tail fibers. This structure reveals an unusual elongated six-stranded antiparallel beta-strand needle domain containing seven iron ions coordinated by histidine residues arranged colinearly along the core of the biological unit. At the end of the tip, the three chains intertwine forming a broader head domain, which contains the putative receptor interaction site. The structure reveals a previously unknown beta-structured fibrous fold, provides insights into the remarkable stability of the fiber, and suggests a framework for mutations to expand or modulate receptor-binding specificity. PMID:21041684

  16. Genetic Analysis of Bacteriophage P22 Lysozyme Structure

    PubMed Central

    Rennell, D.; Poteete, A. R.

    1989-01-01

    The suppression patterns of 11 phage P22 mutants bearing different amber mutations in the gene encoding lysozyme (19) were determined on six different amber suppressor strains. Of the 60 resulting single amino acid substitutions, 18 resulted in defects in lysozyme activity at 30°; an additional seven were defective at 40°. Revertants were isolated on the ``missuppressing'' hosts following UV mutagenesis; they were screened to distinguish primary- from second-site revertants. It was found that second-site revertants were recovered with greater efficiency if the UV-irradiated phage stocks were passaged through an intermediate host in liquid culture rather than plated directly on the nonpermissive host. Eleven second-site revertants (isolated as suppressors of five deleterious substitutions) were sequenced: four were intragenic, five extragenic; three of the extragenic revertants were found to have alterations near and upstream from gene 19, in gene 13. Lysozyme genes from the intragenic revertant phages were introduced into unmutagenized P22, and found to confer the revertant plating phenotype. PMID:2599364

  17. Time-resolved fluorescence of the bacteriophage T4 capsid protein gp23

    Microsoft Academic Search

    Aike Stortelder; Joost B. Buijs; Jaap Bulthuis; Saskia M. van der Vies; Cees Gooijer; Gert van der Zwan

    2005-01-01

    The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated. The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera. Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting.

  18. Expression of the bacteriophage T4 denV structural gene in Escherichia coli

    SciTech Connect

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

    1986-11-01

    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.

  19. ShineDalgarno sequence of bacteriophage T4: GAGG prevails in early genes

    Microsoft Academic Search

    Naglis Malys

    Translation initiation is governed by a limited number of mRNA sequence motifs within the translation initiation region (TIR).\\u000a In bacteria and bacteriophages, one of the most important determinants is a Shine-Dalgarno (SD) sequence that base pairs with\\u000a the anti-SD sequence GAUCACCUCCUUA localized in the 3? end of 16S rRNA. This work assesses a diversity of TIR features in\\u000a phage T4,

  20. Rates of Spontaneous Mutation in Bacteriophage T4 Are Independent of Host Fidelity Determinants

    PubMed Central

    Santos, M. E.; Drake, J. W.

    1994-01-01

    Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity. PMID:7851754

  1. Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

    PubMed Central

    2010-01-01

    Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms. PMID:21129202

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

    NASA Astrophysics Data System (ADS)

    Zheng, Wenjun; Glenn, Paul

    2015-01-01

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

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

    SciTech Connect

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

    1985-08-01

    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.

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

    SciTech Connect

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

    2007-10-25

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

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

    SciTech Connect

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

    1987-03-01

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

  6. Capsid Size and Deoxyribonucleic Acid Length: the Petite Variant of Bacteriophage T4

    PubMed Central

    Eiserling, Frederick A.; Geiduschek, E. Peter; Epstein, Richard H.; Metter, E. Jeffrey

    1970-01-01

    A mutant which produces a small-headed (“petite”) variant of bacteriophage T4 is described. The mutation (E920g) maps in a new gene (66) between genes 23 and 24. Petite phage particles composed up to 70% of the phage yield. The petite phage was nonviable upon single infection but produced progeny when two or more infected a cell. Its genome was shortened by a random deletion of about 30%, and deoxyribonucleic acid (DNA) extracted from the particles was 0.68 the length of normal T4 DNA. The reduction in DNA length was accompanied by a proportional reduction in head volume. Double mutants between E920g and head-defective mutants in gene 21 produced unusually high frequencies of spherical capsidlike structures (?-particles). Images PMID:4924630

  7. Shine-Dalgarno sequence of bacteriophage T4: GAGG prevails in early genes.

    PubMed

    Malys, Naglis

    2012-01-01

    Translation initiation is governed by a limited number of mRNA sequence motifs within the translation initiation region (TIR). In bacteria and bacteriophages, one of the most important determinants is a Shine-Dalgarno (SD) sequence that base pairs with the anti-SD sequence GAUCACCUCCUUA localized in the 3' end of 16S rRNA. This work assesses a diversity of TIR features in phage T4, focusing on the SD sequence, its spacing to the start codon and relationship to gene expression and essentiality patterns. Analysis shows that GAGG is predominant of all core SD motifs in T4 and its related phages, particularly in early genes. Possible implication of the RegB activity is discussed. PMID:21533668

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

    PubMed Central

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

    2002-01-01

    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

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

    PubMed

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

    2002-10-01

    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

  10. Direct Participation of dCMP Hydroxymethylase in Synthesis of Bacteriophage T4 DNA

    PubMed Central

    Wovcha, Merle G.; Tomich, Paul K.; Chiu, Che-Shen; Greenberg, G. Robert

    1973-01-01

    In order to retain in an in situ system the control mechanisms involved in synthesis of bacteriophage T4 DNA, infected cells were made permeable to nucleotides by plasmolysis with concentrated sucrose. Such preparations use exogenous deoxyribonucleotides to synthesize T4 phage DNA. As has been observed with in vivo studies, DNA synthesis was drastically reduced in plasmolyzed preparations from cells infected by amber mutants of genes 1, 32, 41, 42, 43, 44, or 45. Added 5-hydroxymethyl dCTP did not bypass either a mutant of gene 42 (dCMP hydroxymethylase) or of gene 1 (phage-induced deoxyribonucleotide kinase). In a phage system lacking deoxycytidine triphosphatase (gene 56) and the gene-46 product, and therefore incorporating dCTP into DNA, dCTP incorporation did not require dCMP hydroxymethylase, in keeping with in vivo results. With a triple amber mutant of genes 1, 46, and 56 only slight incorporation of dCTP occurred. By contrast, in experiments performed in vivo the synthesis of cytosine-containing DNA was unaffected by an amber mutation in gene 1. These studies provide evidence that dCMP hydroxymethylase, in addition to its known catalytic function, has a second, more direct, role in phage T4 DNA synthesis, apparently in recognition of hydroxymethyl dCTP. The role of the phage-induced deoxyribonucleotide kinase in T4 DNA synthesis in the plasmolyzed system remains unresolved. PMID:4525160

  11. Structure, Assembly, and DNA Packaging of the Bacteriophage T4 Head

    PubMed Central

    Black, Lindsay W.; Rao, Venigalla B.

    2014-01-01

    The bacteriophage T4 head is an elongated icosahedron packed with 172 kb of linear double-stranded DNA and numerous proteins. The capsid is built from three essential proteins: gp23*, which forms the hexagonal capsid lattice; gp24*, which forms pentamers at 11 of the 12 vertices; and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. Intensive work over more than half a century has led to a deep understanding of the phage T4 head. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as numerous other icosahedral bacteriophages. However, phage T4 displays an unusual membrane and portal initiated assembly of a shape determining self-sufficient scaffolding core. Folding of gp23 requires the assistance of two chaperones, the Escherichia coli chaperone GroEL acting with the phage-coded gp23-specific cochaperone, gp31. The capsid also contains two nonessential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. Through binding to adjacent gp23 subunits, Soc reinforces the capsid structure. Hoc and Soc have been used extensively in bipartite peptide display libraries and to display pathogen antigens, including those from human immunodeficiency virus (HIV), Neisseria meningitides, Bacillus anthracis, and foot and mouth disease virus. The structure of Ip1*, one of a number of multiple (>100) copy proteins packed and injected with DNA from the full head, shows it to be an inhibitor of one specific restriction endonuclease specifically targeting glycosylated hydroxymethyl cytosine DNA. Extensive mutagenesis, combined with atomic structures of the DNA packaging/terminase proteins gp16 and gp17, elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. The cryoelectron microscopy structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at the highest rate known and can package multiple segments. Förster resonance energy transfer–fluorescence correlation spectroscopy studies indicate that DNA gets compressed in the stalled motor and that the terminase-to-portal distance changes during translocation. Current evidence suggests a linear two-component (large terminase plus portal) translocation motor in which electrostatic forces generated by ATP hydrolysis drive DNA translocation by alternating the motor between tensed and relaxed states. PMID:22420853

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

    SciTech Connect

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

    1985-07-01

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

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

    SciTech Connect

    Childs, J.D.; Pilon, R.

    1983-05-01

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

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

    SciTech Connect

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

    2009-07-22

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  16. Synthesis of T4 DNA and bacteriophage in the absence of dCMP hydroxymethylase.

    PubMed Central

    Morton, D; Kutter, E M; Guttman, B S

    1978-01-01

    Several lines of research have suggested that the dCMP hydroxymethylase (HMase) coded by bacteriophage T4 is an essential protein in a DNA replication complex, as well as a supplier of hydroxymethyl dCMP for phage DNA synthesis. We show that a mutant [HMase, dCTPase, endonuclease II, endonuclease IV] which lacked this enzyme made cytosine-containing DNA at about two-thirds of the normal rate. When coupled with an alc mutation which permitted synthesis of late proteins, a small burst of phage was produced whose DNA contained no hydroxymethylcytosine. This pentuple mutant made both early and late proteins with abnormal kinetics, whereas the HMase+ parent showed normal kinetics. However, intracellular phage DNA showed no gross abnormalities in alkaline sucrose gradients. We conclude that HMase is not required for DNA synthesis when hydroxymethyl dCMP is not needed as a substrate; however, its absence still impairs both replication and transcription. Images PMID:212605

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

    SciTech Connect

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

    1991-08-01

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

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

    PubMed Central

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

    2014-01-01

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

  19. Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4*

    PubMed Central

    Branagan, Amy M.; Klein, Jenny A.; Jordan, Christian S.; Morrical, Scott W.

    2014-01-01

    The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates. PMID:24338568

  20. Characterization of the flexible lip regions in bacteriophage lambda lysozyme using MD simulations.

    PubMed

    Smith, Lorna J; van Gunsteren, Wilfred F; Hansen, Niels

    2015-05-01

    The upper and lower lip regions in lysozyme from bacteriophage lambda (?-lysozyme) are flexible in solution and exhibit two different conformations in crystal structures of the protein. MD simulations have been used to characterize the structure and dynamics of these lip regions, which surround the active site. Ten different simulations have been run including those with restraining to experimental NOE distance and (1)H-(15)N order parameter data. The simulations show that the lower lip region, although undergoing considerable backbone fluctuations, contains two persistent ?-strands. In the upper lip region, a wide range of conformations are populated and it is not clear from the available data whether some helical secondary structure is present. The work provides a clear example of the advantages of combining MD simulations with experimental data to obtain a structural interpretation of the latter. In this case, time-averaged order parameter restraining has played an essential role in enabling convergence between two different starting structures and identifying the extent to which flexible regions in solution can contain persistent secondary structure. PMID:25820531

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

    PubMed Central

    Chiu, Che-Shen; Greenberg, G. Robert

    1973-01-01

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

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

    SciTech Connect

    Rao, V.B.

    1997-09-01

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

  3. Bacteriophage T4 alc gene product: general inhibitor of transcription from cytosine-containing DNA.

    PubMed Central

    Kutter, E M; Bradley, D; Schenck, R; Guttman, B S; Laiken, R

    1981-01-01

    The alc gene of bacteriophage T4 was originally defined on the basis of mutations which allow late protein synthesis directed by T4 DNA containing cytosine rather than hydroxymethylcytosine. The question remained whether the normal alc gene product (gpalc) also blocks the transcription of early genes from cytosine-containing DNA. Complementation experiments were performed between hydroxymethylcytosine-containing phage which direct gpalc synthesis but carry mutations in a given gene(s) and cytosine-containing phage carrying that gene(s). The required protein would then have to be directed by the cytosine-containing DNA: it is looked for directly on polyacrylamide gels or through its physiological effects or both. For all early proteins examined in this way, no synthesis was observed when 95 to 100% of the hydroxymethylcytosine was substituted by cytosine in the infecting DNA, whereas there was significant synthesis with 75% substitution or less. The results indicate that gpalc is carried in with the infecting DNA or is made very early to block transcription of all cytosine-containing DNA. Images PMID:7321103

  4. Bacteriophage T5 structure reveals similarities with HK97 and T4 suggesting evolutionary relationships.

    PubMed

    Effantin, G; Boulanger, P; Neumann, E; Letellier, L; Conway, J F

    2006-09-01

    Evolutionary relationships between viruses may be obscure by protein sequence but unmasked by structure. Analysis of bacteriophage T5 by cryo-electron microscopy and protein sequence analysis reveals analogies with HK97 and T4 that suggest a mosaic of such connections. The T5 capsid is consistent with the HK97 capsid protein fold but has a different geometry, incorporating three additional hexamers on each icosahedral facet. Similarly to HK97, the T5 major capsid protein has an N-terminal extension, or Delta-domain that is missing in the mature capsid, and by analogy with HK97, may function as an assembly or scaffold domain. This Delta-domain is predicted to be largely coiled-coil, as for that of HK97, but is approximately 70% longer correlating with the larger capsid. Thus, capsid architecture appears likely to be specified by the Delta-domain. Unlike HK97, the T5 capsid binds a decoration protein in the center of each hexamer similarly to the "hoc" protein of phage T4, suggesting a common role for these molecules. The tail-tube has unusual trimeric symmetry that may aid in the unique two-stage DNA-ejection process, and joins the tail-tip at a disk where tail fibers attach. This intriguing mix of characteristics embodied by phage T5 offers insights into virus assembly, subunit function, and the evolutionary connections between related viruses. PMID:16876823

  5. Systematic analysis of domain motions in proteins from conformational change: new results on citrate synthase and T4 lysozyme.

    PubMed

    Hayward, S; Berendsen, H J

    1998-02-01

    Methods developed originally to analyze domain motions from simulation [Proteins 27:425-437, 1997] are adapted and extended for the analysis of X-ray conformers and for proteins with more than two domains. The method can be applied as an automatic procedure to any case where more than one conformation is available. The basis of the methodology is that domains can be recognized from the difference in the parameters governing their quasi-rigid body motion, and in particular their rotation vectors. A clustering algorithm is used to determine clusters of rotation vectors corresponding to main-chain segments that form possible dynamic domains. Domains are accepted for further analysis on the basis of a ratio of interdomain to intradomain fluctuation, and Chasles' theorem is used to determine interdomain screw axes. Finally residues involved in the interdomain motion are identified. The methodology is tested on citrate synthase and the M6I mutant of T4 lysozyme. In both cases new aspects to their conformational change are revealed, as are individual residues intimately involved in their dynamics. For citrate synthase the beta sheet is identified to be part of the hinging mechanism. In the case of T4 lysozyme, one of the four transitions in the pathway from the closed to the open conformation, furnished four dynamic domains rather than the expected two. This result indicates that the number of dynamic domains a protein possesses may not be a constant of the motion. PMID:9489922

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

    PubMed

    Arnold, G E; Ornstein, R L

    1997-04-15

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

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

    PubMed Central

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

    2013-01-01

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

  8. Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages*

    PubMed Central

    Gao, Song; Rao, Venigalla B.

    2011-01-01

    Tailed bacteriophages use powerful molecular motors to package the viral genome into a preformed capsid. Packaging at a rate of up to ?2000 bp/s and generating a power density twice that of an automobile engine, the phage T4 motor is the fastest and most powerful reported to date. Central to DNA packaging are dynamic interactions among the packaging components, capsid (gp23), portal (gp20), motor (gp17, large “terminase”), and regulator (gp16, small terminase), leading to precise orchestration of the packaging process, but the mechanisms are poorly understood. Here we analyzed the interactions between small and large terminases of T4-related phages. Our results show that the gp17 packaging ATPase is maximally stimulated by homologous, but not heterologous, gp16. Multiple interaction sites are identified in both gp16 and gp17. The specificity determinants in gp16 are clustered in the diverged N- and C-terminal domains (regions I–III). Swapping of diverged region(s), such as replacing C-terminal RB49 region III with that of T4, switched ATPase stimulation specificity. Two specificity regions, amino acids 37–52 and 290–315, are identified in or near the gp17-ATPase “transmission” subdomain II. gp16 binding at these sites might cause a conformational change positioning the ATPase-coupling residues into the catalytic pocket, triggering ATP hydrolysis. These results lead to a model in which multiple weak interactions between motor and regulator allow dynamic assembly and disassembly of various packaging complexes, depending on the functional state of the packaging machine. This might be a general mechanism for regulation of the phage packaging machine and other complex molecular machines. PMID:21127059

  9. Structure-Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

    PubMed Central

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

    2013-01-01

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

  10. Time-resolved fluorescence of the bacteriophage T4 capsid protein gp23.

    PubMed

    Stortelder, Aike; Buijs, Joost B; Bulthuis, Jaap; van der Vies, Saskia M; Gooijer, Cees; van der Zwan, Gert

    2005-01-14

    The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated. The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera. Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting. A red-shift of the emission maximum within the first nanosecond of decay is observed, which can be explained by the different decay-associated spectra of fluorescence lifetimes of the protein in combination with dipolar relaxation. In addition, iodide quenching experiments are performed, to study the degree of exposure of the various tryptophan residues. A model for the origin of the observed lifetimes of 0.032 +/- 0.003, 0.39 +/- 0.06, 2.1 +/- 0.1 and 6.8 +/- 0.8 ns is presented: the 32 ps lifetime can be assigned to the emission of a buried tryptophan residue, the 0.4 and 2.1 ns lifetimes to two partly buried residues, and the 6.8 ns lifetime to a single tryptophan outside the bulk of the folded gp23. PMID:15629249

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

    PubMed Central

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

    2012-01-01

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

  12. Isolation and Genomic Characterization of the T4-Like Bacteriophage PM2 Infecting Pectobacterium carotovorum subsp. carotovorum

    PubMed Central

    Lim, Jeong-A; Lee, Dong Hwan; Heu, Sunggi

    2015-01-01

    In order to control Pectobacterium carotovorum subsp. carotovorum, a novel virulent bacteriophage PM2 was isolated. Bacteriophage PM2 can infect 48% of P. carotovorum subsp. carotovorum and 78% of P. carotovorum subsp. brasilliensis but none of atrosepticum, betavasculorum, odoriferum and wasabiae isolates had been infected with PM2. PM2 phage belongs to the family Myoviridae, and contains a large head and contractile tail. It has a 170,286 base pair genome that encodes 291 open reading frames (ORFs) and 12 tRNAs. Most ORFs in bacteriophage PM2 share a high level of homology with T4-like phages including IME08, RB69, and JS98. Phylogenetic analysis based on the amino acid sequence of terminase large subunits confirmed that PM2 is classified as a T4-like phage. It contains no integrase- or no repressor-coding genes related to the lysogenic cycle, and lifestyle prediction using PHACT software suggested that PM2 is a virulent bacteriophage. PMID:25774115

  13. Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates

    Microsoft Academic Search

    Pernilla Lagerback; Evalena Andersson; Christer Malmberg; Karin Carlson

    2009-01-01

    The oligomerization state and mode of binding to DNA of the GIY-YIG endonuclease II (EndoII) from bacteriophage T4 was studied using gel filtration and electrophoretic mobility shift assays with a set of mutants previously found to have altered enzyme activity. At low enzyme\\/DNA ratios all mutants except one bound to DNA only as tetramers to two DNA substrates. The putatively

  14. Properties of the nonlethal recombinational repair x and y mutants of bacteriophage T4. II. DNA synthesis.

    PubMed Central

    Melamede, R J; Wallace, S S

    1977-01-01

    The bacteriophage T4 recombination-deficient mutants x and y exhibited decreased rates of DNA synthesis as compared to wild-type T4. Mutant-induced DNA synthesis was more sensitive to mitomycin C than was wild-type synthesis. However, DNA synthesis in mutant- and wild-type-infected cells exhibited the same sensitivity to UV light and X-irradiation. When high-specific-activity label was administered at various times postinfection, mutant DNA synthesis resembled that of wild type for 12 min. after which time mutant-induced incorporation was greatly decreased and sensitive to mitomycin C as compared to that of the wild type. Rifampin and chloramphenicol studies indicated that the gene products necessary for synthesis measured at 15 min postinfection, including those of x+ and y+ were transcribed within 2 min and translated within 8 min postinfection. Administration of chloramphenicol to mutant x- or mutant y-infected cells exactly 8 min postinfection, however, allowed for increased synthesis at 15 min that was sensitive to mitomycin C. Cells coinfected with T4+ and T4x or T4x and T4y retained a reduced mutant-type synthesis, whereas cells coinfected with T4+ and T4y exhibited a synthesis more closely resembling that of wild type. PMID:904025

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

    PubMed

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

    2013-07-01

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

  16. Alanine Scanning Mutagenesis of the ?-Helix 115-123 of Phage T4 Lysozyme: Effects on Structure, Stability and the Binding of Solvent

    Microsoft Academic Search

    Michael Blaber; Walter A. Baase; Nadine Gassner; Brian W. Matthews

    1995-01-01

    A series of individual alanine mutations has been constructed in the helical region 115 to 123 in phage T4 lysozyme in order to evaluate the contribution to protein stability of the different side-chains within this region. Pairwise alanine mutations and a combination mutant with seven alanine substitutions were constructed to evaluate the additive effects upon structure and stability. Only three

  17. Expression of bacteriophage T4 gene 25 is regulated via RNA secondary structure in the translational initiation region.

    PubMed

    Nivinskas, R; Vaiskunaite, R; Raudonikiene, A

    1993-04-01

    Analysis of the nucleotide sequence in the 5' flanking region of bacteriophage T4 gene 25 revealed three potential Shine and Dalgarno sequences, SD1, SD2 and SD3, with a spacing of 8, 17 and 27 nucleotides from the initiation codon of this gene, respectively. Results of our experiments in the bacteriophage T7 expression system clearly demonstrate that the SD3 sequence is required for efficient expression of gene 25. We propose the existence of a stem-loop structure that includes SD1 and SD2 sequences and brings the SD3 sequence to a favourable spacing with the initiation codon of gene 25. Since the predicted secondary structure in the translational initiation region of gene 25 is relatively unstable and the SD3 sequence, GAGG, is more typical than the SD1 sequence, GAG, we suggest that this structure could control the level of gene expression. PMID:8478927

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

    PubMed

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

    1979-01-01

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

  19. Use of a non-rigid region in T4 lysozyme to design an adaptable metal-binding site.

    PubMed

    Wray, J W; Baase, W A; Ostheimer, G J; Zhang, X J; Matthews, B W

    2000-05-01

    It is not easy to find candidate sites within a given protein where the geometry of the polypeptide chain matches that of metal-binding sites in known protein structures. By choosing a location in T4 lysozyme that is inherently flexible, it was possible to engineer a two-histidine site that binds different divalent cations. Crystallographic analysis shows that the geometry of binding of zinc is distorted tetrahedral while that of cobalt and nickel is octahedral. Insofar as spectroscopic data can be measured, they indicate that similar modes of coordination are retained in solution. The two substitutions, Thr21 --> His and Thr142 --> His, lie, respectively, on the surface of the N- and C-terminal domains on opposite sides of the active site cleft. The design takes advantage of hinge-bending motion which allows the binding site to adapt to the most favorable ligand geometry for the metal. Introduction of the two histidines increases the melting temperature of the protein by 2.0 degrees C at pH 7.4. Metal binding further increases the melting temperature, but only by a small amount (up to 1.5 degrees C). A third substitution, Gln141 --> His, which could act as a third ligand in principle, does not do so, demonstrating the difficulty in mimicking naturally occurring metal-binding sites. PMID:10835104

  20. Reconstruction of Bacteriophage T4 DNA Replication Apparatus from Purified Components: Rolling Circle Replication Following de novo Chain Initiation on a Single-Stranded Circular DNA Template

    Microsoft Academic Search

    Charles F. Morris; Navin K. Sinha; Bruce M. Alberts

    1975-01-01

    The protein products of T4 bacteriophage genes 41, 43, 45, 44, and 62 have been purified to near homogeneity using an assay which measures their stimulation of DNA synthesis in a crude lysate of Escherichia coli cells infected by an appropriate mutant phage. When all of these proteins and T4 gene 32 protein are incubated in the presence of deoxyribonucleoside

  1. Detection of Escherichia coli in the sewage influent by fluorescent labeled T4 phage

    Microsoft Academic Search

    Kazuhiko Miyanaga; Tomonori Hijikata; Chiaki Furukawa; Hajime Unno; Yasunori Tanji

    2006-01-01

    For a precise estimation of sanitary condition, Escherichia coli detection system using E. coli-specific bacteriophage T4 was constructed. To facilitate E. coli detection, T4e? phage which did not produce the lysozyme was constructed and green fluorescent protein (GFP) was displayed on T4e? small outer capside (SOC) protein. This T4e?\\/GFP can detect E. coli K12 without cell lysis. In this study,

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

    SciTech Connect

    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

    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.

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

    PubMed Central

    2010-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  5. An ORFan no more: the bacteriophage T4 39.2 gene product, NwgI, modulates GroEL chaperone function.

    PubMed

    Ang, Debbie; Georgopoulos, Costa

    2012-03-01

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

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

    SciTech Connect

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

    2011-09-16

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

  7. Binding of the bacteriophage T4 regA protein to mRNA targets: an initiator AUG is required.

    PubMed Central

    Unnithan, S; Green, L; Morrissey, L; Binkley, J; Singer, B; Karam, J; Gold, L

    1990-01-01

    Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein. Images PMID:2263467

  8. Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex.

    PubMed Central

    Song, H K; Sohn, S H; Suh, S W

    1999-01-01

    Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 2.1.2.8), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyl-dCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 A resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (approximately 20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built. PMID:10064578

  9. Simultaneous initiation of synthesis of bacteriophage T4 DNA and of deoxyribonucleotides.

    PubMed Central

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

    1976-01-01

    In earlier reports we have suggested that bacteriophate T4 DNA replication occurs in a complex composed of the proteins required for polymerization and the system of enzymes synthesizing the deoxyribonucleoside triphosphate precursors of DNA. T4-induced dCMP hydroxymethylase and dTMP synthetase, though demonstrable in extracts soon after infection, are not active in vivo until about 5 min. The in vivo activities increase exponentially for approximately 15 min and then become constant. We have suggested that the exponential period represents the formation of the complexes. This paper shows that the initiation of DNA synthesis and of the two deoxyribonucleotide-synthesizing activities occurs simultaneously and with coinciding exponential kinetics. The in vivo activities of the two enzymes were tested after infection by a number of T4 amber Dna- mutants. Their activities were essentially unchanged compared to the wild-type phage, except on infection by mutants of gene 43 (T4 DNA nucleotidyltransferase or DNA polymerase). With these mutants the rate of increase of dTMP synthetase and dCMP hydroxymethylase activities was always substantially lower than after infection by wild-type phage. It is proposed that an intimate interaction occurs between T4-induced DNA polymerase and the complex of enzymes forming 5-hydroxymethyl-dCMP and dTMP. PMID:1062786

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2012-01-01

    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. PMID:22207623

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

    PubMed Central

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

    1976-01-01

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

  13. Partial complementation of the UV sensitivity of E. coli and yeast excision repair mutants by the cloned denV gene of bacteriophage T4

    Microsoft Academic Search

    Janet M. Chenevert; Louie Naumovski; Roger A. Schultz; Errol C. Friedberg

    1986-01-01

    The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors. The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E. coli and in yeast. Induction of the TAC promoter with IPTG resulted in overexpression of the gene,

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

    SciTech Connect

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

    2009-03-06

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

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

    SciTech Connect

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

    2009-07-21

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

  16. 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

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

    1990-02-01

    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.

  17. Bacteriophage

    NSDL National Science Digital Library

    Gene Mayer

    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.

  18. Mutants in a Nonessential Gene of Bacteriophage T4 Which Are Defective in the Degradation of Escherichia coli Deoxyribonucleic Acid 1

    PubMed Central

    Hercules, Kitty; Munro, Judith L.; Mendelsohn, Steven; Wiberg, John S.

    1971-01-01

    Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine (3H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the 3H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4. Images PMID:5543437

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

    SciTech Connect

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

    1994-12-31

    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.

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

    PubMed Central

    Esvelt, Kevin M.; Church, George M.

    2015-01-01

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

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

    PubMed Central

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

    2002-01-01

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Cavity as a source of conformational fluctuation and high-energy state: high-pressure NMR study of a cavity-enlarged mutant of T4 lysozyme.

    PubMed

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

    2015-01-01

    Although the structure, function, conformational dynamics, and controlled thermodynamics of proteins are manifested by their corresponding amino acid sequences, the natural rules for molecular design and their corresponding interplay remain obscure. In this study, we focused on the role of internal cavities of proteins in conformational dynamics. We investigated the pressure-induced responses from the cavity-enlarged L99A mutant of T4 lysozyme, using high-pressure NMR spectroscopy. The signal intensities of the methyl groups in the (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

  4. A species barrier between bacteriophages T2 and T4: exclusion, join-copy and join-cut-copy recombination and mutagenesis in the dCTPase genes.

    PubMed Central

    Gary, T P; Colowick, N E; Mosig, G

    1998-01-01

    Bacteriophage T2 alleles are excluded in crosses between T2 and T4 because of genetic isolation between these two virus species. The severity of exclusion varies in different genes, with gene 56, encoding an essential dCT(D)Pase/dUT(D)Pase of these phages, being most strongly affected. To investigate reasons for such strong exclusion, we have (1) sequenced the T2 gene 56 and an adjacent region, (2) compared the sequence with the corresponding T4 DNA, (3) constructed chimeric phages in which T2 and T4 sequences of this region are recombined, and (4) tested complementation, recombination, and exclusion with gene 56 cloned in a plasmid and in the chimeric phages in Escherichia coli CR63, in which growth of wild-type T2 is not restricted by T4. Our results argue against a role of the dCTPase protein in this exclusion and implicate instead DNA sequence differences as major contributors to the apparent species barrier. This sequence divergence exhibits a remarkable pattern: a major heterologous sequence counter-clockwise from gene 56 (and downstream of the gene 56 transcripts) replaces in T2 DNA the T4 gene 69. Gene 56 base sequences bordering this substituted region are significantly different, whereas sequences of the dam genes, adjacent in the clockwise direction, are similar in T2 and in T4. The gene 56 sequence differences can best be explained by multiple compensating frameshifts and base substitutions, which result in T2 and T4 dCTPases whose amino acid sequences and functions remain similar. Based on these findings we propose a model for the evolution of multiple sequence differences concomitant with the substitution of an adjacent gene by foreign DNA: invasion by the single-stranded segments of foreign DNA, nucleated from a short DNA sequence that was complementary by chance, has triggered recombination-dependent replication by "join-copy" and "join-cut-copy" pathways that are known to operate in the T-even phages and are implicated in other organisms as well. This invasion, accompanied by heteroduplex formation between partially similar sequences, and perhaps subsequent partial heteroduplex repair, simultaneously substituted T4 gene 69 for foreign sequences and scrambled the sequence of the dCTPase gene 56. We suggest that similar mechanisms can mobilize DNA segments for horizontal transfer without necessarily requiring transposase or site-specific recombination functions. PMID:9560366

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

    PubMed

    Chang, P L; Yen, T F

    1984-03-01

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

  6. Hexamerization of the bacteriophage T4 capsid protein gp23 and its W13V mutant studied by time-resolved tryptophan fluorescence.

    PubMed

    Stortelder, Aike; Hendriks, Johnny; Buijs, Joost B; Bulthuis, Jaap; Gooijer, Cees; van der Vies, Saskia M; van der Zwan, Gert

    2006-12-14

    The bacteriophage T4 capsid protein gp23 was studied using time-resolved and steady-state fluorescence of the intrinsic protein fluorophore tryptophan. In-vitro gp23 consists mostly of monomers at low temperature but forms hexamers at room temperature. To extend our knowledge of the structure and hexamerization characteristics of gp23, the temperature-dependent fluorescence properties of a tryptophan mutant (W13V) were compared to those of wild-type gp23. The W13V mutation is located in the N-terminal part of the protein, which is cleaved off after prohead formation in the live bacteriophage. Results show that W13 plays a role in the hexamerization process but is not needed to stabilize the hexamer once it is formed. Furthermore, besides the monomer-to-hexamer temperature transition (15-23 degrees C and 12-43 degrees C for wild-type and W13V gp23, respectively), we were able to observe denaturation of the N-terminus in hexameric wild-type gp23 around 40 degrees C. In addition, with the aid of a recently published homology model of gp23, the lifetimes obtained from time-resolved fluorescence measurements could tentatively be assigned to specific tryptophan residues. PMID:17149929

  7. Role of primary photoacceptors in low-power laser effects: action of He-Ne laser radiation on bacteriophage T4-Escherichia coli interaction

    SciTech Connect

    Tiphlova, O.; Karu, T.

    1989-01-01

    The effect of He-Ne laser radiation (lambda = 632.8 nm) on bacteriophage T4-Escherichia coli WP2 interactions was studied. Irradiation of bacteria having respiratory chain components as primary photoacceptors accelerated their division in a dose-dependent manner, but irradiation had no effect on the properties of the phage (measured as its ability to infect host cells). At the same time, exposure of bacteria to stimulating doses of He-Ne laser radiation (from 10(3) to 6 x 10(4) J/m2) increased their ability to promote the growth of unexposed phages. These results clearly indicate that low-power laser effects require primary photoacceptors (phage contains no chromophores for red light).

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

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

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

    2002-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  11. Bacteriophage T4 nrdA and nrdB genes, encoding ribonucleotide reductase, are expressed both separately and coordinately: characterization of the nrdB promoter.

    PubMed Central

    Tseng, M J; He, P; Hilfinger, J M; Greenberg, G R

    1990-01-01

    We examined the expression of the bacteriophage T4 nrdA and nrdB genes, which encode the alpha 2 and beta 2 subunits, respectively, of ribonucleoside diphosphate reductase, the first committed enzyme in the pathway of synthesis of the deoxyribonucleoside triphosphates. T4 nrdA, located 700 bp upstream from nrdB, has been shown previously to be transcribed by two major transcripts: a prereplicative, polycistronic message, TU, orginating at an immediate-early promoter, PE, that is 3.5 kb upstream from nrdA, and a postreplicative message commencing from a late promoter in its 5' flank. We have found a third promoter initiating a transcript at 159 nucleotides upstream from the reading frame of nrdB. PnrdB functions only in the presence of the T4 motA gene product, which is required for middle (time) promoters, and therefore the onset of nrdB transcription is delayed more than 2 min after infection. Because of the distance of nrdA from PE, the inception of nrdA transcription (delayed early) coincides closely with that of nrdB. An apparent termination site, tA, occurs about 80 bp downstream from nrdA. Some of the polycistronic mRNA reading through the site after 5 min contributes to nrdB transcription. nrdA and nrdB genes in an uninfected host have been reported to be transcribed only coordinately. In contrast, T4 nrdA and nrdB are initially transcribed separately onto the PE and PnrdB transcripts, respectively, but at about 5 min after infection are transcribed both coordinately and on separate transcripts. Evidence is presented that TU coordinately transcribes a deoxyribonucleotide operon in the order: frd, td, gene 'Y,' nrdA, nrdB. Since the beta 2 subunit is known to be formed after the alpha 2 subunit, the expression of the nrdB gene determines the onset of deoxyribonucleoside triphosphate synthesis and thus of T4 DNA replication. Images PMID:2228963

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

    PubMed Central

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

    2013-01-01

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

  13. Optical tweezers studies of viral DNA packaging: Motor function and DNA confinement in Bacteriophages phi29, lambda, and T4

    NASA Astrophysics Data System (ADS)

    Smith, Douglas

    2007-03-01

    In the assembly of many viruses a powerful molecular motor translocates the genome into a pre-assembled capsid. We use optical tweezers to directly measure translocation of a single DNA molecule into the viral capsid. Improved techniques allow us to measure initiation and early stages of packaging. With phi29 the DNA terminal protein was found to cause large variations in the starting point of packaging. Removal of this protein results in terminal initiation, permitting more accurate assessment of motor function and DNA confinement forces. We investigated the role of electrostatic repulsion by varying ionic screening of the DNA. The observed trends are in accord with those theoretically expected considering counter-ion competition; however the forces are larger than expected in comparison with recent theories and DNA ejection measurements. We have recently succeeded in extending our methods to study two other phages: lambda and T4. These systems have unique structural and functional features, presenting an opportunity for comparative studies in this family of molecular motors. Initial measurements show that lambda and T4 translocate DNA several times faster than the phi29 motor, but are more sensitive to applied load.

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

    PubMed Central

    Watts, N R; Coombs, D H

    1989-01-01

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

  15. denV gene of bacteriophage T4 restores DNA excision repair to mei-9 and mus201 mutants of Drosophila melanogaster

    SciTech Connect

    Banga, S.S.; Boyd, J.B.; Valerie, K.; Harris, P.V.; Kurz, E.M.; de Riel, J.K. (Univ. of California, Davis (USA))

    1989-05-01

    The denV gene of bacteriophage T4 was fused to a Drosophila hsp70 (70-kDa heat shock protein) promoter and introduced into the germ line of Drosophila by P-element-mediated transformation. The protein product of that gene (endonuclease V) was detected in extracts of heat-shocked transformants with both enzymological and immunoblotting procedures. That protein restores both excision repair and UV resistance to mei-9 and mus201 mutants of this organism. These results reveal that the denV gene can compensate for excision-repair defects in two very different eukayotic mutants, in that the mus201 mutants are typical of excision-deficient mutants in other organisms, whereas the mei-9 mutants exhibit a broad pleiotropism that includes a strong meiotic deficiency. This study permits an extension of the molecular analysis of DNA repair to the germ line of higher eukaryotes. It also provides a model system for future investigations of other well-characterized microbial repair genes on DNA damage in the germ line of this metazoan organism.

  16. Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

    Microsoft Academic Search

    Umender K. Sharma; Dipankar Chatterji

    2008-01-01

    physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent

  17. The non-enzymatic microbicidal activity of lysozymes

    Microsoft Academic Search

    Petra Porsch; Andreas Mahn; Olaf Brinkmann; Werner Gieffers

    1999-01-01

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

  18. An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50*

    PubMed Central

    De la Rosa, Metzere Bierlein; Nelson, Scott W.

    2011-01-01

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

  19. DNA "melting" proteins. II. Effects of bacteriophage T4 gene 32-protein binding on the conformation and stability of nucleic acid structures.

    PubMed

    Jensen, D E; Kelly, R C; von Hippel, P H

    1976-11-25

    Bacteriophage T4-coded gene 32-protein is an essential component of the T4 replication and recombination systems. Alberts and co-workers (Alberts, B.M., Amodio, F.J., Jenkins, M., Gutmann, E.D., and Ferris, F.L. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, 289-305) have shown that the major physiological activity of the protein involves preferential and cooperative binding to single-stranded DNA. In this paper, the physiochemical parameters characterizing this "melting" protein system are quantitatively determined. Boundary sedimentation velocity experiments are used to measure the interaction of gene 32-protein with native DNA. The binding is shown to be non-cooperative and involves an overlapping site size (nh) of approximately 10 nucleotide residues (or approximately 5 nucleotide pairs). In analogy with the ribonuclease results (Jensen, D.E., and von Hippel, P.H. (1976) J. Biol. Chem. 251, 7198-7214), the logarithm of the association constant (Kh) is found to be linerarly related to log [Na+]. The binding of gene 32-protein to denatured (single-stranded) DNA involves appreciable distortion of the polynucleotide backbone from the unliganded conformation; binding totally unstacks the bases of both ribose- and deoxyribose-containing polynucleotides at 10 degrees, and results in a hyperchromic change exceeding that which can be induced by heating. This hyperchromism induced in poly(dA) on binding gene 32-protein under low salt (tight binding) conditions is used to determine a value of nc (the single-stranded DNA site size) of approximately 6.7 nucldotide residues per protein. In addition, gene 32-protein binding to single-stranded polynucleotide induces an unusual circular dichroic spectrum characterized principally by a marked decrease in the magnitude of the positive CD band centered at approximately 265 nm. This spectral change is attributed to significant uncoupling of the transition moments of the vicinal bases of the single-stranded polynucleotide on gene 32-protein binding, in accord with the ultraviolet hyperchromism observed. Binding of gene 32-protein to double helical DNA has virtually no effect on the spectral properties of this conformation... PMID:791945

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

    SciTech Connect

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

    2006-02-05

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

  1. Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

    PubMed Central

    Masschalck, Barbara; Van Houdt, Rob; Van Haver, Ellen G. R.; Michiels, Chris W.

    2001-01-01

    We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 ?g of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides. PMID:11133464

  2. Naturally resident and exogenously applied T4-like and T5-like bacteriophages can reduce Escherichia coli O157:H7 levels in sheep guts

    PubMed Central

    Raya, Raul R; Oot, Rebecca A; Moore-Maley, Ben; Wieland, Serena; Callaway, Todd R; Kutter, Elizabeth M

    2011-01-01

    In preparing sheep for an in vivo Escherichia coli O157:H7 eradication trial, we found that 20/39 members of a single flock were naturally colonized by O157:H7-infecting phages. Characterization showed these were all one phage type (subsequently named CEV2) infecting 15/16 O157:H7, 7/72 ECOR and common lab strains. Further characterization by PFGE (genome?120 kb), restriction enzyme digest (DNA appears unmodified), receptor studies (FhuA but not TonB is required for infection) and sequencing (>95% nucleotide identity) showed it is a close relative of the classically studied coliphage T5. Unlike T5, CEV2 infects O157:H7 in vitro, both aerobically and anaerobically, rapidly adsorbing and killing, but resistant mutants regrew within 24 h. When used together with T4-like CEV1 (MOI ?2 per phage), bacterial killing was longer lasting. CEV2 did not reproduce when co-infecting the same cell as CEV1, presumably succumbing to CEV1's ability to shut off transcription of cytosine-containing DNA. In vivo sheep trials to remove resident O157:H7 showed that a cocktail of CEV2 and CEV1 (?1011 total PFU) applied once orally was more effective (>99.9% reduction) than CEV1 alone (?99%) compared to the untreated phage-free control. Those sheep naturally carrying CEV2, receiving no additional phage treatment, had the lowest O157:H7 levels (?99.99% reduction). These data suggest that phage cocktails are more effective than individual phage in removing O157:H7 that have taken residence if the phage work in concert with one another and that naturally resident O157:H7-infecting phages may prevent O157:H7 gut colonization and be one explanation for the transient O157:H7 colonization in ruminants. PMID:21687531

  3. Single Molecule Recordings of Lysozyme Activity

    PubMed Central

    Choi, Yongki; Weiss, Gregory A.

    2013-01-01

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

  4. Transcription of the T4 late genes

    Microsoft Academic Search

    E Peter Geiduschek; George A Kassavetis

    2010-01-01

    This article reviews the current state of understanding of the regulated transcription of the bacteriophage T4 late genes, with a focus on the underlying biochemical mechanisms, which turn out to be unique to the T4-related family of phages or significantly different from other bacterial systems. The activator of T4 late transcription is the gene 45 protein (gp45), the sliding clamp

  5. Protein engineering with biosynthesized libraries from Bordetella bronchiseptica bacteriophage.

    PubMed

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

    2013-01-01

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

  6. Protein Engineering with Biosynthesized Libraries from Bordetella bronchiseptica Bacteriophage

    PubMed Central

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

    2013-01-01

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

  7. [Bactericidal effect of lysozyme].

    PubMed

    Podborodov, V M; Shchelkanov, M Iu; Smirnova, I P; Burenkova, L A; Novikova, V P; Aristova, V A; Novikova, E L; Moskvitina, G G; Ioffe, A M

    2013-01-01

    Isolation of lysozyme from hemolymph of Alveonasus lahorensis (Acari: Parasitiformes, Argasidae) and Hyalomma marginatum (Acari: Parasitiformes, Ixodidae) with using ultrasound is described. It was shown that the bactericidal effect of the ultrasound-extracted lysozyme against Staphylococcus aureus and Micrococcus luteus significantly exceeded that of the chicken egg lysozyme and lysozyme from ticks without ultrasound exposure. Disintegration of the hemolymph cells increased lysozyme production. PMID:24640148

  8. Molecular Structure of Lysozyme

    NSDL National Science Digital Library

    2003-01-23

    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).

  9. THE KINETICS OF PARENTAL DEOXYRIBONUCLEIC ACID (DNA) REPLICATION, DEOXYGUANYLATE KINASE FORMATION, AND P*SUP 32\\/INACTIVATION OF THE PARENTAL VIRUS IN ESCHERICHIA COLI INFECTED WITH BACTERIOPHAGE T4

    Microsoft Academic Search

    A. W. Kozinski; M. J. Bessman

    1961-01-01

    The kinetics of deoxyguanylate kinase formation and DNA replication were ; followed in E. coli grown in a medium containing 5-bromouracil and after ; infection of the cells with P³²-labeled T4 phage. Transfer of ; radioactivity to DNA of high mol wt, estimated by ultracentrifugation in CsCl ; density-gradient solution, was taken as an indication of replication. Three min ;

  10. Antiviral effect of cationic compounds on bacteriophages

    PubMed Central

    Ly-Chatain, Mai H.; Moussaoui, Saliha; Vera, Annabelle; Rigobello, Véronique; Demarigny, Yann

    2013-01-01

    The antiviral activity of several cationic compounds – cetyltrimethylammonium bromide (CTAB), chitosan, nisin, and lysozyme – was investigated on the bacteriophage c2 (DNA head and non-contractile tail) infecting Lactococcus strains and the bacteriophage MS2 (F-specific RNA) infecting E. coli. Firstly, these activities were evaluated in a phosphate buffer pH 7 – 10 mM. The CTAB had a virucidal effect on the Lactococcus bacteriophages, but not on the MS2. After 1 min of contact with 0.125 mM CTAB, the c2 population was reduced from 6 to 1.5 log(pfu)/mL and completely deactivated at 1 mM. On the contrary, chitosan inhibited the MS2 more than it did the bacteriophages c2. No antiviral effect was observed for the nisin or the lysozyme on bacteriophages after 1 min of treatment. A 1 and 2.5 log reduction was respectively observed for nisin and lysozyme when the treatment time increased (5 or 10 min). These results showed that the antiviral effect depended both on the virus and structure of the antimicrobial compounds. The antiviral activity of these compounds was also evaluated in different physico-chemical conditions and in complex matrices. The antiviral activity of CTAB was impaired in acid pH and with an increase of the ionic strength. These results might be explained by the electrostatic interactions between cationic compounds and negatively charged particles such as bacteriophages or other compounds in a matrix. Milk proved to be protective suggesting the components of food could interfere with antimicrobial compounds. PMID:23487495

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

    E-print Network

    Kihara, Daisuke

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

  12. Lytic bacteriophages

    PubMed Central

    Sharma, Manan

    2013-01-01

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

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

    E-print Network

    Sparks, Donald L.

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

  14. Molecular Biology and Biotechnology of Bacteriophage

    NASA Astrophysics Data System (ADS)

    Onodera, Kazukiyo

    The development of the molecular biology of bacteriophage such as T4, lambda and filamentous phages was described and the process that the fundamental knowledge obtained in this field has subsequently led us to the technology of phage display was introduced.

  15. Deciphering Lysis and its Regulation in Bacteriophage T4

    E-print Network

    Moussa, Samir

    2012-10-19

    it to undergo signal peptidase II processing, N-terminal lipoylation and sorting to the inner leaflet of the outer membrane, and it also has a C-terminal TMD (172). In effect, gp11 encodes a single component spanin, although its protomeric topology is very...

  16. Lysozymes in the animal kingdom

    Microsoft Academic Search

    Lien Callewaert; Chris W. Michiels

    2010-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Goodridge, Lawrence (Inventor)

    2007-01-01

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

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

    Microsoft Academic Search

    K. E. Hopper; H. A. McKenzie

    1974-01-01

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

  19. t4 Workshop Report

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-11-01

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

  1. Microneedle-mediated transdermal bacteriophage delivery

    PubMed Central

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

    2012-01-01

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

  2. Biophysical aspects of lysozyme adduct with monocrotophos.

    PubMed

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

    2014-09-01

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

  3. Bacteriophages Infecting Propionibacterium acnes

    PubMed Central

    2013-01-01

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

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

    SciTech Connect

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

    2010-07-22

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

  5. Bypass of a Nick by the Replisome of Bacteriophage T7* Received for publication,April 18, 2011, and in revised form, June 14, 2011 Published, JBC Papers in Press, June 23, 2011, DOI 10.1074/jbc.M111.252023

    E-print Network

    Richardson, Charles C.

    Bypass of a Nick by the Replisome of Bacteriophage T7* Received for publication,April 18, 2011. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encoun- tering, bacteriophage T4- and T7-infected cells, and mito- chondria (2­9). In general, association of the two proteins

  6. Release of lysozyme from electrospun PVA\\/lysozyme-gelatin scaffolds

    Microsoft Academic Search

    Dong-zhi Yang; Yu-hua Long; Jun Nie

    2008-01-01

    This article describes an electrospinning process in fabricating ultra fine fibers with core-shell structure. A biodegradable\\u000a polymer, poly(vinyl alcohol) (PVA), was used as the shell; lysozyme was a kind of antioxidant; and gelatin were used as the\\u000a core.Morphology and microstructure of the ultra fine fibers were characterized by scanning electron microscope (SEM), transmission\\u000a electron microscopy (TEM) and X-ray photoelectron spectroscopy

  7. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

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

  8. Enhanced Human Lysozyme Production by Kluyveromyces lactis

    Microsoft Academic Search

    Eric Lu Huang; Ali Demirci

    2009-01-01

    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

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

    PubMed Central

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

    2008-01-01

    Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme). A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme) of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria interacting with an animal host. PMID:18369469

  10. Intracellular lysozyme and lactoferrin in myeloproliferative disorders

    Microsoft Academic Search

    D Y Mason

    1977-01-01

    Samples from 49 cases of myeloproliferative diseases were tested by an immunocytochemical technique for leucocyte lysozyme and lactoferrin. The presence of these constituents in myeloid precursors from cases of acute and chronic myeloid leukaemia reflected the degree of cellular maturation, lysozyme appearing (as it does in normal myeloid cells) at the stage of primary granule production (in promyelocytes), while lactoferrin

  11. Fluorescence Studies of Lysozyme Nucleation

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Smith, Lori

    1998-01-01

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

  12. Immunoassay of tear lysozyme in conjunctival diseases.

    PubMed Central

    Sen, D. K.; Sarin, G. S.

    1982-01-01

    The tear lysozyme content in 111 normal subjects and in 159 patients with various conjunctival diseases was determined by a single radial immunodiffusion technique. Tear lysozyme level in normal people was 1.33/mg/ml. (SI conversion: mg/ml = g/l.) The mean tear lysozyme levels in patients with chronic irritative conjunctivitis (0.97 mg/ml) and nutritional deficiency with epithelial xerosis (0.76 mg/ml) were significantly lower than in the normal controls. The mean tear lysozyme levels in tears from patients with vernal conjuctivitis (1.20 mg/ml), phlyctenular conjunctivitis (1.10 mg/ml), and acute bacterial conjunctivitis (1.48 mg/ml) were not significantly different from those in the normal controls. Superimposition of acute bacterial conjunctivitis on trachoma did not alter the low tear lysozyme level that existed before in these patients. PMID:6812619

  13. BACTERIOPHAGE THERAPY AND CAMPYLOBACTER

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Division M: Bacteriophage

    NSDL National Science Digital Library

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

  15. Division M: Bacteriophage

    NSDL National Science Digital Library

    American Society for Microbiology. Division M.

    1998-01-01

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

  16. Bacteriophages of Erwinia amylovora

    Microsoft Academic Search

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

    2003-01-01

    Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms.

  17. Coevolutionary arms races between bacteria and bacteriophage

    E-print Network

    Weitz, Joshua S.

    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

  18. A phage T4 site-specific endonuclease, SegE, is responsible for a non-reciprocal genetic exchange between T-even-related phages

    Microsoft Academic Search

    Farid A Kadyrov; Michael G Shlyapnikov; Valentine M Kryukov

    1997-01-01

    The bacteriophage T4 segE gene encoding site-specific endonuclease lies between the hoc.1 and uvsW genes. The similar region of T-even-related phage RB30 lacks the segE gene. Here we demonstrate that the phage T4 segE gene is inherited preferably by progeny of mixed infection with RB30. The preferred inheritance of the segE gene depends on its own expression and is based

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

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

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

  20. The lysozyme of the starfish Asterias rubens. A paradygmatic type i lysozyme.

    PubMed

    Bachali, Sana; Bailly, Xavier; Jollès, Jacqueline; Jollès, Pierre; Deutsch, Jean S

    2004-01-01

    On the basis of a partial N-terminal sequence, Jollès and Jollès previously proposed that the lysozyme from the starfish Asterias rubens represents a new form of lysozyme, called type i (invertebrate) lysozyme. Indeed, it differed from both the types c (chicken) and g (goose) known in other animals, as well as from plant and phage lysozymes. Recently, several proteins belonging to the same family have been isolated from protostomes. Here we report the complete mature protein sequence and cDNA sequence of the lysozyme from Asterias. These sequences vindicate the previously proposed homology between the starfish, a deuterostome, and protostome lysozymes. In addition, we present a structural analysis that allows us to postulate upon the function of several conserved residues. PMID:14717691

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

    PubMed Central

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

    2012-01-01

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

  2. Innate and acquired bacteriophage-mediated immunity

    PubMed Central

    Barr, Jeremy J.; Youle, Merry; Rohwer, Forest

    2013-01-01

    We recently described a novel, non-host-derived, phage-mediated immunity active at mucosal surfaces, the main site of pathogen entry in metazoans. In that work, we showed that phage T4 adheres to mucus glycoproteins via immunoglobulin-like domains displayed on its capsid. This adherence positions the phage in mucus surfaces where they are more likely to encounter and kill bacteria, thereby benefiting both the phage and its metazoan host. We presented this phage-metazoan symbiosis based on an exclusively lytic model of phage infection. Here we extend our bacteriophage adherence to mucus (BAM) model to consider the undoubtedly more complex dynamics in vivo. We hypothesize how mucus-adherent phages, both lytic and temperate, might impact the commensal microbiota as well as protect the metazoan epithelium from bacterial invasion. We suggest that BAM may provide both an innate and an acquired antimicrobial immunity. PMID:24228227

  3. Cytoplasmic bacteriophage display system

    DOEpatents

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

    1998-06-16

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

  4. Cytoplasmic bacteriophage display system

    DOEpatents

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

    1998-06-16

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

  5. Bacteriophage-Based Biosensors

    Microsoft Academic Search

    Mohammed Zourob; Steven Ripp

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

  6. Bacteriophages of Listeria

    Microsoft Academic Search

    Steven Hagens; Martin J. Loessner

    Bacteriophages have been shown to influence the evolution of their host and, in several cases, have a major effect on pathogenicity\\u000a and\\/or virulence of bacterial pathogens. Several mechanisms allow phages to change the biology and associated phenotypes of\\u000a their host. This chapter aims at explaining these mechanisms in the context of Listeria evolution and pathogenesis, using examples from other pathogens.

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

    Microsoft Academic Search

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

    2008-01-01

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

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

    DOEpatents

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

    1998-01-01

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

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

    DOEpatents

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

    1998-11-03

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

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

    DOEpatents

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

    1998-01-01

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

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

    DOEpatents

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

    1998-10-20

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

  12. Direct observation of stalled fork restart via fork regression in the T4 replication system

    PubMed Central

    Manosas, Maria; Perumal, Senthil K.; Croquette, Vincent; Benkovic, Stephen J.

    2013-01-01

    The restart of a stalled replication fork is a major challenge for DNA replication. Depending upon the nature of the damage, different repair processes might be triggered; one is template switching that is bypass of a leading strand lesion via fork regression. Using magnetic tweezers to study the T4 bacteriophage enzymes, we have reproduced in vitro the complete process of template switching. We show that the UvsW DNA helicase in cooperation with the T4 holoenzyme can overcome leading strand lesion damage by a pseudo stochastic process periodically forming and migrating a four ways Holliday junction. The initiation of the repair process requires partial replisome disassembly via the departure of the replicative helicase. The results support the role of fork regression pathways in DNA repair. PMID:23197534

  13. The bacteriophage f29 portal motor can package DNA

    E-print Network

    Croquette, Vincent

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

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

    E-print Network

    Tran, Tram Anh Thi

    2009-05-15

    transmembrane domain with its Nterminus (TNTD) in the cytoplasm and C-terminus in the periplasm (TCTD). In contrast, the N-terminus of RI (RINTD) is predicted to function as a cleavable signal sequence allowing the secretion of the RI C-terminal domain (RICTD...

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

    EPA Science Inventory

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

  16. The structure of bacteriophage T4 gene product 9: the trigger for tail contraction

    E-print Network

    Rossmann, Michael G.

    -terminal domain generates a triple coiled coil; the middle domain is a mixed, seven-stranded sandwich and genetic analyses show that the C-terminal domain is bound to the baseplate, and the N-terminal coiled-coil in an interaction of the baseplate hub with the cell, pene- tration of the tail tube across the cell wall

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

    Microsoft Academic Search

    Craig Tuerk; Larry Gold

    1990-01-01

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

  18. Dynamics of Protein–ssDNA Interactions in the Bacteriophage T4 Homologous Recombination System

    Microsoft Academic Search

    Jie Liu; Scott W. Morrical

    \\u000a Homologous recombination plays critical roles in maintaining genetic diversity and genome stability through processes such\\u000a as meiosis and DNA double-strand break repair. The central process in homologous recombination is DNA strand exchange, in\\u000a which single-stranded DNA (ssDNA) from the resected, broken end of one chromosome invades the homologous double-stranded DNA\\u000a (dsDNA) of a sister chromosome. This reaction is catalyzed by

  19. Enterococcus faecalis Constitutes an Unusual Bacterial Model in Lysozyme Resistance

    Microsoft Academic Search

    Laurent Hebert; Pascal Courtin; Riccardo Torelli; Maurizio Sanguinetti; Marie-Pierre Chapot-Chartier; Yanick Auffray; Abdellah Benachour

    2007-01-01

    Lysozyme is an important and widespread compound of the host constitutive defense system, and it is assumed that Enterococcus faecalis is one of the few bacteria that are almost completely lysozyme resistant. On the basis of the sequence analysis of the whole genome of E. faecalis V583 strain, we identified two genes that are potentially involved in lysozyme resistance, EF_0783

  20. Coacervates of lysozyme and ?-casein.

    PubMed

    Anema, Skelte G; de Kruif, C G Kees

    2013-05-15

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Microsoft Academic Search

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

    1970-01-01

    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

  4. Generation of nanostructures of mica supported lysozyme and lysozyme-nanogold conjugates by diving tip nanowriting.

    PubMed

    Kolb, H-A; Leisten, Frank; Wiechmann, Matthias; Enders, Oliver

    2005-01-01

    Nanostructures of lysozyme molecules and lysozyme-nanogold conjugates were generated by atomic force microscopy in contact-, tapping- and force-distance- mode on mica in aqueous solution. In contact mode at high ionic strength, adjusted lysozyme concentration and lower loading force a monolayer of defined structure and orientation of lysozyme can be formed by the scan process of the tip. A lateral resolution of the monolayer of about 80 nm could be achieved. At larger loading forces besides a lysozyme monolayer also 3D- aggregates could be generated in parallel. In force-distance mode the volume of 3D-aggregates was studied as function of lysozyme concentration, loading force and number or frequency of up- and down-movement of the tip. Also in tapping mode 3D-aggregates were generated at the selected incubation conditions. Application of the linescan mode for solutions of nanogold or lysozyme-nanogold conjugates allowed the formation of monlayers of linear shape with lateral resolution of about 35 nm on mica. Nanogold line-structures could be connected to macroscopic gold contacts. It is postulated that adjustment of electrostatic interaction between lysozyme and substrate and the applied loading force is critical for monolayer formation. Different to the underlying mechanism of the well-established dip-pen nanolithography (DPN) (Piner et al., 1999) for the presented method of diving tip nanowriting (DTN) adsorption of the molecules from the aqueous bulk phase to the tip and thereafter the flow to the mica surface is discussed. DTN could be used to either contact proteins electrically or to form preaggregates for protein crystallization (Wiechmann et al. PMID:17282229

  5. Morphogenesis of the T4 tail and tail fibers

    PubMed Central

    2010-01-01

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

  6. Widespread genetic exchange among terrestrial bacteriophages

    E-print Network

    Hartl, Daniel L.

    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

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

    Microsoft Academic Search

    A. D. Hershey; MARTHA CHASE

    1952-01-01

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

  8. Evolution of T4-related phages

    Microsoft Academic Search

    Elizabeth Kutter; Ketevan Gachechiladze; Alexandr Poglazov; Elena Marusich; Mikhail Shneider; Pia Aronsson; Alberto Napuli; Darlene Porter; Vadim Mesyanzhinov

    1995-01-01

    Much progress has been made in understanding T-even phage biology in the last 50 years. We now know the entire sequence of T4, encoding nearly 300 genes, only 69 of which have been shown to be essential under standard laboratory conditions; no specific function is yet known for about 140 of them. The origin of most phage genes is unclear,

  9. Novel Bacteriophages in Enterococcus spp.

    PubMed

    Mazaheri Nezhad Fard, Ramin; Barton, Mary D; Heuzenroeder, Michael W

    2010-06-01

    Most of the bacteriophages (phages) currently reported in Enterococcus spp. belong to tailed families of bacteriophages Podoviridae, Siphoviridae, and Myoviridae. There is a little information on non-tailed bacteriophages isolated from enterococci. Samples of sewage and piggery effluents were tested on pig and chicken isolates of Enterococcus faecalis, E. faecium and E. gallinarum for lytic phages. In addition, isolates were exposed to mitomycin C to induce lysogenic phages. Bacteriophages that were detected were visualized by electron microscopy. Ten bacteriophages were of isometric shape with long flexible or non-flexible tails, while one had a long head with a long flexible tail; all contained double-stranded DNA molecules. Seven Polyhedral, filamentous, and pleomorphic-shaped phages containing DNA or RNA were also observed. The pleomorphic phages were droplet- or lemon-shaped in morphology. This study is the first report on polyhedral phages in Enterococcus spp. of animal origin and also the first report of filamentous and pleomorphic phages in enterococci. PMID:19967374

  10. Lysogenic bacteriophage isolated from acidophilium

    DOEpatents

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

    1992-01-01

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

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

    SciTech Connect

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

    2012-07-11

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

  12. Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

    PubMed

    Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

    2008-04-01

    Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages. PMID:18281701

  13. Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage

    PubMed Central

    Brok-Volchanskaya, Vera S.; Kadyrov, Farid A.; Sivogrivov, Dmitry E.; Kolosov, Peter M.; Sokolov, Andrey S.; Shlyapnikov, Michael G.; Kryukov, Valentine M.; Granovsky, Igor E.

    2008-01-01

    Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3? 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA T?C stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages. PMID:18281701

  14. T4 DNA ligase synthesizes dinucleoside polyphosphates

    Microsoft Academic Search

    Olga Madrid; Daniel Martín; Eva Ana Atencia; Antonio Sillero; María A. Günther Sillero

    1998-01-01

    T4 DNA ligase (EC 6.5.1.1), one of the most widely used enzymes in genetic engineering, transfers AMP from the E-AMP complex to tripolyphosphate, ADP, ATP, GTP or dATP producing p4A, Ap3A, Ap4A, Ap4G and Ap4dA, respectively. Nicked DNA competes very effectively with GTP for the synthesis of Ap4G and, conversely, tripolyphosphate (or GTP) inhibits the ligation of DNA by the

  15. Evolution of T4-related phages.

    PubMed

    Kutter, E; Gachechiladze, K; Poglazov, A; Marusich, E; Shneider, M; Aronsson, P; Napuli, A; Porter, D; Mesyanzhinov, V

    1995-01-01

    Much progress has been made in understanding T-even phage biology in the last 50 years. We now know the entire sequence of T4, encoding nearly 300 genes, only 69 of which have been shown to be essential under standard laboratory conditions; no specific function is yet known for about 140 of them. The origin of most phage genes is unclear, and only 42 genes in T4 have significant similarity to anything currently included in GenBank. Comparative analysis of related phages is now being used to gain insight into both the evolutionary origins and interrelationships of these phage genes, and the functions of their protein products. The genomes of phages isolated from Tbilisi hospitals, Long Island sewage plants, the Denver zoo, and Khabarovsk show basic similarity. However, these phages show substantial insertions and deletions in a number of regions relative to each other, and closer investigation of specific sequences often reveals much more complex relationships. There are only a few cases in T4-related phages in which there is evidence for evolution through DNA duplication. These include the fibrous products of genes 12, 34, and 37; head proteins gp23 and gp24; and the Alt enzyme and its downstream neighbors. T4 also contains 13 apparent relatives of group I and group II intron homing endonucleases. Distal portions of the tail fibers of various T-even phages contain segments closely related to tail-fiber regions of other DNA coliphages, such as Mu, P1, P2, and lambda. Horizontal gene transfer clearly emerges as a major factor in the evolution of at least the tail-fiber regions, where site-specific recombination probably is involved in the exchange of host-range determinants. PMID:8828153

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

    PubMed

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

    2015-05-01

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

  17. Light scattering and phase behavior of Lysozyme-PEG mixtures

    E-print Network

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

    2005-12-14

    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.

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

    SciTech Connect

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

    2009-08-28

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

  19. Enumeration of bacteriophage particles

    PubMed Central

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

    2011-01-01

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

  20. Mechanics of bacteriophage maturation

    PubMed Central

    Roos, Wouter H.; Gertsman, Ilya; May, Eric R.; Brooks, Charles L.; Johnson, John E.; Wuite, Gijs J. L.

    2012-01-01

    Capsid maturation with large-scale subunit reorganization occurs in virtually all viruses that use a motor to package nucleic acid into preformed particles. A variety of ensemble studies indicate that the particles gain greater stability during this process, however, it is unknown which material properties of the fragile procapsids change. Using Atomic Force Microscopy-based nano-indentation, we study the development of the mechanical properties during maturation of bacteriophage HK97, a ?-like phage of which the maturation-induced morphological changes are well described. We show that mechanical stabilization and strengthening occurs in three independent ways: (i) an increase of the Young’s modulus, (ii) a strong rise of the capsid’s ultimate strength, and (iii) a growth of the resistance against material fatigue. The Young’s modulus of immature and mature capsids, as determined from thin shell theory, fit with the values calculated using a new multiscale simulation approach. This multiscale calculation shows that the increase in Young’s modulus isn’t dependent on the crosslinking between capsomers. In contrast, the ultimate strength of the capsids does increase even when a limited number of cross-links are formed while full crosslinking appears to protect the shell against material fatigue. Compared to phage ?, the covalent crosslinking at the icosahedral and quasi threefold axes of HK97 yields a mechanically more robust particle than the addition of the gpD protein during maturation of phage ?. These results corroborate the expected increase in capsid stability and strength during maturation, however in an unexpected intricate way, underlining the complex structure of these self-assembling nanocontainers. PMID:22308333

  1. Lysozyme crystallization rates controlled by anomalous fluctuations

    NASA Astrophysics Data System (ADS)

    Pullara, F.; Emanuele, A.; Palma-Vittorelli, M. B.; Palma, M. U.

    2005-02-01

    Nucleation of protein aggregates and crystals is a process activated by statistical fluctuations of concentration. Nucleation rates may change by several orders of magnitude upon apparently minor changes in the multidimensional space of parameters (temperature, pH, protein concentration, salt type and concentrations, additives). We use available data on hen egg lysozyme crystal induction times in different solution conditions. We measure by static and dynamic light scattering the amplitudes and lifetimes of anomalously ample and long-lived fluctuations occurring in proximity of the liquid-liquid demixing region of the given lysozyme solutions. This allows determining the related spinodal temperatures TS and ?=(T-TS)/TS. Experimental induction times appear to depend solely upon ? over many orders of magnitude. This is quantitatively accounted for in terms of an extended two-stage nucleation model, which jointly takes into consideration amplitudes, lifetimes and scaling properties of anomalous fluctuations. One and the same relation describes quantitatively and equally well the present case of lysozyme crystallization (the best studied case of protein crystallization) and that of sickle hemoglobin fiber formation (the best studied case of protein fiber formation). Comparison with other recent models shows that taking into account lifetimes of anomalous fluctuations allows capturing the essence of the observed behavior.

  2. The optical activity of lysozyme crystals.

    PubMed

    Kobayashi, J; Asahi, T; Sakurai, M; Kagomiya, I; Asai, H; Asami, H

    1998-09-01

    The components of the gyration tensor of the enzyme lysozyme were measured by using the HAUP method: g11 = -0.90 x 10(-5) and g33 = 1.05 x 10(-5) at 303.4 K and a wavelength of 4880 A. The optical rotatory powers along the a and c axes in the same conditions were calculated: rho a = -21.3 and rho c = -24.8 degrees cm-1. The optically active property of lysozyme is strange in that, although it contains a considerable quantity of alpha-helices (about 30%), the rotatory powers are unexpectedly small in magnitude, one order of magnitude less than those of quartz and with very large anisotropy. A conceptual consideration of this phenomenon is given. In order to assess the difference between the structures in both crystalline and solution states, the chirality index r was calculated to be 0.16. This value indicates that the structural change of lysozyme from the solution into the crystalline state is expressed by an increase of 19% in optical activity. From the NMR results [Smith et al. (1993), J. Mol. Biol. 229, 930-944], it is anticipated that the r value reflects the increased constraint in atomic motion in the side chains of exposed amino acid residues in the crystalline state. PMID:9764466

  3. Functional assay for T4 lysozyme-engineered G Protein-Coupled Receptors with an ion channel reporter

    PubMed Central

    Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J.

    2013-01-01

    Summary: Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains in order to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that Ion Channel-Coupled Receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. PMID:24268646

  4. Bacteriophage therapy in animal production

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Lysis Timing and Bacteriophage Fitness

    Microsoft Academic Search

    Ing-Nang Wang

    2005-01-01

    The effect of lysis timing on bacteriophage (phage) fitness has received little theoretical or experi- mental attention. Previously, the impact of lysis timing on phage fitness was studied using a theoretical model based on the marginal value theorem from the optimal foraging theory. An implicit conclusion of the model is that, for any combination of host quantity and quality, an

  6. Lysozyme pattern formation in evaporating droplets

    NASA Astrophysics Data System (ADS)

    Gorr, Heather Meloy

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

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

    PubMed

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

    1999-01-01

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

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

    SciTech Connect

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

    2009-06-30

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

  9. Genetic Dissection of T4 Lysis

    PubMed Central

    Moussa, Samir H.; Lawler, Jessica L.

    2014-01-01

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

  10. Genetic dissection of T4 lysis.

    PubMed

    Moussa, Samir H; Lawler, Jessica L; Young, Ry

    2014-06-01

    t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an N(in)-C(out) topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; N(out)-C(in)) of the coliphage lambda and S68 (2 TMDs; N(in)-C(in)) 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

  11. Lysozyme purification with dye-affinity beads under magnetic field.

    PubMed

    Ba?ar, Nilgün; Uzun, Lokman; Güner, Ali; Denizli, Adil

    2007-08-01

    Magnetic poly(2-hydroxyethyl methacrylate) mPHEMA beads carrying Cibacron Blue F3GA were prepared by suspension polymerization of HEMA in the presence of Fe3O4 nano-powder. Average size of spherical beads was 80-120 microm. The beads had a specific surface area of 56.0m(2)/g. The characteristic functional groups of dye-attached mPHEMA beads were analyzed by Fourier transform infrared spectrometer (FTIR) and Raman spectrometer. mPHEMA with a swelling ratio of 68% and carrying 28.5 micromol CibacronBlueF3GA/g were used for the purification of lysozyme. Adsorption studies were performed under different conditions in a magnetically stabilized fluidized bed (i.e., pH, protein concentration, flow-rate, temperature, and ionic strength). Lysozyme adsorption capacity of mPHEMA and mPHEMA/Cibacron Blue F3GA beads were 0.8 mg/g and 342 mg/g, respectively. It was observed that after 20 adsorption-desorption cycle, mPHEMA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 87.4% with recovery about 79.6%. The specific activity of the desorbed lysozyme was high as 41.586 U/mg. PMID:17418399

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

    PubMed

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

    2005-09-30

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

  13. Investigation of the interaction between chlorophenols and lysozyme in solution.

    PubMed

    Zhang, Hong-Mei; Xu, Yi-Qiang; Zhou, Qiu-Hua; Wang, Yan-Qing

    2011-09-01

    The binding interactions of lysozyme with 2-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol were investigated by UV-vis absorption, CD, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques under physiological pH 7.40. The binding constants, quenching mechanism, and the number of binding sites were determined by the quenching of lysozyme fluorescence in presence of chlorophenols. H-bonds and hydrophobic interactions played major roles in stabilizing the chlorophenols-lysozyme complex. The distances r between chlorophenols and lysozyme were calculated to be 1.94nm, 2.75nm, 3.54nm, and 3.76nm for 2-CP, 2,4-DCP, 2,4,6-TCP, and PCP, respectively. The effects of chlorophenols on the conformation of lysozyme were analyzed using CD, synchronous fluorescence and three-dimensional fluorescence spectra. PMID:21596581

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

    PubMed Central

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

    2015-01-01

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

  15. Recombinant bacteriophage lysins as antibacterials

    PubMed Central

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

    2010-01-01

    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

  16. Bacteriophage-Based Pathogen Detection

    Microsoft Academic Search

    Steven Ripp

    2010-01-01

    \\u000a Considered the most abundant organism on Earth, at a population approaching 1031, bacteriophage, or phage for short, mediate interactions with myriad bacterial hosts that has for decades been exploited\\u000a in phage typing schemes for signature identification of clinical, food-borne, and water-borne pathogens. With over 5,000 phage\\u000a being morphologically characterized and grouped as to susceptible host, there exists an enormous cache

  17. Sewage bacteriophage inactivation by cationic porphyrins: influence of light parameters.

    PubMed

    Costa, Liliana; Carvalho, Carla M B; Faustino, Maria A F; Neves, Maria G P M S; Tomé, João P C; Tomé, Augusto C; Cavaleiro, José A S; Cunha, Angela; Almeida, Adelaide

    2010-08-01

    Photodynamic therapy has been used to inactivate microorganisms through the use of targeted photosensitizers. Although the photoinactivation of microorganisms has already been studied under different conditions, a systematic evaluation of irradiation characteristics is still limited. The goal of this study was to test how the light dose, fluence rate and irradiation source affect the viral photoinactivation of a T4-like sewage bacteriophage. The experiments were carried out using white PAR light delivered by fluorescent PAR lamps (40 W m(-2)), sun light (600 W m(-2)) and an halogen lamp (40-1690 W m(-2)). Phage suspensions and two cationic photosensitizers (Tetra-Py(+)-Me, Tri-Py(+)-Me-PF) at concentrations of 0.5, 1.0 and 5.0 microM were used. The results showed that the efficacy of the bacteriophage photoinactivation is correlated not only with the sensitizer and its concentration but also with the light source, energy dose and fluence rate applied. Both photosensitizers at 5.0 microM were able to inactivate the T4-like phage to the limit of detection for each light source and fluence rate. However, depending of the light parameters, different irradiation times are required. The efficiency of photoinactivation is dependent on the spectral emission distribution of the light sources used. Considering the same light source and a fixed light dose applied at different fluence rates, phage inactivation was significantly higher when low fluence rates were used. In this way, the light source, fluence rate and total light dose play an important role in the effectiveness of the antimicrobial photodynamic therapy and should always be considered when establishing an optimal antimicrobial protocol. PMID:20563346

  18. Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

    PubMed Central

    Volozhantsev, Nikolay V.; Oakley, Brian B.; Morales, Cesar A.; Verevkin, Vladimir V.; Bannov, Vasily A.; Krasilnikova, Valentina M.; Popova, Anastasia V.; Zhilenkov, Eugeni L.; Garrish, Johnna K.; Schegg, Kathleen M.; Woolsey, Rebekah; Quilici, David R.; Line, J. Eric; Hiett, Kelli L.; Siragusa, Gregory R.; Svetoch, Edward A.; Seal, Bruce S.

    2012-01-01

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ?CPV4 and ?ZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ?CP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ?CP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage ?29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae. PMID:22666499

  19. Genome Landscapes and Bacteriophage Codon Usage Julius B. Lucks1

    E-print Network

    Plotkin, Joshua B.

    Genome Landscapes and Bacteriophage Codon Usage Julius B. Lucks1 , David R. Nelson1,2 , Grzegorz R and simple eukaryotes. Here we analyze patterns of codon usage across 74 diverse bacteriophages that infect E of bacteriophages. Citation: Lucks JB, Nelson DR, Kudla GR, Plotkin JB (2008) Genome Landscapes and Bacteriophage

  20. Use of Bacteriophages to control bacterial pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lytic bacteriophages can provide a natural method and an effective alternative to antibiotics to reduce bacterial pathogens in animals, foods, and other environments. Bacteriophages (phages) are viruses which infect bacterial cells and eventually kill them through lysis, and represent the most abun...

  1. Surface plasmon resonance detection of E. coli and methicillin-resistant S. aureus using bacteriophages.

    PubMed

    Tawil, Nancy; Sacher, Edward; Mandeville, Rosemonde; Meunier, Michel

    2012-01-01

    Early diagnosis and appropriate treatment of Escherichia coli (E. coli) O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) are key elements in preventing resultant life-threatening illnesses, such as hemorrhagic colitis, hemolytic uremic syndrome, and septicemia. In this report, we describe the use of surface plasmon resonance (SPR) for the biodetection of pathogenic bacteria, using bacteriophages as the recognition elements. T4 bacteriophages were used to detect E. coli, while a novel, highly specific phage was used to detect MRSA. We found that the system permits label-free, real-time, specific, rapid and cost-effective detection of pathogens, for concentrations of 10(3) colony forming units/milliliter, in less than 20 min. This system promises to become a diagnostic tool for bacteria that cause major public concern for food safety, bioterrorism, and nosocomial infections. PMID:22609555

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

    SciTech Connect

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

    2008-04-02

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

  3. The Effects of Acetate Buffer Concentration on Lysozyme Solubility

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Pusey, Marc L.

    1996-01-01

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

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

    PubMed Central

    Storms, Zachary J.; Sauvageau, Dominic

    2014-01-01

    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

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

    PubMed Central

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

    2002-01-01

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

  6. Nucleation and Growth According to Lysozyme

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  7. Single molecule studies of DNA packaging by bacteriophages

    NASA Astrophysics Data System (ADS)

    Fuller, Derek Nathan

    The DNA packaging dynamics of bacteriophages ?29, gamma, and T4 were studied at the single molecule level using a dual trap optical tweezers. Also, a method for producing long DNA molecules by PCR for optical tweezers studies of protein DNA interactions is presented and thoroughly characterized. This DNA preparation technique provided DNA samples for the ?29 and T4 studies. In the studies of ?29, the role of charge was investigated by varying the ionic conditions of the packaging buffer. Ionic conditions in which the DNA charge was highly screened due to divalent and trivalent cations showed the lowest resistance to packaging of the DNA to high density. This confirmed the importance of counterions in shielding the DNA interstrand repulsion when packaged to high density. While the ionic nature of the packaging buffer had a strong effect on packaging velocities, there was no clear trend between the counterion-screened charge of the DNA and the maximum packaging velocity. The packaging studies of lambda and T4 served as systems for comparative studies with ?29. Each system showed similarities to the ?29 system and unique differences. Both the lambda and T4 packaging motors were capable of generating forces in excess of 50 pN and showed remarkably high processivity, similar to ?29. However, dynamic structural transitions were observed with lambda that are not observed with ?29. The packaging of the lambda genome showed capsid expansion at approximately 30 percent of the genome packaged and capsid rupture at 90 percent of the genome packaged in the absence of capsid stabilizing protein gpD. Unique to the T4 packaging motor, packaging dynamics showed a remarkable amount of variability in velocities. This variability was seen both within individual packaging phages and from one phage to the next. This is possibly due to different conformational states of the packaging machinery. Additionally, lambda and T4 had average packaging velocities under minimal load of 600 bp/s and 700 bp/s, respectively, as compared to 140 bp/s for ?29.

  8. Intrinsic Mean Square Displacement in Lysozyme

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

    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.

  9. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  13. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  14. Effect of Lysozyme on Resting Spores of Bacillus Megaterium

    PubMed Central

    Suzuki, Yahiko; Rode, L. J.

    1969-01-01

    Resting spores of Bacillus megaterium ATCC 9885 were found to be markedly affected by lysozyme. Exposure to as little as 1.5 ?g of lysozyme per ml caused the spores to lose refractility, the darkened spores to shed their coat structures, and the spore central bodies to lyse. The spores of seven other strains of B. megaterium and seven other Bacillus species were not similarly affected by lysozyme. Proteolytic enzymes such as pronase, trypsin, pepsin, and subtilisin did not induce the change. The action of lysozyme differed in certain important respects from that of common “physiological” germinants. Its action was considered to be direct via its enzymatic attack on exposed sites directly accessible in the resting spores of B. megaterium ATCC 9885. Images PMID:4977688

  15. Hofmeister effect and the phase diagram of lysozyme

    NASA Astrophysics Data System (ADS)

    Lettieri, S.; Li, Xiaofei; Gunton, J. D.

    2008-07-01

    The phase diagrams for lysozyme are calculated for two different precipitant salts, NaCl and NaSCN, using a potential of mean force that takes into account contributions from ion-dispersion forces [M. Boström , J. Phys. Chem. B 110, 24757 (2006)]. Our results are consistent with a recent perturbation theory calculation (referenced above) in that the phase diagram for lysozyme with NaCl is quite different than for lysozyme with NaSCN for the same molar concentration (0.2M) . However, in contrast to the perturbation theory calculation, we find that the lysozyme phase diagram with NaCl has a metastable fluid-fluid coexistence curve and that the metastability gap in the case of NaSCN is much larger than predicted by perturbation theory.

  16. Hofmeister effect and the phase diagram of lysozyme

    NASA Astrophysics Data System (ADS)

    Lettieri, Steven; Li, Xiaofei; Gunton, James

    2009-03-01

    The phase diagrams for lysozyme are calculated for two different precipitant salts, NaCl and NaSCN, using a potential of mean force that takes into account contributions from ion-dispersion forces (J.Phys.Chem.B, 110, 24757). Our results are consistent with a recent perturbation theory calculation (J.Phys.Chem.B, 110, 24757) in that the phase diagram for lysozyme with NaCl is quite different than for lysozyme with NaSCN for the same molar concentration (0.2M). However, in contrast to the perturbation theory calculation, we find that the lysozyme phase diagram with NaCl has a metastable fluid-fluid coexistence curve and that the metastability gap in the case of NaSCN is much larger than predicted by perturbation theory.

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

    NASA Technical Reports Server (NTRS)

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

    1990-01-01

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

  18. Inferring bacteriophage infection strategies from genome sequence: analysis of bacteriophage 7-11 and related phages

    PubMed Central

    2015-01-01

    Background Analyzing regulation of bacteriophage gene expression historically lead to establishing major paradigms of molecular biology, and may provide important medical applications in the future. Temporal regulation of bacteriophage transcription is commonly analyzed through a labor-intensive combination of biochemical and bioinformatic approaches and macroarray measurements. We here investigate to what extent one can understand gene expression strategies of lytic phages, by directly analyzing their genomes through bioinformatic methods. We address this question on a recently sequenced lytic bacteriophage 7 - 11 that infects bacterium Salmonella enterica. Results We identify novel promoters for the bacteriophage-encoded ? factor, and test the predictions through homology with another bacteriophage (phiEco32) that has been experimentally characterized in detail. Interestingly, standard approach based on multiple local sequence alignment (MLSA) fails to correctly identify the promoters, but a simpler procedure that is based on pairwise alignment of intergenic regions identifies the desired motifs; we argue that such search strategy is more effective for promoters of bacteriophage-encoded ? factors that are typically well conserved but appear in low copy numbers, which we also verify on two additional bacteriophage genomes. Identifying promoters for bacteriophage encoded ? factors together with a more straightforward identification of promoters for bacterial encoded ? factor, allows clustering the genes in putative early, middle and late class, and consequently predicting the temporal regulation of bacteriophage gene expression, which we demonstrate on phage 7-11. Conclusions While MLSA algorithms proved highly useful in computational analysis of transcription regulation, we here established that a simpler procedure is more successful for identifying promoters that are recognized by bacteriophage encoded ? factor/RNA polymerase. We here used this approach for predicting sequence specificity of a novel (bacteriophage encoded) ? factor, and consequently inferring phage 7-11 transcription strategy. Therefore, direct analysis of bacteriophage genome sequences is a plausible first-line approach for efficiently inferring phage transcription strategies, and may provide a wealth of information on transcription initiation by diverse ? factors/RNA polymerases. PMID:25708710

  19. Effects of purification on the crystallization of lysozyme

    Microsoft Academic Search

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

    1996-01-01

    We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20°C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had

  20. Destroying activity of magnetoferritin on lysozyme amyloid fibrils

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  1. Movable Finite Automata (MFA) models for biological systems. I: Bacteriophage assembly and operation.

    PubMed

    Thompson, R L; Goel, N S

    1988-04-01

    A new class of models, called Movable Finite Automata (MFA) models, is introduced. MFA models are physically realistic, but still share some of the features of cellular automata that make the latter easy to handle mathematically and computationally. They are found to be quite versatile in modeling the self-organization of biological systems. Their use in simulating the interaction of protein molecules in the self-assembly and operation of the T4 bacteriophage is described. The results of these simulations carried out on a microcomputer, are given. PMID:3193776

  2. Presence and characterization of multiple mantle lysozymes in the Pacific oyster, Crassostrea gigas.

    PubMed

    Itoh, Naoki; Okada, Yuki; Takahashi, Keisuke G; Osada, Makoto

    2010-07-01

    Mantle tissue extracts from the Pacific oyster, Crassostrea gigas, exhibited anti-Gram-positive bacterial and lysozyme activities over a wide pH range, suggesting that multiple defensive mantle lysozymes were present. Degenerated reverse-transcription PCR detected the expression of two mantle lysozymes, CGL-1 and a novel lysozyme CGL-3, confirming the presence of multiple lysozymes in the mantle. Since CGL-3 is a cognate protein of the digestive lysozyme CGL-2, it is assumed that CGL-3 has evolved specifically a defensive function. Functional assays using recombinant CGL-1 and CGL-3 suggested that CGL-1 and CGL-3 play a major defensive role in the mantle tissue, and that they are responsible for lysozyme activity under different pH, ionic strength and temperature conditions. Based on these observations, we conclude that multiple mantle lysozymes in the Pacific oyster are better for host-defense under broader conditions than a single lysozyme. PMID:20211734

  3. Strong and Selective Adsorption of Lysozyme on Graphene Oxide

    PubMed Central

    2015-01-01

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

  4. Lytic Clostridium perfringens Bacteriophage 39-O Genomic

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Preparation of endotoxin-free bacteriophages.

    PubMed

    Boraty?ski, Janusz; Syper, Danuta; Weber-Dabrowska, Beata; ?usiak-Szelachowska, Marzanna; Po?niak, Gryzelda; Górski, Andrzej

    2004-01-01

    Bacteriophages (phages) are bacterial viruses that interact with bacterial walls and invade bacterial cells. Moreover, they disturb bacterial metabolism and lead to bacteria lysis. In the case of Gram-negative bacteria crude phage cultures, apart from the phages themselves, the bacterial debris, bacterial proteins and nucleic acids contain endotoxins. These endotoxins (lipopolysaccharides) posses a high degree of toxicity in vitro and in vivo, and their removal is essential for safety in antibacterial bacteriophage therapy. An effective, scaleable purification of bacteriophages from endotoxins was accomplished by sequential ultrafiltration through polysulfone membrane (30 nm) followed by chromatography on sepharose 4B and Matrex Cellulofine Sulfate. The phage fraction after gel filtration chromatography routinely contained endotoxins in the 150-2500 EU/ml range. The procedure yielded bacteriophages contaminated with as little as 0.4-7 EU/ml (Limulus assay). This value lies within the permitted level for intravenous applications (5 EU/kg/h by European Pharmacopoeia, 1997). PMID:15213806

  6. Interrupted thymidylate synthase gene of bacteriophages T2 and T6 and other potential self-splicing introns in the T-even bacteriophages

    SciTech Connect

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

    1987-09-01

    Southern hybridization analyses of procaryotic DNA from Escherchia coli, lambda bacteriophage, and T1 to T7 phages were carried out. The hybridization probes used consisted of DNA restriction fragments derived from the T4 phage intron-containing thymidylate synthase gene (td) and short synthetic oligodeoxynucleotides defining specific exon and intron regions of the gene. It was shown that intact as well as restricted DNA from the T-even phages hybridized not only to both T4 phage td intron- and exon-specific probes but also to probes defining the td 5' (exon I-intron) and 3' (intron-exon II) presplice junctions. These data strongly suggest that, analogous to the T4 phage, only the T2 and T6 phages among the procaryotes tested contain interrupted td genes. The td intervening sequence in each phage is roughly 1 kilobase pair (kb) in size and interrupts the td gene at a site analogous to that in the T4 phage. This was confirmed by data from Northern (RNA) hybridization analysis of td-specific in vitro transcripts of these phage DNAs. (..cap alpha..-/sup 32/P)GTP in vitro labeling of total RNA from T4 phage-infected cells produced five species of labeled RNAs that were 1, 0.9, 0.83, 0.75, and 0.6 kb in size. Only the 1-, 0.9-, and 0.75-kb species were labeled in RNA from T2- or T6-infected cells. The commonly present 1-kb RNA is the excised td intron, which exists in both linear and circular forms in the respective T-even-phage-infected cells, while the 0.6-kb RNA unique to T4 may be the excised intron derived from the ribonucleotide reductase small subunit gene (nrdB) of the phage. The remaining labeled RNA species are likely candidates for other self-splicing introns.

  7. Homotypic interactions among bacteriophage ?KMV early proteins

    Microsoft Academic Search

    B. Roucourt; A. Chibeu; E. Lecoutere; R. Lavigne; G. Volckaert; K. Hertveldt

    2007-01-01

    Summary  Little is known about the bacteriophage proteins expressed immediately after infection of the host cell. Most of these early\\u000a proteins are probably involved in bacteriophage-host interactions redirecting the bacterial metabolism to phage production.\\u000a Interaction analysis of the first 16 ?KMV gene products (gp) identified homotypic interactions of gp7, gp9 and gp15. Two related\\u000a yeast two-hybrid procedures, a matrix and a

  8. Non-canonical RNA arrangement in T4-even phages: accommodated ribosome binding site at the gene 26-25 intercistronic junction.

    PubMed

    Malys, Naglis; Nivinskas, Rimas

    2009-09-01

    Translational initiation region of bacteriophage T4 gene 25 contains three potential Shine and Dalgarno sequences: SD1, SD2 and SD3. Mutational analysis has predicted that an mRNA stem-loop structure may include SD1 and SD2, bringing the most typical sequence SD3, GAGG, to the initiation codon. Here, we report physical evidence demonstrating that previously predicted mRNA stem-loop structure indeed exists in vivo during gene 25 expression in T4-infected Escherichia coli cells. The second mRNA stem-loop structure is identified 14 nucleotides upstream of the stem-loop I, while the SD3 sequence, as well as the start codon of the gene, are proved to be within an unfolded stretch of mRNA. Phylogenetic comparison of 38 T4-like phages reveals that the T-even and some pseudoT-even phages evolve a similar structural strategy for the translation initiation of 25, while pseudoT-even, schizoT-even and exoT-even phages use an alternative mRNA arrangement. Taken together, the results indicate that a specific mRNA fold forms the split ribosome binding site at the gene 26-25 intercistronic junction, which is highly competent in the translational initiation. We conclude that this ribosome binding site has evolved after T-even diverged from other T4-like phages. Additionally, we determine that the SD sequence GAGG is most widespread in T4. PMID:19708923

  9. Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification

    PubMed Central

    Schaerli, Yolanda; Stein, Viktor; Spiering, Michelle M.; Benkovic, Stephen J.; Abell, Chris; Hollfelder, Florian

    2010-01-01

    In vitro reconstitution of the bacteriophage T4 replication machinery provides a novel system for fast and processive isothermal DNA amplification. We have characterized this system in two formats: (i) in circular nicking endonuclease-dependent amplification (cNDA), the T4 replisome is supplemented with a nicking endonuclease (Nb.BbvCI) and a reverse primer to generate a well-defined uniform double-stranded linear product and to achieve up to 1100-fold linear amplification of a plasmid in 1?h. (ii) The T4 replisome with its primase (gp61) can also support priming and exponential amplification of genomic DNA in primase-based whole-genome amplification (T4 pWGA). Low amplification biases between 4.8 and 9.8 among eight loci for 0.3–10?ng template DNA suggest that this method is indeed suitable for uniform whole-genome amplification. Finally, the utility of the T4 replisome for isothermal DNA amplification is demonstrated in various applications, including incorporation of functional tags for DNA labeling and immobilization; template generation for in vitro transcription/translation and sequencing; and colony screening and DNA quantification. PMID:20921065

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

    SciTech Connect

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

    2009-12-20

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

  11. Bacteriophage Lysins as Effective Antibacterials

    PubMed Central

    Fischetti, Vincent A.

    2008-01-01

    Summary Lysins are highly evolved enzymes produced by bacteriophage ( phage for short) to digest the bacterial cell wall for phage progeny release. In gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Thus, lysins may be a much needed anti-infective in an age of mounting antibiotic resistance. PMID:18824123

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

    PubMed Central

    2010-01-01

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

  13. Properties of r Mutants of Bacteriophage T4 Photodynamically Induced in the Presence of Thiopyronin and Psoralen

    PubMed Central

    Drake, John W.; McGuire, Janice

    1967-01-01

    About 4 × 10?4r mutants were induced per lethal hit, a frequency characteristic of weak mutagens. Collections of mutants produced in the presence of either dye were indistinguishable in most of their properties. The rII mutants differed sharply from spontaneous mutants in their mutational spectra (fine scale map distribution) and their reversion responses to specific mutagens. Few or none of the induced mutants were induced to revert with proflavine (sign mutants; reading frame shift mutants). A majority were induced to revert with base analogues (base pair substitution mutants), and about half of these also responded to the hydroxymethylcytosine-specific agent hydroxylamine. A large minority of the mutants reverted spontaneously but failed to respond either to proflavine or to base analogues. We believe these mutants, as well as some of the mutants which did respond to base analogues, to be transversions (base pair substitutions which reverse the purine-pyrimidine orientation). PMID:5623962

  14. Comparative Genomics of the T4Like Escherichia coli Phage JS98: Implications for the Evolution of T4 Phages

    Microsoft Academic Search

    Sandra Chibani-Chennoufi; Carlos Canchaya; Anne Bruttin; Harald Brussow

    2004-01-01

    About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh. The DNA shared up to 81% base pair identity with phage T4. The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform. The head genes showed the

  15. Effects of Purification on the Crystallization of Lysozyme

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  16. Surfactant copolymers prevent aggregation of heat denatured lysozyme.

    PubMed

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

    2006-07-01

    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 degrees 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  18. 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...

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

    E-print Network

    Harrison, Stephen C.

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

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

    E-print Network

    Collins, James J.

    Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy Timothy K. Lua, 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

  1. Membrane-Anchoring Interactions of Bacteriophage Major Coat Proteins

    E-print Network

    Hemminga, Marcus A.

    Membrane-Anchoring Interactions of Bacteriophage Major Coat Proteins A.B. Meijer #12;#12;Membrane-Anchoring Interactions of Bacteriophage Major Coat Proteins A.B. Meijer #12;promotor: prof. dr. T.J. Schaafsma, Laboratoriumvoor Microbiologie,Universiteit van Ljubljana #12;Membrane-Anchoring Interactions of Bacteriophage

  2. Modeling and analysis of a marine bacteriophage infection

    E-print Network

    Kuang, Yang

    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

  3. 2006 Nature Publishing Group Structure of epsilon15 bacteriophage reveals

    E-print Network

    Jiang, Wen

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

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

    E-print Network

    Phillips, Rob

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

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

    E-print Network

    Bordenstein, Seth

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

  6. Equine lysozyme: The molecular basis of folding, self-assembly and innate amyloid toxicity

    Microsoft Academic Search

    Ludmilla A. Morozova-Roche

    2007-01-01

    Calcium-binding equine lysozyme (EL) combines the structural and folding properties of c-type lysozymes and ?-lactalbumins, connecting these two most studied subfamilies. The structural insight into its native and partially folded states is particularly illuminating in revealing the general principles of protein folding, amyloid formation and its inhibition. Among lysozymes EL forms one of the most stable molten globules and shows

  7. Effects of surfactants on emulsification and secondary structure of lysozyme in aqueous solutions

    Microsoft Academic Search

    Liu Huizhou; Yang Weijing; Chen Jiayong

    1998-01-01

    This paper reports our investigations on the secondary structure of lysozyme in aqueous solution with D2O and comparing systems emulsified with ionic and non-ionic amphiphiles, respectively. The opposite effect of concentration of surfactants on the lysozyme aqueous system was studied and the activity of lysozyme by the turbidimetric assay with different kinds of surfactant was compared. The effect of different

  8. Analysis of Monomer Aggregation and Crystal Growth Rates of Lysozyme

    NASA Technical Reports Server (NTRS)

    Nadarajah, Arunan

    1996-01-01

    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.

  9. Effect of Chaperonin Encoded by Gene 146 on Thermal Aggregation of Lytic Proteins of Bacteriophage EL Pseudomonas aeruginosa.

    PubMed

    Semenyuk, P I; Orlov, V N; Kurochkina, L P

    2015-02-01

    Investigation of the chaperonin encoded by gene 146 of bacteriophage EL Pseudomonas aeruginosa that we characterized earlier has been continued. To reveal the mechanism of its functioning, new recombinant substrate proteins, fragments of gene product (gp) 183 containing the lysozyme domain were prepared. Their interaction with gp146 was studied. The influence of the phage chaperonin on the thermal aggregation of one of these gp183 fragments and endolysin (gp188) was investigated in both the presence and the absence of ATP by dynamic light scattering. In the absence of ATP, the phage chaperonin forms stable complexes with substrate proteins, thereby protecting them against thermal aggregation. Experimental data obtained for different substrate proteins are analyzed. PMID:25756532

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

    NASA Astrophysics Data System (ADS)

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

    2012-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  12. The solubility of hen egg-white lysozyme

    NASA Technical Reports Server (NTRS)

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

    1988-01-01

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

  13. Thyroid Hormones, T3 and T4, in the Brain

    PubMed Central

    Schroeder, Amy C.; Privalsky, Martin L.

    2014-01-01

    Thyroid hormones (THs) are essential for fetal and post-natal nervous system development and also play an important role in the maintenance of adult brain function. Of the two major THs, T4 (3,5,3?,5?-tetraiodo-l-thyronine) is classically viewed as an pro-hormone that must be converted to T3 (3,5,3?-tri-iodo-l-thyronine) via tissue-level deiodinases for biological activity. THs primarily mediate their effects by binding to thyroid hormone receptor (TR) isoforms, predominantly TR?1 and TR?1, which are expressed in different tissues and exhibit distinctive roles in endocrinology. Notably, the ability to respond to T4 and to T3 differs for the two TR isoforms, with TR?1 generally more responsive to T4 than TR?1. TR?1 is also the most abundantly expressed TR isoform in the brain, encompassing 70–80% of all TR expression in this tissue. Conversion of T4 into T3 via deiodinase 2 in astrocytes has been classically viewed as critical for generating local T3 for neurons. However, deiodinase-deficient mice do not exhibit obvious defectives in brain development or function. Considering that TR?1 is well-established as the predominant isoform in brain, and that TR?1 responds to both T3 and T4, we suggest T4 may play a more active role in brain physiology than has been previously accepted. PMID:24744751

  14. Bacteriophage Protein–Protein Interactions

    PubMed Central

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

    2012-01-01

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

  15. Genome of Bacteriophage P1†

    PubMed Central

    ?obocka, Ma?gorzata B.; Rose, Debra J.; Plunkett, Guy; Rusin, Marek; Samojedny, Arkadiusz; Lehnherr, Hansjörg; Yarmolinsky, Michael B.; Blattner, Frederick R.

    2004-01-01

    P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from ?70 promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication. PMID:15489417

  16. Bacteriophage-Based Pathogen Detection

    NASA Astrophysics Data System (ADS)

    Ripp, Steven

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

  17. Inducible bacteriophages from ruminal bacteria.

    PubMed Central

    Klieve, A V; Hudman, J F; Bauchop, T

    1989-01-01

    The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria. Images PMID:2504111

  18. My Life with Bacteriophage ?29

    PubMed Central

    Salas, Margarita

    2012-01-01

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

  19. Inducible bacteriophages from ruminal bacteria.

    PubMed

    Klieve, A V; Hudman, J F; Bauchop, T

    1989-06-01

    The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria. PMID:2504111

  20. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    Letarov, A V; Krisch, H M

    2013-01-01

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

  2. Functional Characterization of a c-type Lysozyme from Indian Shrimp Fenneropenaeus indicus.

    PubMed

    Karthik, Viswanathan; Kamalakannan, Vijayan; Thomas, Ancy; Sudheer, Naduvilamuriparambu Saidumuhammed; Singh, Issac S Bright; Narayanan, Rangarajan Badri

    2014-06-01

    Lysozyme gene from Fenneropenaeus indicus was cloned, expressed in Escherichia coli and characterized. The cDNA consists of 477 base pairs and encodes amino acid sequence of 159 residues. F. indicus lysozyme had high identity (98 %) with Fenneropenaeus merguiensis and Fenneropenaeus chinensis and exhibits low to moderate identities with lysozymes of other invertebrates and vertebrates. This lysozyme is presumed to be chicken types as it possesses two catalytic and eight cysteine residues that are conserved across c-type lysozymes and a c-terminal extension, which is a characteristic of lysozymes from marine invertebrates. Further, the antimicrobial properties of the recombinant lysozyme from F. indicus were determined in comparison with recombinant hen egg white lysozyme. This exhibited high activity against a Gram-negative pathogenic bacterium Salmonella typhimurium and two fungal strains Pichia pastoris and Saccharomyces cerevisiae in turbidimetric assay. Distribution of lysozyme gene and protein in tissues of shrimps infected with white spot syndrome virus revealed that the high levels of lysozyme are correlated with low and high viral load in abdominal muscle and tail, respectively. In conclusion, lysozyme from F. indicus has a broad spectrum of antimicrobial properties, which once again emphasizes its role in shrimp innate immune response. PMID:24676722

  3. Bacteriophages with the Ability to Degrade Uropathogenic Escherichia Coli Biofilms

    PubMed Central

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

    2012-01-01

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

  4. Yak (Bos grunniens) stomach lysozyme: molecular cloning, expression and its antibacterial activities.

    PubMed

    Jiang, Mingfeng; Chen, Yan; Wang, Yong; Loor, Juan J; Ye, Yuhui; Wen, Yongli; Zi, Xiangdong; Cai, Yingfan; Drackley, James K

    2010-01-01

    The cDNA coding for stomach lysozyme in yak was cloned. The cloned cDNA contains a 432 bp open reading frame and encodes 143 amino acids (16.24 KDa) with a signal peptide of 18 amino acids. Further analysis revealed that its amino acid sequence shares many common properties with cow milk lysozyme. Expression of this gene was also detected in mammary gland tissue by RT-PCR. Phylogenetic relationships among yak stomach lysozyme and 8 cow lysozymes indicated that the yak enzyme is more closely related to both cow milk lysozyme and the pseudogene PsiNS4 than cow stomach lysozyme. Recombinant yak lysozyme purified by Ni(2+)-column showed a molecular weight of 33.78 kDa and exhibited lytic activity against Staphylococcus aureus, providing evidence of its antibacterial activities. PMID:20024784

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

    NASA Astrophysics Data System (ADS)

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

    2005-03-01

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

  6. Locations of Bromide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  7. 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...

  8. 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...

  9. Bacteriophage Ecology in Commercial Sauerkraut Fermentations

    Microsoft Academic Search

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

    2003-01-01

    Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total

  10. Biochemical and Genetic Characterization of Bacteriophage Holins

    E-print Network

    To, Kam Ho

    2013-11-06

    Materials, strains, bacteriophages, plasmids, and growth media ............. 43 Mutagenesis of P2 Y ................................................................................ 47 Growth conditions, inductions and TCA precipitation... .............................................................................. 52 Requirement for C-terminal cytoplasmic domain .................................... 58 Sizing the Y hole ...................................................................................... 60 Lysis deficiency of Y mutants is associated...

  11. 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...

  12. Antimicrobial drug discovery through bacteriophage genomics

    Microsoft Academic Search

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

    2004-01-01

    Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of

  13. Long-Circulating Bacteriophage as Antibacterial Agents

    Microsoft Academic Search

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

    1996-01-01

    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

  14. A stochastic model for bacteriophage therapies

    E-print Network

    Bardina, Xavier; Rovira, Carles; Tindel, Samy

    2011-01-01

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

  15. Immobilization of bacteriophages on modified silica particles

    Microsoft Academic Search

    Rebecca Cademartiri; Hany Anany; Isabelle Gross; Rahul Bhayani; Mansel Griffiths; Michael A. Brook

    2010-01-01

    Bacteriophages are selective anti-bacterial agents, which are receiving increasing acceptance by regulatory agencies for use both in the food industry and in clinical settings for biocontrol. While immobilized phage could be particularly useful to create antimicrobial surfaces, current immobilization strategies require chemical bioconjugation to surfaces or more difficult processes involving modification of their head proteins to express specific binding moieties,

  16. Optimal bacteriophage mutation rates for phage therapy

    Microsoft Academic Search

    David T. Kysela; Paul E. Turner

    2007-01-01

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

  17. Bacteriophages and Diffusion of ?-lactamase Genes

    PubMed Central

    Muniesa, Maite; García, Aurora; Miró, Elisenda; Mirelis, Beatriz; Prats, Guillem; Jofre, Juan

    2004-01-01

    We evaluated the presence of various ?-lactamase genes within the bacteriophages in sewage. Results showed the occurrence of phage particles carrying sequences of blaOXA-2, blaPSE-1 or blaPSE-4 and blaPSE-type genes. Phages may contribute to the spread of some ?-lactamase genes. PMID:15207070

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

    SciTech Connect

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

    2006-01-01

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

  19. Effectiveness of bacteriophages in the sputum of cystic fibrosis patients.

    PubMed

    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

    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

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

    E-print Network

    Hall, Sarah Alice

    2011-11-24

    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...

  1. 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...

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

    E-print Network

    Jiang, Wen

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

  3. 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 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...

  4. 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 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...

  5. 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 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...

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

    PubMed

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

    2014-12-12

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

  7. Enzyme characterisation and gene expression profiling of Atlantic salmon chicken- and goose-type lysozymes.

    PubMed

    Myrnes, Bjørnar; Seppola, Marit; Johansen, Audny; Overbø, Kersti; Callewaert, Lien; Vanderkelen, Lise; Michiels, Chris W; Nilsen, Inge W

    2013-05-01

    Lysozymes represent important innate immune components against bacteria. In this study, Atlantic salmon (Salmo salar) goose (g-) and chicken (c-) types of lysozyme were subjected to protein characterisations and tissue expression analyses. Specific bacterial protein inhibitors of g- and c-type lysozymes were employed to discriminate between respective enzyme activities. Blood, gills and liver contained activities exclusive for the g-type lysozyme. Only haematopoietic organs (head kidney and spleen) contained enzyme activities of both g- and c-lysozyme enzymes and c-type activity was not found outside these organs. Gene transcript levels proportional to enzyme activity levels were detected for the g-type lysozyme but not for the c-type. In vitro studies revealed significant induction of c-type gene expression and enzyme activity in macrophages after incubation with lipopolysaccharide (LPS) while expression of the g-type lysozyme gene was unaffected. The activity of purified native c-type enzyme was profoundly reduced by divalent cations and displayed low tolerance to monovalent cations, while the native g-type lysozyme was stimulated by monovalent cations and tolerated low concentrations of divalent cations. Activities of both enzymes increased with temperature elevations up to 60°C. The native g-type lysozyme responses to temperature in particular are in apparent conflict to the ones for the recombinant salmon g-lysozyme. Our results imply separate expression regulations and different functions of c- and g-type lysozymes in salmon. LPS-induced expression of c-type lysozyme and broad constitutive tissue distribution of g-type lysozyme in salmon is different from findings in other studied fish species. PMID:23396098

  8. Inhibition of amyloid fibrillation of lysozyme by phenolic compounds involves quinoprotein formation.

    PubMed

    Feng, Shuang; Song, Xiu-Huan; Zeng, Cheng-Ming

    2012-11-16

    Numerous phenolic compounds have been reported to have an inhibitory role on amyloid formation of proteins. The present study, utilizing lysozyme as a model system, examined the anti-amyloidogenic effects of phenol and three diphenol epimers. The results indicated that catechol and hydroquinone dose-dependently inhibited lysozyme fibrillation and covalently bound to the peptide chains to form quinoproteins, showing a similar effect to benzoquinone. In contrast, phenol and resorcinol did not modify the peptide with a quinone moiety, showing no effect on lysozyme fibrillation. We suggest that quinone intermediates are the active form for a phenolic compound to inhibit lysozyme fibrillation. The modification of lysozyme with quinone moieties alters the interacting forces between peptide chains and consequently interrupts the process of lysozyme fibrillation. PMID:23058288

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

    PubMed

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

    2008-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  12. Photoprocesses of merocyanine 540 bound to serum albumin and lysozyme

    NASA Astrophysics Data System (ADS)

    Zhang, Yazhou; Görner, Helmut

    2012-03-01

    The binding of merocyanine 540 to either lysozyme or bovine serum albumin (BSA) in aqueous solution and the related photodecomposition processes were studied. Absorption, fluorescence and trans ? cis photoisomerization demonstrate a shift from free dimers to monomers upon binding to BSA, in contrast to lysozyme, where the binding appears spectroscopically less pronounced. The quantum yield (?red) of merocyanine damage is generally small (?0.0004). However, ?red is markedly enhanced upon binding and was found to be comparable to the quantum yields of protein oxidation, which are ca. 0.002. The mechanisms of protein oxidation were discussed. The major effect is electron transfer from aromatic amino acid residues of the protein to the triplet state of merocyanine 540.

  13. Locations of Halide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  14. Actin - Lysozyme Interactions in Model Cystic Fibrosis Sputum

    NASA Astrophysics Data System (ADS)

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

    2003-03-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  16. Evolution and the complexity of bacteriophages

    PubMed Central

    Serwer, Philip

    2007-01-01

    Background The genomes of both long-genome (> 200 Kb) bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. Hypothesis Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection). (3) The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection). (4) The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. Testing the hypothesis I propose testing this hypothesis by controlled evolution in microbial communities to (1) determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2) find the environmental conditions that select for the presence of cellular gene homologs, (3) determine which, if any, bacteriophage genes were selected for maintaining the homologs and (4) determine the dynamics of homolog evolution. Implications of the hypothesis This hypothesis is an explanation of evolutionary leaps in general. If accurate, it will assist both understanding and influencing the evolution of microbes and their communities. Analysis of evolutionary complexity increase for at least prokaryotes should include analysis of genomes of long-genome bacteriophages. PMID:17355641

  17. Adsorption of Lysozyme Using Citric Acid Modified Waste Beer Yeast

    Microsoft Academic Search

    Haixia Guo; Xiaomei Sun; Buhai Li

    2012-01-01

    To prepare a novel biosorbent, we modified waste beer yeast with citric acid using NaH2PO4 as catalyst in N, N-dimethylformamide. We characterized the absorbent using Fourier transform infrared and X-ray photoelectron spectroscopy. We examined the factors of pH, temperature, adsorption time, initial lysozyme concentration and NaCl ionic strength on the adsorption capacity. Based on the Langmuir isotherm, the adsorption capacity

  18. Functional relationship between bacteriophages G4 and phi X174.

    PubMed Central

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

    1979-01-01

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

  19. 21 CFR 866.2050 - Staphylococcal typing bacteriophage.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

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

    PubMed Central

    Cherepanov, A V; de Vries, S

    2001-01-01

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

  1. Identification and expression analysis of three c-type lysozymes in Oreochromis aureus.

    PubMed

    Gao, Feng-ying; Qu, Lan; Yu, Shao-guo; Ye, Xing; Tian, Yuan-yuan; Zhang, Li-li; Bai, Jun-jie; Lu, Maixin

    2012-05-01

    Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. Three genes encoding chicken-type (c-type) lysozymes, C1-, C2-, C3-type, were obtained from tilapia Oreochromis aureus by RT-PCR and the RACE method. Catalytic and other conserved structure residues required for functionality were identified. The amino acid sequence identities between C1- and C2-type, C1- and C3-type, C2- and C3-type were 67.8%, 65.7% and 63.9%, respectively. Phylogenetic tree analyze indicated the three genes were firstly grouped to those of higher teleosteans, Pleuronectiformes and Tetraodontiformes fishes, and then clustered to those of lower teleosteans, Cypriniformes fishes. Bioinformatic analysis of mature peptide showed that the three genes possess typical sequence characteristics, secondary and tertiary structure of c-type lysozymes. The three tilapia c-type lysozymes mRNAs were mainly expressed in liver and muscle, and C1-type lysozyme also highly expressed in intestine. C1-type lysozyme mRNA was weakly expressed in stomach, C2- and C3-type mRNAs were weakly expressed in intestine. After bacterial challenge, up-regulation was obvious in kidney and spleen for C1-type lysozyme mRNA, while for C2- and C3-type lysozyme obvious increase were observed in stomach and liver, suggesting that C1-type lysozyme may mainly play roles in defense, while C2- and C3-type lysozyme mainly conduct digestive function against bacteria infection. All the three c-type recombinant lysozymes displayed lytic activity against Gram-negative and Gram-positive bacteria. These results indicated that three c-type lysozymes play important roles in the defense of O. aureus against bacteria infections. PMID:22343107

  2. Analysis of two lysozyme genes and antimicrobial functions of their recombinant proteins in Asian seabass.

    PubMed

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

    2013-01-01

    Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass (Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu(50) and Asp(67)) 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 (Glu(71), Asp(84), Asp(95)) 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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Jay, James M.

    1966-01-01

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

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

    Microsoft Academic Search

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

    2004-01-01

    Lysozyme was purified from the plasma of eastern oysters (Crassostrea virginica) using a combination of ion exchange and gel filtration chromatographies. The molecular mass of purified lysozyme was estimated at 18.4 kDa by SDS-PAGE, and its isoelectric point was greater than 10. Mass spectrometric analysis of the purified enzyme revealed a high-sequence homology with i-type lysozymes. No similarity was found

  6. Investigation of Lysozyme–Antilysozyme Interactions in a Model Tetrahymena–EscherichiaCommunity

    Microsoft Academic Search

    O. V. Bukharin; N. V. Nemtseva

    2001-01-01

    Lysozyme and antilysozyme activities present in a wide range of microorganisms determine the so-called lysozyme–antilysozyme system of hydrobionts, which greatly contribute to the formation of aquatic biocenoses. However, the mechanism of the functioning of this system in natural freshwater communities remains obscure. The experimental investigation of lysozyme–antilysozyme interactions in a model Tetrahymena–Escherichiacommunity showed that the antilysozyme activity of Escherichia colileads

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

    NASA Astrophysics Data System (ADS)

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

    2012-05-01

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

  8. Levels of salivary lysozyme, lactoperoxidase, and lactoferrin in diabetic hamsters.

    PubMed Central

    Muratsu, K; Morioka, T

    1985-01-01

    In an attempt to clarify the mechanism(s) of increased susceptibility to oral infection in diabetics, we examined the levels of salivary antibacterial factors, including lysozyme, lactoperoxidase, and lactoferrin, in diabetic hamsters whose condition was induced with streptozotocin. Saliva was collected from these hamsters periodically for 19 weeks after the administration of streptozotocin. Diabetes persisted with significant hyperglycemia throughout the experiment after a single injection of streptozotocin. There was no significant difference between groups in the amount of saliva secreted. In diabetic hamsters, lysozyme activity decreased by 56% and lactoperoxidase activity decreased by 53% compared with the control hamsters 19 weeks after the administration of streptozotocin. There was no significant difference between groups in the amount of salivary lactoferrin. However, the ratio of lactoferrin to total protein increased to approximately double the amount of that of the control hamsters. Insulin treatment had a significant effect on lysozyme and lactoperoxidase activity, recovering 73 and 74% those of the controls, respectively, and the ratio of lactoferrin to total salivary protein reverted to normal values. Growth inhibition of Lactobacillus plantarum ATCC 8014 with whole saliva and amylase activity significantly decreased in diabetic hamsters. The position of each protein band of whole saliva on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was almost the same for control and diabetic hamsters; however, there was some variability in band intensity. Images PMID:2580790

  9. Stability of crystallised and spray-dried lysozyme.

    PubMed

    Elkordy, Amal A; Forbes, Robert T; Barry, Brian W

    2004-07-01

    Moisture and temperature promote protein degradation. The stabilities of commercial, crystallised and spray-dried lysozyme, a model protein, were assessed under these stresses to explore whether a crystalline protein had better storage stability than a conventionally produced one. Samples were maintained at different relative humidities (RH) and temperatures for 20 weeks and stabilities estimated in solid and aqueous states. Differential scanning calorimetry (DSC) and thermogravimetry (TGA) characterised solid samples. Fourier transform Raman (FT-Raman) spectroscopy analysed solid material and aqueous solutions. High sensitivity differential scanning calorimetry (HSDSC) and enzymatic assays were used to monitor solutions. DSC and HSDSC data revealed that crystals maintained thermal stability at high RH; spray drying appreciably changed melting characteristics. These results correlated with enzymatic assays that demonstrated good activity retention for crystals but less so for spray-dried material (e.g. 95 and 87% relative to fresh samples after 20 weeks at 40 degrees C/75% RH). FT-Raman analysis showed that crystallised lysozyme better-maintained protein conformational integrity compared to spray-dried samples in accelerated stability studies. Based on TGA data, spray-dried protein absorbed water on storage under humid conditions, which induced instability. Thus, crystallisation enhanced storage stability of lysozyme with negligible loss of activity. PMID:15196626

  10. A phage T4 site-specific endonuclease, SegE, is responsible for a non-reciprocal genetic exchange between T-even-related phages.

    PubMed

    Kadyrov, F A; Shlyapnikov, M G; Kryukov, V M

    1997-09-22

    The bacteriophage T4 segE gene encoding site-specific endonuclease lies between the hoc.1 and uvsW genes. The similar region of T-even-related phage RB30 lacks the segE gene. Here we demonstrate that the phage T4 segE gene is inherited preferably by progeny of mixed infection with RB30. The preferred inheritance of the segE gene depends on its own expression and is based on a non-reciprocal homologous recombination event providing the transfer of the gene from the segE-containing to the segE-lacking allele. The SegE endonuclease cleaves DNA in a site located at the 5' end of the uvsW gene in the RB30 genome. The T4 DNA is also cleaved by the enzyme, but less efficiently. The cleavage at the RB30 site appears to initiate the observed conversion, which is stimulated by DNA homology and accompanied by co-conversion of flanking markers. Our findings provide a novel example of endonuclease-dependent generation of genetic variation in prokaryotes. PMID:9326373

  11. Bacteriophage exclusion, a new defense system.

    PubMed

    Barrangou, Rodolphe; van der Oost, John

    2015-01-13

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

  12. Studies on bacteriophages of Propionibacterium acnes

    Microsoft Academic Search

    E. C. Jong; H. L. Ko; G. Pulverer

    1975-01-01

    With the help of adaptation experiments, 61 phage preparations out of 36Propionibacterium acnes bacteriophages (32 isolated by us and 4 sent from abroad) were established. On the basis of their stability, spectrum of activity, and virulence, 13 phages were selected for phagetyping. 58 well-classifiedP. acnes strains were grouped into 7 phage-types. 7 strains ofP. granulosum, two strains ofP. avidum, and

  13. Splint ligation of RNA with T4 DNA ligase

    PubMed Central

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

    2014-01-01

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

  14. Isolation, growth and genome of the Rhodothermus RM378 thermophilic bacteriophage.

    PubMed

    Hjorleifsdottir, Sigridur; Aevarsson, Arnthor; Hreggvidsson, Gudmundur O; Fridjonsson, Olafur H; Kristjansson, Jakob K

    2014-03-01

    Several bacteriophages that infect different strains of the thermophilic bacterium Rhodothermus marinus were isolated and their infection pattern was studied. One phage, named RM378 was cultivated and characterized. The RM378 genome was also sequenced and analyzed. The phage was grouped as a member of the Myoviridae family with A2 morphology. It had a moderately elongated head, with dimensions of 85 and 95 nm between opposite apices and a 150 nm long tail, attached with a connector to the head. RM378 showed a virulent behavior that followed a lytic cycle of infection. It routinely gave lysates with 10(11) pfu/ml, and sometimes reached titers as high as 10(13) pfu/ml. The titer remained stable up to 65 °C but the phage lost viability when incubated at higher temperatures. Heating for 30 min at 96 °C lowered the titer by 10(4). The RM378 genome consisted of ds DNA of 129.908 bp with a GC ratio of 42.0% and contained about 120 ORFs. A few structural proteins, such as the major head protein corresponding to the gp23 in T4, could be identified. Only 29 gene products as probable homologs to other proteins of known function could be predicted, with most showing only low similarity to known proteins in other bacteriophages. These and other studies based on sequence analysis of a large number of phage genomes showed RM378 to be distantly related to all other known T4-like phages. PMID:24318108

  15. Bacteriophage P70: unique morphology and unrelatedness to other Listeria bacteriophages.

    PubMed

    Schmuki, Martina M; Erne, Doris; Loessner, Martin J; Klumpp, Jochen

    2012-12-01

    Listeria monocytogenes is an important food-borne pathogen, and its bacteriophages find many uses in detection and biocontrol of its host. The novel broad-host-range virulent phage P70 has a unique morphology with an elongated capsid. Its genome sequence was determined by a hybrid sequencing strategy employing Sanger and PacBio techniques. The P70 genome contains 67,170 bp and 119 open reading frames (ORFs). Our analyses suggest that P70 represents an archetype of virus unrelated to other known Listeria bacteriophages. PMID:22993158

  16. Cloning, expression and stable maintenance of a bacteriophage DNA repair gene in E. coli

    SciTech Connect

    Recinos, A. III; Lloyd, R.S.

    1986-05-01

    Efforts to clone the bacteriophage T4 DNA repair gene, denV, have met with repeated problems of gene instability. Therefore, oligonucleotide site-directed mutagenesis was used to create new DNA restriction sites closely flanking the structural gene, facilitating the removal of adjacent deleterious T4 sequences. The sequence which encodes the DNA repair enzyme, endonuclease V, was then reconstructed behind the hybrid lambda promoter O/sub L/P/sub R/ and its associated ribosome-binding site in a plasmid vector for expression in E. coli. Transformants harboring the expression plasmid bearing the correct reconstruction of denV were screened and selected by colony hybridization to a synthetic bridge oligonucleotide and by restriction analyses. The denV structural gene was found inserted at an equal frequency in both orientations relative to the promoter. These plasmids were transformed into an E. coli strain which is defective for UV excision recombination and repair (uvrA/sup -/, recA/sup -/). The defective hosts were assayed for survival after UV irradiation, and expression of the denV gene was found to enhance UV survival to levels indicative of full complementation of the uvrA/sup -/ deficiency. Correspondingly, Western blot analyses of cellular protein extracts from the defective host harboring the denV/sup +/ plasmid showed a protein which was immunoreactive with an anti-endonuclease V antibody. Additionally, in infections using a UV-irradiated T4 mutant (T4 denV1, defective for active endonuclease V), these cells fully complemented the mutant phage to wild type survival levels.

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

    E-print Network

    Bordenstein, Seth

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

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

    E-print Network

    Priscu, John C.

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

  19. 2d-LC-MS/MS Method-ORNL Developed for Bacteriophage

    E-print Network

    Sullivan, Matthew B.

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. iBioSeminar: Bacteriophages: Genes and Genomes

    NSDL National Science Digital Library

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

    2011-06-06

    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.

  2. Complete Genome Sequences of Five Paenibacillus larvae Bacteriophages

    PubMed Central

    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

    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

  3. Complete Genome Sequences of Five Paenibacillus larvae Bacteriophages.

    PubMed

    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

    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

  4. Modeling and analysis of a marine bacteriophage infection

    Microsoft Academic Search

    Edoardo Beretta; Yang Kuang

    1998-01-01

    A mathematical model for the marine bacteriophage infection is proposed and its essential mathematical features are analyzed. Since bacteriophage infection induces bacterial lysis which releases into the marine environment, on the average, `b' viruses per cell, the parameter b?(1,+?) or `virus replication factor' is chosen as the main parameter on which the dynamics of the infection depends. We proved that

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Targeting Antibacterial Agents by Using Drug-Carrying Filamentous Bacteriophages

    Microsoft Academic Search

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

    2006-01-01

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

  7. 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...

  8. Bacteriophage sensitivity patterns among bacteria isolated from marine waters

    Microsoft Academic Search

    K. Moebus; H. Nattkemper

    1981-01-01

    Phage-host cross-reaction tests were performed with 774 bacterial strains and 298 bacteriophages. The bacteria (bacteriophages) were isolated at different times from water samples collected in the Atlantic Ocean between the European continental shelf and the Sargasso Sea: 733 (258) strains; in the North Sea near Helgoland: 31 (31) strains; and in the Bay of Biscay: 10 (9) strains. Of the

  9. The effects of bacteriophage and nanoparticles on microbial processes

    NASA Astrophysics Data System (ADS)

    Moody, Austin L.

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

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

    SciTech Connect

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

    2006-01-01

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

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

    PubMed

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

    2015-03-31

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

  12. Protist-Type Lysozymes of the Nematode Caenorhabditis elegans Contribute to Resistance against Pathogenic Bacillus thuringiensis

    PubMed Central

    Boehnisch, Claudia; Wong, Daniel; Habig, Michael; Isermann, Kerstin; Michiels, Nicolaas K.; Roeder, Thomas; May, Robin C.; Schulenburg, Hinrich

    2011-01-01

    Pathogens represent a universal threat to other living organisms. Most organisms express antimicrobial proteins and peptides, such as lysozymes, as a protection against these challenges. The nematode Caenorhabditis elegans harbours 15 phylogenetically diverse lysozyme genes, belonging to two distinct types, the protist- or Entamoeba-type (lys genes) and the invertebrate-type (ilys genes) lysozymes. In the present study we characterized the role of several protist-type lysozyme genes in defence against a nematocidal strain of the Gram-positive bacterium Bacillus thuringiensis. Based on microarray and subsequent qRT-PCR gene expression analysis, we identified protist-type lysozyme genes as one of the differentially transcribed gene classes after infection. A functional genetic analysis was performed for three of these genes, each belonging to a distinct evolutionary lineage within the protist-type lysozymes (lys-2, lys-5, and lys-7). Their knock-out led to decreased pathogen resistance in all three cases, while an increase in resistance was observed when two out of three tested genes were overexpressed in transgenic lines (lys-5, lys-7, but not lys-2). We conclude that the lysozyme genes lys-5, lys-7, and possibly lys-2 contribute to resistance against B. thuringiensis, thus highlighting the particular role of lysozymes in the nematode's defence against pathogens. PMID:21931778

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

    ERIC Educational Resources Information Center

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

    2010-01-01

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

  14. Absence of Allergic Reactions to Egg White Lysozyme Additive in Grana Padano Cheese

    Microsoft Academic Search

    Amerigo Iaconelli; Lucia Fiorentini; Sara Bruschi; Filippo Rossi; Geltrude Mingrone; Gianfranco Piva

    2008-01-01

    Objective: The negative effects on cheese quality of milk contaminated by spores of Clostridium butyricum and Cl.tyrobutyricum is prevented by the use of egg white lysozyme as additive. Since the presence of lysozyme in cheese could be possibly risky in allergic subjects, we aimed at investigating its absorption as well as serum IgE antibody titers after ingestion of Grana Padano

  15. Sensitization of heat-treated Listeria monocytogenes to added lysozyme in milk.

    PubMed Central

    Kihm, D J; Leyer, G J; An, G H; Johnson, E A

    1994-01-01

    Listeria monocytogenes was highly resistant to hen egg white lysozyme in whole milk but was sensitive in media and in phosphate buffer. Methods to sensitize the pathogen to lysozyme in milk were investigated. Treatment of whole milk by cation exchange to remove minerals, particularly Ca2+ and Mg2+, slightly promoted inactivation of L. monocytogenes by lysozyme at 4 degrees C over a period of 6 days. Heat treatment (62.5 degrees C for 15 s) strongly sensitized L. monocytogenes to lysozyme in demineralized milk and in MES [2-(N-morpholino)ethanesulfonic acid] buffer. Addition of Ca2+ or Mg2+ to the demineralized milk restored resistance to lysozyme. Cells were more rapidly heat inactivated at 55 degrees C in demineralized milk containing lysozyme, and addition of Ca2+ to the demineralized milk restored the resistance to heat. The results indicate that minerals or mineral-associated components protect L. monocytogenes from inactivation by lysozyme and heat in milk, probably by increasing cell surface stability. The heat treatment of foods containing added lysozyme can probably play a significant role in producing microbiologically safe foods. Images PMID:7986052

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

    PubMed

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

    2014-10-14

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

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

    Microsoft Academic Search

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

    2000-01-01

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

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

    Microsoft Academic Search

    Sirlei Daffre; Per Kylsten; Christos Samakovlis; Dan Hultmark

    1994-01-01

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

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

    PubMed

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

    2014-09-01

    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

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Volpato, Roberto

    2014-08-01

    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.

  2. T4 DNA condensation in water-alcohol media

    E-print Network

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

    2011-07-21

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

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

    PubMed Central

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

    2015-01-01

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

  4. Polar solvation dynamics of lysozyme from molecular dynamics studies

    NASA Astrophysics Data System (ADS)

    Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy

    2012-05-01

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

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

    Microsoft Academic Search

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

    1994-01-01

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

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

    SciTech Connect

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

    2001-10-01

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

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

    PubMed

    Beyer, C

    2013-09-01

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

  8. M13 Bacteriophage Based Protein Sensors

    NASA Astrophysics Data System (ADS)

    Lee, Ju Hun

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

  9. Bacteriophages as pathogens and immune modulators?

    PubMed

    Lengeling, A; Mahajan, A; Gally, D L

    2013-01-01

    While Shiga toxins (Stx) are key determinants of enterohemorrhagic Escherichia coli (EHEC) pathophysiology in humans, their dissemination to target organs following gastrointestinal EHEC infection is still poorly understood. Most types of Stx target cells with globotriaosylceramide (Gb3) receptors, which are expressed on endothelial cells. According to current theory, Stx is trafficked on the surface of peripheral blood cells, and transfer of toxin from these trafficking cells to endothelial cells results in microvascular damage to target organs, including the kidneys and brain. Inside the cell, Stx inhibits protein synthesis, resulting in cell death. Host "repair" responses can lead to microthrombus formation, erythrocyte damage, and reduced oxygen supply, potentially resulting in organ failure. A recent study [L. V. Bentancor et al., mBio 4(5):e00501-13, 2013, doi:10.1128/mBio.00501-13] indicates that another mechanism for Stx "dissemination" needs to be considered. Bentancor et al. demonstrated that high-pressure injection of a plasmid encoding the "prokaryotic" Stx2 sequence into mice can lead to mortality, with pathology indicative of Stx activity and antibody responses to Stx. While the plasmid levels and injection methodology were extreme, the study indicates that these sequences are potentially taken up into eukaryotic cells, transcribed, and translated, producing active Stx. Stx genes are present on integrated bacteriophage genomes in EHEC, and Stx-encoding phages are released following bacterial lysis in the gastrointestinal tract. We therefore need to consider whether bacteriophage sequences can be expressed in eukaryotic cells, what the wider implications are for our understanding of many "bacterial" diseases, and the possibility of developing novel interventions that target bacteriophages. PMID:24222490

  10. Studies on bacteriophages of Propionibacterium acnes.

    PubMed

    Jong, E C; Ko, H L; Pulverer, G

    1975-09-19

    With the help of adaptation experiments, 61 phage preparations out of 36 Propionibacterium acnes bacteriophages (32 isolated by us and 4 sent from abroad) were established. On the basis of their stability, spectrum of activity, and virulence, 13 phages were selected for phagetyping. 58 well-classified P. acnes strains were grouped into 7 phage-types. 7 strains of P. granulosum, two strains of P. avidum, and one yet ungroupable microserophilic propionibacterium strain were resistant to all 61 phages, even when tested in 100 X RTD. A combination of phagetyping with biotyping resulted in data especially useful for the differentiation of P. acnes. PMID:1196261

  11. Bacteriophages as biocontrol agents of food pathogens.

    PubMed

    Mahony, Jennifer; McAuliffe, Olivia; Ross, R Paul; van Sinderen, Douwe

    2011-04-01

    Bacteriophages have long been recognized for their potential as biotherapeutic agents. The recent approval for the use of phages of Listeria monocytogenes for food safety purposes has increased the impetus of phage research to uncover phage-mediated applications with activity against other food pathogens. Areas of emerging and growing significance, such as predictive modelling and genomics, have shown their potential and impact on the development of new technologies to combat food pathogens. This review will highlight recent advances in the research of phages that target food pathogens and that promote their use in biosanitation, while it will also discuss its limitations. PMID:21115341

  12. Bacteriophage lambda display systems: developments and applications.

    PubMed

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

    2014-04-01

    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

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

    PubMed

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

    2015-05-15

    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

  14. Printed in Great Britain A bacteriophage of Rhodopseudomonas

    E-print Network

    H. Gorhamt; C. S. Dow

    A bacteriophage, 4BHG1, was isolated from a small eutrophic pond from which its host, Rhodopseudomonas blastica, was originally obtained. It is a lytic bacteriophage specific for R. blastica which also causes non-specific lysis of Rhodobacter sphaeroides 8253. 4BHGl has an icosahedral head of 62 nm diameter and a short 39 nm tail. Caesium chloride density gradient centrifugation of infected cell lysates gave a single bacteriophage band at a density of 1.385 g ~ m-~, but also occasionally a second band was observed at a lower density. No differences were apparent between bacteriophage taken from either of the two bands. 4BHG1 contained double-stranded DNA with a size of 48 kb and a G +C content of 50.6 mol%. The bacteriophage adsorbed to both photosynthetically and chemoheterotrophically grown R. blastica at an identical rate of 1-39 x ml-1 mi+. One-step growth curves and kinetic studies of the bacteriophage under these physiological regimes showed no differences in the latent and rise periods and only slight changes in the burst size. Adsorption of this bacteriophage is cell-surface specific and attachment only occurs to the 'older ' pole of the budding reproductive cell.

  15. Genetically enhanced lysozyme evades a pathogen derived inhibitory protein.

    PubMed

    Dostal, Sarah M; Fang, Yongliang; Guerrette, Jonathan C; Scanlon, Thomas C; Griswold, Karl E

    2015-04-17

    The accelerating spread of drug-resistant bacteria is creating demand for novel antibiotics. Bactericidal enzymes, such as human lysozyme (hLYZ), are interesting drug candidates due to their inherent catalytic nature and lack of susceptibility to the resistance mechanisms typically directed toward chemotherapeutics. However, natural antibacterial enzymes have their own limitations. For example, hLYZ is susceptible to pathogen derived inhibitory proteins, such as Escherichia coli Ivy. Here, we describe proof of concept studies demonstrating that hLYZ can be effectively redesigned to evade this potent lysozyme inhibitor. Large combinatorial libraries of hLYZ were analyzed using an innovative screening platform based on microbial coculture in hydrogel microdroplets. Isolated hLYZ variants were orders of magnitude less susceptible to E. coli Ivy yet retained high catalytic proficiency and inherent antibacterial activity. Interestingly, the engineered escape variants showed a disadvantageous increase in susceptibility to the related Ivy ortholog from Pseudomonas aeruginosa as well as an unrelated E. coli inhibitory protein, MliC. Thus, while we have achieved our original objective with respect to escaping E. coli Ivy, engineering hLYZ for broad-spectrum evasion of proteinaceous inhibitors will require consideration of the complex and varied determinants that underlie molecular recognition by these emerging virulence factors. PMID:25607237

  16. Influence of inhibitor binding on the internal motions of lysozyme.

    PubMed Central

    Cross, A J; Fleming, G R

    1986-01-01

    Time-resolved laser-induced fluorescence depolarization measurements of internal motions in lysozyme are presented. The fluorescent dye eosin binds in a one-to-one complex with the enzyme, and is used both to measure the overall tumbling time constants and to probe the motions of residues in the region of binding. The precision and accuracy of the present method for determining the overall tumbling time constants compare favorably with those from other methods used in the literature. The extent of the internal motions, as described by a model independent order parameter, S2, is temperature dependent, and changes when the inhibitor N,N',N"-triacetylchitotriose, (GlcNAc)3, is bound to the active site of the enzyme. The observed temperature dependence and changes in S2 upon binding of (GlcNAc)3 are interpreted in terms of a nonharmonic model of the effective potential that is consistent with the picture of concerted motions in the protein. The values of the parameters of the potential that reproduce the data with and without the bound inhibitor imply that (GlcNAc)3 binding causes an increase in the rigidity of the protein, which agree qualitatively with other results on the lysozyme-(GlcNAc)3 system. PMID:3756301

  17. Lysozyme Protein Solution with an Intermediate Range Order Structure

    SciTech Connect

    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

    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.

  18. The Effect of Protein Impurities on Lysozyme Crystal Growth

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

    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.

  19. Bacteriophages and Their Role in Food Safety

    PubMed Central

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

    2012-01-01

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

  20. Immobilization of bacteriophages on modified silica particles.

    PubMed

    Cademartiri, Rebecca; Anany, Hany; Gross, Isabelle; Bhayani, Rahul; Griffiths, Mansel; Brook, Michael A

    2010-03-01

    Bacteriophages are selective anti-bacterial agents, which are receiving increasing acceptance by regulatory agencies for use both in the food industry and in clinical settings for biocontrol. While immobilized phage could be particularly useful to create antimicrobial surfaces, current immobilization strategies require chemical bioconjugation to surfaces or more difficult processes involving modification of their head proteins to express specific binding moieties, for example, biotin or cellulose binding domains; procedures that are both time and money intensive. We report that morphologically different bacteriophages, active against a variety of food-borne bacteria: Escherichia coli; Salmonella enterica; Listeria monocytogenes; and Shigella boydii, will effectively physisorb to silica particles, prepared by silica surface modification with poly(ethylene glycol), carboxylic acid groups, or amines. The phages remain infective to their host bacteria while adsorbed on the surface of the silica particles. The number of infective phage bound to the silica is enhanced by the presence of ionic surfaces, with greater surface charge - to a maximum - correlating with greater concentration of adsorbed phage. Above the maximum charge concentration, the number of active phage drops. PMID:19945158

  1. Understanding Bacteriophage Specificity in Natural Microbial Communities

    PubMed Central

    Koskella, Britt; Meaden, Sean

    2013-01-01

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

  2. Bacteriophage recombination systems and biotechnical applications.

    PubMed

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    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

  3. HyShot-T4 Supersonic Combustion Experiments

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

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

    PubMed

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

    2013-07-10

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

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

    PubMed

    Yokooji, Tomoharu; Hamura, Koh; Matsuo, Hiroaki

    2013-08-16

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

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

    SciTech Connect

    Goven, A.J.; Chen, S.C.; Fitzpatrick, L.C. (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

    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.

  7. Fecal lysozyme in assessment of disease activity in inflammatory bowel disease.

    PubMed

    van der Sluys Veer, A; Brouwer, J; Biemond, I; Bohbouth, G E; Verspaget, H W; Lamers, C B

    1998-03-01

    This study was undertaken to determine whether measurement of fecal lysozyme is helpful in determining disease activity in inflammatory bowel disease. In 112 patients with Crohn's disease, 46 patients with ulcerative colitis, and 40 controls, fecal lysozyme concentration was measured. Results were correlated with CDAI and AI in Crohn's disease and with Truelove and Witts' grading in ulcerative colitis. Fecal lysozyme concentration (mean +/- SEM) was significantly (P < 0.001) higher in Crohn's disease (75 +/- 14 microg/g) and ulcerative colitis (238 +/- 33 microg/g) than in controls (6 +/- 1 microg/g). There was only a weak correlation between fecal lysozyme concentration and CDAI (r = 0.32; P = 0.001) and AI (r = 0.38; P < 0.0005) in patients with Crohn's disease and with Truelove and Witts' grading (r = 0.47; P = 0.001) in ulcerative colitis. When CDAI > or = 150 or AI > or = 100 were used as the standard for active disease, fecal lysozyme concentration was elevated in 78% of patients with active colonic Crohn's disease. In ulcerative colitis fecal lysozyme concentration was increased in active disease (95% in grade II and 94% in grade III) as compared 33% in grade I. Measurement of fecal lysozyme is of little help in diagnosing and determining disease activity of inflammatory bowel disease as whole, but it may be of help for diagnosis and assessment of activity of colonic IBD. PMID:9539656

  8. Use of serum antibody and lysozyme levels for diagnosis of leprosy and tuberculosis.

    PubMed Central

    Near, K A; Lefford, M J

    1992-01-01

    Active tuberculosis (TB) and leprosy are difficult to diagnose early because there are few organisms to detect and the specific immune response does not distinguish between active and inactive disease. We developed an immunoassay for lysozyme to see whether serum lysozyme levels could be used to identify individuals with clinical leprosy or TB. The immunoassay for lysozyme proved superior to standard enzyme assays that were less sensitive and reliable. The lysozyme assay was compared with assays for antibodies to Mycobacterium tuberculosis lipoarabinomannan (LAM) and M. leprae phenolic glycolipid-1. The sera tested were from Ethiopian leprosy (paucibacillary and multibacillary) and TB patients and from healthy Ethiopian and U.S. controls. The lysozyme assay was able to detect more of the individuals with TB (sensitivity, 100% for 19 patients) or leprosy (sensitivity, 86% for 36 patients) than either antibody assay. In particular, lysozyme levels were raised in a higher proportion of the paucibacillary leprosy patients (83% of 17), for whom the antibody assays were less sensitive; the LAM IgG and the phenolic glycolipid-1 IgM levels were raised in only 62 and 44% of 16 patients, respectively. The data suggest that lysozyme measurements may be useful in the diagnosis of mycobacterial infections and other chronic infectious granulomatoses. Images PMID:1583106

  9. Immobilization of lysozyme-CLEA onto electrospun chitosan nanofiber for effective antibacterial applications.

    PubMed

    Park, Jae-Min; Kim, Mina; Park, Hyun-Sung; Jang, Am; Min, Jiho; Kim, Yang-Hoon

    2013-03-01

    Chitosan (CS) nanofibers with a diameter of 150-200nm were fabricated from a mixed chitosan/poly (vinyl alcohol) (PVA) solution by the electrospinning method. The nascent CS/PVA nanofibers were treated with 0.5M NaOH solution to make stable CS nanofibers by removing PVA under aqueous conditions. Hen egg-white lysozyme was immobilized on electrospun CS nanofibers via cross-linked enzyme aggregates (CLEAs) and used for effective and continuous antibacterial applications. The maximum amount of lysozyme immobilized on the CS nanofibers was determined to be 62.3mg/g of nanofibers under the optimum conditions. The immobilized lysozyme-CLEA retained more than 75.4% of its initial activity after 80 days of storage at room temperature, while the free lysozyme lost all of its activity under the same conditions. In addition, the immobilized lysozyme-CLEA retained more than 76% of its activity after 100 consecutive uses. Finally, the durability of the lysozyme-CLEA immobilized CS nanofibers showed bacteriostasis ratios of 82.4%, 79.8%, 83.4%, and 84.1% after 10 cycles against 4 pathogenic bacteria, viz. Staphylococcus aureus, Bacillus subtilis, Shigella flexneri, and Psedomonas aeruginosa, respectively. These results demonstrated that lysozyme-CLEA immobilized CS nanofibers could be used as a promising material for enhanced and continuous antibacterial applications. PMID:23201775

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

    E-print Network

    Baker, Timothy S.

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

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

    PubMed

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

    1999-05-01

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

  12. Activity of lysozyme on Lactobacillus hilgardii strains isolated from Port wine.

    PubMed

    Dias, Rita; Vilas-Boas, Eduardo; Campos, Francisco M; Hogg, Tim; Couto, José António

    2015-08-01

    This work evaluated the effect of lysozyme on lactobacilli isolated from Port wine. Bacterial growth experiments were conducted in MRS/TJ medium and inactivation studies were performed in phosphate buffer (KH2PO4), distilled water and wine supplemented with different concentrations of lysozyme. The response of bacteria to lysozyme was found to be highly strain dependent. Some strains of Lactobacillus hilgardii together with Lactobacillus collinoides and Lactobacillus fructivorans were found to be resistant to concentrations of lysozyme as high as 2000 mg/L. It was observed that among the L. hilgardii taxon the resistant strains possess an S-layer coat. Apparently, the strains of L. collinoides and L. fructivorans studied are also S-layer producers as suggested by the total protein profile obtained by SDS-PAGE. Thus, the hypothetical protective role of the S-layer against the action of lysozyme was investigated. From the various treatments used to remove the protein from the surface of the cells, the one employing LiCl (5 M) was the most effective. LiCl pre-treated cells exposed to lysozyme (2000 mg/L) in KH2PO4 buffer maintained its resistance. However, when cells were suspended in distilled water an increased sensitivity to lysozyme was observed. Moreover, it was found that the addition of ethanol (20% v/v) to the suspension medium (distilled water) triggered a strong inactivation effect especially on cells previously treated with LiCl (reduction of >6 CFU log cycles). The results suggest that the S-layer exerts a protective effect against lysozyme and that the cell suspension medium influences the bacteriolysis efficiency. It was also noted that ethanol enhances the inactivation effect of lysozyme. PMID:25846910

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

    SciTech Connect

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

    2008-07-11

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

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

    SciTech Connect

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

    2009-06-04

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

  15. The Antibacterial Protein Lysozyme Identified as the Termite Egg Recognition Pheromone

    PubMed Central

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

    2007-01-01

    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

  16. Effects of environmental and clinical interferents on the host capture efficiency of immobilized bacteriophages.

    PubMed

    Dixon, Daniel V; Hosseinidoust, Zeinab; Tufenkji, Nathalie

    2014-03-25

    Bacteriophage-functionalized surfaces are a new class of advanced functional material and have been demonstrated to be applicable for use as antimicrobial surfaces in medical applications (e.g., indwelling medical devices or wound dressings) or as biosensors for bacterial capture and detection. However, the complex composition of many real life samples (e.g., blood, natural waters, etc.) can potentially interfere with the interaction of phage and its bacterial host, leading to a decline in the efficiency of the phage-functionalized surface. In this study, the bacterial capture efficiency of two model phage-functionalized surfaces was assessed in the presence of potential environmental and biomedical interferents. The two phage-bacteria systems used in this study are PRD1 with Salmonella Typhimurium and T4 with Escherichia coli. The potential interferents tested included humic and fulvic acids, natural groundwater, colloidal latex microspheres, host extracellular polymeric substances (EPS), albumin, fibrinogen, and human serum. EPS and human serum decreased the host capture efficiency for immobilized PRD1 and T4, and also impaired the infectivity of the nonimmobilized (planktonic) phage. Interestingly, humic and fulvic acids reduced the capture efficiency of T4-functionalized surfaces, even though they did not lead to inactivation of the suspended virions. Neither humic nor fulvic acids affected the capture efficiency of PRD1. These findings demonstrate the inadequacy of traditional phage selection methods (i.e., infectivity of suspended phage toward its host in clean buffer) for designing advanced functional materials and further highlight the importance of taking into account the environmental conditions in which the immobilized phage is expected to function. PMID:24617341

  17. Nitric oxide and lysozyme production as an impact to Clostridium perfringens mastitis.

    PubMed

    Osman, Kamelia M; El-Enbaawy, Mona I; Ezzeldin, Nashwa A; Hussein, Hussein M G

    2010-12-01

    The anaerobic mastitis incidence was used to study the bovine udder response in anaerobic bacterial mastitis caused by the Gram-positive bacterial strain of Clostridium perfringens. Milk samples positive for C. perfringens were assayed for NO and lysozyme. The model produced a strong NO and lysozyme response which correlated positively with the severity and outcome of the disease (subclinical and clinical stages). This study is, to our knowledge, the first to suggest a possible link between NO and lysozyme and bovine mastitis caused by C. perfringens. The results raise the possibility that interfering with NO production during mastitis may help to prevent tissue damage. PMID:19783303

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

    E-print Network

    Lee, Soo-Kwan

    2006-01-01

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

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

    EPA Science Inventory

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

  20. Structural characterization of bacteriophage M13 solubilization by amphiphiles

    E-print Network

    Hemminga, Marcus A.

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

  1. Bacteriophage ms2 l protein: genetic and biochemical characterization

    E-print Network

    McIntosh, Brenley Kathleen

    2009-05-15

    BACTERIOPHAGE MS2 L PROTEIN: GENETIC AND BIOCHEMICAL CHARACTERIZATION A Dissertation by BRENLEY KATHLEEN M c INTOSH Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment... of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2008 Major Subject: Microbiology ii BACTERIOPHAGE MS2 L PROTEIN: GENETIC AND BIOCHEMICAL CHARACTERIZATION A Dissertation by BRENLEY KATHLEEN M c INTOSH...

  2. Bacteriophage-Triggered Defense Systems: Phage Adaptation and Design Improvements

    Microsoft Academic Search

    G. M. DJORDJEVIC; T. R. KLAENHAMMER

    1997-01-01

    A novel bacteriophage defense system, based on an inducible suicide gene, was challenged with a lactococcal bacteriophage to investigate the potential for phage adaptation. The defense system was encoded by pTRK414H, a high-copy-number replicon encoding a tightly regulated f31p trigger promoter fused to the lethal LlaIR 1 restriction endonuclease cassette. Repeated transfers of Lactococcus lactis NCK690(pTRK414H) in the pres- ence

  3. Crystallization of Chicken Egg White Lysozyme from Sulfate Salts

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth; Pusey, Marc

    1998-01-01

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

  4. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

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

    SciTech Connect

    Favi, Pelagie M [ORNL; Zhang, Qiu [ORNL; O'Neill, Hugh Michael [ORNL; Mamontov, Eugene [ORNL; Omar Diallo, Souleymane [ORNL; Palmer, Jeremy [North Carolina State University

    2014-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  7. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel; Pusey, Marc

    1998-01-01

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

  8. Simulated hatchery system to assess bacteriophage efficacy against Vibrio harveyi.

    PubMed

    Raghu Patil, J; Desai, Srividya Narayanamurthy; Roy, Panchali; Durgaiah, Murali; Saravanan, R Sanjeev; Vipra, Aradhana

    2014-12-01

    Vibriosis caused by luminous Vibrio harveyi commonly contributes to poor survival in shrimp hatcheries and aquaculture ponds. Lytic bacteriophages pathogenic for V. harveyi are currently being investigated as an alternative to antibiotics to prevent vibriosis. Here, 8 bacteriophages were isolated from oysters and clams using V. harveyi strains as baiting hosts. Among these bacteriophages, 1 strain (VHP6b) identified as broadly pathogenic for 27 V. harveyi strains examined was further characterized by electron microscopy and genome sequence analysis. Phage VHP6b possessed a tail and morphology consistent with it being a member of the family Siphoviridae, and its genome and proteome were most closely related to the Vibrio phages SSP02 and MAR10. An integrase gene essential for lysogeny was not evident. The ability of bacteriophage VHP6b to protect shrimp postlarvae against vibriosis caused by V. harveyi strain VH6 was demonstrated in a model system designed to simulate typical hatchery conditions. Bacteriophage treatment improved survival of postlarvae by 40 to 60% under these conditions, so therapies based on this or other bacteriophages may be useful in shrimp hatcheries. PMID:25449322

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  11. Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes.

    PubMed

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

    2014-11-01

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

  12. A suggested new bacteriophage genus: "Viunalikevirus".

    PubMed

    Adriaenssens, Evelien M; Ackermann, Hans-Wolfgang; Anany, Hany; Blasdel, Bob; Connerton, Ian F; Goulding, David; Griffiths, Mansel W; Hooton, Steven P; Kutter, Elizabeth M; Kropinski, Andrew M; Lee, Ju-Hoon; Maes, Martine; Pickard, Derek; Ryu, Sangryeol; Sepehrizadeh, Zargham; Shahrbabak, S Sabouri; Toribio, Ana L; Lavigne, Rob

    2012-10-01

    We suggest a bacteriophage genus, "Viunalikevirus", as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ?SH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed. PMID:22707043

  13. Comparison of Several Methods for Preserving Bacteriophages

    PubMed Central

    Clark, William A.

    1962-01-01

    A wide variety of bacteriophages were processed and stored under different conditions to compare methods for long-term preservation. Specimens were stored for 2 years at room temperature (24 to 28 C) and at 4 C as broth lysates in 50% glycerol, dried, and freeze-dried. Titers determined after processing indicated that, of the broth, glycerol, and freeze-dry methods, freeze-drying was most damaging to the phages tested, glycerol less damaging, and the broth method least damaging. After 2 years, titers of broth lysates were generally higher than those of glycerol or freeze-dried preparations. Dried preparations generally did not prove satisfactory. Preparations stored at 4 C showed better titers than those kept at room temperature. All titers declined with time regardless of the conditions of preservation. PMID:14021544

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

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

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

    NASA Astrophysics Data System (ADS)

    Bonincontro, A.; Onori, G.

    2004-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  17. Production of recombinant human lysozyme in the milk of transgenic pigs.

    PubMed

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

    2011-04-01

    In the swine industry pathogenic infections have a significant negative impact on neonatal survival. Piglets fed with human lysozyme, a natural antibiotic, might be more resistant to gastrointestinal infections. Here we describe the generation of transgenic swine expressing recombinant human lysozyme by somatic cell nuclear transfer. Three cloned female pigs were born, one of which expressed rhLZ at 0.32 ± 0.01 ?g/ml in milk, 50-fold higher than that of the pig native lysozyme. Both the transgenic gilts and their progeny appear healthy. Introducing human lysozyme into pigs' milk has a potential to benefit the piglets by enhancing immune function and defending against pathogenic bacteria, thereby increasing the new born survival rate. This advance could be of great value to commercial swine producers. PMID:20549346

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

    PubMed Central

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

    2014-01-01

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

  19. Bacteriophages of Leuconostoc, Oenococcus, and Weissella

    PubMed Central

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

    2014-01-01

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

  20. Montmorillonite-induced Bacteriophage ?6 Disassembly

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  1. Regulation of T cell autocrine growth. T4+ cells become refractory to interleukin 2

    PubMed Central

    1986-01-01

    During the course of investigating the regulation of IL-2-dependent T cell proliferation, we found that the subset of human T cells expressing the T4 surface glycoprotein become refractory to IL-2 growth promotion earlier than T8+ cells. Since T4+ cells proliferate in an autocrine fashion to endogenous IL-2, whereas most T8+ cells respond in a paracrine fashion to IL-2 derived from T4+ cells, we thought it likely that a unique mechanism was operative to restrict T4+ cell IL-2- dependent autocrine proliferation. Moreover, we anticipated that the T4+ cell IL-2-refractory state related either to suppression by T8+ cells, or to expression of T4+ cell IL-2-R. However, several experimental approaches did not support either of these mechanisms as being responsible for the loss of T4+ cell IL-2 responsiveness. Isolated T4+ cells ceased to respond to IL-2 well before T8+ cells, and before the disappearance of adequate levels of IL-2-R. Moreover, a detailed comparison of IL-2-R expression by T4+ vs. T8+ cells revealed no differences in the number, affinity, rate of expression, or functional activity of high-affinity IL-2-R expressed by the two subsets. Accordingly, T4+ cell autocrine IL-2 responsiveness is restricted by a mechanism that is independent of IL-2-R, and which ultimately results in cessation of both T4+ and T8+ cell IL-2-dependent clonal expansion. PMID:2935591

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

    PubMed

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

    1985-02-01

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

  3. Influence of different restorative materials on lysozyme and amylase activity of the salivary pellicle in situ.

    PubMed

    Hannig, Christian; Wasser, Mathias; Becker, Klaus; Hannig, Matthias; Huber, Karin; Attin, Thomas

    2006-09-15

    Lysozyme and amylase are the most abundant enzymatic components in the salivary pellicle. The purpose of the present study was to determine the influence of different substrata on amylase and lysozyme activity in salivary pellicles formed in situ. Slabs (5 mm diameter) of bovine dentine and enamel, of titanium, gold alloy, resin composite, PMMA, amalgam, and feldspar ceramic were fixed on the buccal sites of individual splints worn by six subjects for 30 min to allow pellicle formation. Thereafter, slabs were removed from the trays and rinsed with running water. Lysozyme activity was determined via lysis of Micrococcus lysodeicticus. Amylase activity was measured with a photometric method using 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosylmaltotriosid (GalG2CNP) as substrate. Both pellicle enzymes were evaluated in the immobilized as well as in the desorbed state. Salivary enzyme activities were also measured. All investigated pellicles exhibited lysozyme and amylase activity. Great intraindividual and interindividual differences were observed. Over all samples, immobilized amylase activity amounted to 0.65 +/- 0.64 mU/cm2. Immobilized lysozyme activity was 5.04 +/- 1.55 U/cm2. There were no major effects of the substratum on pellicle-bound amylase and lysozyme activity. Immobilized and desorbed enzyme activities revealed a strong correlation (lysozyme: r = 0.700; amylase: r = 0.990). Salivary enzyme activities had only little impact on pellicle-bound enzyme activities. Amylase and lysozyme are incorporated in the acquired in situ pellicle on different solid surfaces in an active conformation. Dental material and enzyme activity in the saliva have only little impact on enzymatic activity in the pellicle in situ. PMID:16739107

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

    PubMed

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

    1992-03-01

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

  5. Expression of Lysozyme in the Life History of the House Fly (Musca domestica L.)

    PubMed Central

    Nayduch, Dana; Joyner, Chester

    2014-01-01

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

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

    Microsoft Academic Search

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

    2007-01-01

    Lysozymes are antimicrobial defences that act as digestive enzymes when expressed in the stomach of herbivores with pre-gastric fermentation. We studied this enzyme in the complex stomach of the three-toed sloth (Bradypus variegatus), a folivore with pre-gastric fermentation. Lysozymes were identified by SDS-PAGE and immunoblotting in all portions: diverticulum, pouch, glandular and muscular prepyloric area with 14.3 kDa of molecular mass.

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

    PubMed

    Nayduch, Dana; Joyner, Chester

    2013-07-01

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

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

    PubMed

    Pridgeon, Julia W; Klesius, Phillip H; Dominowski, Paul J; Yancey, Robert J; Kievit, Michele S

    2013-09-01

    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 A. hydrophila by bath immersion. Quantitative PCR revealed that the transcription levels of Lys-c in infected catfish were significantly (P < 0.05) induced in all five tissues tested as well as in blood cells. Recombinant CC-Lys-c produced in Escherichia coli expression system (R-CC-Lys-c) exhibited significant (P < 0.05) lytic activity to Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-c (pcDNA-Lys-c) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-c offered significant (P < 0.05) protection to G1B against A. hydrophila infection. When channel catfish were intraperitoneally injected with QCDCR adjuvant formulated pcDNA-Lys-c and challenged with a highly virulent A. hydrophila strain AL-09-71 at 1-, 2-, 14-, and 28-days post treatment, pcDNA-Lys-c offered 75%, 100%, 60%, and 77% protection to channel catfish, respectively. Macrophages of fish treated with pcDNA-Lys-c produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish treated with pcDNA vector alone. Taken together, our results suggest that pcDNA-Lys-c could be used as a novel immunostimulant to protect channel catfish against A. hydrophila infection. PMID:23732847

  9. Selective Bacteriophage Screening Targeting GqQ209L Protein Abdul Karim Khan1

    E-print Network

    Zhou, Yaoqi

    Selective Bacteriophage Screening Targeting GqQ209L Protein Abdul Karim Khan1 , Kamakshi Sishtila1 melanoma in majority of all oncogenic cases. The method of bacteriophage display was used to find a peptide

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

    E-print Network

    Levitt, Michael

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

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

    E-print Network

    Podgornik, Rudolf

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

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

    E-print Network

    Bordenstein, Seth

    Temperature Affects the Tripartite Interactions between Bacteriophage WO, Wolbachia 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

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

    E-print Network

    Shi, Amy (Amy J.)

    2007-01-01

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

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

    E-print Network

    Citorik, Robert James

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Hinds Vaughan, Nichole; Smith, Sylvia L

    2013-12-01

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

  17. Protein-Protein Interaction on Lysozyme Crystallization Revealed by Rotational Diffusion Analysis

    PubMed Central

    Takahashi, Daisuke; Nishimoto, Etsuko; Murase, Tadashi; Yamashita, Shoji

    2008-01-01

    Intermolecular interactions between protein molecules diffusing in various environments underlie many biological processes as well as control protein crystallization, which is a crucial step in x-ray protein structure determinations. Protein interactions were investigated through protein rotational diffusion analysis. First, it was confirmed that tetragonal lysozyme crystals containing fluorescein-tagged lysozyme were successfully formed with the same morphology as that of native protein. Using this nondisruptive fluorescent tracer system, we characterized the effects of sodium chloride and ammonium sulfate concentrations on lysozyme-lysozyme interactions by steady-state and time-resolved fluorescence anisotropy measurements and the introduction of a novel interaction parameter, krot. The results suggested that the specific attractive interaction, which was reflected in the retardation of the protein rotational diffusion, was induced depending on the salt type and its concentration. The change in the attractive interactions also correlated with the crystallization/precipitation behavior of lysozyme. Moreover, we discuss the validity of our rotational diffusion analysis through comparison with the osmotic second virial coefficient, B22, previously reported for lysozyme and those estimated from krot. PMID:18310245

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

    PubMed

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

    2007-07-01

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

  19. The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

    PubMed Central

    Sotelo-Mundo, Rogerio R.; Lopez-Zavala, Alonso A.; Garcia-Orozco, Karina D.; Arvizu-Flores, Aldo A.; Velazquez-Contreras, Enrique F.; Valenzuela-Soto, Elisa M.; Rojo-Dominguez, Arturo; Kanost, Michael R.

    2008-01-01

    Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of ?-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5?30 °C. These results together with measured thermodynamical activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins. PMID:17979817

  20. Effect of Fe3O4 magnetic nanoparticles on lysozyme amyloid aggregation

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

    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.

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

    SciTech Connect

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

    2007-01-01

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

  2. [Friedrich Mauz: T4 assessor and military psychiatrist].

    PubMed

    Silberzahn-Jandt, G; Schmuhl, H-W

    2012-03-01

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

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

    PubMed

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

    2015-01-01

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

  4. Bacteriophage sensitivity patterns among bacteria isolated from marine waters

    NASA Astrophysics Data System (ADS)

    Moebus, K.; Nattkemper, H.

    1981-09-01

    Phage-host cross-reaction tests were performed with 774 bacterial strains and 298 bacteriophages. The bacteria (bacteriophages) were isolated at different times from water samples collected in the Atlantic Ocean between the European continental shelf and the Sargasso Sea: 733 (258) strains; in the North Sea near Helgoland: 31 (31) strains; and in the Bay of Biscay: 10 (9) strains. Of the Atlantic Ocean bacteria 326 were found to be susceptible to one or more Atlantic Ocean bacteriophage(s). The bacteriophage sensitivity patterns of these bacteria vary considerably, placing 225 of them in two large clusters of bacteriophage-host systems. Taking all into account, 250 of the 326 Atlantic Ocean bacteria are different from each other. This high degree of variation among the bacteria distinguishes microbial populations derived from widely separated eastern and western regions of the Atlantic Ocean. It also sets apart from each other the populations derived from samples collected at successive stations some 200 miles apart, although to a lesser degree. With bacterial populations found from samples collected on the way back and forth between Europe and the Sargasso Sea a gradual change was observed from "western" phage sensitivity patterns to "eastern" ones. Sixty-nine Atlantic Ocean bacteria are sensitive to bacteriophages isolated from the North Sea and the Bay of Biscay; of these only 26 strains are also susceptible to Atlantic Ocean phages. The interpretation of the results is based on the hydrographical conditions prevailing in the northern Atlantic Ocean including the North Sea, and on the assumption that the microbial populations investigated have undergone genetic changes while being transported within water masses from west to east.

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

    Microsoft Academic Search

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

    2009-01-01

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

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

    E-print Network

    Weitz, David

    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

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

    E-print Network

    Turner, Paul

    2007-01-01

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

  8. Detailed Architecture of a DNA Translocating Machine: The High-resolution Structure of the Bacteriophage

    E-print Network

    Rossmann, Michael G.

    of the Bacteriophage fff29 Connector Particle Alicia Guasch1 {, Joan Pous1 {, Borja Ibarra2 , F. Xavier Gomis-RuÈth1-dimensional crystal structure of the bacteriophage f29 connec- tor has been solved and re®ned to 2.1 AÊ resolution axis is coupled to connector spinning. # 2002 Academic Press Keywords: bacteriophage phi29; X

  9. OmpAandOmpCarecritical host factorsfor bacteriophage Sf6entryinShigella

    E-print Network

    Baker, Timothy S.

    OmpAandOmpCarecritical host factorsfor bacteriophage Sf6entryinShigella Kristin N. Parent,1 with bacteriophage Sf6, implicating both outer membrane proteins as poten- tial host receptors. We determined-permeable pores facilitate genome translocation as occurs in diverse viral systems such as tailed bacteriophage

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

    E-print Network

    Bordenstein, Seth

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

  11. 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

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

    E-print Network

    Hudspeth, A. James

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

  13. Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts

    E-print Network

    Richardson, Charles C.

    Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts with T7 bacteriophage growth. We show here that inhibition of T7 phage growth by udk overexpression can-studied bacteriophage T7 have no known function. In addition, the function of many genetic elements, such as the re

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

    E-print Network

    Bohannan, Brendan

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

  15. Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 in model membranes

    E-print Network

    Hemminga, Marcus A.

    Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 in model membranes M Abstract Gene 9 minor coat protein from bacteriophage M13 is known to be located in the inner membrane.V. All rights reserved. Keywords: Spontaneous insertion; Bacteriophage M13; Model membranes 1

  16. Edinburgh Research Explorer Identification of Bacteriophage-Encoded Anti-sRNAs in

    E-print Network

    Millar, Andrew J.

    Edinburgh Research Explorer Identification of Bacteriophage-Encoded Anti-sRNAs in Pathogenic 2014, 'Identification of Bacteriophage- Encoded Anti-sRNAs in Pathogenic Escherichia coli' Molecular date: 15. Sep. 2014 #12;Molecular Cell Article Identification of Bacteriophage-Encoded Anti

  17. A Single-stranded DNA-binding Protein of Bacteriophage T7 Defective in DNA Annealing*

    E-print Network

    Richardson, Charles C.

    A Single-stranded DNA-binding Protein of Bacteriophage T7 Defective in DNA Annealing* Received during the process of DNA replication, recom- bination, and repair. In bacteriophage T7-infected cells bacteriophage lack- ing gene 2.5. Purified gene 2.5 protein-R82C binds sin- gle-stranded DNA with a greater

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

    E-print Network

    Kuang, Yang

    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

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

    E-print Network

    Hillis, David

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

  20. Proteinlipid interactions of bacteriophage M13 major coat protein David Stopara

    E-print Network

    Hemminga, Marcus A.

    Review Protein­lipid interactions of bacteriophage M13 major coat protein David Stopara , Ruud B understanding of the replication cycle of bacteriophage M13 and the molecular details that enable phage proteins of the major coat protein (gp8) that may play a role during the infection of Escherichia coli by bacteriophage

  1. 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...

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

    E-print Network

    Straight, Aaron

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

  3. 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

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

    E-print Network

    Augustine, Mathew P.

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

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

    E-print Network

    Ely, Bert

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

  6. 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

  7. MODELING AND ANALYSIS OF A MARINE BACTERIOPHAGE INFECTION WITH LATENCY PERIOD

    E-print Network

    Kuang, Yang

    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

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

    E-print Network

    Erickson, Harold P.

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

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

    E-print Network

    Baker, Timothy S.

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

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

    E-print Network

    Jiang, Wen

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

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

    E-print Network

    Harrison, Stephen C.

    1983-01-01

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

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

    E-print Network

    Phillips, Rob

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

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

    E-print Network

    Straight, Aaron

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

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

    E-print Network

    Ryan, Joe

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

  15. SOLID-STATE 3'P NMR SPECTROSCOPY OF BACTERIOPHAGE MI3 AND

    E-print Network

    Hemminga, Marcus A.

    SOLID-STATE 3'P NMR SPECTROSCOPY OF BACTERIOPHAGE MI3 AND TOBACCO MOSAIC VIRUS Pieter Magusin #12;#12;SOLID-STATE 31P NMR SPECTROSCOPY OF BACTERIOPHAGE MI3 AND TOBACCO MOSAIC VIRUS #12;Promotor: dr. T vakgroep Moleculaire Fysica #12;Pieter Magusin SOLID-STATE 31P NMR SPECTROSCOPY OF BACTERIOPHAGE MI3

  16. An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity.

    PubMed

    Yue, Xin; Liu, Baozhong; Xue, Qinggang

    2011-02-01

    Lysozymes function in animal immunity. Three types of lysozyme have been identified in animal kingdom and most lysozymes identified from bivalve molluscs belong to the invertebrate (i) type. In this research, we cloned and sequenced a new i-type lysozyme, named MmeLys, from the Asiatic hard clam Meretrix meretrix. MmeLys cDNA was constituted of 552 bp, with a 441 bp open reading frame encoding a 146 amino acid polypeptide. The encoded polypeptide was predicted to have a 15 amino acid signal peptide, and a 131 amino acid mature protein with a theoretical mass of 14601.44 Da and an isoelectric point (pI) of 7.14. MmeLys amino acid sequence bore 64% identity with the Manila clam (Venerupis philippinarum) i-type lysozyme and was grouped with other veneroid i-type lysozymes in a bivalve lysozyme phylogenetic tree predicted using Neighbor-Jointing method. Recombinantly expressed MmeLys showed lysozyme activity and strong antibacterial activity against Gram positive and Gram negative bacteria. MmeLys mRNA and protein were detected to be mainly produced in hepatopancreas and gill by the methods of semi-quantitative RT-PCR and western blotting. In addition, MmeLys gene expression increased following Vibrio parahaemolyticus challenge. Results of this research indicated that MmeLys represents a new i-type lysozyme that likely functions in M. meretrix immunity. PMID:21134465

  17. Recombinant, truncated CD4 molecule (rT4) binds IgG.

    PubMed

    Lederman, S; Yellin, M J; Cleary, A M; Gulick, R; Chess, L

    1990-01-01

    CD4 is a cell surface glycoprotein that identifies the subset of human T lymphocytes that induces sIg+ B lymphocytes to differentiate and secrete Ig after intimate T-B cell contact. In the course of studying a recombinant, truncated form of CD4 (rT4) we noticed that goat antibodies of apparently irrelevant specificities bound to immobilized rT4. To directly study whether rT4 interacts with Ig molecules, purified human IgG was added to rT4-coated wells and a dose-dependent interaction between IgG and rT4 was observed by ELISA. Purified myeloma IgG proteins bound to immobilized rT4 with the same avidity as polyclonal IgG that suggests that rT4-IgG binding was not due to the presence of anti-rT4 antibodies in the IgG fraction. IgG from 6 sera bound to rT4 in concentration dependent manner similar to purified IgG. Immobilized rT4 specifically bound IgG, and not IgM, IgA, IgD, or beta 2-microglobulin. The specific interaction of rT4 and IgG was also observed when IgG or IgM were immobilized, demonstrating that IgG binding was not a unique property of immobilized rT4. As with low affinity receptors for IgG, rT4 bound heat aggregated IgG with increased avidity. Neither anti-CD4 mAb nor dextran sulfate inhibited rT4-IgG binding. rT4 bound Fc but not F(ab)2 fragments. Each of the purified IgG subclasses; IgG1, 2, 3, and 4 bound to rT4 with similar avidity. Taken together, these data suggest that rT4 specifically interacts with a public structure on IgG Fc. PMID:2295793

  18. Protein-lipid interactions of bacteriophage M13 gene 9 minor coat protein M. Chantal Houbiers and Marcus A. Hemminga*

    E-print Network

    Hemminga, Marcus A.

    Protein-lipid interactions of bacteriophage M13 gene 9 minor coat protein (Review) M. Chantal- ol; SDS, Sodium dodecyl sulphate; TFE, Trifluoroethanol. Introduction Bacteriophage M13 Bacteriophage- teria. Bacteriophage M13 belongs to the F-specific filamen- tous phages (Ff group), which only infect

  19. Bacteriophage Infection of Model Metal Reducing Bacteria

    NASA Astrophysics Data System (ADS)

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

    2008-12-01

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

  20. Formation of lysozyme oligomers at model cell membranes monitored with sum frequency generation spectroscopy.

    PubMed

    Rze?nicka, I I; Pandey, R; Schleeger, M; Bonn, M; Weidner, T

    2014-07-01

    A growing number of studies suggest that the formation of toxic oligomers, precursors of amyloid fibrils, is initiated at the cell membrane and not in the cytosolic compartments of the cell. Studies of membrane-induced protein oligomerization are challenging due to the difficulties of probing small numbers of proteins present at membrane surfaces. Here, we employ surface-sensitive vibrational sum frequency generation (VSFG) to investigate the secondary structure of lysozyme at the surface of lipid monolayers. We investigate lysozyme aggregation at negatively charged 1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-1-glycerol) (DPPG) lipid monolayers under different pH conditions. The changes in the molecular vibrations of lipids, proteins, and water as a function of pH and surface pressure allow us to simultaneously monitor details of the conformation state of lysozyme, the organization of lipids, and the state of lipid-bound water. At pH = 6 lysozyme induces significant disordering of the lipid layer, and it exists in two states: a monomeric state with a predominantly ?-helix content and an oligomeric (za-mer) state. At pH ? 3, all membrane-bound lysozyme self-associates into oligomers characterized by an antiparallel ?-sheet structure. This is different from the situation in bulk solution, for which circular dichroism (CD) shows that the protein maintains an ?-helix conformation, under both neutral and acidic pH conditions. The transition from monomers to oligomers is also associated with a decreased hydration of the lipid monolayer resulting in an increase of the lipid acyl chains ordering. The results indicate that oligomerization requires cooperative action between lysozyme incorporated into the lipid membrane and peripherally adsorbed lysozyme and is associated with the membrane dehydration and lipid reorganization. Membrane-bound oligomers with antiparallel ?-sheet structure are found to destabilize lipid membranes. PMID:24941083

  1. Complexation of lysozyme with poly(sodium(sulfamate-carboxylate)isoprene).

    PubMed

    Karayianni, Maria; Pispas, Stergios; Chryssikos, Georgios D; Gionis, Vassilis; Giatrellis, Sarantis; Nounesis, George

    2011-05-01

    The complexation between hen egg white lysozyme (HEWL) and a novel pH-sensitive and intrinsically hydrophobic polyelectrolyte poly(sodium(sulfamate-carboxylate)isoprene) (SCPI), was investigated by means of dynamic, static, and electrophoretic light scattering and isothermal titration calorimetry measurements. The complexation process was studied at both pH 7 and 3 (high and low charge density of the SCPI, respectively) and under low ionic strength conditions for two polyelectrolyte samples of different molecular weights. The solution behavior, structure, and effective charge of the formed complexes proved to be dependent on the pH, the [-]/[+] charge ratio, and the molecular weight of the polyelectrolyte. Increasing the ionic strength of the solution led to vast aggregation and eventually precipitation of the complexes. The interaction between HEWL and SCPI was found to be mainly electrostatic, associated with an exothermic enthalpy change. The structural investigation of the complexed protein by fluorescence, infrared, circular dichroism spectroscopic, and differential scanning calorimetric measurements revealed no signs of denaturation upon complexation. PMID:21410146

  2. Myricetin prevents fibrillogenesis of hen egg white lysozyme.

    PubMed

    He, Jianwei; Wang, Yu; Chang, Alan K; Xu, Linan; Wang, Na; Chong, Xiaoying; Li, Hui; Zhang, Bing; Jones, Gary W; Song, Youtao

    2014-10-01

    Myricetin is a natural flavonol found in many grapes, berries, fruits, vegetables, and herbs as well as other plants. Recent studies have identified potential antiamyloidogenic activity for this compound. In this study, the kinetics of amyloid fibril formation by hen egg white lysozyme (HEWL) and the antifibril-forming activity of myricetin were investigated. We demonstrate that myricetin significantly inhibits the fibrillation of HEWL and the inhibitory effect is dose-dependent. Interestingly, the inhibitory effect toward HEWL fibrillation was stronger than that exerted by the previously characterized fibril-forming inhibitor quercetin, which has high structural similarity with myricetin. Spectrofluorometric and computational studies suggest that the mechanism underlying the inhibitory action of myricetin at a molecular level is to reduce the population of partially unfolded HEWL intermediates. This action is achieved by the tight binding of myricetin to the aggregation-prone region of the ?-domain of HEWL and linking to the relatively stable ?-domain, thus resulting in the inhibition of amyloid fibril formation. PMID:25196984

  3. Crystallization of Chicken Egg-White Lysozyme from Ammonium Sulfate

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Snell, Edward H.; Pusey, Marc L.

    1997-01-01

    Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg/ml. Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(sub 3)2(sub 1)2 space group with crystals grown at ph 4.4 having unit-cell dimensions of a = b = 78.7 1, c=38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(sub 1)2(sub 1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

    Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Four different crystal morphologies have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed, Crystals grown at 15 C were generally tetragonal, with space group P43212. Crystallization at 20 C typically resulted in the formation of orthorhombic crystals, space group P21212 1. The tetragonal much less than orthorhombic morphology transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20 C and between 100 and 125 mg/ml protein concentration. Crystallization from 0.8 -1.2M magnesium sulfate at pH 7.6 - 8.0 gave a hexagonal (trigonal) crystal form, space group P3121, which diffracted to 2.8 A. Ammonium sulfate was also found to result in a monoclinic form, space group C2. Small twinned monoclinic crystals of approx. 0.2 mm on edge were grown by dialysis followed by seeded sitting drop crystallization.

  5. Exons encode functional and structural units of chicken lysozyme.

    PubMed Central

    Jung, A; Sippel, A E; Grez, M; Schütz, G

    1980-01-01

    The nucleotide sequence was determined for the chicken egg white lysozyme mRNA and for the exons of the gene together with their flanking intron regions. The exon pattern is to some degree related to the structural subdivision of the final protein product. However, the relationship of exons to functional units of the enzyme is better established. Exon 2 codes for amino acids 28-82, which include the catalytically active residues and a cluster of amino acids which bind rings C, D, E, and F of the oligosaccharide substrate. Exon 3 codes for amino acids 82-108, which give additional substrate specificity, determine the cleavage frame for the alternating N-acetylglucosamine/N-acetylmuramic acid chain, and increase the catalytic efficiency of the active center. Exons 1 and 4, respectively, code for translational signal sequences on the mRNA, for the signal peptide of prelysozyme, and for the amino- and carboxy-terminal regions of the enzyme. These regions increase the stability of the molecule but are not directly involved in the catalytic function. Images PMID:6934509

  6. Hydroanalysis of Animal Lysozymes c and Human Defensins a

    E-print Network

    J. C. Phillips

    2008-08-17

    Proteins appear to be the most dramatic natural example of self-organized criticality (SOC), a concept that explains many otherwise apparently unlikely phenomena. Protein functionality is dominated by long range hydro(phobic/philic) interactions which both drive protein compaction and mediate protein-protein interactions. In contrast to previous reductionist short range hydrophobicity scales, the holistic Moret-Zebende hydrophobicity scale represents a hydroanalytic tool that bioinformatically quantifies SOC in a way fully compatible with evolution. Hydroprofiling identifies chemical trends in the activities and substrate binding abilities of model enzymes and antibiotic animal lysozymes c and antibiotic human defensins, which have been the subject of tens of thousands of experimental studies. The analysis is simple and easily performed, and immediately yields insights not obtainable by traditional methods based on short-range real-space interactions, as described either by classical force fields (CFF) used in molecular dynamics simulations (MDS), or hydrophobicity scales based on transference energies from water to organic solvents.

  7. Elastic constants in orthorhombic hen egg-white lysozyme crystals.

    PubMed

    Kitajima, N; Tsukashima, S; Fujii, D; Tachibana, M; Koizumi, H; Wako, K; Kojima, K

    2014-01-01

    The ultrasonic sound velocities of cross-linked orthorhombic hen egg-white lysozyme (HEWL) crystals, including a large amount of water in the crystal, were measured using an ultrasonic pulse-echo method. As a result, seven elastic constants of orthorhombic crystals were observed to be C11 = 5.24 GPa, C22 = 4.87 GPa, C12 = 4.02 GPa, C33 = 5.23 GPa, C44 = 0.30 GPa, C55 = 0.40 GPa, and C66 = 0.43 GPa, respectively. However, C13 and C23 could not be observed because the suitable crystal planes could not be cut from bulk crystals. We conclude that the observed elastic constants of the cross-linked crystals are coincident with those of the intrinsic crystals without cross-linking. Moreover, the characteristics of the elastic constants in orthorhombic HEWL crystals are due to the fact that the shear elastic constants, C44, C55, and C66, are softer than in tetragonal crystals. That is, the shear components, C44, C55, and C66, are one half of those of the tetragonal crystals. PMID:24580264

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

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

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

    Microsoft Academic Search

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

    2012-01-01

    Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as

  10. Characterization of Bdellovibrio bacteriovorus bacteriophage MAC-1.

    PubMed

    Roberts, R C; Keefer, M A; Ranu, R S

    1987-11-01

    The bacteriophage MAC-1, which specifically infects Bdellovibrio bacteriovorus, was plaque purified and raised to high titre. The phage was purified by NaCl/polyethylene glycol precipitation, followed by two cycles of isopycnic density gradient centrifugation in CsCl. The purified phage exhibited a density of 1.363 g cm-3 and a sedimentation coefficient of 94S. Nucleic acid isolated from purified phage was resistant to hydrolysis under alkaline conditions and to digestion with RNAase, but it was hydrolysed by DNAase, providing evidence that the phage genome is made up of DNA. The lack of hyperchromic effect upon denaturation, hydrolysis of phage DNA by S1 nuclease, characteristic fluorescent staining with acridine orange, and resistance to digestion with a variety of restriction endonucleases are consistent with the DNA being single-stranded. A buoyant density of 1.722 g cm-3 and a sedimentation coefficient of 17.9S were obtained for the phage DNA. The molecular mass of phage DNA was determined as 1.58 MDa by agarose gel electrophoresis with single-stranded DNA as standards. Electron microscopy of the DNA showed that the genome is circular in nature. In addition, using Southern blots, the two replicative forms, RF1 (supercoiled) and RF2 (circular) have been identified and isolated from infected cell extracts. PMID:3446745

  11. Complex kinetics of the ? Bacteriophage genetic switch

    NASA Astrophysics Data System (ADS)

    Finzi, Laura; Manzo, Carlo; Zurla, Chiara; Dunlap, David

    2010-03-01

    The kinetics of the ? bacteriophage repressor-mediated DNA loop formation and breakdown were characterized by Tethered Particle Microscopy (TPM). A generalized likelihood ratio test was first applied to determine the location of change points (cp) in the TPM trace. Expectation-maximization (EM) clustering and the Bayesian information criterion were then used for accurate determination of the number of states accessible to the system. This procedure (cp-EM) allows objective and quantitative determination of TPM change points without the artificial time resolution limitations that arise from filtering and thresholding. Only two states were identified, which corresponded to the looped to the unlooped DNA configurations. The probability distribution function of the looped and unlooped DNA state dwell times revealed a complex kinetics. In particular, it was found that a stretched exponential provided a satisfactory fitting for the probability distribution of the unlooped state dwell times, while the dwell time distribution for the looped DNA state could not be fitted with a standard pdf; we observed, however, that a power law decay fits well the long dwell times. A mechanism is proposed to explain this kinetic behavior, where ? repressor non-specific binding to DNA may play an important physiological role.

  12. Bacteriophage biocontrol in animals and meat products

    PubMed Central

    Atterbury, R. J.

    2009-01-01

    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

  13. The DNA ejection process in bacteriophage lambda

    NASA Astrophysics Data System (ADS)

    Grayson, Paul

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

  14. Development of a bacteriophage displayed peptide library and biosensor

    NASA Astrophysics Data System (ADS)

    Chin, Robert C.; Salazar, Noe; Mayo, Michael W.; Villavicencio, Victor I.; Taylor, Richard B.; Chambers, James P.; Valdes, James J.

    1996-04-01

    A miniaturized, handheld biosensor for identification of hazardous biowarfare agents with high specificity is being developed. An innovative biological recognition system based on bacteriophage displayed peptide receptors will be utilized in conjunction with the miniature biosensor technology being developed. A bacteriophage library has been constructed to provide the artificial receptors. The library can contain millions of bacteriophage with randomly displayed peptide sequences in the phage outer protein coat which act as binding sites for the agents of interest. This library will be used to 'bio-pan' for phages that bind to a number of toxins and infectious agents and can, thus, provide an endless supply of low cost, reliable, specific, and stable artificial receptors. The biosensor instrument will utilize evanescent wave, planar waveguide, far-red dyes, diode laser and miniature circuit technologies for performance and portability.

  15. Different approaches for using bacteriophages against antibiotic-resistant bacteria

    PubMed Central

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

    2014-01-01

    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

  16. Bacteriophage-based biocontrol of biological sludge bulking in wastewater.

    PubMed

    Choi, Jeongdong; Kotay, Shireen Meher; Goel, Ramesh

    2011-01-01

    In a previous paper, the first ever application of lytic bacteriophage (virus)-mediated biocontrol of biomass bulking in the activated sludge process using Haliscomenobacter hydrossis as a model filamentous bacterium was demonstrated. In this work we extended the biocontrol application to another predominant filamentous bacterium, Sphaerotilus natans, notoriously known to cause filamentous bulking in wastewater treatment systems. Very similar to previous study, one lytic bacteriophage was isolated from wastewater that could infect S. natans and cause lysis. Significant reduction in sludge volume index and turbidity of the supernatant was observed in batches containing S. natans biomass following addition of lytic phages. Microscopic examination confirmed that the isolated lytic phage can trigger the bacteriolysis of S. natans. This extended finding further strengthens our hypothesis of bacteriophage-based biocontrol of overgrowth of filamentous bacteria and the possibility of phage application in activated sludge processes, the world's widely used wastewater treatment processes. PMID:21829092

  17. Bacteriophage as instructional organisms in introductory biology labs

    PubMed Central

    Hyman, Paul

    2014-01-01

    Designing lab exercises for introductory biology classes requires balancing the need for students to obtain results with a desire to provide unpredictable outcomes to better approximate actual research. Bacteriophage are particularly well suited for this as many species are well-understood but, with their hosts, represent a relatively complex interacting system. I have designed a seven week series of lab exercises that allow students to select bacteriophage resistant mutant hosts, isolate and sequence the corresponding receptor gene to identify the specific bacterial mutation from a large number of potential mutations. I also examined the possibility of collecting useful mutant strains for other studies. After two semesters, the lab series is working well with over 90% of students successfully isolating mutant bacteria and about half identifying the specific mutation. Here I discuss the advantages of using bacteriophage in an introductory class, the specific labs in this series and future plans. PMID:24478938

  18. Effects of aminoglycosides on glomerular permeability, tubular reabsorption, and intracellular catabolism of the cationic low-molecular-weight protein lysozyme.

    PubMed

    Cojocel, C; Dociu, N; Maita, K; Sleight, S D; Hook, J B

    1983-03-30

    Gentamicin and other aminoglycoside antibiotics in high doses may produce proteinuria and other signs of nephrotoxicity. Proteinuria may result from general renal damage or may reflect alterations in specific steps in the renal handling of proteins. To distinguish between these two possibilities, experiments were designed to quantify the effects of nephrotoxic doses of several aminoglycosides on the renal handling of proteins in the isolated perfused rat kidney with the cationic low-molecular-weight protein lysozyme as a representative protein. Each aminoglycoside was administered ip to male Wistar rats (30 mg/kg/day) for 7 days. Lysozyme and 125I-lysozyme were added to the perfusate to achieve a lysozyme perfusate concentration of about 100 mg/liter. Clearances of inulin and lysozyme, release of tyrosine and trichloroacetic acid-soluble radioactive metabolites into the perfusate, and the glomerular sieving coefficient of lysozyme were determined. Scanning and transmission electron microscopy indicated that gentamicin and tobramycin decreased the number and diameter of the endothelial fenestrae of the glomerular capillaries. Concurrently, gentamicin and tobramycin decreased the glomerular sieving coefficient of lysozyme from 0.8 to 0.6 and 0.5, respectively. Netilmicin did not affect the percentage reabsorption of lysozyme whereas gentamicin and tobramycin decreased lysozyme reabsorption from 71.7 to 35.4 and 34.4% of the filtered load, respectively. Lysozyme degradation, estimated by the release of tyrosine into the perfusate during a 150-min perfusion period, was decreased from a control value of 12 mumol/liter to 4.43 and 4.65 mumol/liter in kidneys from rats treated with gentamicin and tobramycin, respectively. This study demonstrates that polycationic aminoglycosides may affect several processes involved in renal handling of lysozyme including glomerular permeability, tubular reabsorption, and intracellular proteolytic degradation. PMID:6845378

  19. Immunoglobulins G and lysozyme concentrations in canine fetal fluids at term of pregnancy.

    PubMed

    Dall'Ara, P; Meloni, T; Rota, A; Servida, F; Filipe, J; Veronesi, M C

    2015-03-01

    In the dog, the endotheliochorial placenta allows only the 5% to 10% transfer of maternal antibodies to the fetus, but the timing and the factors influencing the immunoglobulin G (IgG) transplacental transport were not fully investigated. The aims of the present study were the following: (1) to assess the presence of both IgG and lysozyme in amniotic and allantoic fluids collected from fully developed and viable newborn puppies born by elective cesarean section at term and possible correlations between amniotic and allantoic IgG and lysozyme levels; (2) to verify possible differences in IgG and lysozyme concentrations between the two fluids; and (3) to detect possible differences in IgG and lysozyme fetal fluid levels in relation to the maternal breed body size and parity, as well as to the neonatal gender. The study, performed on 41 purebred bitches submitted to elective cesarean section at term, enrolled 142 puppies, 74 males and 68 females, born mature, viable, without gross malformations, and with a normal weight. At surgery, a total of 129 amniotic and 84 allantoic samples were collected for IgG and lysozyme analysis. Class G immunoglobulins and lysozyme were detected in both fluids, but IgG concentrations were higher (P < 0.01) in amniotic fluid. Moreover, a significant positive correlation (P < 0.01) between IgG amniotic and allantoic levels, but not for lysozyme, was observed. A significant effect of the maternal parity (P < 0.05), but not of the breed body size, on the amniotic IgG concentrations was found, whereas the newborn gender was not associated to different IgG or lysozyme amniotic or allantoic levels. Given the significant contributions of fetal fluids to fetal and neonatal health, the results reported that the amniotic and allantoic fluids play a role in the immune protection of the fetus/newborn also in canine species. However, additional research is needed to better elucidate both the origin of IgG and lysozyme and the factors influencing the wide interindividual variations. PMID:25488792

  20. Considerations for using bacteriophages for plant disease control

    PubMed Central

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

    2012-01-01

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