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Sample records for bacteriophage t4 lysozyme

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

    SciTech Connect

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

    2010-07-19

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

  2. Bacteriophage T4 Genome†

    PubMed Central

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

    2003-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. A novel thermophilic lysozyme from bacteriophage phiIN93.

    PubMed

    Matsushita, Isao; Yanase, Hideshi

    2008-12-01

    The lysozyme of bacteriophage phiIN93 was purified to apparent homogeneity with Carboxymethyl Sepharose and Hydroxyapatie columns from lysates of the phage grown on Thermus aquaticus TZ2. The enzyme is a single polypeptide chain with a molecular weight of 33,000. From the determined N-terminal amio acids of the enzyme, the locus of the gene was specified on a phiIN93 genome. The enzyme was not similar to egg white lysozyme, T4 phage lysozyme, or lambda phage lysozyme. The enzyme, phiIN93 lysozyme, was found to be a novel type of thermophilic lysozyme, which lyses specifically Thermus sp. cells, and exhibited conspicuous thermal stability at 95 degrees C for 1h in the presence of beta-mercaptoethanol. PMID:18831965

  5. Structure and function of bacteriophage T4

    PubMed Central

    Yap, Moh Lan; Rossmann, Michael G

    2014-01-01

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

  6. Analysis of the solution conformations of T4 lysozyme by paramagnetic NMR spectroscopy.

    PubMed

    Chen, Jia-Liang; Yang, Yin; Zhang, Lin-Lin; Liang, Haobo; Huber, Thomas; Su, Xun-Cheng; Otting, Gottfried

    2016-02-17

    A large number of crystal structures of bacteriophage T4 lysozyme (T4-L) have shown that it contains two subdomains, which can arrange in a compact conformation (closed state) or, in mutants of T4-L, more extended structures (open state). In solution, wild-type T4-L displays only a single set of nuclear magnetic resonance (NMR) signals, masking any conformational heterogeneity. To probe the conformational space of T4-L, we generated a site-specific lanthanide binding site by attaching 4-mercaptomethyl dipicolinic acid via a disulfide bond to Cys44 in the triple-mutant C54T/C97A/S44C of T4-L and measured pseudocontact shifts (PCS) and magnetically induced residual dipolar couplings (RDC). The data indicate that, in solution and in the absence of substrate, the structure of T4-L is on average more open than suggested by the closed conformation of the crystal structure of wild-type T4-L. A slightly improved fit was obtained by assuming a population-weighted two-state model involving an even more open conformation and the closed state, but paramagnetic relaxation enhancements measured with Gd(3+) argue against such a conformational equilibrium. The fit could not be improved by including a third conformation picked from the hundreds of crystal structures available for T4-L mutants. PMID:26680012

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

    PubMed Central

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

    1984-01-01

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

  8. REDOR NMR Characterization of DNA Packaging in Bacteriophage T4

    PubMed Central

    Yu, Tsyr-Yan; Schaefer, Jacob

    2008-01-01

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

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

    PubMed Central

    Yirdaw, RobelB.; Mchaourab, HassaneS.

    2012-01-01

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

  10. 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

  11. Structure and assembly of bacteriophage T4 head.

    PubMed

    Rao, Venigalla B; Black, Lindsay W

    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

  12. 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

  13. Coordinated DNA Replication by the Bacteriophage T4 Replisome.

    PubMed

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

    2015-06-01

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

  14. A helix initiation signal in T4 lysozyme identified by polyalanine mutagenesis.

    PubMed

    Zhang, Xue-jun; Baase, Walter A; Matthews, Brian W

    2002-12-10

    To better understand the relation between sequence and structure, and in an attempt to simplify the protein folding problem, a series of alanine substitutions was introduced into bacteriophage T4 lysozyme. In contrast to previous studies in this system, which were restricted to single alpha-helices, the present analysis included a helix-turn-helix region, a loop-helix region, and two alpha-helices that were well separated in the three-dimensional structure. It was shown previously that T4 lysozyme is very tolerant of alanine substitutions within alpha-helices, especially at solvent-exposed sites. The present study shows that the protein is also tolerant of such substitutions in turn and loop regions, although less than in helices. The results confirm that the structural information in the amino acid sequence is highly redundant. For example, the protein with the sequence 127AAAAAALAAAAWAAA141 folds normally, has melting temperature only 0.8 degrees C lower than wildtype, and has a crystal structure that is also very similar to wildtype. Polyalanine substitutions within turns or loops can, however, lead to differences in structure and in folding. In one example the triple substitution K35A/S36A/P37A caused this region of the molecule to change to a more helical conformation. In a second case the mutant with the sequence 34AAAAALAAAKAALAAA49, which spans a loop-helix region, had a dramatically altered thermal unfolding transition, suggesting that this region may tend to form a single, uninterrupted, helix. Substitution of Ala38 in the above construct with aspartic acid caused the unfolding to be more like wildtype, suggesting that residue 38, which is at a helix-capping position in the wildtype structure, provides an initiation signal that is essential in the polyalanine mutant for the correct formation of alpha-helix 39-50. In a typical protein, the information that codes for the 3D structure is presumably distributed over many amino acids. The present results suggest that in simplified sequences the key folding information may be restricted to a subset of critical residues, and so be more readily accessible to experimental analysis. PMID:12487988

  15. 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

  16. A promiscuous DNA packaging machine from bacteriophage T4.

    PubMed

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

    2011-01-01

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

  17. Adsorption of T4 bacteriophages on planar indium tin oxide surface via controlled surface tailoring.

    PubMed

    Liana, Ayu Ekajayanthi; Chia, Ed Win; Marquis, Christopher P; Gunawan, Cindy; Gooding, J Justin; Amal, Rose

    2016-04-15

    The work investigates the influence of surface physicochemical properties of planar indium tin oxide (ITO) as a model substrate on T4 bacteriophage adsorption. A comparative T4 bacteriophage adsorption study shows a significant difference in bacteriophage adsorption observed on chemically modified planar ITO when compared to similarly modified particulate ITO, which infers that trends observed in virus-particle interaction studies are not necessarily transferrable to predict virus-planar surface adsorption behaviour. We also found that ITO surfaces modified with methyl groups, (resulting in increased surface roughness and hydrophobicity) remained capable of adsorbing T4 bacteriophage. The adsorption of T4 onto bare, amine and carboxylic functionalised planar ITO suggests the presence of a unique binding behaviour involving specific functional groups on planar ITO surface beyond the non-specific electrostatic interactions that dominate phage to particle interactions. The paper demonstrates the significance of physicochemical properties of surfaces on bacteriophage-surface interactions. PMID:26851452

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

    PubMed

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

    2016-01-01

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

  19. Excision repair and patch size in UV-irradiated bacteriophage T4

    SciTech Connect

    Yarosh, D.B.; Rosenstein, B.S.; Setlow, R.B.

    1981-11-01

    We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control, experiments with the denV/sub 1/ excision-deificient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

  20. Excision repair and patch size in UV-irradiated bacteriophage T4

    SciTech Connect

    Yarosh, D.B.; Rosenstein, B.S.; Setlow, R.B.

    1981-11-01

    We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

  1. T4 phage lysozyme: a protein designed for understanding tryptophan photophysics

    NASA Astrophysics Data System (ADS)

    Hudson, Bruce S.; Harris, Dan

    1990-05-01

    Bacteriophage T4 lysozyme in its wild type form contains three tryptophan residues (at sequence postions 126, 138 and 158). These three residues are in rather different environments in the protein: 126 and 158 are near the protein surface while residue 138 is more buried. T4 lysozyme has been genetically engineered to prepare all possible variants in which one or more of the tryptophan residues have been replaced by tyrosine. The available data supports the hypothesis that this substitution has, at most, a very minor effect on the structure of the protein. The three species with single tryptophan residues have been investigated in detail. The surface location of residue 126 compared to the buried location of residue 138 is reflected in the difference in collisional quenching observed with added potassium iodide. It is found that the spectral and radiative properties of the three proteins are very similar but that their radiationless decay properties are quite distinct. This is apparently due to short-range collisional quenching by neighboring side chains. Comparison with solution quenching measurements permits the identification of the specific quenching groups involved for each tryptophan residue and provides a semi-quantitative rationale for the radiationless decay rate. This collisional quenching interpretation is supported by mutational effects on fluorescence quantum yield. This simple picture of the behavior of these single-tryptophan proteins is clearly revealed in this particular case because of the unambiguous choice of collisional quenching groups. The time dependence of the fluorescence decay of each of these single-tryptophan proteins is quite complex. Several methods of analysis are presented and discussed in terms of their underlying physical basis. Internal collisional quenching, as suggested from the comparative studies, is expected to lead to non-exponential behavior. This is consistent with the observed time dependence. Analysis of the temporal nature of the fluorescence as a function of emission wavelength is also revealing. Such data can be used to test discrete component, distribution and relaxation models of the time decay. It is found, in agreement with previous studies for other proteins, that the average lifetime for the emission increases with increasing emission wavelength. Analysis of the overall emission wavelength dependence of the time dependent data in a global sense based on a discrete population model shows acceptable agreement with the data in only one of the three cases. Application of several continuous distribution models to this data at each emission wavelength reveals that as the emission is moved to the red, a negative component appears in the distribution of decay components. This is a characteristic feature of relaxation behavior resulting in emission from kinetic species that are not present at the time of excitation. This negative preexponential character is not revealed by discrete component analyses since these do not have sufficient flexibility to describe the underlying complexity of the relaxing distribution. Finally, examination of the three proteins containing two tryptophan residues indicates that there is energy transfer between these residues in these cases and in the wild type protein. The order of energy transfer is in accord with the variation of the magnitude of the ratio k2/R6 controlling the efficiency of Forster energy transfer.

  2. The effect of bacteriophages T4 and HAP1 on in vitro melanoma migration

    PubMed Central

    2009-01-01

    Background The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans). Bacteriophages are constantly present in mammalian bodies and the environment in great amounts. On the other hand, the perspective of the possible use of bacteriophage preparations for antibacterial therapies in cancer patients generates a substantial need to investigate the effects of phages on cancer processes. Results In these studies the migration of human and mouse melanoma on fibronectin was inhibited by purified T4 and HAP1 bacteriophage preparations. The migration of human melanoma was also inhibited by the HAP1 phage preparation on matrigel. No response of either melanoma cell line to lipopolysaccharide was observed. Therefore the effect of the phage preparations cannot be attributed to lipopolysaccharide. No differences in the effects of T4 and HAP1 on melanoma migration were observed. Conclusion We believe that these observations are of importance for any further attempts to use bacteriophage preparations in antibacterial treatment. The risk of antibiotic-resistant hospital infections strongly affects cancer patients and these results suggest the possibility of beneficial phage treatment. We also believe that they will contribute to the general understanding of bacteriophage biology, as bacteriophages, extremely ubiquitous entities, are in permanent contact with human organisms. PMID:19154575

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

    NASA Astrophysics Data System (ADS)

    Sims, Patrick Craig

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

  4. Control of Gene Function in Bacteriophage T4

    PubMed Central

    Sauerbier, Walter; Schweiger, Manfred; Herrlich, Peter

    1971-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. The structure of the ATPase that powers DNA packaging into bacteriophage T4 procapsids.

    PubMed

    Sun, Siyang; Kondabagil, Kiran; Gentz, Petra M; Rossmann, Michael G; Rao, Venigalla B

    2007-03-23

    Packaging the viral genome into empty procapsids, an essential event in the life cycle of tailed bacteriophages and some eukaryotic viruses, is a process that shares features with chromosome assembly. Most viral procapsids possess a special vertex containing a dodecameric portal protein that is used for entry and exit of the viral genome. The portal and an ATPase are parts of the genome-packaging machine. The ATPase is required to provide energy for translocation and compaction of the negative charges on the genomic DNA. Here we report the atomic structure of the ATPase component in a phage DNA-packaging machine. The bacteriophage T4 ATPase has the greatest similarity to monomeric helicases, suggesting that the genome is translocated by an inchworm mechanism. The similarity of the packaging machines in the double-stranded DNA (dsDNA) bacteriophage T4 and dsRNA bacteriophage varphi12 is consistent with the evolution of many virions from a common ancestor. PMID:17386269

  7. Pushing single molecule techniques to microsecond resolution proves that T4 Lysozyme is a Brownian ratchet

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    Single-molecule techniques can monitor conformational dynamics of proteins, but such methods usually lack the resolution to directly observe conformational pathways or intermediate conformational states. We have recently described a single-molecule electronic technique that breaks this barrier. Using a 1 MHz-bandwidth carbon nanotube transistor, the transition pathways between open and closed conformations of T4 lysozyme have been recorded with a microsecond resolution. We directly resolve a smooth, continuous transition with an average duration of 37 microseconds. Unexpectedly, the mechanical closing and re-opening of the enzyme have identical distributions of transition durations, and the motion is independent of the enzyme catalyzing the substrate. These results illustrate the principle of microscopic reversibility applied to a Brownian ratchet, with lysozyme tracing a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity.

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

    PubMed Central

    Ahrenholtz, Ingrid; Harms, Klaus; de Vries, Johann; Wackernagel, Wilfried

    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 associate the cells of Bacillus subtilis with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Dsire and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed B. subtilis cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5- to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growth-sustaining effects of plants on bacteria. PMID:10788351

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

    PubMed

    Korehei, Reza; Kadla, John F

    2014-01-16

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

  10. 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.

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

    PubMed Central

    Conley, M P; Wood, W B

    1975-01-01

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

  12. Assembly and infection process of bacteriophage T4

    NASA Astrophysics Data System (ADS)

    Arisaka, Fumio

    2005-12-01

    Bacterophage T4 consists of three parts, namely, a head, a tail, and six tail fibers, each of which is assembled along an independent pathway and then joined. In contrast to simple plant viruses such as tobacco mosaic virus, disassembly and reassembly of the virion is not possible. This is due mainly to the fact that the assembly involves not only irreversible steps such as cleavage of covalent bonds of some constituent proteins, but also that it requires a scaffold and involves the inner membrane of the host cell. Another unique feature of the assembly as a biological nanomachine is the involvement of specific protein devices such as a "ruler molecule," which determines the length of the tail, an ATP-driven DNA packaging protein complex, and phage-encoded molecular chaperones. Recent structural biological studies of the phage started to unveil the molecular mechanics of structural transformation of the tail upon infection.

  13. 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 30 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

  14. Complete genome sequence of T4-Like Escherichia coli bacteriophage HX01.

    PubMed

    Tang, Fang; Li, Yanzhe; Zhang, Wei; Lu, Chengping

    2012-12-01

    Phage T4 is among the best-characterized biological systems (S. Kanamaru and F. Arisaka, Seikagaku 74:131-135, 2002; E. S. Miller et al., Microbiol. Mol. Biol. Rev. 67:86-156, 2003; W. B. Wood and H. R. Revel, Bacteriol. Rev. 40:847-868, 1976). To date, several genomes of T4-like bacteriophages are available in public databases but without any APEC bacteriophages (H. Jiang et al., Arch. Virol. 156:1489-1492, 2011; L. Kaliniene, V. Klausa, A. Zajanckauskaite, R. Nivinskas, and L. Truncaite, Arch. Virol. 156:1913-1916, 2011; J. H. Kim et al., Vet. Microbiol. 157:164-171, 2012; W. C. Liao et al., J. Virol. 85:6567-6578, 2011). We isolated a bacteriophage from a duck factory, named HX01, that infects avian pathogenic Escherichia coli (APEC). Sequence and morphological analyses revealed that phage HX01 is a T4-like bacteriophage and belongs to the family Myoviridae. Here, we announce the complete genome sequence of phage HX01 and report the results of our analysis. PMID:23166268

  15. Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4.

    PubMed

    Bullard, Desmond R; Bowater, Richard P

    2006-08-15

    The genome of bacteriophage T4 encodes three polynucleotide ligases, which seal the backbone of nucleic acids during infection of host bacteria. The T4Dnl (T4 DNA ligase) and two RNA ligases [T4Rnl1 (T4 RNA ligase 1) and T4Rnl2] join a diverse array of substrates, including nicks that are present in double-stranded nucleic acids, albeit with different efficiencies. To unravel the biochemical and functional relationship between these proteins, a systematic analysis of their substrate specificity was performed using recombinant proteins. The ability of each protein to ligate 20 bp double-stranded oligonucleotides containing a single-strand break was determined. Between 4 and 37 degrees C, all proteins ligated substrates containing various combinations of DNA and RNA. The RNA ligases ligated a more diverse set of substrates than T4Dnl and, generally, T4Rnl1 had 50-1000-fold lower activity than T4Rnl2. In assays using identical conditions, optimal ligation of all substrates was at pH 8 for T4Dnl and T4Rnl1 and pH 7 for T4Rnl2, demonstrating that the protein dictates the pH optimum for ligation. All proteins ligated a substrate containing DNA as the unbroken strand, with the nucleotides at the nick of the broken strand being RNA at the 3'-hydroxy group and DNA at the 5'-phosphate. Since this RNA-DNA hybrid was joined at a similar maximal rate by T4Dnl and T4Rnl2 at 37 degrees C, we consider the possibility that this could be an unexpected physiological substrate used during some pathways of 'DNA repair'. PMID:16671895

  16. Scanning Tunneling and Atomic Force Microscopy of T4 Bacteriophage and Tobacco Mosaic Virus

    NASA Astrophysics Data System (ADS)

    Imai, Kensaku; Yoshimura, Kosei; Tomitori, Masahiko; Nishikawa, Osamu; Kokawa, Ryouhei; Yamamoto, Mitsuhiko; Kobayashi, Masato; Ikai, Atsushi

    1993-06-01

    Bacteriophage T4 and tobacco mosaic virus were imaged by scanning tunneling microscopy after metal coating of the specimens and by atomic force microscopy without coating, and their topographies were examined. A metal coating approximately 5 nm in thickness assured easy and reproducible imaging of samples but obscured their detailed structural features. AFM gave a better resolution on this scale, revealing the presence of tail fibers 2 nm in diameter of T4 phage, which is the best resolution so far reported for isolated biological structures. Sample topographies were in good agreement with the results of previous electron microscopic analysis.

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

    PubMed

    Deep, Shashank; Ahluwalia, J C

    2003-06-01

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

  18. 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 variantwhile 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.

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

    SciTech Connect

    Zheng, Wenjun Glenn, Paul

    2015-01-21

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

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

    PubMed Central

    Wachsman, Joseph T.; Drake, John W.

    1987-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  2. On the Direction of Reading of Bacteriophage T4 Gene 43 (Deoxyribonucleic Acid Polymerase)

    PubMed Central

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

    1972-01-01

    Amber (am) mutants of the two closely linked sites, B22 and C125, in bacteriophage T4 gene 43 [deoxyribonucleic acid (DNA) polymerase] synthesize in the nonpermissive (su?) Escherichia coli host gene 43 products which are devoid of DNA polymerase activity, but which retain a 3?-exonuclease activity. Diethylaminoethyl-cellulose chromatographic analysis of DNA polymerase and deoxyribonuclease activities from extracts of su? cells infected with single- and double-am mutants of T4 gene 43 showed that the exonuclease activity which is observed with amB22 is not seen with double mutants carrying, in addition to amB22, am mutations which map to the clockwise side of the B22 site on the circular genetic map of T4. Similarly, am mutations which map to the clockwise side of the C125 site abolish the exonuclease activity which is observed with an am mutant (amE4335) of this site. It was concluded that in these double mutants termination signals to the clockwise side of amB22 and amE4335 are encountered before the amB22 and amE4335 signals during translation of the messenger ribonucleic acid from T4 gene 43. Thus, it seems that the T4 DNA polymerase is synthesized in vivo in a direction which corresponds to a counterclockwise reading of gene 43. PMID:4556512

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

    SciTech Connect

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

    2007-10-25

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1973-01-01

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

  6. Structure, assembly, and DNA packaging of the bacteriophage T4 head.

    PubMed

    Black, Lindsay W; Rao, Venigalla B

    2012-01-01

    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

  7. 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. Frster resonance energy transferfluorescence 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

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

    PubMed Central

    2010-01-01

    The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature. PMID:21029436

  9. 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.

  10. Bacteriophage T4 Mutants Hypersensitive to an Antitumor Agent That Induces Topoisomerase-DNA Cleavage Complexes

    PubMed Central

    Woodworth, D. L.; Kreuzer, K. N.

    1996-01-01

    Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex. A pathway of recombination can apparently repair this DNA damage. In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4. Substantial increases in sensitivity to the antitumor agent m-AMSA [4'-(9-acridinyl-amino) methanesulfon-m-anisidide] were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), ?-gt (?-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (? subunit of ribonucleotide reductase). The role of the rnh gene in drug sensitivity was further characterized. First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA. Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase. Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage. Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage. PMID:8807283

  11. Structural remodeling of bacteriophage T4 and host membranes during infection initiation

    PubMed Central

    Hu, Bo; Margolin, William; Molineux, Ian J.; Liu, Jun

    2015-01-01

    The first stages of productive bacteriophage infections of bacterial host cells require efficient adsorption to the cell surface followed by ejection of phage DNA into the host cytoplasm. To achieve this goal, a phage virion must undergo significant structural remodeling. For phage T4, the most obvious change is the contraction of its tail. Here, we use skinny E. coli minicells as a host, along with cryo-electron tomography and mutant phage virions, to visualize key structural intermediates during initiation of T4 infection. We show for the first time that most long tail fibers are folded back against the tail sheath until irreversible adsorption, a feature compatible with the virion randomly walking across the cell surface to find an optimal site for infection. Our data confirm that tail contraction is triggered by structural changes in the baseplate, as intermediates were found with remodeled baseplates and extended tails. After contraction, the tail tube penetrates the host cell periplasm, pausing while it degrades the peptidoglycan layer. Penetration into the host cytoplasm is accompanied by a dramatic local outward curvature of the cytoplasmic membrane as it fuses with the phage tail tip. The baseplate hub protein gp27 and/or the ejected tape measure protein gp29 likely form the transmembrane channel for viral DNA passage into the cell cytoplasm. Building on the wealth of prior biochemical and structural information, this work provides new molecular insights into the mechanistic pathway of T4 phage infection. PMID:26283379

  12. Impaired expression of certain prereplicative bacteriophage T4 genes explains impaired T4 DNA synthesis in Escherichia coli rho (nusD) mutants.

    PubMed Central

    Stitt, B L; Mosig, G

    1989-01-01

    The Escherichia coli rho 026 mutation that alters the transcription termination protein Rho prevents growth of wild-type bacteriophage T4. Among the consequences of this mutation are delayed and reduced T4 DNA replication. We show that these defects can be explained by defective synthesis of certain T4 replication-recombination proteins. Expression of T4 gene 41 (DNA helicase/primase) is drastically reduced, and expression of T4 genes 43 (DNA polymerase), 30 (DNA ligase), 46 (recombination nuclease), and probably 44 (DNA polymerase-associated ATPase) is reduced to a lesser extent. The compensating T4 mutation goF1 partially restores the synthesis of these proteins and, concomitantly, the synthesis of T4 DNA in the E. coli rho mutant. From analyzing DNA synthesis in wild-type and various multiply mutant T4 strains, we infer that defective or reduced synthesis of these proteins in rho 026-infected cells has several major effects on DNA replication. It impairs lagging-strand synthesis during the primary mode of DNA replication; it delays and depresses recombination-dependent (secondary mode) initiation; and it inhibits the use of tertiary origins. All three T4 genes whose expression is reduced in rho 026 cells and whose upstream sequences are known have a palindrome containing a CUUCGG sequence between the promoter(s) and ribosome-binding site. We speculate that these palindromes might be important for factor-dependent transcription termination-antitermination during normal T4 development. Our results are consistent with previous proposals that the altered Rho factor of rho 026 may cause excessive termination because the transcription complex does not interact normally with a T4 antiterminator encoded by the wild-type goF gene and that the T4 goF1 mutation restores this interaction. Images PMID:2544560

  13. 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.

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

    SciTech Connect

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

    1991-08-01

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

  15. 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

  16. Structure of the bacteriophage T4 baseplate as determined by chemical cross-linking.

    PubMed Central

    Watts, N R; Coombs, D H

    1990-01-01

    We have carried out a series of reversible chemical cross-linking experiments using the reagent ethylene glycol-bis(succinimidylsuccinate) with the goal of determining the three-dimensional structure of the bacteriophage T4 baseplate. In a previous report, we investigated the near-neighbor contacts in baseplate precursors and substructures (N.R.M. Watts and D.H. Coombs, J. Virol. 63:2427-2436, 1989). Here we report completion of the analysis by examining finished baseplates and tails. Most of the previous contacts were confirmed, and we report several new contacts, including those within the central hub (gp5-gptd2, gp26-gptd), between the hub and the outer wedges (gp6-gp27(2], between baseplate and sheath (gp54-gp18), and between sheath and core (gp19-gp18). On the basis of this and other available information, a partial three-dimensional model of the baseplate is proposed. Images PMID:2403438

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

    SciTech Connect

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

    2009-07-22

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

  18. 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

  19. Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain.

    PubMed

    Barfoot, Tasida; Herdendorf, Timothy J; Behning, Bryanna R; Stohr, Bradley A; Gao, Yang; Kreuzer, Kenneth N; Nelson, Scott W

    2015-09-25

    Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn(2+) cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn(2+) is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision. PMID:26242734

  20. Control of helicase loading in the coupled DNA replication and recombination systems of bacteriophage T4.

    PubMed

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

    2014-01-31

    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

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

    SciTech Connect

    Hu, Dehong; Lu, H PETER.

    2004-07-01

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

  2. 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 IIII). 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 3752 and 290315, 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

  3. Specificity of interactions among the DNA-packaging machine components of T4-related bacteriophages.

    PubMed

    Gao, Song; Rao, Venigalla B

    2011-02-01

    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

  4. 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.

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

    PubMed Central

    Hulen, C; Legault-Demare, J

    1975-01-01

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

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

    SciTech Connect

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

    2014-06-19

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

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

    PubMed

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

    2014-03-01

    Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging. PMID:24126213

  8. Coordination of DNA ends during double-strand-break repair in bacteriophage T4.

    PubMed Central

    Stohr, Bradley A; Kreuzer, Kenneth N

    2002-01-01

    The extensive chromosome replication (ECR) model of double-strand-break repair (DSBR) proposes that each end of a double-strand break (DSB) is repaired independently by initiating extensive semiconservative DNA replication after strand invasion into homologous template DNA. In contrast, several other DSBR models propose that the two ends of a break are repaired in a coordinated manner using a single repair template with only limited DNA synthesis. We have developed plasmid and chromosomal recombinational repair assays to assess coordination of the broken ends during DSBR in bacteriophage T4. Results from the plasmid assay demonstrate that the two ends of a DSB can be repaired independently using homologous regions on two different plasmids and that extensive replication is triggered in the process. These findings are consistent with the ECR model of DSBR. However, results from the chromosomal assay imply that the two ends of a DSB utilize the same homologous repair template even when many potential templates are present, suggesting coordination of the broken ends during chromosomal repair. This result is consistent with several coordinated models of DSBR, including a modified version of the ECR model. PMID:12454052

  9. Multiple mechanisms for degradation of bacteriophage T4 soc mRNA.

    PubMed Central

    Kai, Toshie; Yonesaki, Tetsuro

    2002-01-01

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

  10. Role of bacteriophage T4 baseplate in regulating assembly and infection.

    PubMed

    Yap, Moh Lan; Klose, Thomas; Arisaka, Fumio; Speir, Jeffrey A; Veesler, David; Fokine, Andrei; Rossmann, Michael G

    2016-03-01

    Bacteriophage T4 consists of a head for protecting its genome and a sheathed tail for inserting its genome into a host. The tail terminates with a multiprotein baseplate that changes its conformation from a "high-energy" dome-shaped to a "low-energy" star-shaped structure during infection. Although these two structures represent different minima in the total energy landscape of the baseplate assembly, as the dome-shaped structure readily changes to the star-shaped structure when the virus infects a host bacterium, the dome-shaped structure must have more energy than the star-shaped structure. Here we describe the electron microscopy structure of a 3.3-MDa in vitro-assembled star-shaped baseplate with a resolution of 3.8 Å. This structure, together with other genetic and structural data, shows why the high-energy baseplate is formed in the presence of the central hub and how the baseplate changes to the low-energy structure, via two steps during infection. Thus, the presence of the central hub is required to initiate the assembly of metastable, high-energy structures. If the high-energy structure is formed and stabilized faster than the low-energy structure, there will be insufficient components to assemble the low-energy structure. PMID:26929357

  11. T4-related bacteriophage LIMEstone isolates for the control of soft rot on potato caused by 'Dickeya solani'.

    PubMed

    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. 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

  13. 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

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

    PubMed

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

    2015-03-01

    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

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

    SciTech Connect

    Gordon, L.K.; Haseltine, W.A.

    1980-12-25

    A comparison was made of the activity of the uv-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultraviolet light-irradiated DNA substrates of defined sequence. The two enzyms cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA.

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

    PubMed Central

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

    1980-01-01

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

  17. Evaluation at atomic resolution of the role of strain in destabilizing the temperature-sensitive T4 lysozyme mutant Arg 96 ? His

    PubMed Central

    Mooers, Blaine H M; Tronrud, Dale E; Matthews, Brian W

    2009-01-01

    Mutant R96H is a classic temperature-sensitive mutant of bacteriophage T4 lysozyme. It was in fact the first variant of the protein to be characterized structurally. Subsequently, it has been studied extensively by a variety of experimental and computational techniques, but the reasons for the loss of stability of the mutant protein remain controversial. In the crystallographic refinement of the mutant structure at 1.9 resolution one of the bond angles at the site of substitution appeared to be distorted by about 11, and it was suggested that this steric strain was one of the major factors in destabilizing the mutant. Different computationally-derived models of the mutant structure, however, did not show such distortion. To determine the geometry at the site of mutation more reliably, we have extended the resolution of the data and refined the wildtype (WT) and mutant structures to be better than 1.1 resolution. The high-resolution refinement of the structure of R96H does not support the bond angle distortion seen in the 1.9 structure determination. At the same time, it does confirm other manifestations of strain seen previously including an unusual rotameric state for His96 with distorted hydrogen bonding. The rotamer strain has been estimated as about 0.8 kcal/mol, which is about 25% of the overall reduction in stability of the mutant. Because of concern that contacts from a neighboring molecule in the crystal might influence the geometry at the site of mutation we also constructed and analyzed supplemental mutant structures in which this crystal contact was eliminated. High-resolution refinement shows that the crystal contacts have essentially no effect on the conformation of Arg96 in WT or on His96 in the R96H mutant. PMID:19384984

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Gordon, L K; Haseltine, W A

    1980-12-25

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

  2. Phospholipase Activity in Bacteriophage-Infected Escherichia coli I. Demonstration of a T4 Bacteriophage-Associated phospholipase

    PubMed Central

    Nelson, E. T.; Buller, C. S.

    1974-01-01

    Phospholipase activity has been found to be associated with T4 phage and T4 ghost particles. The attachment of the phospholipase to the phage persists during purification through cesium chloride gradients and dialysis, indicating that it is firmly bound. The presence of the enzymatic activity on T4 ghosts suggests that it is not normally packaged within the head of the virus. The enzyme has specificity for phosphatidylglycerol and its activity is stimulated by 0.1% Triton X-100 and 20% methanol. It does not have a requirement for Ca2+ and is inactivated at temperatures above 60 C. The association of the phospholipase with T4 phage grown in a phospholipase-deficient host and its absence on unsuppressed T4amtA3 suggests that it may be phage gene specific. PMID:4604905

  3. 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

  4. 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

  5. Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme

    PubMed Central

    Yousef, Mohammad S.; Baase, Walter A.; Matthews, Brian W.

    2004-01-01

    We have designed a molecular switch in a T4 lysozyme construct that controls a large-scale translation of a duplicated helix. As shown by crystal structures of the construct with the switch on and off, the conformational change is triggered by the binding of a ligand (guanidinium ion) to a site that in the wild-type protein was occupied by the guanidino head group of an Arg. In the design template, a duplicated helix is flanked by two loop regions of different stabilities. In the on state, the N-terminal loop is weakly structured, whereas the C-terminal loop has a well defined conformation that is stabilized by means of nonbonded interactions with the Arg head group. The truncation of the Arg to Ala destabilizes this loop and switches the protein to the off state, in which the duplicated helix is translocated ?20 . Guanidinium binding restores the key interactions, restabilizes the C-terminal loop, and restores the on state. Thus, the presence of an external ligand, which is unrelated to the catalytic activity of the enzyme, triggers the inserted helix to translate 20 away from the binding site. The results illustrate a proposed mechanism for protein evolution in which sequence duplication followed by point mutation can lead to the establishment of new function. PMID:15286283

  6. 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

  7. Phospholipase activity in bacteriophage-infected Escherichia. II. Activation of phospholipase by T4 ghost infection.

    PubMed Central

    Buller, C S

    1975-01-01

    The release of free fatty acids from the phospholipids of Escherichia coli is initiated immediately after the attachment of T4 ghosts. A similar accumulation of free fatty acids is observed if the cells are infected with T4 phage in the presence of chloramphenicol or puromycin. An early accumulation of free fatty acids, however, is not observed in T4 infections in which chloramphenicol or puromycin are not present, nor does it occur if the E. coli are infected with T4 phage before ghost infection, suggesting that phage products can prevent the phospholipid deacylation. If E. coli is infected with T4 ghosts before T4 phage infection, the accumulation of free fatty acids is not suppressed. When phospholipase-deficient E, coli are infected with T4 ghosts the appearance of free fatty acids is not observed, suggesting that T4 ghost attachment can activate the phospholipase of wild-type E. coli. Although the formation of free fatty acid apparently is a consequence of activation of the detergent-resistant phospholipase of the outer membrane, it is not observed in mutants deficient in the detergent-sensitive phospholipase. PMID:1095777

  8. Bacteriophage T4 rnh (RNase H) Null Mutations: Effects on Spontaneous Mutation and Epistatic Interaction with rII Mutations

    PubMed Central

    Bebenek, Anna; Smith, Leslie A.; Drake, John W.

    1999-01-01

    The bacteriophage T4 rnh gene encodes T4 RNase H, a relative of a family of flap endonucleases. T4 rnh null mutations reduce burst sizes, increase sensitivity to DNA damage, and increase the frequency of acriflavin resistance (Acr) mutations. Because mutations in the related Saccharomyces cerevisiae RAD27 gene display a remarkable duplication mutator phenotype, we further explored the impact of rnh mutations upon the mutation process. We observed that most Acr mutants in an rnh+ strain contain ac mutations, whereas only roughly half of the Acr mutants detected in an rnh? strain bear ac mutations. In contrast to the mutational specificity displayed by most mutators, the DNA alterations of ac mutations arising in rnh? and rnh+ backgrounds are indistinguishable. Thus, the increase in Acr mutants in an rnh? background is probably not due to a mutator effect. This conclusion is supported by the lack of increase in the frequency of rI mutations in an rnh? background. In a screen that detects mutations at both the rI locus and the much larger rII locus, the r frequency was severalfold lower in an rnh? background. This decrease was due to the phenotype of rnh rII double mutants, which display an r+ plaque morphology but retain the characteristic inability of rII mutants to grow on ? lysogens. Finally, we summarize those aspects of T4 forward-mutation systems which are relevant to optimal choices for investigating quantitative and qualitative aspects of the mutation process. PMID:10322013

  9. The asiA gene of bacteriophage T4 codes for the anti-sigma 70 protein.

    PubMed Central

    Orsini, G; Ouhammouch, M; Le Caer, J P; Brody, E N

    1993-01-01

    The anti-sigma 70 factor of bacteriophage T4 is a 10-kDa (10K) protein which inhibits the sigma 70-directed initiation of transcription by Escherichia coli RNA polymerase holoenzyme. We have partially purified the anti-sigma 70 factor and obtained the sequence of a C-terminal peptide of this protein. Using reverse genetics, we have identified, at the end of the lysis gene t and downstream of an as yet unassigned phage T4 early promoter, an open reading frame encoding a 90-amino-acid protein with a predicted molecular weight of 10,590. This protein has been overproduced in a phage T7 expression system and partially purified. It shows a strong inhibitory activity towards sigma 70-directed transcription (by RNA polymerase holoenzyme), whereas it has no significant effect on sigma 70-independent transcription (by RNA polymerase core enzyme). At high ionic strength, this inhibition is fully antagonized by the neutral detergent Triton X-100. Our results corroborate the initial observations on the properties of the phage T4 10K anti-sigma 70 factor, and we therefore propose that the gene which we call asiA, identified in the present study, corresponds to the gene encoding this T4 transcriptional inhibitor. Images PMID:8416914

  10. Characterization of the major capsid genes (g23) of T4-type bacteriophages in the wetlands of northeast China.

    PubMed

    Zheng, Chunyu; Wang, Guanghua; Liu, Junjie; Song, Changchun; Gao, Hongxing; Liu, Xiaobing

    2013-04-01

    To obtain genetic information and to evaluate the composition of T4-type bacteriophage (phage) communities in wetlands, environmental soil and water DNAs were obtained from two natural wetlands dominated by Carex lasiocarpa and Deyeuxia angustifolia plant species, and a neighboring paddy field in Sanjiang plain of northeast China. The biomarker gene of g23, which encodes the major capsid protein of T4-type phages, was amplified with primers MZIA1bis and MZIA6, and the PCR products were cloned and sequenced. In total, 96 and 50 different g23 clones were obtained from natural wetlands and a paddy field, respectively. A larger number of clones with low levels of identity to known sequences were found in water than in soil both in the natural wetlands and the paddy field, suggesting that many of T4-type phages in wetland water and paddy floodwater in Sanjiang plain are uncharacterized. Phylogenetic analyses showed that the g23 clones in natural wetlands, irrespective of water and soil, were distinctly different from those in marine waters, lake waters, and upland black soils, but were similar to those in paddy fields. The UniFrac analysis of g23 assemblages indicated that T4-type phage community compositions were different between soils and waters, and also were different between the natural wetlands and the paddy field. In general, the global analysis of g23 clone assemblages demonstrated that T4-type phage community compositions were different among natural wetlands, marines, lakes, paddy fields, and upland black soils. PMID:23306393

  11. Repair of Double-Strand Breaks in Bacteriophage T4 by a Mechanism That Involves Extensive DNA Replication

    PubMed Central

    George, J. W.; Kreuzer, K. N.

    1996-01-01

    We investigated double-strand break (dsb) repair in bacteriophage T4 using a physical assay that involves a plasmid substrate with two inverted DNA segments. A dsb introduced into one repeat during a T4 infection induces efficient dsb repair using the second repeat as a template. This reaction is characterized by the following interesting features. First, the dsb induces a repair reaction that is directly coupled to extensive plasmid replication; the repaired/replicated product is in the form of long plasmid concatemers. Second, repair of the dsb site is frequently associated with exchange of flanking DNA. Third, the repair reaction is absolutely dependent on the products of genes uvsX, uvsY, 32, 46, and 59, which are also required for phage genomic recombination-dependent DNA replication. Fourth, the coupled repair/replication reaction is only partly dependent on endonuclease VII (gp49), suggesting that either another Holliday-junction-cleaving activity or an alternate resolution pathway is active during T4 infections. Because this repair reaction is directly coupled to extensive replication, it cannot be explained by the SZOSTAK et al. model. We present and discuss a model for the coupled repair/replication reaction, called the extensive chromosome replication model for dsb repair. PMID:8844141

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

    PubMed

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

    2013-04-01

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

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

    SciTech Connect

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

    2011-09-16

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

  14. The Bacteriophage T4 Rapid-Lysis Genes and Their Mutational Proclivities ▿

    PubMed Central

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

    2011-01-01

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

  15. Characterization of the novel T4-like Salmonella enterica bacteriophage STP4-a and its endolysin.

    PubMed

    Li, Meng; Li, Mengzhe; Lin, Hong; Wang, Jingxue; Jin, Yanqiu; Han, Feng

    2016-02-01

    While screening for new antimicrobial agents for multidrug-resistant Salmonella enterica, the novel lytic bacteriophage STP4-a was isolated and characterized. Phage morphology revealed that STP4-a belongs to the family Myoviridae. Bacterial challenge assays showed that different serovars of Salmonella enterica were susceptible to STP4-a infection. The genomic characteristics of STP4-a, containing 159,914 bp of dsDNA with an average GC content of 36.86 %, were determined. Furthermore, the endolysin of STP4-a was expressed and characterized. The novel endolysin, LysSTP4, has hydrolytic activity towards outer-membrane-permeabilized S. enterica and Escherichia coli. These results provide essential information for the development of novel phage-based biocontrol agents against S. enterica. PMID:26563319

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

    PubMed Central

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

    2014-01-01

    The phage-proximal part of the long tail fibres of bacteriophage T4 consists of a trimer of the 1289 amino-acid gene product 34 (gp34). Different carboxy-terminal parts of gp34 have been produced and crystallized. Crystals of gp34(7261289) diffracting X-rays to 2.9? resolution, crystals of gp34(7811289) diffracting to 1.9? resolution and crystals of gp34(8941289) diffracting to 3.0 and 2.0? resolution and belonging to different crystal forms were obtained. Native data were collected for gp34(7261289) and gp34(8941289), while single-wavelength anomalous diffraction data were collected for selenomethionine-containing gp34(7811289) and gp34(8941289). For the latter, high-quality anomalous signal was obtained. PMID:25005101

  17. Nucleotide sequence and analysis of the 58.3 to 65.5-kb early region of bacteriophage T4.

    PubMed Central

    Valerie, K; Stevens, J; Lynch, M; Henderson, E E; de Riel, J K

    1986-01-01

    The complete 7.2-kb nucleotide sequence from the 58.3 to 65.5-kb early region of bacteriophage T4 has been determined by Maxam and Gilbert sequencing. Computer analysis revealed at least 20 open reading frames (ORFs) within this sequence. All major ORFs are transcribed from the left strand, suggesting that they are expressed early during infection. Among the ORFs, we have identified the ipIII, ipII, denV and tk genes. The ORFs are very tightly spaced, even overlapping in some instances, and when ORF interspacing occurs, promoter-like sequences can be implicated. Several of the sequences preceding the ORFs, in particular those at ipIII, ipII, denV, and orf61.9, can potentially form stable stem-loop structures. PMID:3024113

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

    PubMed

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

    2014-10-21

    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

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

    SciTech Connect

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

    2012-04-04

    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.

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

    PubMed

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

    2012-01-17

    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

  1. 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

  2. Bacteriophage T4 rnh (RNase H) null mutations: effects on spontaneous mutation and epistatic interaction with rII mutations.

    PubMed

    Bebenek, A; Smith, L A; Drake, J W

    1999-05-01

    The bacteriophage T4 rnh gene encodes T4 RNase H, a relative of a family of flap endonucleases. T4 rnh null mutations reduce burst sizes, increase sensitivity to DNA damage, and increase the frequency of acriflavin resistance (Acr) mutations. Because mutations in the related Saccharomyces cerevisiae RAD27 gene display a remarkable duplication mutator phenotype, we further explored the impact of rnh mutations upon the mutation process. We observed that most Acr mutants in an rnh+ strain contain ac mutations, whereas only roughly half of the Acr mutants detected in an rnhDelta strain bear ac mutations. In contrast to the mutational specificity displayed by most mutators, the DNA alterations of ac mutations arising in rnhDelta and rnh+ backgrounds are indistinguishable. Thus, the increase in Acr mutants in an rnhDelta background is probably not due to a mutator effect. This conclusion is supported by the lack of increase in the frequency of rI mutations in an rnhDelta background. In a screen that detects mutations at both the rI locus and the much larger rII locus, the r frequency was severalfold lower in an rnhDelta background. This decrease was due to the phenotype of rnh rII double mutants, which display an r+ plaque morphology but retain the characteristic inability of rII mutants to grow on lambda lysogens. Finally, we summarize those aspects of T4 forward-mutation systems which are relevant to optimal choices for investigating quantitative and qualitative aspects of the mutation process. PMID:10322013

  3. Suppressors of Mutations in the rII Gene of Bacteriophage T4 Affect Promoter Utilization

    PubMed Central

    Hall, Dwight H.; Snyder, Ronald D.

    1981-01-01

    Homyk, Rodriguez and Weil (1976) have described T4 mutants, called sip, that partially suppress the inability of T4rII mutants to grow in ? lysogens. We have found that mutants sip1 and sip2 are resistant to folate analogs and overproduce FH2 reductase. The results of recombination and complementation studies indicate that sip mutations are in the mot gene. Like other mot mutations (Mattson, Richardson and Goodin 1974; Chace and Hall 1975; Sauerbier, Hercules and Hall 1976), the sip2 mutation affects the expression of many genes and appears to affect promoter utilization. The mot gene function is not required for T4 growth on most hosts, but we have found that it is required for good growth on E. coli CTr5X. Homyk, Rodriguez and Weil (1976) also described L mutations that reverse the effects of sip mutations. L2 decreases the folate analog resistance and the inability of sip2 to grow on CTr5X. L2 itself is partially resistant to a folate analog, and appears to reverse the effects of sip2 on gene expression. These results suggest that L2 affects another regulatory gene related to the mot gene. PMID:7262547

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-06-01

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

  7. DNA packaging and the pathway of bacteriophage T4 head assembly.

    PubMed Central

    Hsiao, C L; Black, L W

    1977-01-01

    A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA. Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature. It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation). The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing. Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging. Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume. Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction. Images PMID:269421

  8. The Small Terminase, gp16, of Bacteriophage T4 Is a Regulator of the DNA Packaging Motor*

    PubMed Central

    Al-Zahrani, Abdulrahman S.; Kondabagil, Kiran; Gao, Song; Kelly, Noreen; Ghosh-Kumar, Manjira; Rao, Venigalla B.

    2009-01-01

    Tailed bacteriophages and herpes viruses use powerful molecular motors to translocate DNA into a preassembled prohead and compact the DNA to near crystalline density. The phage T4 motor, a pentamer of 70-kDa large terminase, gp17, is the fastest and most powerful motor reported to date. gp17 has an ATPase activity that powers DNA translocation and a nuclease activity that cuts concatemeric DNA and generates the termini of viral genome. An 18-kDa small terminase, gp16, is also essential, but its role in DNA packaging is poorly understood. gp16 forms oligomers, most likely octamers, exhibits no enzymatic activities, but stimulates the gp17-ATPase activity, and inhibits the nuclease activity. Extensive mutational and biochemical analyses show that gp16 contains three domains, a central oligomerization domain, and N- and C-terminal domains that are essential for ATPase stimulation. Stimulation occurs not by nucleotide exchange or enhanced ATP binding but by triggering hydrolysis of gp17-bound ATP, a mechanism reminiscent of GTPase-activating proteins. gp16 does not have an arginine finger but its interaction with gp17 seems to position a gp17 arginine finger into the catalytic pocket. gp16 inhibits DNA translocation when gp17 is associated with the prohead. gp16 restricts gp17-nuclease such that the putative packaging initiation cut is made but random cutting is inhibited. These results suggest that the phage T4 packaging machine consists of a motor (gp17) and a regulator (gp16). The gp16 regulator is essential to coordinate the gp17 motor ATPase, translocase, and nuclease activities, otherwise it could be suicidal to the virus. PMID:19561086

  9. The small terminase, gp16, of bacteriophage T4 is a regulator of the DNA packaging motor.

    PubMed

    Al-Zahrani, Abdulrahman S; Kondabagil, Kiran; Gao, Song; Kelly, Noreen; Ghosh-Kumar, Manjira; Rao, Venigalla B

    2009-09-01

    Tailed bacteriophages and herpes viruses use powerful molecular motors to translocate DNA into a preassembled prohead and compact the DNA to near crystalline density. The phage T4 motor, a pentamer of 70-kDa large terminase, gp17, is the fastest and most powerful motor reported to date. gp17 has an ATPase activity that powers DNA translocation and a nuclease activity that cuts concatemeric DNA and generates the termini of viral genome. An 18-kDa small terminase, gp16, is also essential, but its role in DNA packaging is poorly understood. gp16 forms oligomers, most likely octamers, exhibits no enzymatic activities, but stimulates the gp17-ATPase activity, and inhibits the nuclease activity. Extensive mutational and biochemical analyses show that gp16 contains three domains, a central oligomerization domain, and N- and C-terminal domains that are essential for ATPase stimulation. Stimulation occurs not by nucleotide exchange or enhanced ATP binding but by triggering hydrolysis of gp17-bound ATP, a mechanism reminiscent of GTPase-activating proteins. gp16 does not have an arginine finger but its interaction with gp17 seems to position a gp17 arginine finger into the catalytic pocket. gp16 inhibits DNA translocation when gp17 is associated with the prohead. gp16 restricts gp17-nuclease such that the putative packaging initiation cut is made but random cutting is inhibited. These results suggest that the phage T4 packaging machine consists of a motor (gp17) and a regulator (gp16). The gp16 regulator is essential to coordinate the gp17 motor ATPase, translocase, and nuclease activities, otherwise it could be suicidal to the virus. PMID:19561086

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

    SciTech Connect

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

    2009-07-21

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

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

    SciTech Connect

    Ogura, Toshihiko

    2009-03-06

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

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

    PubMed

    Lagerbck, Pernilla; Andersson, Evalena; Malmberg, Christer; Carlson, Karin

    2009-10-01

    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 catalytic E118 residue actually interfered with DNA binding (possibly due to steric hindrance or repulsion between the glutamate side chain and DNA), as shown by the ability of E118A to bind stably also as monomer or dimer to a single substrate. The tetrameric structure of EndoII in the DNA-protein complex is surprising considering the asymmetry of the recognized sequence and the predominantly single-stranded nicking. Combining the results obtained here with those from our previous in vivo studies and the recently obtained crystal structure of EndoII E118A, we suggest a model where EndoII translocates DNA between two adjacent binding sites and either nicks one strand of one or both substrates bound by the tetramer, or nicks both strands of one substrate. Thus, only one or two of the four active sites in the tetramer is catalytically active at any time. PMID:19666720

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

    PubMed

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

    1994-04-01

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

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

    SciTech Connect

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

    1994-04-26

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

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

    PubMed

    Golec, Piotr; Karczewska-Golec, Joanna; Łoś, Marcin; Węgrzyn, Grzegorz

    2014-02-01

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

  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. )

    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. Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 DAS mutation

    SciTech Connect

    Mickelson, C.; Wiberg, J.S.

    1981-10-01

    Lethal, amber mutations in T4 genes 46 and 47 cause incomplete degradation of host DNA, premature arrest of phage DNA synthesis, accumulation of abnormal DNA replication intermediates, and defective recombination. These phenotypes can be explained by the hypothesis that genes 46 and 47 control a DNA exonuclease, but in vitro demonstration of such a nuclease has not yet been reported. Membrane and supernatant fractions from 46/sup -/ and 47/sup -/ mutant-infected and 46/sup +/ 47/sup +/ control-infected cells were assayed for the presence of the protein products of these genes (i.e., gp46 and gp47) and for the ability to degrade various DNA substrates to acid-soluble products in vitro. The two proteins were found only on membranes. The membrane fraction from 46/sup -/ 47/sup -/ mutant-infected cells digested native or heavily nicked Escherichia coli DNA to acid-soluble products three to four times slower than the membrane fraction from control-infected cells. No such effect was found in the cytoplasmic fractions. The effect on nuclease activity in membranes was the same whether 47/sup -/ and 47/sup -/ mutants were present singly or together. NaClO/sub 4/, a chaotropic agent, released both gp46 and gp47 from 46/sup +/ 47/sup +/ membranes, as well as the DNase activity controlled by genes 46 and 47. DNA cellulose chromatography of proteins released from membranes by NaClO/sub 4/ showed that gp46 and gp47 bound to the native DNAs of both E. coli and T4. Thus, the overall enrichment of gp46 and gp47 relative to total T4 protein was 600-fold (10-fold in membranes, 2-fold more upon release from membranes by NaClO/sub 4/, and 30-fold more upon elution from DNA cellulose). T4 das mutations, which partially suppress the defective phenotype of 46/sup -/ and 47/sup -/ mutants, caused a considerable increase in in vitro DNase activity in both membrane and cytoplasmic fractions.

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

    PubMed Central

    Bustamante, Noem; Campillo, Nuria E.; Garca, Ernesto; Gallego, Cristina; Pera, Benet; Diakun, Gregory P.; Siz, Jos Luis; Garca, Pedro; Daz, J. Fernando; Menndez, Margarita

    2010-01-01

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

  19. Cpl-7, a lysozyme encoded by a pneumococcal bacteriophage with a novel cell wall-binding motif.

    PubMed

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

    2010-10-22

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

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

    PubMed Central

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

    1993-01-01

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

  1. 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.

    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.

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

    SciTech Connect

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

    1988-05-01

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

  3. An ADP-ribosyltransferase Alt of bacteriophage T4 negatively regulates the Escherichia coli?MazF toxin of a toxin-antitoxin module.

    PubMed

    Alawneh, Abdulraheem M; Qi, Dan; Yonesaki, Tetsuro; Otsuka, Yuichi

    2016-01-01

    Prokaryotic toxin-antitoxin (TA) systems are linked to many roles in cell physiology, such as plasmid maintenance, stress response, persistence and protection from phage infection, and the activities of toxins are tightly regulated. Here, we describe a novel regulatory mechanism for a toxin of Escherichia coli?TA systems. The MazF toxin of MazE-MazF, which is one of the best characterized type II TA systems, was modified immediately after infection with bacteriophage T4. Mass spectrometry demonstrated that the molecular weight of this modification was 542?Da, corresponding to a mono-ADP-ribosylation. This modification disappeared in cells infected with T4 phage lacking Alt, which is one of three ADP-ribosyltransferases encoded by T4 phage and is injected together with phage DNA upon infection. In vivo and in vitro analyses confirmed that T4 Alt ADP-ribosylated MazF at an arginine residue at position 4. Finally, the ADP-ribosylation of MazF by Alt resulted in the reduction of MazF RNA cleavage activity in vitro, suggesting that it may function to inactivate MazF during T4 infection. This is the first example of the chemical modification of an E.?coli toxin in TA systems to regulate activity. PMID:26395283

  4. 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...

  5. Multiple self-splicing introns in bacteriophage T4: evidence from autocatalytic GTP labeling of RNA in vitro.

    PubMed

    Gott, J M; Shub, D A; Belfort, M

    1986-10-10

    RNA from T4-infected cells yielded multiple end-labeled species when incubated with alpha-32P-GTP under self-splicing conditions. One of these corresponds to the previously identified intron from the td gene of T4, while others appear to represent additional group I introns in T4. Two loci distinct from the td gene were found to hybridize to a mixed alpha-32P-GTP-labeled T4 RNA probe. These mapped in or near the unlinked genes nrdB and nrdC. A fragment from the nrdB region that contains the intron has been cloned and shown to generate characteristic group I splice products with RNA synthesized in vivo and in vitro. Multiple introns, and the prospect that these occur within several genes in the same metabolic pathway, suggest a possible regulatory role for splicing in T4. PMID:3757035

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

    SciTech Connect

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

    2007-01-01

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

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

    SciTech Connect

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

    2008-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  9. 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

  10. Genes 55, alpha gt, 47 and 46 of bacteriophage T4: the genomic organization as deduced by sequence analysis.

    PubMed Central

    Gram, H; Rüger, W

    1985-01-01

    The nucleotide sequence of T4 genes 55, alpha gt, 47 and 46 was determined by a combination of 'classical' procedures and a shotgun approach. Small DNA fragments generated by frequent cleavage with restriction enzymes or by sonication of restriction fragments were cloned in phage M13 vectors and sequenced by the dideoxy method. The positions of the genes were determined by marker rescue between the corresponding T4 amber mutants and the cloned T4 DNA fragments used in the sequencing experiments. The sequence gives an insight into the organization of this 7.1-kb early region of the T4 genome and shows that genetically 'silent' portions within this region are not void of genetic information. PMID:4018026

  11. Study of bacteriophage T4-encoded Dam DNA (adenine-N6)-methyltransferase binding with substrates by rapid laser UV cross-linking.

    PubMed

    Evdokimov, Alexey A; Sclavi, Bianca; Zinoviev, Victor V; Malygin, Ernst G; Hattman, Stanley; Buckle, Malcolm

    2007-09-01

    DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e. no exogenous cross-linking reagents) laser UV cross-linking as a universal non-perturbing approach for studying the characteristics of T4Dam binding with substrates in the equilibrium and transient modes of interaction. UV irradiation of the enzyme.substrate complexes using an Nd(3+):yttrium aluminum garnet laser at 266 nm resulted in up to 3 and >15% yields of direct T4Dam cross-linking to DNA and AdoMet, respectively. Consequently, we were able to measure equilibrium constants and dissociation rates for enzyme.substrate complexes. In particular, we demonstrate that both reaction substrates, specific DNA and AdoMet (or product AdoHcy), stabilized the ternary complex. The improved substrate affinity for the enzyme in the ternary complex significantly reduced dissociation rates (up to 2 orders of magnitude). Several of the parameters obtained (such as dissociation rate constants for the binary T4Dam.AdoMet complex and for enzyme complexes with a nonfluorescent hemimethylated DNA duplex) were previously inaccessible by other means. However, where possible, the results of laser UV cross-linking were compared with those of fluorescence analysis. Our study suggests that rapid laser UV cross-linking efficiently complements standard DNA methyltransferase-related tools and is a method of choice to probe enzyme-substrate interactions in cases in which data cannot be acquired by other means. PMID:17630395

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

    PubMed Central

    Chang, Philip L.; Yen, Teh Fu

    1984-01-01

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

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

    PubMed Central

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

    2015-01-01

    Although the structure, function, conformational dynamics, and controlled thermodynamics of proteins are manifested by their corresponding amino acid sequences, the natural rules for molecular design and their corresponding interplay remain obscure. In this study, we focused on the role of internal cavities of proteins in conformational dynamics. We investigated the pressure-induced responses from the cavity-enlarged L99A mutant of T4 lysozyme, using high-pressure NMR spectroscopy. The signal intensities of the methyl groups in the 1H/13C 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. 13C and 1H NMR line-shape simulations showed that the pressure-induced loss in the peak intensity could be explained by the increase in the high-energy state population. In this high-energy state, the aromatic side chain of F114 gets flipped into the enlarged cavity. The accommodation of the phenylalanine ring into the efficiently packed cavity may decrease the partial molar volume of the high-energy state, relative to the ground state. We suggest that the enlarged cavity is involved in the conformational transition to high-energy states and in the volume fluctuation of the ground state. PMID:25564860

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  17. 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

  18. 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).

  19. Two New Early Bacteriophage T4 Genes, repEA and repEB, That Are Important for DNA Replication Initiated from Origin E

    PubMed Central

    Vaiskunaite, Rita; Miller, Andrew; Davenport, Laura; Mosig, Gisela

    1999-01-01

    Two new, small, early bacteriophage T4 genes, repEA and repEB, located within the origin E (oriE) region of T4 DNA replication, affect functioning of this origin. An important and unusual property of the oriE region is that it is transcribed at early and late periods after infection, but in opposite directions (from complementary DNA strands). The early transcripts are mRNAs for RepEA and RepEB proteins, and they can serve as primers for leading-strand DNA synthesis. The late transcripts, which are genuine antisense RNAs for the early transcripts, direct synthesis of virion components. Because the T4 genome contains several origins, and because recombination can bypass a primase requirement for retrograde synthesis, neither defects in a single origin nor primase deficiencies are lethal in T4 (Mosig et al., FEMS Microbiol. Rev. 17:8398, 1995). Therefore, repEA and repEB were expected and found to be important for T4 DNA replication only when activities of other origins were reduced. To investigate the in vivo roles of the two repE genes, we constructed nonsense mutations in each of them and combined them with the motA mutation sip1 that greatly reduces initiation from other origins. As expected, T4 DNA synthesis and progeny production were severely reduced in the double mutants as compared with the single motA mutant, but early transcription of oriE was reduced neither in the motA nor in the repE mutants. Moreover, residual DNA replication and growth of the double mutants were different at different temperatures, suggesting different functions for repEA and repEB. We surmise that the different structures and protein requirements for functioning of the different origins enhance the flexibility of T4 to adapt to varied growth conditions, and we expect that different origins in other organisms with multiorigin chromosomes might differ in structure and function for similar reasons. PMID:10559179

  20. Studies of viral DNA packaging motors with optical tweezers: a comparison of motor function in bacteriophages φ29, λ, and T4

    NASA Astrophysics Data System (ADS)

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

    2007-09-01

    A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the capsid against resisting forces. In addition to studying bacteriophage φ29, we have recently extended these methods to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three systems have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and dynamic disorder, responses to load, and pausing and slipping dynamics.

  1. Involvement of envelope-bound calcium in the transient depolarization of the Escherichia coli cytoplasmic membrane induced by bacteriophage T4 and T5 adsorption.

    PubMed Central

    Letellier, L; Labedan, B

    1984-01-01

    We previously showed that adsorption of bacteriophages T4 and T5 to their respective outer membrane receptors induced a partial depolarization of the cytoplasmic membrane. As these membrane potential changes were independent of phage properties, we proposed that phage adsorption triggered the emission of a signal which must be transmitted between the two membranes. We show here that these two phages use different mechanisms of transmission of this stimulation signal. In the case of T4, but not of T5, a specific requirement for envelope-bound calcium was found. Indeed, addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the membrane potential changes induced by T4. This envelope-bound calcium became accessible to the chelator only as a consequence of phage adsorption and remained in this state during the depolarization and repolarization. Membrane potential changes again occurred if calcium was added after the addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phage. The same concentration (300 microM) of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the T4-induced depolarization between multiplicities of infection of 6 and 30. This suggests that phage adsorption triggers both a conformational change of membrane components, the number of which reflects the number of stimuli (phages), and the liberation of a definite amount of calcium. This liberated calcium would, in turn, activate these modified membrane components to induce the depolarization. The fact that depolarization may be induced several times after a unique adsorption implies that these membrane components remain irreversibly modified. PMID:6421800

  2. Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas

    SciTech Connect

    Ling, S.; Vogelbacker, H.H.; Restifo, L.L.; Mattson, T.; Kozinski, A.W.

    1981-11-01

    Upon infection of Escherichia coli with bormodeoxyuridine-labeled T4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, non-bromo-deoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.

  3. 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

  4. 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.

  5. The spectrum of acridine resistant mutants of bacteriophage T4 reveals cryptic effects of the tsL141 DNA polymerase allele on spontaneous mutagenesis.

    PubMed Central

    Wang, F J; Ripley, L S

    1998-01-01

    Mutations in the ac gene of bacteriophage T4 confer resistance to acridine-inhibition of phage development. Previous studies had localized the ac gene region; we show that inactivation of T4 Open Reading Frame 52.2 confers the Acr phenotype. Thus, 52.2 is ac. The resistance mechanism is unknown. The ac gene provides a convenient forward mutagenesis assay. Its compact size (156 bp) simplifies mutant sequencing and diverse mutant types are found: base substitutions leading to missense or nonsense codons, in-frame deletions or duplications within the coding sequence, deletion or duplication frameshifts, insertions, complex mutations, and large deletions extending into neighboring sequences. Comparisons of spontaneous mutagenesis between phages bearing the wild-type or tsL141 alleles of DNA polymerase demonstrate that the impact of the mutant polymerase is cryptic when total spontaneous mutant frequencies are compared, but the DNA sequences of the ac mutants reveal a substantial alteration of fidelity by the mutant polymerase. The patterns of base substitution mutagenesis suggest that some site-specific mutation rate effects may reflect hotspots for mutagenesis arising by different mechanisms. A new class of spontaneous duplication mutations, having sequences inconsistent with misaligned pairing models, but consistent with nick-processing errors, has been identified at a hotspot in ac. PMID:9560385

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

    PubMed Central

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

    2015-01-01

    Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these macromolecular machines. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3?-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 23 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3?-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and DNA map) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex. PMID:26275775

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

    PubMed Central

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

    2001-01-01

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

  8. Protein induced by bacteriophage T4 which is absent in Escherichia coli infected with nuclear disruption-deficient phage mutants.

    PubMed Central

    Koerner, J F; Thies, S K; Snustad, D P

    1979-01-01

    A protein induced by wild-type T4 phage which is absent in Escherichia coli infected with nuclear disruption-deficient phage (with mutations in gene ndd) was identified by polacrylamide gel electrophoresis. This protein was synthesized at maximum rate at 3 to 6 min after infection. It had a molecular weight of 15,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was associated with sedimentable fractions of the cell from which it can be dissociated with 1 M guanidine-hydrochloride. The dissociated protein can be partly recovered in a form soluble in dilute buffer after partial purification and dialysis. The occurrence of this protein in a particulate cell fraction is of interest because of the postulated role of the bacterial cell membrane in nuclear disruption. Images PMID:384022

  9. Structure of bacteriophage T4 endonuclease II mutant E118A, a tetrameric GIY-YIG enzyme.

    PubMed

    Andersson, C Evalena; Lagerbck, Pernilla; Carlson, Karin

    2010-04-01

    Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16 kDa, 136 aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P2(1) with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context. PMID:20156453

  10. Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4

    SciTech Connect

    Liuzzi, M.; Weinfeld, M.; Paterson, M.C.

    1987-06-16

    The UV endonucleases from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. The authors have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV-treated, (/sup 3/H)thymine-labeled poly(dA) x poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical-(5 kJ/m/sup 2/, 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. The data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. The results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies.

  11. A critical coiled coil motif in the small terminase, gp16, from bacteriophage T4: insights into DNA packaging initiation and assembly of packaging motor.

    PubMed

    Kondabagil, Kiran R; Rao, Venigalla B

    2006-04-21

    Double-stranded DNA packaging in bacteriophages is driven by one of the most powerful force-generating molecular motors reported to date. The phage T4 motor is composed of the small terminase protein, gpl6 (18kDa), the large terminase protein, gp17 (70kDa), and the dodecameric portal protein gp20 (61kDa). gp16, which exists as an oligomer in solution, is involved in the recognition of the viral DNA substrate, the very first step in the DNA packaging pathway, and stimulates the ATPase and packaging activities associated with gp17. Sequence analyses using COILS2 revealed the presence of coiled coil motifs (CCMs) in gp16. Sixteen T4-family and numerous phage small terminases show CCMs in the corresponding region of the protein, suggesting a common structural and functional theme. Biochemical properties such as reversible thermal denaturation and analytical gel filtration data suggest that the central CCM-1 is critical for oligomerization of gp16. Mutations in CCM-1 that change the hydrophobicity of key residues, or pH 6.0, destabilized coiled coil interactions, resulting in a loss of gp16 oligomerization. The gp16 oligomers are in a dynamic equilibrium with lower M(r) intermediate species and monomer. Monomeric gp16 is unable to stimulate gp17-ATPase, an activity essential for DNA packaging, while conversion back into oligomeric form restored the activity. These data for the first time defined a CCM that is critical for structure and function of the small terminase. We postulate a packaging model in which the gp16 CCM is implicated in the regulation of packaging initiation and assembly of a supramolecular DNA packaging machine on the viral concatemer. PMID:16513134

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

    PubMed Central

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

    2015-01-01

    The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000 bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH? nucleophile. The ATPase Walker B motif, thus, may be redefined as ?-strand (46 hydrophobic-rich amino acids)DE-hydrophobic/nonpolar amino acid. PMID:25443668

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

    PubMed

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

    2008-05-01

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

  14. Elasticity theory for self-assembled protein lattices with application to the martensitic phase transition in bacteriophage T4 tail sheath

    NASA Astrophysics Data System (ADS)

    Falk, Wayne; James, Richard D.

    2006-01-01

    We propose an elasticity theory for one- and two-dimensional arrays of globular proteins for which the free energy is affected by relative position and relative rotation between neighboring molecules. The kinematics of such assemblies is described, the conditions of compatibility are found, a form of the free energy is given, and formulas for applied forces and moments are developed. It is shown that fully relaxed states of sheets consist of helically deformed sheets which themselves are composed of helical chains of molecules in rational directions. We apply the theory to the fascinating contractile deformation that occurs in the tail sheath of the virus bacteriophage T4, which aids its invasion of its bacterial host. Using electron density maps of extended and contracted sheaths, we approximate the domains of each molecule by ellipsoids and then evaluate our formulas for the position and orientation of each molecule. We show that, with the resulting kinematic description, the configurations of extended and contracted tail sheaths are generated by a simple formula. We proposed a constrained version of the theory based on measurements on extended and contracted sheath. Following a suggestion of Pauling [Discuss. Faraday Soc. 13, 170 (1953)], we develop a simple model of the molecular interaction. The resulting free energy is found to have a double-well structure. Certain simple deformations are studied (tension, torsion inflation); the theory predicts a first-order Poynting effect and some unexpected relations among moduli. Finally, the force of penetration is given, and a possibly interesting program of epitaxial growth and patterning of such sheets is suggested.

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

    PubMed

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

    2012-04-01

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

  16. Portal-Large Terminase Interactions of the Bacteriophage T4 DNA Packaging Machine Implicate a Molecular Lever Mechanism for Coupling ATPase to DNA Translocation

    PubMed Central

    Hegde, Shylaja; Padilla-Sanchez, Victor; Draper, Bonnie

    2012-01-01

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

  17. Double-strand break repair and recombination-dependent replication of DNA in bacteriophage T4 in the absence of UvsX recombinase: replicative resolution pathway.

    PubMed

    Shcherbakov, Victor P; Plugina, Lidia; Shcherbakova, Tamara; Kudryashova, Elena; Sizova, Svetlana

    2012-05-01

    The effects of mutations in bacteriophage T4 genes uvsX and 49 on the double-strand break (DSB)-promoted recombination were studied in crosses, in which DSBs were induced site-specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type iets1 and in three-factor crosses of the type iets1 a6, where ets1 is an insertion in the rIIB gene carrying the cleavage site for SegC; i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site, and a6 is rIIA point mutation located at 2040 bp from ets1. The frequency/distance relationships were obtained in crosses of the wild-type phage and of the amber mutant S17 (gene uvsX) and the double mutant S17 E727 (genes uvsX and 49). These data provide information about the frequency and distance distribution of the single-exchange (splices) and double-exchange (patches) events. The extended variant of the splice/patch coupling (SPC) model of recombination, which includes transition to the replication resolution (RR) alternative is substantiated and used for interpretation of the frequency/distance relationships. We conclude that the uvsX mutant executes recombination-dependent replication but does it by a qualitatively different way. In the absence of UvsX function, the DSB repair runs largely through the RR subpathway because of inability of the mutant to form a Holliday junction. In the two-factor crosses, the double uvsX 49- is recombinationally more proficient than the single uvsX mutant (partial suppression of the uvsX deficiency), while the patch-related double exchanges are virtually eliminated in this background. PMID:22365497

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2012-02-17

    Recently the use of engineered viral scaffolds in biotechnology and medical applications has been increasing dramatically. T4 phage capsid derived nanoparticles (NPs) have potential advantages as sensors and in biotechnology. These applications require that the physical properties and cellular uptake of these NPs be understood. In this study we used a T4 deletion mutant to investigate the effects of removing both the Hoc and Soc proteins from the capsid surface on T4 tailless NPs. The surface charge, zeta potential, size, and cellular uptake efficiencies for both the T4 NP and T4ΔHocΔSoc NP mutant were measured and compared using dynamic light scattering and flow cytometry and significant differences were detected. PMID:22285187

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

    PubMed Central

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

    2014-01-01

    Recently the use of engineered viral scaffolds in biotechnology and medical applications has been increasing dramatically. T4 phage capsid derived nanoparticles (NPs) have potential advantages as sensors and in biotechnology. These applications require that the physical properties and cellular uptake of these NPs be understood. In this study we used a T4 deletion mutant to investigate the effects of removing both the Hoc and Soc proteins from the capsid surface on T4 tailless NPs. The surface charge, zeta potential, size, and cellular uptake efficiencies for both the T4 NP and T4ΔHocΔSoc NP mutant were measured and compared using dynamic light scattering and flow cytometry and significant differences were detected. PMID:22285187

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

    SciTech Connect

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

    2006-02-05

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

  2. The gene e.1 (nudE.1) of T4 bacteriophage designates a new member of the Nudix hydrolase superfamily active on flavin adenine dinucleotide, adenosine 5'-triphospho-5'-adenosine, and ADP-ribose.

    PubMed

    Xu, WenLian; Gauss, Peter; Shen, JianYing; Dunn, Christopher A; Bessman, Maurice J

    2002-06-28

    The T4 bacteriophage gene e.1 was cloned into an expression vector and expressed in Escherichia coli, and the purified protein was identified as a Nudix hydrolase active on FAD, adenosine 5'-triphospho-5'-adenosine (Ap(3)A), and ADP-ribose. Typical of members of the Nudix hydrolases, the enzyme has an alkaline pH optimum (pH 8) and requires a divalent cation for activity that can be satisfied by Mg(2+) or Mn(2+). For all substrates, AMP is one of the products, and unlike most of the other enzymes active on Ap(3)A, the T4 enzyme hydrolyzes higher homologues including Ap(4-6)A. This is the first member of the Nudix hydrolase gene superfamily identified in bacterial viruses and the only one present in T4. Although the protein was predicted to be orthologous to E. coli MutT on the basis of a sequence homology search, the properties of the gene and of the purified protein do not support this notion because of the following. (a) The purified enzyme hydrolyzes substrates not acted upon by MutT, and it does not hydrolyze canonical MutT substrates. (b) The e.1 gene does not complement mutT1 in vivo. (c) The deletion of e.1 does not increase the spontaneous mutation frequency of T4 phage. The properties of the enzyme most closely resemble those of Orf186 of E. coli, the product of the nudE gene, and we therefore propose the mnemonic nudE.1 for the T4 phage orthologue. PMID:11976345

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    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 (∼10(11) 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

  6. 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

  7. Photochemical crosslinking of bacteriophage T4 single-stranded DNA-binding protein (gp32) to oligo-p(dT)8: identification of phenylalanine-183 as the site of crosslinking

    SciTech Connect

    Shamoo, Y.; Williams, K.R.; Konigsberg, W.H.

    1988-01-01

    Using ultraviolet light, both the 33,000-dalton single-stranded DNA-binding protein from T4 bacteriophage (gp32) as well as a 25,000-dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single-stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion-exchange high-performance liquid chromatography. Gas-phase sequencing of this modified peptide gave the following sequence: Gln-Val-Ser-Gly-(X)-Ser-Asn-Tyr-Asp-Glu-Ser-Lys, which corresponds to residues 179-190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:p(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.

  8. Models for the Binary Complex of Bacteriophage T4 Gp59 Helicase Loading Protein. GP32 Single-Stranded DNA-Binding Protein and Ternary Complex with Pseudo-Y Junction DNA

    SciTech Connect

    Hinerman, Jennifer M.; Dignam, J. David; Mueser, Timothy C.

    2012-04-05

    The bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Moreover, fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596–18607).

  9. 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 circuits conductance helped elucidate unexplored and previously invisible aspects of lysozymes 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

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

    SciTech Connect

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

    2008-04-01

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

  11. T4 test

    MedlinePLUS

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

  12. Lysozyme Crystal

    NASA Technical Reports Server (NTRS)

    2004-01-01

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

  13. Differential Bacteriophage Mortality on Exposure to Copper ▿

    PubMed Central

    Li, Jinyu; Dennehy, John J.

    2011-01-01

    Many studies report that copper can be used to control microbial growth, including that of viruses. We determined the rates of copper-mediated inactivation for a wide range of bacteriophages. We used two methods to test the effect of copper on bacteriophage survival. One method involved placing small volumes of bacteriophage lysate on copper and stainless steel coupons. Following exposure, metal coupons were rinsed with lysogeny broth, and the resulting fluid was serially diluted and plated on agar with the corresponding bacterial host. The second method involved adding copper sulfate (CuSO4) to bacteriophage lysates to a final concentration of 5 mM. Aliquots were removed from the mixture, serially diluted, and plated with the appropriate bacterial host. Significant mortality was observed among the double-stranded RNA (dsRNA) bacteriophages Φ6 and Φ8, the single-stranded RNA (ssRNA) bacteriophage PP7, the ssDNA bacteriophage ΦX174, and the dsDNA bacteriophage PM2. However, the dsDNA bacteriophages PRD1, T4, and λ were relatively unaffected by copper. Interestingly, lipid-containing bacteriophages were most susceptible to copper toxicity. In addition, in the first experimental method, the pattern of bacteriophage Φ6 survival over time showed a plateau in mortality after lysates dried out. This finding suggests that copper's effect on bacteriophage is mediated by the presence of water. PMID:21841029

  14. 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

  15. 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.

  16. Rate of Lysozyme Crystallization

    NASA Astrophysics Data System (ADS)

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

    1997-03-01

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

  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. Lysozymes in the animal kingdom.

    PubMed

    Callewaert, Lien; Michiels, Chris W

    2010-03-01

    Lysozymes (EC 3.2.1.17) are hydrolytic enzymes, characterized by their ability to cleave the beta-(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 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 distribution of these lysozymes reveals that c-type lysozymes are predominantly present in the phylum of the Chordata and in different classes of the Arthropoda. Moreover, g-type lysozymes (or at least their corresponding genes) are found in members of the Chordata, as well as in some bivalve mollusks belonging to the invertebrates. In general, the latter animals are known to produce i-type lysozymes. Although the homology in primary structure for representatives of these three lysozyme types is limited, their three-dimensional structures show striking similarities. Nevertheless, some variation exists in their catalytic mechanisms and the genomic organization of their genes. Regarding their biological role, the widely recognized function of lysozymes is their contribution to antibacterial defence but, additionally, some lysozymes (belonging to different types) are known to function as digestive enzymes. PMID:20413917

  19. Lysozyme mediated calcium carbonate mineralization.

    PubMed

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

    2009-04-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    SciTech Connect

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

    2010-07-22

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

  2. Bacteriophage prehistory

    PubMed Central

    Thomas-Abedon, Cameron; Thomas, Anne; Mazure, Hubert

    2011-01-01

    We identified 30 actual or presumptive “bacteriophage” references dating between the years 1895 and 1917 and have further explored one of the oldest: Hankin's 1896 study of a bactericidal action associated with the waters of the Ganges and Jumna rivers in India. As Hankin's work took place approximately 20 years prior to the actual discovery of bacteriophages, no claims were made as to a possible phage nature of the phenomenon. Here we suggest that it may be imprudent to assume nevertheless that it represents an early observation of phagemediated bactericidal activity. Our principal argument is that the antibacterial aspect of these river waters was able to retain full potency following “heating” for one-half hour in hermetically sealed tubes, where heating in “open” tubes resulted in loss of antibacterial activity. We also suggest that environmental phage counts would have had to have been unusually high—greater than 106/ml impacting a single host strain—to achieve the rates of bacterial loss that Hankin observed. PMID:22164351

  3. Unravelling the structure of the pneumococcal autolytic lysozyme.

    PubMed

    Monterroso, Begoa; Lpez-Zumel, Consuelo; Garca, Jos L; Siz, Jos L; Garca, Pedro; Campillo, Nuria E; Menndez, Margarita

    2005-10-01

    The LytC lysozyme of Streptococcus pneumoniae forms part of the autolytic system of this important pathogen. This enzyme is composed of a C-terminal CM (catalytic module), belonging to the GH25 family of glycosyl hydrolases, and an N-terminal CBM (choline-binding module), made of eleven homologous repeats, that specifically recognizes the choline residues that are present in pneumococcal teichoic and lipoteichoic acids. This arrangement inverts the general assembly pattern of the major pneumococcal autolysin, LytA, and the lytic enzymes encoded by pneumococcal bacteriophages that place the CBM (made of six repeats) at the C-terminus. In the present paper, a three-dimensional model of LytC built by homology modelling of each module and consistent with spectroscopic and hydrodynamic studies is shown. In addition, the putative catalytic-pair residues are identified. Despite the inversion in the modular arrangement, LytC and the bacteriophage-encoded Cpl-1 lysozyme most probably adopt a similar global fold. However, the distinct choline-binding ability and their substrate-binding surfaces may reflect a divergent evolution directed by the different roles played by them in the host (LytC) or in the bacteriophage (Cpl-1). The tight binding of LytC to the pneumococcal envelope, mediated by the acquisition of additional choline-binding repeats, could facilitate the regulation of the potentially suicidal activity of this autolysin. In contrast, a looser attachment of Cpl-1 to the cell wall and the establishment of more favourable interactions between its highly negatively charged catalytic surface and the positively charged chains of pneumococcal murein could enhance the lytic activity of the parasite-encoded enzyme and therefore liberation of the phage progeny. PMID:15943581

  4. Unravelling the structure of the pneumococcal autolytic lysozyme

    PubMed Central

    Monterroso, Begoa; Lpez-Zumel, Consuelo; Garca, JosL.; Siz, JosL.; Garca, Pedro; Campillo, NuriaE.; Menndez, Margarita

    2005-01-01

    The LytC lysozyme of Streptococcus pneumoniae forms part of the autolytic system of this important pathogen. This enzyme is composed of a C-terminal CM (catalytic module), belonging to the GH25 family of glycosyl hydrolases, and an N-terminal CBM (choline-binding module), made of eleven homologous repeats, that specifically recognizes the choline residues that are present in pneumococcal teichoic and lipoteichoic acids. This arrangement inverts the general assembly pattern of the major pneumococcal autolysin, LytA, and the lytic enzymes encoded by pneumococcal bacteriophages that place the CBM (made of six repeats) at the C-terminus. In the present paper, a three-dimensional model of LytC built by homology modelling of each module and consistent with spectroscopic and hydrodynamic studies is shown. In addition, the putative catalytic-pair residues are identified. Despite the inversion in the modular arrangement, LytC and the bacteriophage-encoded Cpl-1 lysozyme most probably adopt a similar global fold. However, the distinct choline-binding ability and their substrate-binding surfaces may reflect a divergent evolution directed by the different roles played by them in the host (LytC) or in the bacteriophage (Cpl-1). The tight binding of LytC to the pneumococcal envelope, mediated by the acquisition of additional choline-binding repeats, could facilitate the regulation of the potentially suicidal activity of this autolysin. In contrast, a looser attachment of Cpl-1 to the cell wall and the establishment of more favourable interactions between its highly negatively charged catalytic surface and the positively charged chains of pneumococcal murein could enhance the lytic activity of the parasite-encoded enzyme and therefore liberation of the phage progeny. PMID:15943581

  5. SELECTIVE TRANSLATION OF T4 TEMPLATE RNA BY RIBOSOMES FROM T4-INFECTED Escherichia coli

    PubMed Central

    Hsu, Wen-Tah; Weiss, Samuel B.

    1969-01-01

    The present studies indicate that T4 infection induces an alteration in host ribosomes which restricts the translation of host and other T4-unrelated template RNAs but permits normal translation of T4 RNA. A heat-labile factor has been isolated from T4-infected cell ribosomes which, when combined with normal cell ribosomes, confers upon the latter the property of selective T4 template RNA translation. PMID:4904642

  6. Microneedle-mediated transdermal bacteriophage delivery

    PubMed Central

    Ryan, Elizabeth; Garland, Martin J.; Singh, Thakur Raghu Raj; Bambury, Eoin; ODea, 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 2.67נ106PFU/ml (plaque forming units per ml) was detected in the receiver compartment when delivered across dermatomed skin and 4.0נ103PFU/ml was detected in the receiver compartment when delivered across full thickness skin. An in vivo study resulted in 4.13נ103PFU/ml being detected in blood 30min following initial MN-mediated phage administration. Clearance occurred rapidly, with phages being completely cleared from the systemic circulation within 24h, which was expected in the absence of infection. We have shown here that MN-mediated delivery allows successful systemic phage absorption. Accordingly, bacteriophage-based therapeutics may now have an alternative route for systemic delivery. Once fully-investigated, this could lead to more widespread investigation of these interesting therapeutic viruses. PMID:22750416

  7. Protein determinants of phage T4 lysis inhibition

    PubMed Central

    Moussa, Samir H; Kuznetsov, Vladimir; Tran, Tram Anh T; Sacchettini, James C; Young, Ry

    2012-01-01

    Genetic studies have established that lysis inhibition in bacteriophage T4 infections occurs when the RI antiholin inhibits the lethal hole-forming function of the T holin. The T-holin is composed of a single N-terminal transmembrane domain and a ∼20 kDa periplasmic domain. It accumulates harmlessly throughout the bacteriophage infection cycle until suddenly causing permeabilization of the inner membrane, thereby initiating lysis. The RI antiholin has a SAR domain that directs its secretion to the periplasm, where it can either be inactivated and degraded or be activated as a specific inhibitor of T. Previously, it was shown that the interaction of the soluble domains of these two proteins within the periplasm was necessary for lysis inhibition. We have purified and characterized the periplasmic domains of both T and RI. Both proteins were purified in a modified host that allows disulfide bond formation in the cytoplasm, due to the functional requirement of conserved disulfide bonds. Analytical centrifugation and circular dichroism spectroscopy showed that RI was monomeric and exhibited ∼80% alpha-helical content. In contrast, T exhibited a propensity to oligomerize and precipitate at high concentrations. Incubation of RI with T inhibits this aggregation and results in a complex of equimolar T and RI content. Although gel filtration analysis indicated a complex mass of 45 kDa, intermediate between the predicted 30 kDa heterodimer and 60 kDa heterotetramer, sedimentation velocity analysis indicated that the predominant species is the former. These results suggest that RI binding to T is necessary and sufficient for lysis inhibition. PMID:22389108

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. 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

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

    PubMed Central

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

    2012-01-01

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

  11. BACTERIOPHAGE: BIOLOGY AND GENETICS

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

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

    2012-01-01

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

  13. 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

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

    SciTech Connect

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

    1980-07-01

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

  15. Enzymatic properties of rhea lysozyme.

    PubMed

    Pooart, Jureerut; Torikata, Takao; Araki, Tomohiro

    2005-01-01

    Rhea lysozyme was analyzed for its enzymatic properties both lytic and oligomer activities to reveal the structural and functional relationships of goose type lysozyme. Rhea lysozyme had the highest lytic activity at pH 6, followed by ostrich and goose at pH 5.5-6, whereas the optimum of cassowary was at pH 5. pH profile was correlated to the net charge of each molecule surface. On the other hand, the pH optimum for oligomer substrate was found to be pH 4, indicating the mechanism of rhea catalysis as a general acid. The time-course of the reaction was studied using beta-1,4-linked oligosaccharide of N-acetylglucosamine (GlcNAc) with a polymerization degree of n ((GlcNAc)n) (n=4, 5, and 6) as the substrate. This enzyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3+(GlcNAc)3 predominating over that to (GlcNAc)2+ (GlcNAc)4. This indicates that the lysozyme hydrolyzed preferentially the third glycosidic linkage from the nonreducing end. Theoretical analysis has shown the highest rate constant value at 1.5 s-1 with (GlcNAc)6. This confirmed six substrate binding subsites as goose lysozyme (Honda, Y., and Fukamizo, T., Biochim. Biophys. Acta, 1388, 53-65 (1998)). The different binding free energy values for subsites B, C, F, and G from goose lysozyme might responsible for the amino acid substitutions, Asn122Ser and Phe123Met, located at the subsite B. PMID:15665474

  16. Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1.

    PubMed Central

    Pontarollo, R A; Rioux, C R; Potter, A A

    1997-01-01

    In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-bp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21. When expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein expressed from pRAP401 but did not detect Orf2 in H. somnus, lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from the serum-resistant preputial strains HS-20P and HS-22P. Elucidation of a potential role for this cryptic prophage in the H. somnus life cycle requires more study. PMID:9068631

  17. Sequence boundary T4, South China Sea

    SciTech Connect

    Thompson, P.R.; Abbott, W.H.

    1995-04-01

    Marginal basins of the western South China Sea were formed by rifting in the Eocene and filled during the Early and Middle Tertiary. Seismic profiling, drilling, and mapping for over a decade has revealed numerous structural and stratigraphic sequences, many bearing gas or oil. Chen et al. (1993) published a sequence stratigraphy for the Qingdongnan Basin, proposing correlations to the global onlap cycles of Haq et al. (1987). Although more than 30 wells have been completed, chronostratigraphy has been largely proprietary. Sequence boundary T4 is a regionally extensive seismic reflector. Major regressive sedimentary facies, shelf margin incision, and basin fan formation indicate that it is a type I sequence boundary. Study of calcareous nannofossils and planktonic foraminifera in wells penetrating T4 away from clastic deposits date the event as early Late Miocene (N14/NN8). In most sections, however, the magnitude of the hiatus varies considerably: erosion below the unconformity may expose units as old as the Late Oligocene (P21/NP24) and non-deposition following the unconformity may continue up to the Early Pliocene (N18/NN12). Microfossil paleobathymetry indicates that basinal sedimentary depositi on following T4 occurred at bathyal or deeper water depths. Because the T4 sequence boundary and subsequent deep-water sediments can be consistently recognized throughout the South China Sea-Phillippines-Indonesia region, T4 can be regarded as a major hydrocarbon seal.

  18. Bacteriophages and Biofilms

    PubMed Central

    Harper, David R.; Parracho, Helena M. R. T.; Walker, James; Sharp, Richard; Hughes, Gavin; Werthén, Maria; Lehman, Susan; Morales, Sandra

    2014-01-01

    Biofilms are an extremely common adaptation, allowing bacteria to colonize hostile environments. They present unique problems for antibiotics and biocides, both due to the nature of the extracellular matrix and to the presence within the biofilm of metabolically inactive persister cells. Such chemicals can be highly effective against planktonic bacterial cells, while being essentially ineffective against biofilms. By contrast, bacteriophages seem to have a greater ability to target this common form of bacterial growth. The high numbers of bacteria present within biofilms actually facilitate the action of bacteriophages by allowing rapid and efficient infection of the host and consequent amplification of the bacteriophage. Bacteriophages also have a number of properties that make biofilms susceptible to their action. They are known to produce (or to be able to induce) enzymes that degrade the extracellular matrix. They are also able to infect persister cells, remaining dormant within them, but re-activating when they become metabolically active. Some cultured biofilms also seem better able to support the replication of bacteriophages than comparable planktonic systems. It is perhaps unsurprising that bacteriophages, as the natural predators of bacteria, have the ability to target this common form of bacterial life.

  19. Sweetness characterization of recombinant human lysozyme.

    PubMed

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

    2015-10-01

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

  20. Sweetness and enzymatic activity of lysozyme.

    PubMed

    Masuda, T; Ueno, Y; Kitabatake, N

    2001-10-01

    Hen egg lysozyme elicits a sweet taste sensation for human beings. Effects of reduction of disulfide bonds, heat treatment, and chemical modification of hen egg lysozyme on both sweetness and hydrolytic activity were investigated. Both the sweetness and enzymatic activities were lost when the intradisulfide linkage in a lysozyme molecule was reduced and S-3-(trimethylated amino) propylated. The sweetness and enzymatic activity of lysozyme were lost on heating at 95 degrees C for 18 h. These facts suggest that tertiary structures of lysozyme are indispensable for eliciting a sweet taste as well as enzymatic activity. Although the modification of carboxyl residues in a lysozyme by glycine methylester or aminomethansulfonic acid resulted in the loss of enzymatic activity by blocking the catalytic residues, the sweetness was fully retained. These results indicate that the sweetness of lysozyme was independent of its enzymatic activity. The lysozyme purified from goose egg white similarly elicited a sweet taste, although goose (g-type) lysozyme is quite different from hen egg lysozyme (c-type) on the basis of structural, immunological, and enzymatic properties. These findings indicate that a specific protein property of lysozyme is required for sweetness elicitation and that the enzymatic activity and carbohydrates produced by enzymatic reaction are not related to the sweet taste. PMID:11600047

  1. 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...

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

    PubMed Central

    Church, George M.

    2014-01-01

    Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether these DNA modifications can similarly impede CRISPR-based defenses. In a bioinformatics search, we found naturally occurring CRISPR spacers that potentially target phages known to modify their DNA. Experimentally, we show that the Cas9 nuclease from the Type II CRISPR system of Streptococcus pyogenes can overcome a variety of DNA modifications in Escherichia coli. The levels of Cas9-mediated phage resistance to bacteriophage T4 and the mutant phage T4 gt, which contains hydroxymethylated but not glucosylated cytosines, were comparable to phages with unmodified cytosines, T7 and the T4-like phage RB49. Our results demonstrate that Cas9 is not impeded by N6-methyladenine, 5-methylcytosine, 5-hydroxymethylated cytosine, or glucosylated 5-hydroxymethylated cytosine. PMID:24886988

  3. 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.

  4. Hyperexpansion of RNA Bacteriophage Diversity

    PubMed Central

    Krishnamurthy, Siddharth R.; Janowski, Andrew B.; Zhao, Guoyan; Barouch, Dan; Wang, David

    2016-01-01

    Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent. PMID:27010970

  5. Bacteriophage and peptidoglycan degrading enzymes with antimicrobial applications.

    PubMed

    Donovan, David M

    2007-01-01

    Peptidoglycan is the major structural component of bacterial cell walls. In this era of increasingly antibiotic resistant pathogens, peptidoglycan hydrolases that degrade this important cell wall structure have emerged as a potential novel source of new antimicrobials. Included in this class are bacteriocins (lysostaphin), lysozyme, and bacteriophage endolysins. Bacteriophage are viruses that infect and utilize bacteria as their host. They can reside in the bacterial genome as a prophage, or enter the lytic phase, take over the bacterial gene expression machinery, synthesize new phage particles, lyse the host, and release up to hundreds of phage progeny. Lysis occurs during the late phase of the lytic cycle when the phage endolysin and a holin molecule are produced. The holin creates holes in the cells lipid bilayer allowing the phage endolysin (peptidoglycan hydrolase) to escape and degrade the structural portion of the cell wall. These (and other phage encoded proteins) have been shown to inhibit bacterial growth. The ability to inhibit growth or kill bacteria make both the bacteriophage and their gene products a rich source of potential antimicrobials. This review summarizes the recent resurgence of these potential antimicrobials as both diagnostic and therapeutic agents and identifies recent patents that describe these technologies. PMID:19075835

  6. Purification of the T4 endonuclease V.

    PubMed

    Higgins, K M; Lloyd, R S

    1987-03-01

    A new purification protocol has been developed for the rapid isolation to physical homogeneity of T4 endonuclease V. The enzyme was purified from an Escherichia coli strain which harbors a plasmid containing the T4 denV structural gene downstream of the lambda rightward promoter. The purification of the enzyme was monitored by pyrimidine dimer-specific nicking activity, Western blot analysis and silver or Coomassie Blue staining of SDS-polyacrylamide gels. Milligram quantities of the enzyme have been purified by the following procedure. After sonication of cells and removal of major cell debris, total protein and nucleic acids were passed over a single-stranded DNA agarose column. Endonuclease V was eluted at 650 mM KCl with a linear salt gradient yielding enzyme of approximately 20% purity and following dialysis, was applied to a chromatofocusing column. The enzyme elutes at pH 9.4 and is greater than 90% homogeneous at this step. The final purification step is CM-Sephadex chromatography which attains greater than 98% homogeneity. PMID:3547104

  7. Chlamydial plasmids and bacteriophages.

    PubMed

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

    2015-01-01

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

  8. 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...

  9. 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

  10. 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…

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

    PubMed

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

    2016-01-10

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

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

    SciTech Connect

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

    2012-07-11

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

  14. Lysozyme-Based Antibacterial Nanomotors.

    PubMed

    Kiristi, Melek; Singh, Virendra V; Esteban-Fernández de Ávila, Berta; Uygun, Murat; Soto, Fernando; Aktaş Uygun, Deniz; Wang, Joseph

    2015-09-22

    An effective and rapid bacterial killing nanotechnology strategy based on lysozyme-modified fuel-free nanomotors is demonstrated. The efficient antibacterial property of lysozyme, associated with the cleavage of glycosidic bonds of peptidoglycans present in the bacteria cell wall, has been combined with ultrasound (US)-propelled porous gold nanowire (p-AuNW) motors as biocompatible dynamic bacteria nanofighters. Coupling the antibacterial activity of the enzyme with the rapid movement of these p-AuNWs, along with the corresponding fluid dynamics, promotes enzyme-bacteria interactions and prevents surface aggregation of dead bacteria, resulting in a greatly enhanced bacteria-killing capability. The large active surface area of these nanoporous motors offers a significantly higher enzyme loading capacity compared to nonporous AuNWs, which results in a higher antimicrobial activity against Gram-positive and Gram-negative bacteria. Detailed characterization studies and control experiments provide useful insights into the underlying factors controlling the antibacterial performance of the new dynamic bacteria nanofighters. Rapid and effective killing of the Gram-positive Micrococcus lysodeikticus bacteria (69-84% within 1-5 min) is demonstrated. PMID:26308491

  15. Innate and acquired bacteriophage-mediated immunity.

    PubMed

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

    2013-07-01

    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

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lysozyme (muramidase) test system. 862.1490... Systems 862.1490 Lysozyme (muramidase) test system. (a) Identification. A lysozyme (muramidase) test system is a device intended to measure the activity of the bacteriolytic enzyme lysozyme (muramidase)...

  17. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Lysozyme (muramidase) test system. 862.1490... Systems 862.1490 Lysozyme (muramidase) test system. (a) Identification. A lysozyme (muramidase) test system is a device intended to measure the activity of the bacteriolytic enzyme lysozyme (muramidase)...

  18. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lysozyme (muramidase) test system. 862.1490... Systems 862.1490 Lysozyme (muramidase) test system. (a) Identification. A lysozyme (muramidase) test system is a device intended to measure the activity of the bacteriolytic enzyme lysozyme (muramidase)...

  19. MOLECULAR CHARACTERIZATION OF LYSOZYME TYPE II GENE IN RAINBOW TROUT.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rainbow trout (Oncorhynchus mykiss) has two types of lysozyme. Type II lysozyme differs from type I by only one amino acid, but only type II lysozyme has significant bactericidal activity. Due to this novel antibacterial property, lysozyme II appears to be a candidate gene for potentially enhancing ...

  20. 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.

  1. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F. William; Rosenberg, Alan 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.

  2. 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

  3. Lysozyme

    NASA Technical Reports Server (NTRS)

    2004-01-01

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

  4. Bacteriophage application to control the contaminated water with Shigella

    PubMed Central

    Jun, Jin Woo; Giri, Sib Sankar; Kim, Hyoun Joong; Yun, Sae Kil; Chi, Cheng; Chai, Ji Young; Lee, Byeong Chun; Park, Se Chang

    2016-01-01

    Shigella is one of the most important waterborne and foodborne pathogens around the world. Emergence of antibiotic-resistant Shigella has made the development of alternatives to conventional antibiotics necessary. In this study, a virulent Myoviridae bacteriophage, pSs-1 was isolated from environmental water in South Korea and showed infectivity to S. flexneri as well as S. sonnei strains. One-step growth analysis showed that pSs-1 has a short latent period (25 min) and a large burst size (97 PFU/cell). According to the genomic analysis, pSs-1 contains 164,999 bp of genome with a G + C content of 35.54% and it is considered as a member of the T4-like bacteriophage group. These results showed that pSs-1 may have potential as a biocontrol agent instead of conventional antibiotics for shigellosis. PMID:26971572

  5. Bacteriophage application to control the contaminated water with Shigella.

    PubMed

    Jun, Jin Woo; Giri, Sib Sankar; Kim, Hyoun Joong; Yun, Sae Kil; Chi, Cheng; Chai, Ji Young; Lee, Byeong Chun; Park, Se Chang

    2016-01-01

    Shigella is one of the most important waterborne and foodborne pathogens around the world. Emergence of antibiotic-resistant Shigella has made the development of alternatives to conventional antibiotics necessary. In this study, a virulent Myoviridae bacteriophage, pSs-1 was isolated from environmental water in South Korea and showed infectivity to S. flexneri as well as S. sonnei strains. One-step growth analysis showed that pSs-1 has a short latent period (25 min) and a large burst size (97 PFU/cell). According to the genomic analysis, pSs-1 contains 164,999 bp of genome with a G + C content of 35.54% and it is considered as a member of the T4-like bacteriophage group. These results showed that pSs-1 may have potential as a biocontrol agent instead of conventional antibiotics for shigellosis. PMID:26971572

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

    ERIC Educational Resources Information Center

    Woodruff, Robert R.; Smith, James F.

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

  7. Purification of lysozyme by multistage affinity filtration.

    PubMed

    He, L Z; Sun, Y

    2002-09-01

    A multistage affinity filtration process was developed for the purification of proteins. An affinity adsorbent was prepared by immobilizing Cibacron Blue 3GA to TSK gel HW-65F. Adsorption equilibrium experiments showed that the blue TSK gel had a high affinity for lysozyme, while its binding to bovine serum albumin (BSA) was weaker. Using a three-stage affinity filtration system, lysozyme was purified from a model system (a mixture of lysozyme and BSA) and a natural source (chicken egg white). From the chicken egg white, the three-stage affinity filtration increased the recovery yield of lysozyme from 61 to 96%, compared with the one-stage process. A mathematical model taking into account the film and interior diffusions of protein and eluant was developed for the modeling and analysis of the experimental data. Both the experimental and modeling results indicate that the multistage affinity filtration technique can be employed for the selective recovery of proteins. PMID:14508673

  8. Crystallogenesis of adenosine A(2A) receptor-T4 lysozyme fusion protein: a practical route for the structure.

    PubMed

    Ashok, Yashwanth; Nanekar, Rahul T; Jaakola, Veli-Pekka

    2013-01-01

    G-protein-coupled receptors (GPCRs) represent a major class of receptors through which a number of signals ranging from photons to large glycoprotein hormones are recognized. Human genome encodes about 800 GPCRs, yet very little structural information is available on this class of receptors. Structural studies provide a wealth of information about not only the activation mechanism of the receptor but also the crucial information about the ligand-binding pocket which could lead to the development of subtype-specific ligands. The crystal structure of human adenosine A(2A) receptor was solved in complex with a high-affinity antagonist ZM241385 at 2.6Å resolution. Here, we describe the methods that were undertaken to solve the fusion protein structure. PMID:23332700

  9. T4 virus-based toolkit for the direct synthesis and 3D organization of metal quantum particles.

    PubMed

    Hou, Li; Gao, Faming; Li, Na

    2010-12-27

    One of the challenges in building superstructures based on small metal particles is producing stable interparticle separation. Herein, we present a novel assembly method based on the use of the T4 bacteriophage capsid as a scaffold for the construction of 3D monodisperse metal-particle arrays. The highly regular and symmetrical protein surface of the T4 capsid allows the site-directed adsorption and subsequent reduction of metal ions, thus permitting the growth of metal particles in situ to enable them to exist at a quantum size with a high degree of monodispersity. Both these characteristics contribute to a great improvement in the electrocatalytic activity of the patterned noble-metal particles. Organized magnetic particles as small as 2-4?nm still maintain an observable ferromagnetic behavior, which makes them promising for a variety of possible biomedical applications. PMID:21089036

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

    SciTech Connect

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

    2009-06-30

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

  11. The Bacillus subtilis extracytoplasmic function ? factor ?(V) is induced by lysozyme and provides resistance to lysozyme.

    PubMed

    Ho, Theresa D; Hastie, Jessica L; Intile, Peter J; Ellermeier, Craig D

    2011-11-01

    Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative ? factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative ? factors are the extracytoplasmic function (ECF) ? factors. In this paper, we demonstrate that the expression of the ECF ? factor ?(V) in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that ?(V) is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon ?(V). Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for ?(V)-dependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF ? factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF ? factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF ? factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme. PMID:21856855

  12. The Bacillus subtilis Extracytoplasmic Function ? Factor ?V Is Induced by Lysozyme and Provides Resistance to Lysozyme?

    PubMed Central

    Ho, Theresa D.; Hastie, Jessica L.; Intile, Peter J.; Ellermeier, Craig D.

    2011-01-01

    Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative ? factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative ? factors are the extracytoplasmic function (ECF) ? factors. In this paper, we demonstrate that the expression of the ECF ? factor ?V in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that ?V is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon ?V. Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for ?V-dependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF ? factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF ? factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF ? factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme. PMID:21856855

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

    PubMed Central

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

    2013-01-01

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

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

    DOEpatents

    Studier, F. William; Dubendorff, John W.

    1998-01-01

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

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

    DOEpatents

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

    1998-11-03

    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.

  16. 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.

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

    DOEpatents

    Studier, F. William; Dubendorff, John W.

    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.

  18. Composite cryogels for lysozyme purification.

    PubMed

    Baydemir, Gzde; Trko?lu, Emir Alper; Anda, Mge; Perin, I??k; Denizli, Adil

    2015-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  20. Lysozyme, an abomasal enzyme in the ruminants.

    PubMed

    Pahud, J J; Schellenberg, D; Monti, J C; Scherz, J C

    1983-01-01

    A strong lytic activity against Micrococcus luteus was demonstrated in abomasal secretions from calf, adult cattle, goat and sheep. This bacteriolytic activity was undetectable in other secretions. Bacteriolysis was caused by a glycosidase displaying endo-N-acetylmuramoylhydrolase specificity (EC 3.2.1.17) and was further characterized in the calf. This lysozyme also displayed significant chitinase activity. Immunofluorescence microscopy confirmed the secretion of lysozyme by abomasal gastric glands exclusively. Electrofocusing revealed multiple molecular forms, the predominant one (more than 80%) being characterized by Mr approx. 15,000, pH optimum 5.0, pl 7.5 and remarkable conformational stability. The lytic activity of lysozyme was ionic strength dependent and competitive inhibition was observed with both N-acetyl glucosamine and N-acetyl-muramic acid. Amino-acid analysis demonstrated common characteristics with known lysozymes, i.e. four disulphide bridges, two proline and N-terminal lysine. Structural homology between the three ruminant lysozymes was established by immunological cross-reactivity. PMID:6677186

  1. X-ray topography of a lysozyme crystal

    SciTech Connect

    Stojanoff,V.; Siddons, D.

    1996-01-01

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

  2. Bacteriophage in polar inland waters.

    PubMed

    Swstrm, Christin; Lisle, John; Anesio, Alexandre M; Priscu, John C; Laybourn-Parry, Johanna

    2008-03-01

    Bacteriophages are found wherever microbial life is present and play a significant role in aquatic ecosystems. They mediate microbial abundance, production, respiration, diversity, genetic transfer, nutrient cycling and particle size distribution. Most studies of bacteriophage ecology have been undertaken at temperate latitudes. Data on bacteriophages in polar inland waters are scant but the indications are that they play an active and dynamic role in these microbially dominated polar ecosystems. This review summarises what is presently known about polar inland bacteriophages, ranging from subglacial Antarctic lakes to glacial ecosystems in the Arctic. The review examines interactions between bacteriophages and their hosts and the abiotic and biotic variables that influence these interactions in polar inland waters. In addition, we consider the proportion of the bacteria in Arctic and Antarctic lake and glacial waters that are lysogenic and visibly infected with viruses. We assess the relevance of bacteriophages in the microbial loop in the extreme environments of Antarctic and Arctic inland waters with an emphasis on carbon cycling. PMID:18188502

  3. 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

  4. Bacteriophages of methanotrophic bacteria

    SciTech Connect

    Tyutikow, F.M.; Bespalova, I.A.; Rebentish, B.A.; Aleksandrushkina, N.N.; Krivisky, A.S.

    1980-10-01

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

  5. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Lipopolysaccharide-deficient, bacteriophage-resistant mutants of Escherichia coli K-12.

    PubMed

    Hancock, R E; Reeves, P

    1976-07-01

    Bacteriophage-resistant mutants isolated and classified in a previous study were examined for alterations in their lipopolysaccharide (LPS) composition, and properties likely to be affected by alterations in LPS composition were studied. It was found that many of the mutants of the Ktw (K2-resistance), Ttk (T2, T4, or K19 resistance), Bar (bacteriophage), Wrm (wide-range mutants), and miscellaneous resistance groups were altered in their response to a series of antibiotics and to two LPS-specific bacteriophages, C21 and U3. Furthermore, many of the bacteriophages to which these mutants were resistant adsorbed to LPS preparations. By direct sugar analysis of the mutant LPS preparations, it was shown that the mutants fitted into six distinct classes, which are readily derived from LPS core with a structure resembling that of Salmonella or Escherichia coli O100. A number of the mutants were shown to map between pyrE and mtl, which has been previously shown to be the site of a cluster of rfa genes in both Salmonella and E. coli. Outer membrane protein composition was studied in the above mutants using polyacrylamide gel electrophoresis. Some strains were shown to have alterations in the amount of major proteins. The nature of the bacteriophage receptors involved and the alterations leading to resistance are discussed. PMID:776951

  8. Monolithic molecularly imprinted cryogel for lysozyme recognition.

    PubMed

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

    2014-10-01

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

  9. Bacteriophages of Bacteroides.

    PubMed

    Booth, S J; Van Tassell, R L; Johnson, J L; Wilkins, T D

    1979-01-01

    Sixty-eight bacteriophages specific for nine species (DNA homology groups) of Bacteroides were isolated from sewage. Four distinct morphological types were isolated, three of which had not previously been described. Attempts to use these phages to transduce Bacteroides fragilis were unsuccessful. A total of 91 phage-susceptible strains of Bacteroides were used with these phages in a study of the feasibility of developing a scheme for identification of Bacteroides species. Blind trials were performed with 10 B. fragilis-specific phages and a collection of over 200 bacterial strains, including 144 strains of B. fragilis. Of the strains of B. fragilis, 78% were identified correctly within 24 hr. A phage-carrier state, or pseudolysogeny, was observed with most of the phage-host systems, and this state was studied in detail with B. fragilis phage Bf-1. The presence of a thick capsule around some cells in a pure culture of a host strain appears to render these cells resistant to phage infection, thus perpetuating the carrier state. It is suggested that such capsules may play a role in the virulence of strains of Bacteroides. PMID:398578

  10. Bacteriophages of Saccharopolyspora erythraea.

    PubMed Central

    Grund, A D; Hutchinson, C R

    1987-01-01

    Five bacteriophages infecting only Saccharopolyspora erythraea (formerly Streptomyces erythreus) among 43 Streptomyces spp. tested were classified into two groups by phage-host relationships, restriction enzyme mapping, cohesive-end determinations, and Southern hybridizations. phi SE6, the most frequently isolated phage, produced clear plaques on all hosts tested, while phi SE45, phi SE57, phi SE60, and phi SE69 produced turbid plaques. phi SE6 DNA was linear, had a molecular weight of (27.6 +/- 1) X 10(6) and, like the DNAs of phi SE45, phi SE57, and phi SE69, lacked cohesive ends. The characteristic patterns of of ClaI and HindIII restriction digests of phi SE6 DNA and the results of Southern hybridizations with three different ClaI fragments of phi SE6 DNA as probes indicated that phi SE6 DNA was partially circularly permuted and terminally redundant, suggesting that it was packaged by a headful packaging mechanism. Southern hybridization data also showed that phi SE45, phi SE57, and phi SE69 were closely related to phi SE6. phi SE60 DNA, in contrast, had cohesive ends, and restriction mapping plus Southern hybridization data showed that phi SE60 was unrelated to the other four phages. Images PMID:3036768

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

    PubMed

    Tellefson, L M; Germaine, G R

    1986-03-01

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

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

    PubMed Central

    Miernikiewicz, Paulina; Owczarek, Barbara; Piotrowicz, Agnieszka; Boczkowska, Barbara; Rzewucka, Kamila; Figura, Grzegorz; Letarov, Andrey; Kulikov, Eugene; Kopciuch, Agnieszka; Świtała-Jeleń, Kinga; Oślizło, Anna; Hodyra, Katarzyna; Gubernator, Jerzy; Dąbrowska, Krystyna

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

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

    PubMed

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

    2012-01-01

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

  14. Characteristics of Bacteriophage N1 and Its Attachment to Cells of Micrococcus lysodeikticus1

    PubMed Central

    Lovett, Paul S.; Shockman, Gerald D.

    1970-01-01

    Bacteriophage N1 was purified by differential and equilibrium gradient centrifugation and characterized with respect to bouyant density in CsCl, one-step growth properties, host range, and morphology by electron microscopy. In a tris (hydroxymethyl) aminomethane-magnesium buffer (pH 7.15), the irreversible adsorption of N1 to cells of Micrococcus lysodeikticus strain 1 (ML-1) followed first-order reaction kinetics with an adsorption-velocity constant of 1.6 × 10−9/min at 32 C. The rate of phage attachment was not significantly altered when adsorption mixtures contained 0.01 m KCN or 1% casein hydrolysate, 0.01 m CaCl2, and 0.001 m tryptophan. The activation energy for the irreversible adsorption reaction was 8.6 kcal. Treatment of ML-1 cells by any of the following procedures reduced the irreversible phage receptor activity over 90%: (i) mechanical disruption, (ii) lysozyme digestion, (iii) incubation in 1% cetyltrimethylammonium bromide, or (iv) incubation of heated cells (100 C, 15 min) with trypsin, Pronase, or lysozyme. The sensitivity of the phage receptor activity of ML-1 cells to lysozyme suggests that the bacterial cell wall is involved in the receptor site for the virus. Destruction of receptor activity by the other treatments cited above implies that, in addition to the cell wall, other cellular components may participate in the irreversible attachment of N1 phage to cells. Images PMID:5471473

  15. Virulence reduction in bacteriophage resistant bacteria

    PubMed Central

    León, Marcela; Bastías, Roberto

    2015-01-01

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

  16. 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.

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

    PubMed

    Oliveira Silva, Catarina; Petersen, Steffen B; Pinto Reis, Catarina; Rijo, Patrcia; Molpeceres, Jess; Vorum, Henrik; Neves-Petersen, Maria Teresa

    2015-01-01

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

  18. 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

  19. Factors influencing recombinant human lysozyme extraction and cation exchange adsorption.

    PubMed

    Wilken, Lisa R; Nikolov, Zivko L

    2006-01-01

    Human lysozyme has numerous potential therapeutic applications to a broad spectrum of human diseases. This glycosidic enzyme is present in tears, saliva, nasal secretions, and milk--sources not amendable for commercial development. Recently, a high expression level of recombinant human lysozyme (0.5% dry weight) was achieved in transgenic rice seed. This paper evaluates the effects of pH and ionic strength on rice protein and lysozyme extractability, as well as their interactions with the strong cation-exchange resin, SP-Sepharose FF. The extraction conditions that maximized lysozyme yield and the ratio of extracted human lysozyme to native rice protein were not optimal for lysozyme adsorption. The conditions that gave the highest extracted lysozyme to native protein ratio were pH 4.5 and 100 mM NaCl in 50 mM sodium acetate buffer. At pH 4.5, salt concentrations above 100 mM NaCl reduced the lysozyme-to-protein ratio. The best conditions for lysozyme adsorption were pH 4.5 and 50 mM sodium acetate buffer. Lysozyme extraction and subsequent adsorption at pH 4.5 and 50 mM NaCl was an acceptable compromise between lysozyme extractability, adsorption, and purity. The primary recovery of human lysozyme from pH 6 extracts, irrespective of ionic strength, was inferior to that using pH 4.5 with unacceptably low saturation capacities and lysozyme purity. High purity was achieved with a single chromatography step by adjusting the pH 4.5 extract to pH 6 before adsorption. The disadvantage of this approach was the drastically lower saturation capacity compared to adsorption at pH 4.5. PMID:16739958

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-11-01

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

  2. 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...

  3. ISOLATION OF LYTIC SALMONELLA BACTERIOPHAGES

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. Bacteriophage endolysins as novel antimicrobials

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. 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

  6. Elasticity and Strength of Biomacromolecular Crystals: Lysozyme

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  7. 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.

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

    SciTech Connect

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

    2009-08-28

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

  9. Bacteriophage-modified microarrays for the direct impedimetric detection of bacteria.

    PubMed

    Shabani, Arghavan; Zourob, Mohammed; Allain, Beatrice; Marquette, Christophe A; Lawrence, Marcus F; Mandeville, Rosemonde

    2008-12-15

    A novel method is presented for the specific and direct detection of bacteria using bacteriophages as recognition receptors immobilized covalently onto functionalized screen-printed carbon electrode (SPE) microarrays. The SPE networks were functionalized through electrochemical oxidation in acidic media of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) by applying a potential of +2.2 V to the working electrode. Immobilization of T4 bacteriophage onto the SPEs was achieved via EDC by formation of amide bonds between the protein coating of the phage and the electrochemically generated carboxylic groups at the carbon surface. The surface functionalization with EDC, and the binding of phages, was verified by time-of-flight secondary ion mass spectrometry. The immobilized T4 phages were then used to specifically detect E. coli bacteria. The presence of surface-bound bacteria was verified by scanning electron and fluorescence microscopies. Impedance measurements (Nyquist plots) show shifts of the order of 10(4) Omega due to the binding of E. coli bacteria to the T4 phages. No significant change in impedance was observed for control experiments using immobilized T4 phage in the presence of Salmonella. Impedance variations as a function of incubation time show a maximum shift after 20 min, indicating onset of lysis, as also confirmed by fluorescence microscopy. Concentration-response curves yield a detection limit of 10(4) cfu/mL for 50-microL samples. PMID:19072262

  10. Classification of Myoviridae bacteriophages using protein sequence similarity

    PubMed Central

    2009-01-01

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

  11. Binding of lysozyme to common pili of Escherichia coli.

    PubMed Central

    McMichael, J C; Ou, J T

    1979-01-01

    Common pili from Escherichia coli were found to bind hen egg white lysozyme. The binding was highly dependent on ionic strength, and the maximum binding occurred near an ionic strength of 0.02. The pili were aggregated by lysozyme, and this process could be followed by optical turbidity, electron microscopy, and coprecipitation. Near the maximum saturation of binding, one lysozyme molecule was bound by two pilus protein subunits. Electron micrographs of this aggregate indicated that they were paracrystalline structures. Piliated bacteria were more readily agglutinated by lysozyme than were nonpiliated bacteria. Since lysozyme is considered to be an antibacterial humoral factor and since pili are considered to be a colonization factor, the binding of lysozyme may represent an important bacterium-host interaction Images PMID:378948

  12. Complete Genome Sequence of vB_EcoM_112, a T-Even-Type Bacteriophage Specific for Escherichiacoli O157:H7

    PubMed Central

    Coffey, Brid; Ross, R. Paul; OFlynn, Gary; OSullivan, Orla; Casey, Aidan; Callanan, Michael; Coffey, Aidan

    2014-01-01

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

  13. Probabilistic approach to lysozyme crystal nucleation kinetics.

    PubMed

    Dimitrov, Ivaylo L; Hodzhaoglu, Feyzim V; Koleva, Dobryana P

    2015-09-01

    Nucleation of lysozyme crystals in quiescent solutions at a regime of progressive nucleation is investigated under an optical microscope at conditions of constant supersaturation. A method based on the stochastic nature of crystal nucleation and using discrete time sampling of small solution volumes for the presence or absence of detectable crystals is developed. It allows probabilities for crystal detection to be experimentally estimated. One hundred single samplings were used for each probability determination for 18 time intervals and six lysozyme concentrations. Fitting of a particular probability function to experimentally obtained data made possible the direct evaluation of stationary rates for lysozyme crystal nucleation, the time for growth of supernuclei to a detectable size and probability distribution of nucleation times. Obtained stationary nucleation rates were then used for the calculation of other nucleation parameters, such as the kinetic nucleation factor, nucleus size, work for nucleus formation and effective specific surface energy of the nucleus. The experimental method itself is simple and adaptable and can be used for crystal nucleation studies of arbitrary soluble substances with known solubility at particular solution conditions. PMID:25749893

  14. Hydration of lysozyme studied by Raman spectroscopy.

    PubMed

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

    2013-05-01

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

  15. Effect of environmental contaminants on nasal lysozyme secretions.

    PubMed

    Noble, Rudolf E

    2002-02-01

    Human nasal secretions are comprised of lysozyme and albumin as their main protein components. Lysozyme, an anti-microbial substance, is produced by nasal serous cells while albumin is obtained, primarily, from increased nasal vasculature permeability. We measured lysozyme levels in nasal secretions following challenge by a variety of non-infectious environmental contaminants. The methodology given presents a simple and rapid method of collecting nasal secretions and determining their lysozyme content, a technique which can be used for a host of environmental irritants. PMID:11846170

  16. 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.

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

    ERIC Educational Resources Information Center

    Woodruff, Robert R.; And Others

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

  18. 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

  19. Mobile DNA elements in T4 and related phages

    PubMed Central

    2010-01-01

    Mobile genetic elements are common inhabitants of virtually every genome where they can exert profound influences on genome structure and function in addition to promoting their own spread within and between genomes. Phage T4 and related phage have long served as a model system for understanding the molecular mechanisms by which a certain class of mobile DNA, homing endonucleases, promote their spread. Homing endonucleases are site-specific DNA endonucleases that initiate mobility by introducing double-strand breaks at defined positions in genomes lacking the endonuclease gene, stimulating repair and recombination pathways that mobilize the endonuclease coding region. In phage T4, homing endonucleases were first discovered as encoded within the self-splicing td, nrdB and nrdD introns of T4. Genomic data has revealed that homing endonucleases are extremely widespread in T-even-like phage, as evidenced by the astounding fact that ~11% of the T4 genome encodes homing endonuclease genes, with most of them located outside of self-splicing introns. Detailed studies of the mobile td intron and its encoded endonuclease, I-TevI, have laid the foundation for genetic, biochemical and structural aspects that regulate the mobility process, and more recently have provided insights into regulation of homing endonuclease function. Here, we summarize the current state of knowledge regarding T4-encoded homing endonucleases, with particular emphasis on the td/I-TevI model system. We also discuss recent progress in the biology of free-standing endonucleases, and present areas of future research for this fascinating class of mobile genetic elements. PMID:21029434

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

    PubMed Central

    Szermer-Olearnik, Bożena; Boratyński, 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

  1. Removal of endotoxins from bacteriophage preparations by extraction with organic solvents.

    PubMed

    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 10(3) and 10(5) 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 10(3)-10(5) EU/10(9) PFU (plaque forming units) down to an average of 2.8 EU/10(9) PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli) and F8 (P. aeruginosa). PMID:25811193

  2. Recombinant bacteriophage lysins as antibacterials.

    PubMed

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

    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

  3. 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

  4. Bacteriophage biocontrol in wastewater treatment.

    PubMed

    Jassim, Sabah A A; Limoges, Richard G; El-Cheikh, Hassan

    2016-04-01

    Waterborne bacterial pathogens in wastewater remains an important public health concern, not only because of the environmental damage, morbidity and mortality that they cause, but also due to the high cost of disinfecting wastewater by using physical and chemical methods in treatment plants. Bacteriophages are proposed as bacterial pathogen indicators and as an alternative biological method for wastewater treatment. Phage biocontrol in large scale treatment requires adaptive and aggressive phages that are able to overcome the environmental forces that interfere with phage-host interactions while targeting unwanted bacterial pathogens and preventing biofilms and foaming. This review will shed light on aspects of using bacteriophage programming technology in wastewater plants to rapidly target and reduce undesirable bacteria without harming the useful bacteria needed for biodegradation. PMID:26941243

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

    PubMed Central

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

    2015-01-01

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

  6. Bacteriophage Transduction in Staphylococcus epidermidis

    PubMed Central

    Olson, Michael E.; Horswill, Alexander R.

    2016-01-01

    The genetic manipulation of Staphylococcus epidermidis for molecular experimentation has long been an area of difficulty. Many of the traditional laboratory techniques for strain construction are laborious and hampered by poor efficiency. The ability to move chromosomal genetic markers and plasmids using bacteriophage transduction has greatly increased the speed and ease of S. epidermidis studies. These molecular genetic advances have advanced the S. epidermidis research field beyond a select few genetically tractable strains and facilitated investigations of clinically relevant isolates. PMID:24222465

  7. Bacteriophage Transduction in Staphylococcus aureus.

    PubMed

    Olson, Michael E

    2016-01-01

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

  8. Immobilization of Active Bacteriophages on Polyhydroxyalkanoate Surfaces.

    PubMed

    Wang, Chanchan; Sauvageau, Dominic; Elias, Anastasia

    2016-01-20

    A rapid, efficient technique for the attachment of bacteriophages (phages) onto polyhydroxyalkanoate (PHA) surfaces has been developed and compared to three reported methods for phage immobilization. Polymer surfaces were modified to facilitate phage attachment using (1) plasma treatment alone, (2) plasma treatment followed by activation by 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), (3) plasma-initiated acrylic acid grafting, or (4) plasma-initiated acrylic acid grafting with activation by EDC and sulfo-NHS. The impact of each method on the surface chemistry of PHA was investigated using contact angle analysis and X-ray photoelectron spectroscopy. Each of the four treatments was shown to result in both increased hydrophilicity and in the modification of the surface functional groups. Modified surfaces were immersed in suspensions of phage T4 for immobilization. The highest level of phage binding was observed for the surfaces modified by plasma treatment alone. The change in chemical bond states observed for surfaces that underwent plasma treatment is suspected to be the cause of the increased binding of active phages. Plasma-treated surfaces were further analyzed through phage-staining and fluorescence microscopy to assess the surface density of immobilized phages and their capacity to capture hosts. The infective capability of attached phages was confirmed by exposing the phage-immobilized surfaces to the host bacteria Escherichia coli in both plaque and infection dynamic assays. Plasma-treated surfaces with immobilized phages displayed higher infectivity than surfaces treated with other methods; in fact, the equivalent initial multiplicity of infection was 2 orders of magnitude greater than with other methods. Control samples - prepared by immersing polymer surfaces in phage suspensions (without prior plasma treatment) - did not show any bacterial growth inhibition, suggesting they did not bind phages from the suspension. PMID:26741170

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

    PubMed Central

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

    2015-01-01

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

  10. Tetragonal Lysozyme, From Monomer to Crystal

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Purification of Lysozyme by Intrinsically Shielded Hydrogel Beads

    NASA Astrophysics Data System (ADS)

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

    2013-07-01

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

  13. Adaptive functional diversification of lysozyme in insectivorous bats.

    PubMed

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

    2014-11-01

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

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

    PubMed

    Başar, 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

  15. 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, Gran; 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

  16. Dynamic clusters in highly concentrated lysozyme solutions

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  17. Splint ligation of RNA with T4 DNA ligase

    PubMed Central

    Kershaw, Christopher J.; OKeefe, 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

  18. Splint ligation of RNA with T4 DNA ligase.

    PubMed

    Kershaw, Christopher J; O'Keefe, Raymond T

    2012-01-01

    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

  19. Topical application of nucleotides increase lysozyme levels in tears.

    PubMed

    Peral, Assumpta; Loma, Patricia; Yerxa, Benjamin; Pintor, Jess

    2008-06-01

    The present work studies the effects of topical application of nucleotides on rabbit tear lysozyme levels. Lysozyme values were determined by the diffusion in Agar method described by van Bijsterveld in 1974, and the protein amount was obtained by measuring the inhibitory halos around a Whatman n degrees 1 paper disc of 5 mm in diameter. The tested nucleotides were UTP, Ap(4)A and Up(4)U. These compounds were topically instilled in a single-dose in one eye (with the contralateral eye as a control) and the lysozyme halos were measured along 5 hours. The obtained results showed an increase in the lysozyme concentrations of 67%, 93%, and 119% for UTP, Ap(4)A, and Up(4)U, respectively, over the basal levels of lysozyme. For this reason, we suggest these molecules as a potential treatment for the reinforcement of the tear film barrier against ocular infection. PMID:19668714

  20. Bioengineered lysozyme in combination therapies for Pseudomonas aeruginosa lung infections.

    PubMed

    Griswold, Karl E; Bement, Jenna L; Teneback, Charlotte C; Scanlon, Thomas C; Wargo, Matthew J; Leclair, Laurie W

    2014-01-01

    There is increasing urgency in the battle against drug-resistant bacterial pathogens, and this public health crisis has created a desperate need for novel antimicrobial agents. Recombinant human lysozyme represents one interesting candidate for treating pulmonary infections, but the wild type enzyme is subject to electrostatic mediated inhibition by anionic biopolymers that accumulate in the infected lung. We have redesigned lysozyme's electrostatic potential field, creating a genetically engineered variant that is less susceptible to polyanion inhibition, yet retains potent bactericidal activity. A recent publication demonstrated that the engineered enzyme outperforms wild type lysozyme in a murine model of Pseudomonas aeruginosa lung infection. Here, we expand upon our initial studies and consider dual therapies that combine lysozymes with an antimicrobial peptide. Consistent with our earlier results, the charge modified lysozyme combination outperformed its wild type counterpart, yielding more than an order-of-magnitude reduction in bacterial burden following treatment with a single dose. PMID:24637705

  1. Determination of monomer concentrations in crystallizing lysozyme solutions

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  2. 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

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

    PubMed

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

    2013-05-21

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

  4. Programming Bacteriophages by Swapping Their Specificity Determinants.

    PubMed

    Goren, Moran G; Yosef, Ido; Qimron, Udi

    2015-12-01

    Bacteriophages, bacteria's natural enemies, may serve as potent antibacterial agents. Their specificity for certain bacterial sub-species limits their effectiveness, but allows selective targeting of bacteria. Lu and colleagues present a platform for such targeting through alteration of bacteriophages' host specificity by swapping specificity domains in their host-recognition ligand. PMID:26526502

  5. 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...

  6. Bacteriophage: laboratorial diagnosis and phage therapy

    PubMed Central

    Silva, Joas L. Da; Hirata, Rosario D.C.; Hirata, Mario H.

    2009-01-01

    Bacteriophages have been researched as a new alternative to antibiotics. These viruses inject their genetic material into bacteria and use their host machinery to multiply themselves. The research of bacteriophages in Brazil will certainly provide low-cost treatment of multidrug resistant bacteria, new microbiological diagnosis and advantages for the Brazilian food industry. PMID:24031398

  7. 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 ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, 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 ΦCP7R 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 Φ29. 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

  8. Production of lysozyme and lysozyme-superoxide dismutase dimers bound by a ditryptophan cross-link in carbonate radical-treated lysozyme.

    PubMed

    Paviani, Vernica; Queiroz, Raphael F; Marques, Emerson F; Di Mascio, Paolo; Augusto, Ohara

    2015-12-01

    Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and in vivo, the products formed from the oxidation of Trp residues remain incompletely understood. Recently, we characterized a ditryptophan cross-link produced by the recombination of hSOD1-tryptophanyl radicals generated from attack of the carbonate radical produced during the bicarbonate-dependent peroxidase activity of the enzyme. Here, we examine whether the ditryptophan cross-link is produced by the attack of the carbonate radical on proteins other than hSOD1. To this end, we treated hen egg white lysozyme with photolytically and enzymatically generated carbonate radical. The radical yields were estimated and the lysozyme modifications were analyzed by SDS-PAGE, western blot, enzymatic activity and MS/MS analysis. Lysozyme oxidation by both systems resulted in its inactivation and dimerization. Lysozyme treated with the photolytic system presented monomers oxidized to hydroxy-tryptophan at Trp(28) and Trp(123) and N-formylkynurenine at Trp(28), Trp(62) and Trp(123). Lysozyme treated with the enzymatic system rendered monomers oxidized to N-formylkynurenine at Trp(28). The dimers were characterized as lysozyme-Trp(28)-Trp(28)-lysozyme and lysozyme-Trp(28)-Trp(32)-hSOD1. The results further demonstrate that the carbonate radical is prone to causing biomolecule cross-linking and hence, may be a relevant player in pathological mechanisms. The possibility of exploring the formation of ditryptophan cross-links as a carbonate radical biomarker is discussed. PMID:26197052

  9. Filamentous Bacteriophage Promote Biofilm Assembly and Function.

    PubMed

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

    2015-11-11

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

  10. Single molecule studies of DNA packaging by bacteriophages

    NASA Astrophysics Data System (ADS)

    Fuller, Derek Nathan

    The DNA packaging dynamics of bacteriophages φ29, 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 φ29 and T4 studies. In the studies of φ29, 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 φ29. Each system showed similarities to the φ29 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 φ29. However, dynamic structural transitions were observed with lambda that are not observed with φ29. 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 φ29.

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

    SciTech Connect

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

    2008-04-02

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

  12. Lysozyme loading and release from Se doped hydroxyapatite nanoparticles.

    PubMed

    Wang, Yanhua; Hao, Hang; Zhang, Shengmin

    2016-04-01

    Element-substituted hydroxyapatite (HA) based nanocomposites have become a promising therapeutic material for improving bone defect repair. Selenium substituted HA nanoparticles can both induce apoptosis of bone tumor cells and enhance osteointegration. However, the effect of selenite ions on the proteins in combination with the HA nanoparticles remains to be elucidated. Here, we investigated the influence of selenium doping concentration on the loading and release of lysozyme (LSM) as a model protein drug. The selenium substituted HA-LSM composites with different doping concentrations were synthesized and characterized. The subsequent delivery of lysozyme was studied in a phosphate buffer solution (PBS). We found that selenium substituted HA-LSM composites with Se:P=10% showed the highest amount of lysozyme loading (41.7%), whereas the amount of lysozyme loaded in undoped HA nanoparticles was the lowest (34.1%). The doped selenium interacts with lysozyme molecules, which leads to the increase of β-sheet and unordered, and the decrease of self-association, α-helix and β-turns in protein structures. Moreover, selenium addition significantly slows the protein release from HA-LSM composites. The composites with Se:P=10% release lysozyme at the slightly slower rate among the samples with different Se doping concentrations. It also shows that the released lysozyme retains most of its enzymatic activity. PMID:26838882

  13. 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.

  14. Phosphorylation of tRNA by T4 polynucleotide kinase.

    PubMed Central

    Lillehaug, J R; Kleppe, K

    1977-01-01

    The phosphorylation of various intact tRNA species by T4 polynucleotide kinase has been studied. The apparent Michaelis constant was on the average found to be 100 times lower than for some single-stranded DNAs previously studied. (J.R. Lillehaug and K. Kleppe, (1975) Biochemistry, 14, 1221). Conditions which result in complete phosphorylation of different tRNA species have also been established. Studies on equilibrium constants and the reversibility of the reaction revealed that the phosphorylation reaction is not a true equilibrium reaction under the conditions used in this work. PMID:190592

  15. Mesoscopic coarse-grained simulations of lysozyme adsorption.

    PubMed

    Yu, Gaobo; Liu, Jie; Zhou, Jian

    2014-05-01

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

  16. 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.

  17. 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.

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

    SciTech Connect

    Mamontov, Eugene; O'Neill, Hugh Michael

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Su, Yan-lei; Li, Chao

    2008-01-01

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

  20. The bacteriophage DNA packaging motor.

    PubMed

    Rao, Venigalla B; Feiss, Michael

    2008-01-01

    An ATP-powered DNA translocation machine encapsidates the viral genome in the large dsDNA bacteriophages. The essential components include the empty shell, prohead, and the packaging enzyme, terminase. During translocation, terminase is docked on the prohead's portal protein. The translocation ATPase and the concatemer-cutting endonuclease reside in terminase. Remarkably, terminases, portal proteins, and shells of tailed bacteriophages and herpes viruses show conserved features. These DNA viruses may have descended from a common ancestor. Terminase's ATPase consists of a classic nucleotide binding fold, most closely resembling that of monomeric helicases. Intriguing models have been proposed for the mechanism of dsDNA translocation, invoking ATP hydrolysis-driven conformational changes of portal or terminase powering DNA motion. Single-molecule studies show that the packaging motor is fast and powerful. Recent advances permit experiments that can critically test the packaging models. The viral genome translocation mechanism is of general interest, given the parallels between terminases, helicases, and other motor proteins. PMID:18687036

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

    PubMed Central

    Seal, Bruce S.

    2014-01-01

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

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

    PubMed

    Seal, Bruce S

    2013-02-01

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

  3. 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.

  4. Bioengineered lysozyme in combination therapies for Pseudomonas aeruginosa lung infections

    PubMed Central

    Griswold, Karl E; Bement, Jenna L; Teneback, Charlotte C; Scanlon, Thomas C; Wargo, Matthew J; Leclair, Laurie W

    2014-01-01

    There is increasing urgency in the battle against drug-resistant bacterial pathogens, and this public health crisis has created a desperate need for novel antimicrobial agents. Recombinant human lysozyme represents one interesting candidate for treating pulmonary infections, but the wild type enzyme is subject to electrostatic mediated inhibition by anionic biopolymers that accumulate in the infected lung. We have redesigned lysozyme’s electrostatic potential field, creating a genetically engineered variant that is less susceptible to polyanion inhibition, yet retains potent bactericidal activity. A recent publication demonstrated that the engineered enzyme outperforms wild type lysozyme in a murine model of Pseudomonas aeruginosa lung infection. Here, we expand upon our initial studies and consider dual therapies that combine lysozymes with an antimicrobial peptide. Consistent with our earlier results, the charge modified lysozyme combination outperformed its wild type counterpart, yielding more than an order-of-magnitude reduction in bacterial burden following treatment with a single dose. PMID:24637705

  5. 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.

  6. Location of Bromide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  7. Immobilization of lysozyme on polyvinylalcohol films for active packaging applications.

    PubMed

    Conte, A; Buonocore, G G; Bevilacqua, A; Sinigaglia, M; Del Nobile, M A

    2006-04-01

    A new technique for the immobilization of lysozyme onto the surface of polyvinylalcohol films is presented. The active compound was sprayed along with a suitable bonding agent onto the surface of the cross-linked polymeric matrix. Active compound release tests determined the amount of lysozyme immobilized on the film surface. With the use of Micrococcus lysodeikticus, the antimicrobial activity of the films was determined and the results correlated with the amount of immobilized lysozyme. This new technique was effective for immobilizing the enzyme, and the developed films were active against the test microorganism. Results were compared with those obtained with a different immobilizing technique, in which the active compound was bound into the bulk of the polymeric film. As expected, the surface-immobilized lysozyme films have a higher antimicrobial activity than bulk-bound films. PMID:16629031

  8. Dynamical changes in hydration water accompanying lysozyme thermal denaturation

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  9. Structural characterization of the PliG lysozyme inhibitor family.

    PubMed

    Leysen, Seppe; Vanderkelen, Lise; Van Asten, Kathleen; Vanheuverzwijn, Steven; Theuwis, Veronique; Michiels, Chris W; Strelkov, Sergei V

    2012-10-01

    Several Gram-negative bacteria protect themselves against the lytic action of host lysozymes by producing specific proteinaceous inhibitors. So far, four different families of lysozyme inhibitors have been identified including Ivy (Inhibitor of vertebrate lysozyme), MliC/PliC (Membrane associated/periplasmic inhibitor of C-type lysozyme), PliI and PliG (periplasmic inhibitors of I- and G-type lysozymes, respectively). Here we provide the first crystallographic description of the PliG family. Crystal structures were obtained for the PliG homologues from Escherichia coli, Salmonella enterica serotype Typhimurium and Aeromonas hydrophila. These structures show that the fold of the PliG family is very distinct from that of all other families of lysozyme inhibitors. Small-angle X-ray scattering studies reveal that PliG is monomeric in solution as opposed to the dimeric PliC and PliI. The PliG family shares a highly conserved SG(x)xY sequence motif with the MliC/PliC and PliI families where it was shown to reside on a loop that blocks the active site of lysozyme leading to inhibition. Surprisingly, we found that in PliG this motif is not well exposed and not involved in the inhibitory action. Instead, we could identify a distinct cluster of surface residues that are conserved across the PliG family and are essential for efficient G-type lysozyme inhibition, as evidenced by mutagenesis studies. PMID:22634186

  10. An intrinsically shielded hydrogel for the adsorptive recovery of lysozyme.

    PubMed

    Wang, Lu; Zhang, Rongsheng; Eisenthal, Robert; Hubble, John

    2006-07-01

    The present paper addresses the selective recovery of lysozyme from egg white using CM-dextran (carboxymethyldextran)-based hydrogels containing Cibacron Blue as an affinity ligand and co-immobilized BSA intended to act as a shielding agent to reduce non-specific adsorption. Initial studies using pure lysozyme were conducted that indicated that the adsorption capacity increased with ligand density and that adsorption was well described by a Langmuir-type isotherm. The inclusion of BSA as a putative shielding agent did not decrease the adsorption capacity for lysozyme in single-adsorbate experiments. To assess the effectiveness of the shielding strategy, subsequent experiments were conducted with both defined lysozyme/ovalbumin mixtures and hen's-egg white. From these studies, the optimal operating conditions for lysozyme recovery have been determined. These include: optimal initial egg-white concentration [a 10% (v/v) solution of native egg white in the chosen buffer], affinity-ligand density (1.86 mM) and ligand-to-shielding-agent ratio (4:1). The purity of lysozyme obtained from egg white was improved from 69% with a non-shielded hydrogel to 94% with an intrinsically shielded hydrogel. Finally, the possibility of using a protein, rather than dextran-backbone-based, hydrogel was investigated. It was found that BSA could take the place of CM-dextran as the gel backbone in a simplified synthesis, producing a gel which also proved effective for lysozyme recovery with a 30% lysozyme in egg-white solution purified to approx. 92% in a single adsorption-desorption cycle. PMID:16674314

  11. 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.

  12. Enterococcus faecalis Constitutes an Unusual Bacterial Model in Lysozyme Resistance?

    PubMed Central

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

    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 and EF_1843. Protein products of these two genes share significant homology with Staphylococcus aureus peptidoglycan O-acetyltransferase (OatA) and Streptococcus pneumoniae N-acetylglucosamine deacetylase (PgdA), respectively. In order to determine whether EF_0783 and EF_1843 are involved in lysozyme resistance, we constructed their corresponding mutants and a double mutant. The ?EF_0783 mutant and ?EF_0783 ?EF_1843 double mutant were shown to be more sensitive to lysozyme than the parental E. faecalis JH2-2 strain and ?EF_1843 mutant were. However, compared to other bacteria, such as Listeria monocytogenes or S. pneumoniae, the tolerance of ?EF_0783 and ?EF_0783 ?EF_1843 mutants towards lysozyme remains very high. Peptidoglycan structure analysis showed that EF_0783 modifies the peptidoglycan by O acetylation of N-acetyl muramic acid, while the EF_1843 deletion has no obvious effect on peptidoglycan structure under the same conditions. Moreover, the EF_0783 and EF_1843 deletions seem to significantly affect the ability of E. faecalis to survive within murine macrophages. In all, while EF_0783 is currently involved in the lysozyme resistance of E. faecalis, peptidoglycan O acetylation and de-N-acetylation are not the main mechanisms conferring high levels of lysozyme resistance to E. faecalis. PMID:17785473

  13. Predicting Tensile Stretchability of Trimmed AA6111-T4 Sheets

    SciTech Connect

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

    2014-02-15

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

  14. 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.

  15. Preparation of endotoxin-free bacteriophages.

    PubMed

    Boratyński, Janusz; Syper, Danuta; Weber-Dabrowska, Beata; Łusiak-Szelachowska, Marzanna; Poźniak, 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

  16. 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 ...

  17. 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 UVvis absorption spectroscopy. PMID:24684375

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

    PubMed

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

    2014-04-23

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

  19. Resistance of rumen bacteria murein to bovine gastric lysozyme

    PubMed Central

    Domnguez-Bello, Mara G; Pacheco, M Andrena; Ruiz, Marie C; Michelangeli, Fabin; Leippe, Matthias; de Pedro, Miguel A

    2004-01-01

    Background Lysozymes, enzymes mostly associated with defence against bacterial infections, are mureinolytic. Ruminants have evolved a gastric c type lysozyme as a digestive enzyme, and profit from digestion of foregut bacteria, after most dietary components, including protein, have been fermented in the rumen. In this work we characterized the biological activities of bovine gastric secretions against membranes, purified murein and bacteria. Results Bovine gastric extract (BGE) was active against both G+ and G- bacteria, but the effect against Gram- bacteria was not due to the lysozyme, since purified BGL had only activity against Gram+ bacteria. We were unable to find small pore forming peptides in the BGE, and found that the inhibition of Gram negative bacteria by BGE was due to an artefact caused by acetate. We report for first time the activity of bovine gastric lysozyme (BG lysozyme) against pure bacterial cultures, and the specific resistance of some rumen Gram positive strains to BGL. Conclusions Some Gram+ rumen bacteria showed resistance to abomasum lysozyme. We discuss the implications of this finding in the light of possible practical applications of such a stable antimicrobial peptide. PMID:15137912

  20. Arthrobacter globiformis and its bacteriophage in soil

    NASA Technical Reports Server (NTRS)

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

    1974-01-01

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

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

    PubMed

    Zhang, Haifeng; Fu, Gang; Zhang, Dawei

    2014-10-01

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

  2. Structures of monoclinic lysozyme iodide at 1.6 A and of triclinic lysozyme nitrate at 1.1 A.

    PubMed

    Steinrauf, L K

    1998-09-01

    Hen egg-white lysozyme is one of the most thoroughly studied of enzymes and has been the subject of study by many methods, including X-ray crystallography. The present work extends the X-ray crystallography to higher resolution, includes the positions of the anions, and examines the contacts of the neighbors in greater detail. Data were collected at room temperature on a Rigaku R-axis area detector with rotating-anode X-ray generator to 1.6 A resolution for monoclinic lysozyme iodide at pH 4.0, to 1.8 A for monoclinic lysozyme iodide at pH 8.0, and to 1.1 A resolution for triclinic lysozyme nitrate at pH 4.5. The structures have been refined by SHELX93 with the expected number of anion sites being accounted for. Two regions of the protein have been found to be variable: residues 65-75 and 99-104. Except for 65-75 and 99-104, lysozyme is a very stable molecule with the crystal forms being held together by the electrostatic contacts of the anions and by layers of water molecules. The anion positions can be described as paired half sites, each half being contributed by a different lysozyme molecule. The many different crystal forms of lysozyme may be due to different combinations of the many such half sites on the surface. A hypothesis is presented for lysozyme in the different crystal forms and which may be extended to behavior in solution. Suggestions for future crystallographic research are proposed, involving anions of different shape and charge. PMID:9757091

  3. Taking bacteriophage therapy seriously: a moral argument.

    PubMed

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

    2014-01-01

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

  4. Taking Bacteriophage Therapy Seriously: A Moral Argument

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    2009-12-20

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

  6. Bacteriophage endolysins as novel antimicrobials.

    PubMed

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

    2012-10-01

    Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology. PMID:23030422

  7. 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

  8. Bacteriophage endolysins as novel antimicrobials

    PubMed Central

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

    2013-01-01

    Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology. PMID:23030422

  9. Tradeoffs in bacteriophage life histories

    PubMed Central

    Keen, Eric C

    2014-01-01

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

  10. Host receptors for bacteriophage adsorption.

    PubMed

    Bertozzi Silva, Juliano; Storms, Zachary; Sauvageau, Dominic

    2016-02-01

    The adsorption of bacteriophages (phages) onto host cells is, in all but a few rare cases, a sine qua non condition for the onset of the infection process. Understanding the mechanisms involved and the factors affecting it is, thus, crucial for the investigation of host-phage interactions. This review provides a survey of the phage host receptors involved in recognition and adsorption and their interactions during attachment. Comprehension of the whole infection process, starting with the adsorption step, can enable and accelerate our understanding of phage ecology and the development of phage-based technologies. To assist in this effort, we have established an open-access resource-the Phage Receptor Database (PhReD)-to serve as a repository for information on known and newly identified phage receptors. PMID:26755501

  11. Selective antibacterial properties of lysozyme for oral microorganisms.

    PubMed

    Iacono, V J; MacKay, B J; DiRienzo, S; Pollock, J J

    1980-08-01

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

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

    SciTech Connect

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

    2006-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Ares, P.; Garcia-Doval, C.; Llaur, A.; Gmez-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.

  14. Droplet hydrodynamics during lysozyme protein crystallization.

    PubMed

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

    2012-11-01

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

  15. Sequence of the T4 recombination gene, uvsX, and its comparison with that of the recA gene of Escherichia coli.

    PubMed Central

    Fujisawa, H; Yonesaki, T; Minagawa, T

    1985-01-01

    We have determined the nucleotide sequence of the uvsX gene of bacteriophage T4 which is involved in DNA recombination and damage repair, and whose product catalyzes in vitro reactions related to recombination process in analogous manners to E. coli recA gene product. The coding region consisted of 1170 nucleotides directing the synthesis of a polypeptide of 390 amino acids in length with a calculated molecular weight of 43,760. Amino acid composition, the sequence of seven NH2-terminal amino acids and molecular weight of the protein deduced from the nucleotide sequence were consistent with the data from the analysis of the purified uvsX protein. The nucleotide sequence and the deduced amino acid sequence were compared with those of the recA gene. Although a significant homology was not found in the nucleotide sequences, the amino acid sequences included 23% of identical and 15% of conservatively substituted residues. Images PMID:2932679

  16. 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

  17. Bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection.

    PubMed

    Khawaldeh, A; Morales, S; Dillon, B; Alavidze, Z; Ginn, A N; Thomas, L; Chapman, S J; Dublanchet, A; Smithyman, A; Iredell, J R

    2011-11-01

    We describe the success of adjunctive bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection in the context of bilateral ureteric stents and bladder ulceration, after repeated failure of antibiotics alone. No bacteriophage-resistant bacteria arose, and the kinetics of bacteriophage and bacteria in urine suggest self-sustaining and self-limiting infection. PMID:21737541

  18. A three-way junction aptasensor for lysozyme detection.

    PubMed

    Xia, Yunfeng; Gan, Siwen; Xu, Qinghao; Qiu, Xiaowen; Gao, Peiyi; Huang, Shasheng

    2013-01-15

    A well-designed three-way junction (TWJ) aptasensor for lysozyme detection was developed based on target-binding-induced conformational change of aptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA is partially hybridized with an anti-lysozyme aptamer to form a folded structure where there is a coaxial stacking of two helices and the third one at an acute angle. In addition, the fabrication of the sensor was achieved via the single-step method, which offered a good condition for sensing. In the absence of lysozyme, electron transfer (eT), through the coaxial two helices called "conductive path", is allowed between Fc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blocks eT, leading to diminished redox signal. This aptasensor with an instinct signal attenuation factor shows a high sensitivity to lysozyme, and the response data is fitted by nonlinear least-squares to Hill equation. Detection limit is 0.2nM with a dynamic range extending to 100nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to be more specific for detection and simpler for regeneration procedure. PMID:22921948

  19. 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.

  20. Surfactant Copolymers Prevent Aggregation of Heat Denatured Lysozyme

    PubMed Central

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

    2007-01-01

    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 90C in the presence and absence of poloxamers with various molecular weights (8.414.3 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

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

    PubMed Central

    Goodrich-Blair, H; Shub, D A

    1994-01-01

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

  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 Wrttemberg 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. Deformation behavior of aluminum alloy 6111-T4

    NASA Astrophysics Data System (ADS)

    Tseng, Carol

    2000-10-01

    Although aluminum alloys have found increasing usage in the automotive industry, their lower tensile elongations as compared with the low carbon steels they replace has raised concern about their lower formability. Lower formability imposes design and economic constraints on the automakers. The cause behind this lower elongation is the primary focus of this research. The specific alloy studied is 6111-T4 (Al-0.76Si-0.61Mg-0.82Cu in w/o), which is used in automobile outer body panels. In order to determine the factors that are limiting the elongation, it is critical to understand the deformation behavior of this alloy. To investigate the deformation behavior of this alloy, uniaxial tensile tests were performed at various temperatures (300K, 77K and 4.2K), strain rates (10-4, 5 x 10-4 , 10-3, 10-2, 10 -1/s) and specimen geometries. The work hardening and deformation behavior were examined both qualitatively and quantitatively. Ex-situ and in-situ observations were made on the tensile samples by using videography and optical microscopy. Several important findings resulted from this study. First, oscillations in the work hardening are due to the formation and propagation of deformation islands and deformation bands. Deformation islands are areas of localized deformation that occur in a cluster of grains. Second, the microstructural feature dominating the formation and propagation of the islands are the clustering of similarly oriented grains and the clustering of large sized grains. Third, the sharp drop in work hardening near the diffuse necking criterion for the 300K, 10-4 is test samples is due to the inhomogeneous deformation arising from these clusters. Finally, diffuse and local necks form before the theoretical predictions. The inhomogeneous microstructures causing the deformation islands and bands to form and propagate, thus leading to strain localization and eventual premature failure.

  4. SecReT4: a web-based bacterial type IV secretion system resource.

    PubMed

    Bi, Dexi; Liu, Linmeng; Tai, Cui; Deng, Zixin; Rajakumar, Kumar; Ou, Hong-Yu

    2013-01-01

    SecReT4 (http://db-mml.sjtu.edu.cn/SecReT4/) is an integrated database providing comprehensive information of type IV secretion systems (T4SSs) in bacteria. T4SSs are versatile assemblages that promote genetic exchange and/or effector translocation with consequent impacts on pathogenesis and genome plasticity. T4SSs have been implicated in conjugation, DNA uptake and release and effector translocation. The effectors injected into eukaryotic target cells can lead to alteration of host cellular processes during infection. SecReT4 offers a unique, highly organized, readily exploreable archive of known and putative T4SSs and cognate effectors in bacteria. It currently contains details of 10 752 core components mapping to 808 T4SSs and 1884 T4SS effectors found in representatives of 289 bacterial species, as well as a collection of more than 900 directly related references. A broad range of similarity search, sequence alignment, phylogenetic, primer design and other functional analysis tools are readily accessible via SecReT4. We propose that SecReT4 will facilitate efficient investigation of large numbers of these systems, recognition of diverse patterns of sequence-, gene- and/or functional conservation and an improved understanding of the biological roles and significance of these versatile molecular machines. SecReT4 will be regularly updated to ensure its ongoing maximum utility to the research community. PMID:23193298

  5. Modeling Tetragonal Lysozyme Crystal Growth Rates

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  6. In vitro synthesis of lysozyme by human and mouse tissues and leucocytes.

    PubMed Central

    McClelland, D B; van Furth, R

    1975-01-01

    Previous studies have shown that lysozyme can be detected in many body fluids, in extracts of tissues, and also in granulocytes, monocytes and macrophages. However, the sites of synthesis of lysozyme have not been defined. In the present report, the synthesis of lysozyme by tissues, and by defined cell populations cultured in vitro has been studied by detecting the incorporation of 14-C-labelled amino acids into lysozyme. This method detects only lysozyme newly synthesized during the incubation of the specimen and therefore shows unequivocally which tissues and cell types are capable of lysozyme synthesis. The validity of the method has been shown by parallel studies using an independent method to detect lysozyme production in vitro. In studies in humans and mice, lysozyme synthesis has been demonstrated in the mucosa of the respiratory and gastrointestinal tracts, and in lymphoid organs. In studies of defined cell populations, monocytes and macrophages (mononuclear phagocytes) have been shown to synthesize lysozyme. Granulocytes from peripheral blood contain lysozyme but do not synthesize it, and lymphocytes neither contain nor synthesize lysozyme. The present findings provide further evidence that lysozyme has an important role in the defence of the host against micro-organisms, and the findings suggest that lysozyme may reach its target by several routes. At an intracellular level it is delivered from lysosomes into the phagocytic granules of granulocytes and macrophages. Local synthesis of the mucous membranes contributes lysozyme to secretions. Synthesis and secretion by mononuclear phagocytes which reach a tissue in response to an inflammatory stimulus contribute lysozyme to the exudate, and the release of lysozyme from breakdown of granulocytes has the same effect. Images FIG. 1 FIG. 2 FIG. 3 FIG. 5 FIG. 6 FIG. 7 PMID:1093971

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

    PubMed

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

    2015-12-01

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

  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. Lysozyme catalysis: kinetics of the hydrolysis of cell wall oligosaccharides.

    PubMed

    Boekelheide, K; Patt, S L; Weisz, G; Baldo, J H; Sykes, B D

    1979-06-01

    The cleavage of cell wall tetrasaccharide, the beta(1 leads to 4)-linked dimer of the basic repeating disaccharide N-acetyl-D-glucosamine-beta(1 leads to 4)-N-acetyl-D-muramic acid, by lysozyme has been studied at various concentrations of lysozyme and over long time ranges. A theoretical analysis of the kinetic results indicates that direct hydrolysis of the tetrasaccharide by binding in subsities CDEF of the active site of lysozyme is significant at long times relative to the transglycosylation pathway. The binding constant for tetrasaccharide in CDEF is shown to be 10(3) times larger than that determined on the basis of an analysis of kinetic data over a more restricted range of times and concentrations. PMID:476521

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

    PubMed

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

    2015-06-17

    A study of lysozyme adsorption below a behenic acid membrane and at the solid-liquid interface between aqueous lysozyme solution and a silicon wafer in the presence of sodium chloride is presented. The salt concentration was varied between 1 mmol L(-1) and 1000 mmol L(-1). X-ray reflectivity data show a clear dependence of the protein adsorption on the salt concentration. Increasing salt concentrations result in a decreased protein adsorption at the interface until a complete suppression at high concentrations is reached. This effect can be attributed to a reduced attractive electrostatic interaction between the positively charged proteins and negatively charged surfaces by charge screening. The measurements at the solid-liquid interfaces show a transition from unoriented order of lysozyme in the adsorbed film to an oriented order with the short protein axis perpendicular to the solid-liquid interface with rising salt concentration. PMID:25992483

  11. Salt induced reduction of lysozyme adsorption at charged interfaces

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

  12. 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.

  13. 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

  14. Bacteriophage typing of Listeria species.

    PubMed Central

    Loessner, M J; Busse, M

    1990-01-01

    A bacteriophage typing scheme for differentiating Listeria isolates from dairy products and various other foodstuffs was developed. Sixteen selected phages isolated from both environmental sources and lysogenic strains were used for typing and, according to their lytic spectra, divided into four groups. Thus far, 41 distinct patterns of lysis were seen when this set was used in typing 57 defined reference strains, representing all five confirmed species and 16 serotypes in addition to 454 Listeria isolates of primarily foodborne origin. Overall, typability was 84.5%; i.e., a strain was lysed by at least one phage at 100x routine test dilution. Strains belonging to serovar 3 were mostly resistant to lysis by the phages employed. The results were highly reproducible, as determined in retyping trials several weeks later. Some phages isolated from environmental sources showed a wider lytic spectrum than did those isolated from lysogenic strains. In accordance with this, the phages were found in different clusters within a computer-generated linkage map. Species specificity and serovar specificity of the lytic reaction were not found. None of the phages was able to lyse strains of Listeria grayi, Listeria murrayi or Jonesia denitrificans. This phage typing system may provide important information for a means of recognizing and eliminating sources of contamination by Listeria spp. within dairy plant equipment. PMID:2116763

  15. T4-thyroid storm after CT-scan with iodinated contrast medium.

    PubMed

    Shimura, H; Takazawa, K; Endo, T; Tawata, M; Onaya, T

    1990-01-01

    A 62-year-old woman had thyroid storm 5 h after a CT examination (contrast enhancement with 65% meglumine amidotrizoate, Angiografin). At the time of admission to our hospital, serum T4 and serum free T4 levels were markedly elevated (29 micrograms/dl and 9.6 ng/dl, respectively); serum T3 and serum free T3 levels were within normal limits (144 ng/dl and 5.9 pg/ml, respectively). These findings suggest that thyroid storm can occur with excess T4 only (T4-thyroid storm), and that T4-thyroid storm can be caused by administration of iodinated contrast medium. PMID:2319111

  16. Insights into the Structure-Function Relationships of Pneumococcal Cell Wall Lysozymes, LytC and Cpl-1*S?

    PubMed Central

    Monterroso, Begoa; Siz, Jos Luis; Garca, Pedro; Garca, Jos Luis; Menndez, Margarita

    2008-01-01

    The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 0.5 sites/monomer) behave as equivalent (Kd = 2.7 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile. PMID:18667432

  17. Insights into the structure-function relationships of pneumococcal cell wall lysozymes, LytC and Cpl-1.

    PubMed

    Monterroso, Begoa; Siz, Jos Luis; Garca, Pedro; Garca, Jos Luis; Menndez, Margarita

    2008-10-17

    The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 degrees C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 degrees C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 +/- 0.5 sites/monomer) behave as equivalent (Kd = 2.7 +/- 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 degrees C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 degrees C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile. PMID:18667432

  18. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    PubMed Central

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

    2012-01-01

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

  19. Characterization of the genome of the Rhodococcus rhodochrous bacteriophage NJL.

    PubMed

    Sunairi, M; Watanabe, T; Oda, H; Murooka, H; Nakajima, M

    1993-01-01

    Temperate bacteriophage NJL of Rhodococcus rhodochrous has a 49-kb linear double-stranded DNA with cohesive ends (cos). NJL DNA has unique target sites for HindIII and SspI, two target sites each for NheI and ScaI, and no cleavage site for AxyI, DraI, EcoRI, SacI, and SphI. The single-stranded regions of cos ends were ligated to each other with T4 DNA ligase, removed with mung bean nuclease, or blunted with the Klenow large fragment of DNA polymerase I; then the sequences of the cos ends were determined. Comparison of these sequences revealed that the single-stranded regions are complementary and 18 bases long and protrude at the 3' ends; they have the following sequences: 5'-TTGGCACCGTGGGAGGAG-3' and 3'-AACCGTGGCAC CCTCCTC-5'. A physical map of NJL was constructed by a cos mapping method based on information about the structure of the cohesive ends and multiple digestions with restriction endonucleases. PMID:8439171

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

    PubMed Central

    Chibeu, Andrew; Lingohr, Erika J.; Masson, Luke; Manges, Amee; Harel, Jose; 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 212 h of incubation. PMID:22590682

  1. 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.

  2. 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.

  3. Sinorhizobium meliloti Phage ΦM9 Defines a New Group of T4 Superfamily Phages with Unusual Genomic Features but a Common T=16 Capsid

    PubMed Central

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

    2015-01-01

    ABSTRACT Relatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of the Sinorhizobium meliloti-infecting T4 superfamily phage ΦM9. This phage and its close relative Rhizobium phage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-like S. meliloti phages of the ΦM12 group. Structurally, ΦM9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the ΦM9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. ΦM9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, ΦM9 and S. meliloti phage ΦM12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide. IMPORTANCE Despite recent advances in the phylogenetic and structural characterization of bacteriophages, only a small number of phages of plant-symbiotic nitrogen-fixing soil bacteria have been studied at the molecular level. The effects of phage predation upon beneficial bacteria that promote plant growth remain poorly characterized. First steps in understanding these soil bacterium-phage dynamics are genetic, molecular, and structural characterizations of these groups of phages. The T4 superfamily phages are among the most complex phages; they have large genomes packaged within an icosahedral head and a long, contractile tail through which the DNA is delivered to host cells. This phylogenetic and structural study of S. meliloti-infecting T4 superfamily phage ΦM9 provides new insight into the diversity of this family. The comparison of structure-related genes in both ΦM9 and S. meliloti-infecting T4 superfamily phage ΦM12, which comes from a completely different lineage of these phages, allows the identification of host infection-related factors. PMID:26311868

  4. Bacteriophage control of foodborne bacteriat.

    PubMed

    Greer, G Gordon

    2005-05-01

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

  5. Bacteriophage lysis: mechanism and regulation.

    PubMed Central

    Young, R

    1992-01-01

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

  6. 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, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (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 λ 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 λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (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 λ 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

  7. The bacteriophage DNA packaging machine.

    PubMed

    Feiss, Michael; Rao, Venigalla B

    2012-01-01

    Large dsDNA bacteriophages and herpesviruses encode a powerful ATP-driven DNA-translocating machine that encapsidates a viral genome into a preformed capsid shell or prohead. The key components of the packaging machine are the packaging enzyme (terminase, motor) and the portal protein that forms the unique DNA entrance vertex of prohead. The terminase complex, comprised of a recognition subunit (small terminase) and an endonuclease/translocase subunit (large terminase), cuts viral genome concatemers. The terminase-viral DNA complex docks on the portal vertex, assembling a motor complex containing five large terminase subunits. The pentameric motor processively translocates DNA until the head shell is full with one viral genome. The motor cuts the DNA again and dissociates from the full head, allowing head-finishing proteins to assemble on the portal, sealing the portal, and constructing a platform for tail attachment. A body of evidence from molecular genetics and biochemical, structural, and biophysical approaches suggests that ATP hydrolysis-driven conformational changes in the packaging motor (large terminase) power DNA motion. Various parts of the motor subunit, such as the ATPase, arginine finger, transmission domain, hinge, and DNA groove, work in concert to translocate about 2 bp of DNA per ATP hydrolyzed. Powerful single-molecule approaches are providing precise delineation of steps during each translocation event in a motor that has a speed as high as a millisecond/step. The phage packaging machine has emerged as an excellent model for understanding the molecular machines, given the mechanistic parallels between terminases, helicases, and numerous motor proteins. PMID:22297528

  8. Goose-Type Lysozyme Inhibitor (PliG) Enhances Survival of Escherichia coli in Goose Egg Albumen ?

    PubMed Central

    Vanderkelen, Lise; Van Herreweghe, Joris M.; Callewaert, Lien; Michiels, Chris W.

    2011-01-01

    The goose-type lysozyme inhibitor PliG enhances the survival of Escherichia coli in goose but not in chicken egg white, which contains goose- and chicken-type lysozymes, respectively. These results indicate that both the type of host lysozyme and the type of bacterial lysozyme inhibitor may affect bacterium-host interactions. PMID:21602367

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

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

    ERIC Educational Resources Information Center

    Boeschen, John; Alderton, Gordon

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

  11. The folding-unfolding transition of equine lysozyme

    NASA Astrophysics Data System (ADS)

    Haezebrouck, P.; Van Dael, H.

    1993-03-01

    A detailed study of the chemical and thermal unfolding transition of equine lysozyme in the presence and in the absence of Ca 2+ gives evidence for a two-step unfolding process. The pretransition can be related to the transfer of exposed Trp groups to the protein interior.

  12. Dye functionalized cryogel columns for reversible lysozyme adsorption.

    PubMed

    Uygun, Murat; Akduman, Begm; Uygun, Deniz Akta?; Akgl, Sinan; Denizli, Adil

    2015-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  14. 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.

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

    PubMed

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

    2015-10-10

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

  16. Lysozyme-loaded, electrospun chitosan-based nanofiber mats for wound healing.

    PubMed

    Charernsriwilaiwat, Natthan; Opanasopit, Praneet; Rojanarata, Theerasak; Ngawhirunpat, Tanasait

    2012-05-10

    In this study, a blend mixture of chitosan-ethylenediaminetetraacetic acid (CS 2 wt%-EDTA) at a weight ratio of 30/70 and polyvinyl alcohol (PVA) solution (10 wt%) was electrospun to produce fibrous mats with lysozyme (10, 20 and 30 wt%) used for wound healing. The morphology and diameter of the electrospun fiber mats with and without lysozyme were analyzed by scanning electron microscopy (SEM). The amount of lysozyme loaded in the nanofiber mats was measured by HPLC. The cell lysis activity of the lysozyme was investigated with Micrococcus lysodeikticus cells as a substrate. The wound healing activity was performed in vivo using male Wistar rats. The SEM images of all lysozyme-loaded fibers show a smooth fiber without beads with an average diameter of 143-209 nm. The amount of lysozyme loaded in the nanofiber mats was slightly decreased when the initial concentration of lysozyme was increased. The rapid lysozyme release from the nanofiber mats was obtained and is dependent on the lysozyme-loading amount. In animal wound healing, lysozyme loaded CS-EDTA nanofiber mats accelerated the rate of wound healing when compared to the controls (gauze). In conclusion, our experiments demonstrated that biomaterials composed of lysozyme loaded CS-EDTA nanofibers have a potential for wound healing. PMID:22353400

  17. 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

  18. 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.

  19. The chemistry of lysozyme and its use as a food preservative and a pharmaceutical.

    PubMed

    Proctor, V A; Cunningham, F E

    1988-01-01

    The chemistry and use of lysozyme as a food preservative and a pharmaceutical are reviewed. Lysozyme inhibits the growth of deleterious organisms, thus prolonging shelf life. Chemicals used to improve the preservative effect of lysozyme and those that inhibit the enzyme are discussed, along with the stability of lysozyme in various chemical environments. Lysozyme has been used to preserve fresh fruits and vegetables, tofu bean curd, seafoods, meats and sausages, potato salad, cooked burdock with soy sauce, and varieties of semihard cheeses such as Edam, Gouda, and some Italian cheeses. Lysozyme added to infant-feeding formulas makes them more closely resemble human milk. Lysozyme has been used clinically in the treatment of periodontitis, administered in chewing gum, and implemented to prevent tooth decay. It has also been administered to patients suffering from cancer for its analgesic effect and has been used as a potentiating agent in antibiotic therapy. PMID:3280250

  20. 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

  1. Bacteriophage ecology in commercial sauerkraut fermentations

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Dispersing biofilms with engineered enzymatic bacteriophage

    PubMed Central

    Lu, Timothy K.; Collins, James J.

    2007-01-01

    Synthetic biology involves the engineering of biological organisms by using modular and generalizable designs with the ultimate goal of developing useful solutions to real-world problems. One such problem involves bacterial biofilms, which are crucial in the pathogenesis of many clinically important infections and are difficult to eradicate because they exhibit resistance to antimicrobial treatments and removal by host immune systems. To address this issue, we engineered bacteriophage to express a biofilm-degrading enzyme during infection to simultaneously attack the bacterial cells in the biofilm and the biofilm matrix, which is composed of extracellular polymeric substances. We show that the efficacy of biofilm removal by this two-pronged enzymatic bacteriophage strategy is significantly greater than that of nonenzymatic bacteriophage treatment. Our engineered enzymatic phage substantially reduced bacterial biofilm cell counts by ?4.5 orders of magnitude (?99.997% removal), which was about two orders of magnitude better than that of nonenzymatic phage. This work demonstrates the feasibility and benefits of using engineered enzymatic bacteriophage to reduce bacterial biofilms and the applicability of synthetic biology to an important medical and industrial problem. PMID:17592147

  3. 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

  4. Subpopulations of CD4+ (T4+) cells in homosexual/bisexual men with persistent generalized lymphadenopathy.

    PubMed Central

    Gupta, S

    1987-01-01

    Using monoclonal antibodies (anti-2H4 and anti-4B4), CD4+ (T4+) T cells are further subdivided into two major subpopulations of inducers of help (T4+4B4+) and inducers of suppression (T4+2H4+). To determine whether human immunodeficiency virus (HIV) infection results in the depletion of a subset of CD4+ cells, peripheral blood mononuclear cells from patients with persistent generalized lymphadenopathy (PGL) were quantified for the subpopulation of CD4+ T cells, using anti-2H4 and anti-4B4 monoclonal antibodies and fluorescence-activated cell sorter analyser. The proportions and numbers of both T4+2H4+ and T4+4B4+ cells were significantly decreased in PGL as compared to healthy controls. This study demonstrates that HIV infection results in the depletion of both subsets of T4 cells to an almost similar extent. PMID:2958183

  5. Does Warming a Lysozyme Solution Cook Ones Data?

    NASA Technical Reports Server (NTRS)

    Pusey, Marc; Burke, Michael; Judge, Russell

    2000-01-01

    Chicken egg white lysozyme has a well characterized thermally driven phase transition. Between pH 4.0 and 5.2, the transition temperature, as defined by the point where the tetragonal and orthorhombic solubility are equal, is a function of the pH, salt (precipitant) type and concentration, and most likely of the buffer concentration as well. This phase transition can be carried out with protein solution alone, prior to the initiation of the crystallization process. We have now measured the kinetics of this process and investigated its reversibility. An aliquot of a stock protein solution is held at a given temperature, and at periodic intervals used to set up batch crystallization experiments. The batch solutions were incubated at 20 C until macroscopic crystals were obtained, at which point the number of crystals in each well were counted. The transition effects increased with temperature, slowly falling off at 30 C with a half time (time to approx. 1/2 the t = 0 number of crystals) of approx. 5 hours, and an estimated half time of approx. 0.5 hours at 43 C. Further, the process was not reversible by simple cooling. After holding a lysozyme solution at 37 C (prior to addition of precipitant) for 16 hours, then cooling and holding it at 4 C, no return to the pre-warmed nucleation kinetics are observed after at least 4 weeks. Thus every thermal excursion above the phase transition point results in a further decrease in the nucleation rate of that solution, the extent being a function of the time and temperature. Orthorhombic lysozyme crystals apparently do not undergo the flow-induced growth cessation of tetragonal lysozyme crystals. We have previously shown that putting the protein in the orthorhombic form does not affect the averaged face growth kinetics, only nucleation, for tetragonal crystals. We may be able to use this differential behavior to elucidate how flow affects tile lysozyme crystal growth process.

  6. The primary structure of cassowary (Casuarius casuarius) goose type lysozyme.

    PubMed

    Thammasirirak, Sompong; Torikata, Takao; Takami, Kazutoshi; Murata, Koichi; Araki, Tomohiro

    2002-01-01

    The complete amino acid sequence of cassowary (Casuarius casuarius) goose type lysozyme was analyzed by direct protein sequencing of peptides obtained by cleavage with trypsin, V8 protease, chymotrypsin, lysyl endopeptidase, and cyanogen bromide. The N-terminal residue of the enzyme was deduced to be a pyroglutamate group by analysis with a LC/MS/MS system equipped with the oMALDI ionization source, and then confirmed by a glutamate aminopeptidase enzyme. The blocked N-terminal is the first reported in this enzyme group. The positions of disulfide bonds in this enzyme were chemically identified as Cys4-Cys60 and Cys18-Cys29. Cassowary lysozyme was proved to consist of 185 amino acid residues and had a molecular mass of 20408 Da calculated from the amino acid sequence. The amino acid sequence of cassowary lysozyme compared to that of reported G-type lysozymes had identities of 90%, 83%, and 81%, for ostrich, goose, and black swan lysozymes, respectively. The amino acid substitutions at PyroGlu1, Glu19, Gly40, Asp82, Thr102, Thr156, and Asn167 were newly detected in this enzyme group. The substituted amino acids that might contribute to substrate binding were found at subsite B (Asn122Ser, Phe123Met). The amino acid sequences that formed three alpha-helices and three beta-sheets were completely conserved. The disulfide bond locations and catalytic amino acid were also strictly conserved. The conservation of the three alpha-helices structures and the location of disulfide bonds were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group. PMID:11866097

  7. Role of Lysozyme Inhibitors in the Virulence of Avian Pathogenic Escherichia coli

    PubMed Central

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

    2012-01-01

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

  8. Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics.

    PubMed

    Singh, Manika Indrajit; Jain, Vikas

    2016-01-26

    Sliding clamp proteins are circular dimers or trimers that encircle DNA and serve as processivity factors during DNA replication. Their presence in all the three domains of life and in bacteriophages clearly indicates their high level of significance. T4 gp45, besides functioning as the DNA polymerase processivity factor, also moonlights as the late promoter transcription determinant. Here we report a detailed biophysical analysis of gp45. The chemical denaturation of gp45 probed by circular dichroism spectroscopy, tryptophan fluorescence anisotropy, and blue-native polyacrylamide gel electrophoresis suggests that the protein follows a three-state denaturation profile and displays an intermediate molten globule-like state. The three-state transition was found to be the result of the sequential unfolding of the two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), of gp45. The experiments involving Trp fluorescence quenching by acrylamide demonstrate that the CTD undergoes substantial changes in conformation during formation of the intermediate state. Further biophysical dissection of the individual domain reveals contrasting properties of the two domains. The NTD unfolds at low urea concentrations and is also susceptible to protease cleavage, whereas the CTD resists urea-mediated denaturation and is not amenable to protease digestion even at higher urea concentrations. These experiments allow us to conclude that the two domains of gp45 differ in their dynamics. While the CTD shows stability and rigidity, we find that the NTD is unstable and flexible. We believe that the asymmetric characteristics of the two domains and the interface they form hold significance in gp45 structure and function. PMID:26735934

  9. 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...

  10. 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...

  11. 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...

  12. [Modulation of T4 to T3 conversion. Comparative aspects (author's transl)].

    PubMed

    Leloup, I; Buscaglia, M; de Luze, A

    1981-01-01

    The peripheral conversion of T4 to T3 has been demonstrated in primitive Vertebrates, larval Lampreys (which do not have thyroid follicles but synthesize T3 and T4 in the endostyle) and in more evolved Vertebrates. Up to now the presence of rT3 has never been demonstrated in fish, its eventual role in Amphibia remains to be established but seems important in chick embryo. The conversion of T4 to T3 is stimulated during amphibian metamorphosis and in salt water fish, concurrently with stimulation of thyroidal secretion. In fish, prolactin stimulates 5-deiodination of T4 directly without the involvement of the pituitary thyroid axis. PMID:7340699

  13. Biogeography of bacteriophages at four hydrothermal vent sites in the Antarctic based on g23 sequence diversity.

    PubMed

    Millard, Andrew D; Pearce, David; Zwirglmaier, Katrin

    2016-04-01

    In this study, which was carried out within the ChEsSO consortium project (Chemosynthetically driven ecosystems south of the Polar Front), we sampled two hydrothermal vent sites on the East Scotia Ridge, Scotia Sea, one in the Kemp Caldera, South Sandwich Arc and one in the Bransfield Strait, north-west of the Antarctic Peninsula, which exhibit strong differences in their chemical characteristics. We compared a subset of their bacteriophage population by Sanger- and 454-sequencing of g23, which codes for the major capsid protein of T4likeviruses. We found that the sites differ vastly in their bacteriophage diversity, which reflects the differences in the chemical conditions and therefore putatively the differences in microbial hosts living at these sites. Comparing phage diversity in the vent samples to other aquatic samples, the vent samples formed a distinct separate cluster, which also included the non-vent control samples that were taken several hundred meters above the vent chimneys. This indicates that the influence of the vents on the microbial population and therefore also the bacteriophage population extends much further than anticipated. PMID:26903011

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

    PubMed

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

    2015-12-01

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

  15. Tear lipocalin, lysozyme and lactoferrin concentrations in postmenopausal women

    PubMed Central

    Careba, I; Chiva, A; Totir, M; Ungureanu, E; Gradinaru, Sinziana

    2015-01-01

    Rationale: Among the most frequently encountered pathologies examined by the ophthalmologist is dry eye syndrome (DE), which can be discovered particularly in the elderly. The initial diagnosis of DE is of high importance, but also challenging. This is because the biochemical changes in the tear film often develop before any detectable signs. Objective: In this study, the possible relationship between ocular symptomatology, tear volume and tear break-up time (TBUT) and lipocalin, lactoferrin and lysozyme concentrations in the tear film were explored in a group of symptomatic dry-eyed postmenopausal (PM) women compared to age-matched controls. Patients and methods: Sixty-six healthy PM females with ages of at least 50 years were grouped in two homogeneous lots (by age, post-menopause, co-morbidities) of 33 females each, one lot presenting mild or moderate dry eye syndrome (DE) and one asymptomatic non-dry eye (NDE), based on their feedback to the Ocular Surface Disease Index (OSDI) questionnaire and noninvasive TBUT and Schirmer test results. Tears were collected via capillary tubes and an eye wash method. Tear lysozyme, lactoferrin and lipocalin concentrations were determined via electrophoresis. Results: OSDI responses revealed 3 mild DE, 30 moderate DE and 33 NDE. The OSDI total score and sub scores for the DE group were significantly greater than for the NDE group (p < 0.001). The mild and moderate DE group exhibited significantly shorter TBUTs compared to NDE (p < 0.001). No difference in tear lysozyme or lipocalin concentrations was found between DE and NDE groups, irrespective of the tear collection method, but a significant difference was found in lactoferrin concentration (p<0.001). No significant correlations were found between symptoms or signs of DE compared to either lipocalin, lysozyme or lactoferrin concentrations. Discussion: In a PM population, lipocalin and lysozyme are invariable, irrespective of the presence and severity of DE symptoms. However, lactoferrin shows a significant decrease. This is a comprehensive study of lipocalin, lactoferrin and lysozyme in dry-eyed PM women and our results suggested that lactoferrin could be used as a biomarker of DE in postmenopausal women. Abbreviations: PM = postmenopausal; DE = dry eye disease; NDE = non-dry eye; ELISA = Enzyme-linked immunosorbent assay PMID:26366224

  16. Campylobacter jejuni Group III Phage CP81 Contains Many T4-Like Genes without Belonging to the T4-Type Phage Group: Implications for the Evolution of T4 Phages?

    PubMed Central

    Hammerl, Jens A.; Jckel, Claudia; Reetz, Jochen; Beck, Sebastian; Alter, Thomas; Lurz, Rudi; Barretto, Caroline; Brssow, Harald; Hertwig, Stefan

    2011-01-01

    CP81 is a virulent Campylobacter group III phage whose linear genome comprises 132,454 bp. At the nucleotide level, CP81 differs from other phages. However, a number of its structural and replication/recombination proteins revealed a relationship to the group II Campylobacter phages CP220/CPt10 and to T4-type phages. Unlike the T4-related phages, the CP81 genome does not contain conserved replication and virion modules. Instead, the respective genes are scattered throughout the phage genome. Moreover, most genes for metabolic enzymes of CP220/CPt10 are lacking in CP81. On the other hand, the CP81 genome contains nine similar genes for homing endonucleases which may be involved in the attrition of the conserved gene order for the virion core genes of T4-type phages. The phage apparently possesses an unusual modification of C or G bases. Efficient cleavage of its DNA was only achieved with restriction enzymes recognizing pure A/T sites. Uncommonly, phenol extraction leads to a significant loss of CP81 DNA from the aqueous layer, a property not yet described for other phages belonging to the T4 superfamily. PMID:21697478

  17. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances § 721.1880 Borate(1-), tris(acetato-.kappa.O)hydro-, sodium... substance identified as borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

  18. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances 721.1880 Borate(1-), tris(acetato-.kappa.O)hydro-, sodium... substance identified as borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

  19. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances 721.1880 Borate(1-), tris(acetato-.kappa.O)hydro-, sodium... substance identified as borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

  20. 40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances 721.1880 Borate(1-), tris(acetato-.kappa.O)hydro-, sodium... substance identified as borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances 721.1880 Borate(1-), tris(acetato-.kappa.O)hydro-, sodium... substance identified as borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...

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

    PubMed

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

    2014-02-01

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

  3. 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

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

    PubMed Central

    Daudn, Mara I.; Martn-Benito, Jaime; Snchez-Ferrero, Juan C.; Pulido-Cid, Mar; Valpuesta, Jos M.; Carrascosa, Jos L.

    2013-01-01

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

  5. One-step assay for the quantification of T4 DNA ligase.

    PubMed

    Franke, Steffi; Kreisig, Thomas; Buettner, Karin; Zuchner, Thole

    2015-02-01

    As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5%) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays. PMID:25503935

  6. Genomic characteristics and environmental distributions of the uncultivated Far-T4 phages

    PubMed Central

    Roux, Simon; Enault, François; Ravet, Viviane; Pereira, Olivier; Sullivan, Matthew B.

    2015-01-01

    Viral metagenomics (viromics) is a tremendous tool to reveal viral taxonomic and functional diversity across ecosystems ranging from the human gut to the world's oceans. As with microbes however, there appear vast swaths of “dark matter” yet to be documented for viruses, even among relatively well-studied viral types. Here, we use viromics to explore the “Far-T4 phages” sequence space, a neighbor clade from the well-studied T4-like phages that was first detected through PCR study in seawater and subsequently identified in freshwater lakes through 454-sequenced viromes. To advance the description of these viruses beyond this single marker gene, we explore Far-T4 genome fragments assembled from two deeply-sequenced freshwater viromes. Single gene phylogenetic trees confirm that the Far-T4 phages are divergent from the T4-like phages, genome fragments reveal largely collinear genome organizations, and both data led to the delineation of five Far-T4 clades. Three-dimensional models of major capsid proteins are consistent with a T4-like structure, and highlight a highly conserved core flanked by variable insertions. Finally, we contextualize these now better characterized Far-T4 phages by re-analyzing 196 previously published viromes. These suggest that Far-T4 are common in freshwater and seawater as only four of 82 aquatic viromes lacked Far-T4-like sequences. Variability in representation across the five newly identified clades suggests clade-specific niche differentiation may be occurring across the different biomes, though the underlying mechanism remains unidentified. While complete genome assembly from complex communities and the lack of host linkage information still bottleneck virus discovery through viromes, these findings exemplify the power of metagenomics approaches to assess the diversity, evolutionary history, and genomic characteristics of novel uncultivated phages. PMID:25852662

  7. 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.

  8. 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.

  9. 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.

  10. Heterogeneous Preferential Solvation of Water and Trifluoroethanol in Homologous Lysozymes

    PubMed Central

    2015-01-01

    Cytoplasmic osmolytes can significantly alter the thermodynamic and kinetic properties of proteins relative to those under dilute solution conditions. Spectroscopic experiments of lysozymes in cosolvents indicate that such changes may arise from the heterogeneous, site-specific hydrophobic interactions between protein surface residues and individual solvent molecules. In pursuit of an accurate and predictive model for explaining biomolecular interactions, we study the averaged structural characteristics of mixed solvents with homologous lysozyme solutes using all-atom molecular dynamics. By observing the time-averaged densities of different aqueous solutions of trifluoroethanol, we deduce trends in the heterogeneous solvent interactions over each protein’s surface, and investigate how the homology of protein structure does not necessarily translate to similarities in solvent structure and composition—even when observing identical side chains. PMID:24823618

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

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

  12. Application of bacteriophages in sensor development.

    PubMed

    Peltomaa, Riikka; López-Perolio, Irene; Benito-Peña, Elena; Barderas, Rodrigo; Moreno-Bondi, María Cruz

    2016-03-01

    Bacteriophage-based bioassays are a promising alternative to traditional antibody-based immunoassays. Bacteriophages, shortened to phages, can be easily conjugated or genetically engineered. Phages are robust, ubiquitous in nature, and harmless to humans. Notably, phages do not usually require inoculation and killing of animals; and thus, the production of phages is simple and economical. In recent years, phage-based biosensors have been developed featuring excellent robustness, sensitivity, and selectivity in combination with the ease of integration into transduction devices. This review provides a critical overview of phage-based bioassays and biosensors developed in the last few years using different interrogation methods such as colorimetric, enzymatic, fluorescence, surface plasmon resonance, quartz crystal microbalance, magnetoelastic, Raman, or electrochemical techniques. PMID:26472318

  13. Modeling tailed bacteriophage adsorption: Insight into mechanisms.

    PubMed

    Storms, Zachary J; Sauvageau, Dominic

    2015-11-01

    The process of a bacteriophage attaching to its host cell is a combination of physical diffusion, biochemical surface interactions, and reaction-induced conformational changes in receptor proteins. Local variations in the physico-chemical properties of the medium, the phage?s mode of action, and the physiology of the host cell also all influence adsorption kinetics. These characteristics can affect a specific phage?s binding capabilities and the susceptibility of the host cell to phage attack. Despite the complexity of this process, describing adsorption kinetics of a population of bacteriophages binding to a culture of cells has been accomplished with relatively simple equations governed by the laws of mass-action. Many permutations and modifications to the basic set of reactions have been suggested through the years. While no single solution emerges as a universal answer, this review provides the fundamentals of current phage adsorption modeling and will guide researchers in the selection of valid, appropriate models. PMID:26331682

  14. How long can bacteriophage ? change its mind?

    PubMed Central

    Semsey, Szabolcs; Campion, Christopher; Mohamed, Abdu; Svenningsen, Sine Lo

    2015-01-01

    A key event in the lifecycle of a temperate bacteriophage is the choice between lysis and lysogeny upon infection of a susceptible host cell. In a recent paper, we showed that a prolonged period exists after the decision to lysogenize, during which bacteriophage ? can abandon the initial decision, and instead develop lytically, as a response to the accumulation of the late lytic regulatory protein Q. Here, we present evidence that expression of Q does not induce replication of ? DNA, suggesting that the DNA to be packaged into the resulting phage progeny was already present at the time of the initial decision to lysogenize. We summarize our findings in a working model of the key determinants of the duration of the post-decision period during which it is possible for the infected cell to switch from the lysogeny decision to successful lytic development. PMID:26459429

  15. Novel alternatives to antibiotics: bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides.

    PubMed

    Parisien, A; Allain, B; Zhang, J; Mandeville, R; Lan, C Q

    2008-01-01

    Extensive research has been conducted on the development of three groups of naturally occurring antimicrobials as novel alternatives to antibiotics: bacteriophages (phages), bacterial cell wall hydrolases (BCWH), and antimicrobial peptides (AMP). Phage therapies are highly efficient, highly specific, and relatively cost-effective. However, precautions have to be taken in the selection of phage candidates for therapeutic applications as some phages may encode toxins and others may, when integrated into host bacterial genome and converted to prophages in a lysogenic cycle, lead to bacterial immunity and altered virulence. BCWH are divided into three groups: lysozymes, autolysins, and virolysins. Among them, virolysins are the most promising candidates as they are highly specific and have the capability to rapidly lyse antibiotic-resistant bacteria on a generally species-specific basis. Finally, AMP are a family of natural proteins produced by eukaryotic and prokaryotic organisms or encoded by phages. AMP are of vast diversity in term of size, structure, mode of action, and specificity and have a high potential for clinical therapeutic applications. PMID:18171378

  16. Role of disulfide bonds in goose-type lysozyme.

    PubMed

    Kawamura, Shunsuke; Ohkuma, Mari; Chijiiwa, Yuki; Kohno, Daiki; Nakagawa, Hiroyuki; Hirakawa, Hideki; Kuhara, Satoru; Torikata, Takao

    2008-06-01

    The role of the two disulfide bonds (Cys4-Cys60 and Cys18-Cys29) in the activity and stability of goose-type (G-type) lysozyme was investigated using ostrich egg-white lysozyme as a model. Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala. No remarkable differences in secondary structure or catalytic activity were observed between the wild-type and mutant proteins. However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild-type. The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state. The effects of deleting each disulfide bond on protein stability were found to be approximately additive, indicating that the individual disulfide bonds contribute to the stability of G-type lysozyme in an independent manner. Under reducing conditions, the thermal stability of the wild-type was decreased to a level nearly equivalent to that of a Cys-free mutant (C4S/C18S/C29S/C60S) in which all Cys residues were replaced by Ser. Moreover, the optimum temperature of the catalytic activity for the Cys-free mutant was downshifted by about 20 degrees C as compared with that of the wild-type. These results indicate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G-type lysozyme. PMID:18430025

  17. 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.

  18. 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.

  19. 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.

  20. THE PREPARATION OF RELATIVELY PURE BACTERIOPHAGE.

    PubMed

    Krueger, A P; Tamada, H T

    1929-11-20

    The method described above, based on the electrophoretic migration of bacteriophage particles into an agar gel and their subsequent re-suspension in a suitable medium, has the following advantages: It is simple and can be readily carried out on a comparatively large scale by merely inserting additional units between the same electrode cups. It requires but one extraction and the resulting phage suspension is strongly lytic, an average sample being capable of completely lysing susceptible bacteria at a dilution of 10(-16). The suspension contains no proteins demonstrable by the biuret, alcohol, xanthoproteic, Millon or Hopkins-Cole reactions and yields but 0.044 mg. N/cc. directly attributable to the phage. Each corpuscle contains no more nitrogen than a single molecule of protein. In addition the method is applicable to determinations of the electric charge carried by biologically active substances of small dimensions, e.g., phage, toxins, and perhaps some viruses. It offers as well a possible means of purification of these substances. The purified bacteriophage obtained by such a procedure or similar ones is relatively unstable. Work now in progress indicates that it does not possess nearly the resistance to chemical agents, drying, etc., that non-purified phage displays. It is suggested that experiments designed to test the therapeutic value of bacteriophage be conducted, when possible, with purified suspensions thereby avoiding any possibility of obscure non-specific reactions due to other constituents of the lysates. PMID:19872513

  1. THE PREPARATION OF RELATIVELY PURE BACTERIOPHAGE

    PubMed Central

    Krueger, A. P.; Tamada, H. T.

    1929-01-01

    The method described above, based on the electrophoretic migration of bacteriophage particles into an agar gel and their subsequent re-suspension in a suitable medium, has the following advantages: It is simple and can be readily carried out on a comparatively large scale by merely inserting additional units between the same electrode cups. It requires but one extraction and the resulting phage suspension is strongly lytic, an average sample being capable of completely lysing susceptible bacteria at a dilution of 1016. The suspension contains no proteins demonstrable by the biuret, alcohol, xanthoproteic, Millon or Hopkins-Cole reactions and yields but 0.044 mg. N/cc. directly attributable to the phage. Each corpuscle contains no more nitrogen than a single molecule of protein. In addition the method is applicable to determinations of the electric charge carried by biologically active substances of small dimensions, e.g., phage, toxins, and perhaps some viruses. It offers as well a possible means of purification of these substances. The purified bacteriophage obtained by such a procedure or similar ones is relatively unstable. Work now in progress indicates that it does not possess nearly the resistance to chemical agents, drying, etc., that non-purified phage displays. It is suggested that experiments designed to test the therapeutic value of bacteriophage be conducted, when possible, with purified suspensions thereby avoiding any possibility of obscure non-specific reactions due to other constituents of the lysates. PMID:19872513

  2. Widespread genetic exchange among terrestrial bacteriophages.

    PubMed

    Silander, Olin K; Weinreich, Daniel M; Wright, Kevin M; O'Keefe, Kara J; Rang, Camilla U; Turner, Paul E; Chao, Lin

    2005-12-27

    Bacteriophages are the most numerous entities in the biosphere. Despite this numerical dominance, the genetic structure of bacteriophage populations is poorly understood. Here, we present a biogeography study involving 25 previously undescribed bacteriophages from the Cystoviridae clade, a group characterized by a dsRNA genome divided into three segments. Previous laboratory manipulation has shown that, when multiple Cystoviruses infect a single host cell, they undergo (i) rare intrasegment recombination events and (ii) frequent genetic reassortment between segments. Analyzing linkage disequilibrium (LD) within segments, we find no significant evidence of intrasegment recombination in wild populations, consistent with (i). An extensive analysis of LD between segments supports frequent reassortment, on a time scale similar to the genomic mutation rate. The absence of LD within this group of phages is consistent with expectations for a completely sexual population, despite the fact that some segments have >50% nucleotide divergence at 4-fold degenerate sites. This extraordinary rate of genetic exchange between highly unrelated individuals is unprecedented in any taxa. We discuss our results in light of the biological species concept applied to viruses. PMID:16365305

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

    PubMed

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

    2016-01-01

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

  4. Comparative insight into surfactants mediated amyloidogenesis of lysozyme.

    PubMed

    Chaturvedi, Sumit K; Khan, Javed M; Siddiqi, Mohammad K; Alam, Parvez; Khan, Rizwan H

    2016-02-01

    Electrostatic and hydrophobic interactions have an important role in the protein aggregation. In this study, we have investigated the effect of charge and hydrophobicity of oppositely charged surfactants i.e., anionic (AOT and SDS) and cationic (CTAB and DTAB) on hen egg white lysozyme at pH 9.0 and 13.0, respectively. We have employed various methods such as turbidity measurements, Rayleigh light scattering, ThT, Congo red and ANS dye binding assays, far-UV CD, atomic force microscopy, transmission electron and fluorescence microscopy. At lower molar ratio, both anionic and cationic surfactants promote amyloid fibril formation in lysozyme at pH 9.0 and 13.0, respectively. The aggregation was proportionally increased with respect to protein concentration and hydrophobicity of surfactant. The morphology of aggregates at both the pH was fibrillar in structure, as visualized by dye binding and microscopic imaging techniques. Initially, the interaction between surfactants and lysozyme was electrostatic and then hydrophobic as investigated by ITC. This study demonstrates the crucial role of charge and hydrophobicity during amyloid fibril formation. PMID:26616452

  5. Magnetic molecularly imprinted nanoparticles for recognition of lysozyme.

    PubMed

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

    2010-10-15

    Molecular imprinting is an attractive technique for preparing mimics of natural, biological receptors. Nevertheless, the imprinting of macromolecule remains a challenge due to their bulkiness and sensitivity to denaturation. In this work, we presented a method for preparing multifunctional lysozyme-imprinted nanoparticles (magnetic susceptibility, molecular recognition and environmental response). The magnetic susceptibility was imparted through the successful encapsulation of Fe3O4 nanoparticles. Selective lysozyme recognition depended on molecularly imprinted film. Moreover, it was also a hydrophilic stimuli-responsive polymer, which could undergo a reversible change of imprinted cavity in response to a small change in the environmental conditions. Thus, magnetic molecularly imprinted nanoparticles had high adsorption capacity (0.11 mg mg(-1)), controlled selectivity and direct magnetic separation (22.1 emicro g(-1)) in crude samples. After preconcentration and purification with magnetic MIPs nanoparticles, a sensitive chemiluminescence method was developed for determination of lysozyme in human serum samples. The results indicated that the spiked recoveries were changed from 92.5 to 113.7%, and the RSD was lower than 11.8%. PMID:20829022

  6. Water in hydrated orthorhombic lysozyme crystal: Insight from atomistic simulations

    NASA Astrophysics Data System (ADS)

    Hu, Zhongqiao; Jiang, Jianwen; Sandler, Stanley I.

    2008-08-01

    Biologically important water in orthorhombic lysozyme crystal is investigated using atomistic simulations. A distinct hydration shell surrounding lysozyme molecules is found from the number distribution of water molecules. While the number of water molecules in the hydration shell increases, the percentage decreases as the hydration level rises. Adsorption of water in the lysozyme crystal shows type-IV behavior. At low hydration levels, water molecules primarily intercalate the minor pores and cavity in the crystal due to the strong affinity between protein and water. At high hydration levels, the major pores are filled with liquidlike water as capillary condensation occurs. A type-H4 hysteresis loop is observed in the adsorption and desorption isotherms. The locations of the water molecules identified from simulation match fairly well with the experimentally determined crystallographic hydration sites. As observed in experiment, water exhibits anomalous subdiffusion because of the geometric restrictions and interactions of protein. With increasing hydration level, this anomaly is reduced and the diffusion of water tends to progressively approach normal Brownian diffusion. The flexibility of protein framework slightly enhances water mobility, but this enhancement decreases with increasing hydration level.

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

    NASA Technical Reports Server (NTRS)

    Nadarajah, Arunan

    1996-01-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2014-04-01

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

  10. Bacteriophage P70: Unique Morphology and Unrelatedness to Other Listeria Bacteriophages

    PubMed Central

    Schmuki, Martina M.; Erne, Doris; Loessner, Martin J.

    2012-01-01

    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

  11. Frequency of ultraviolet radiation-induced mutation at the hprt locus in repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4.

    PubMed

    Kusewitt, D F; Budge, C L; Anderson, M M; Ryan, S L; Ley, R D

    1993-09-01

    The frequency of spontaneous and ultraviolet radiation (UVR)-induced mutation at the hprt locus was determined in control and denV-transfected, repair-proficient murine fibroblasts. Control cells removed an average of 25% of pyrimidine dimers induced by exposure to 150 J/m2 UVR from an FS40 sunlamp within 24 h; under the same conditions of induction and repair, denV-transfected cells removed an average of 71% of pyrimidine dimers. Control cells were somewhat more resistant than denV-transfected cells to killing by UVR. The average frequency of spontaneous mutation at the hprt locus for control and denV-transfected cells was 3 and 15 6-thioguanine (6-TG)-resistant colonies per 10(6) surviving cells, respectively; there was no statistically significant difference between control and denV-transfected cells. However, after exposure to 75 or 150 J/m2 UVR, denV-transfected cells had a significantly lower frequency of mutation to 6-TG resistance. After exposure to a fluence of 75 J/m2, the average frequency of UVR-induced mutation at the hprt locus was 166 mutant colonies per 10(6) surviving cells for control cells and 92 mutant colonies for denV-transfected cells; after 150 J/m2, control cells had 205 6-TG-resistant colonies per 10(6) cells, while denV-transfected cells had 61 mutant colonies. These results demonstrate that UVR-induced pyrimidine dimers are mutagenic photoproducts in mammalian cells. PMID:8234481

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 c...

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

    PubMed

    Yu, Shuang; Luo, Ai-Qin; Biswal, Dipti; Hilt, J Zach; Puleo, David A

    2010-11-15

    Molecular imprinting is a method to fabricate a polymeric material (molecularly imprinted polymer or MIP) capable of selectively recognizing template molecules. Molecular imprinting of small molecules has been studied widely. Less common, however, is the imprinting of biological macromolecules, including proteins, among which lysozyme is an important molecule in the food, pharmaceutical, and diagnostic sciences. In this study, lysozyme MIP was fabricated in two steps. First, lysozyme, PEG600DMA, and methacrylic acid were used as the template molecule, cross-linking monomer, and the functional monomer, respectively, in a UV free-radical polymerization process to synthesize a polymeric gel. Second, lysozyme was removed by enzymatic digestion. Non-imprinted polymer (NIP) was synthesized without lysozyme addition. To evaluate the preferential binding capability of MIP, lysozyme, RNase A, or a 50:50 mixture of lysozyme and RNase A was added to MIP and NIP and then released by digestion. It was found that when more lysozyme was added to the reaction mixture, the quantity of protein released from the polymer increased, reflecting more potential binding sites. Tests of MIP with a competitive binding mixture of lysozyme and RNase A showed the MIP preferentially bound a greater amount of lysozyme, up to 20 times more than RNase A. NIP bound only small amounts of both proteins and did not show a preference for binding either lysozyme or RNase A. These results demonstrate that lysozyme was successfully imprinted into the MIP by UV free-radical polymerization, and the fabricated MIP was able to preferentially bind its template protein. PMID:21035657

  14. 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

  15. Antibacterial activity of hen egg white lysozyme against Listeria monocytogenes Scott A in foods.

    PubMed Central

    Hughey, V L; Wilger, P A; Johnson, E A

    1989-01-01

    Egg white lysozyme killed or prevented growth of Listeria monocytogenes Scott A in several foods. Lysozyme was more active in vegetables than in animal-derived foods that we tested. For maximum activity in certain foods, EDTA was required in addition to lysozyme. Lysozyme with EDTA effectively killed inoculated populations of 10(4) L. monocytogenes per g in fresh corn, fresh green beans, shredded cabbage, shredded lettuce, and carrots during storage at 5 degrees C. Control incubations without lysozyme supported growth of L. monocytogenes to 10(6) to 10(7)/g. Lysozyme had less activity in animal-derived foods, including fresh pork sausage (bratwurst) and Camembert cheese. In bratwurst, lysozyme with EDTA prevented L. monocytogenes from growing for 2 to 3 weeks but did not kill significant numbers of cells and did not prevent eventual growth. The control sausages not containing lysozyme supported rapid and heavy growth, which indicated that lysozyme was bacteriostatic for 2 to 3 weeks in fresh pork sausage. We also prepared Camembert cheese containing 10(4) L. monocytogenes cells per g and investigated the changes during ripening in cheeses supplemented with lysozyme and EDTA. Cheeses with lysozyme by itself or together with EDTA reduced the L. monocytogenes population by approximately 10-fold over the first 3 to 4 weeks of ripening. In the same period, the control cheese wheels without added lysozyme with and without chelator slowly started to grown and eventually reached 10(6) to 10(7) CFU/g after 55 days of ripening.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2494938

  16. 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

  17. 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

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

    PubMed

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

    2013-07-01

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

  19. Plasma Membrane Proteomics Identifies Biomarkers Associated with MMSET Overexpression in T(4;14) Multiple Myeloma

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-06-21

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

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

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.

    1991-01-01

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

  2. Engineering Escherichia coli for soluble expression and single step purification of active human lysozyme.

    PubMed

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

    2013-03-10

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

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

    PubMed

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

    2014-01-01

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

  4. 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...

  5. Expression of a bioactive bacteriophage endolysin in Nicotiana benthamiana plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The emergence and spread of antibiotic-resistant pathogens has led to an increased interest in alternative antimicrobial treatments, such as bacteriophage, bacteriophage-encoded peptidoglycan hydrolases (endolysins) and antimicrobial peptides. In our study, the antimicrobial activity of the CP933 en...

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

    PubMed

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

    2015-01-01

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

  7. 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...

  8. 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.

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

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

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

  10. Lipoprotein LprI of Mycobacterium tuberculosis Acts as a Lysozyme Inhibitor.

    PubMed

    Sethi, Deepti; Mahajan, Sahil; Singh, Chaahat; Lama, Amrita; Hade, Mangesh Dattu; Gupta, Pawan; Dikshit, Kanak L

    2016-02-01

    Mycobacterium tuberculosis executes numerous defense strategies for the successful establishment of infection under a diverse array of challenges inside the host. One such strategy that has been delineated in this study is the abrogation of lytic activity of lysozyme by a novel glycosylated and surface-localized lipoprotein, LprI, which is exclusively present in M. tuberculosis complex. The lprI gene co-transcribes with the glbN gene (encoding hemoglobin (HbN)) and both are synchronously up-regulated in M. tuberculosis during macrophage infection. Recombinant LprI, expressed in Escherichia coli, exhibited strong binding (Kd ? 2 nm) with lysozyme and abrogated its lytic activity completely, thereby conferring protection to fluorescein-labeled Micrococcus lysodeikticus from lysozyme-mediated hydrolysis. Expression of the lprI gene in Mycobacterium smegmatis (8-10-fold) protected its growth from lysozyme inhibition in vitro and enhanced its phagocytosis and survival during intracellular infection of peritoneal and monocyte-derived macrophages, known to secrete lysozyme, and in the presence of exogenously added lysozyme in secondary cell lines where lysozyme levels are low. In contrast, the presence of HbN enhanced phagocytosis and intracellular survival of M. smegmatis only in the absence of lysozyme but not under lysozyme stress. Interestingly, co-expression of the glbN-lprI gene pair elevated the invasion and survival of M. smegmatis 2-3-fold in secondary cell lines in the presence of lysozyme in comparison with isogenic cells expressing these genes individually. Thus, specific advantage against macrophage-generated lysozyme, conferred by the combination of LprI-HbN during invasion of M. tuberculosis, may have vital implications on the pathogenesis of tuberculosis. PMID:26589796

  11. Structure of T4moF, the Toluene 4-Monooxygenase Ferredoxin Oxidoreductase.

    PubMed

    Acheson, Justin F; Moseson, Hannah; Fox, Brian G

    2015-09-29

    The 1.6 Å crystal structure of toluene 4-monooxygenase reductase T4moF is reported. The structure includes ferredoxin, flavin, and NADH binding domains. The position of the ferredoxin domain relative to the other two domains represents a new configuration for the iron-sulfur flavoprotein family. Close contacts between the C8 methyl group of FAD and [2Fe-2S] ligand Cys36-O represent a plausible pathway for electron transfer between the redox cofactors. Energy-minimized docking of NADH and calculation of hingelike motions between domains suggest how simple coordinated shifts of residues at the C-terminus of the enzyme could expose the N5 position of FAD for productive interaction with the nicotinamide ring. The domain configuration revealed by the T4moF structure provides an excellent steric and electrostatic match to the obligate electron acceptor, Rieske-type [2Fe-2S] ferredoxin T4moC. Protein-protein docking and energy minimization of the T4moFC complex indicate that T4moF [2Fe-2S] ligand Cys41 and T4moC [2Fe-2S] ligand His67, along with other electrostatic interactions between the protein partners, form the functional electron transfer interface. PMID:26309236

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

    PubMed

    Araki, T; Kuramoto, M; Torikata, T

    1990-09-01

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

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

    PubMed

    Brzozowska, Ewa; Śmietana, 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. 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

  15. 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.

  16. DMSO-induced denaturation of hen egg white lysozyme.

    PubMed

    Voets, Ilja K; Cruz, Willemberg A; Moitzi, Christian; Lindner, Peter; Aras, Elizabeth P G; Schurtenberger, Peter

    2010-09-16

    We report on the size, shape, structure, and interactions of lysozyme in the ternary system lysozyme/DMSO/water at low protein concentrations. Three structural regimes have been identified, which we term the "folded" (0 < ?(DMSO) < 0.7), "unfolded" (0.7 ? ?(DMSO) < 0.9), and "partially collapsed" (0.9 ? ?(DMSO) < 1.0) regime. Lysozyme resides in a folded conformation with an average radius of gyration of 1.3 0.1 nm for ?(DMSO) < 0.7 and unfolds (average R(g) of 2.4 0.1 nm) above ?(DMSO) > 0.7. This drastic change in the protein's size coincides with a loss of the characteristic tertiary structure. It is preceded by a compaction of the local environment of the tryptophan residues and accompanied by a large increase in the protein's overall flexibility. In terms of secondary structure, there is a gradual loss of ?-helix and concomitant increase of ?-sheet structural elements toward ?(DMSO) = 0.7, while an increase in ?(DMSO) at even higher DMSO volume fractions reduces the presence of both ?-helix and ?-sheet secondary structural elements. Protein-protein interactions remain overall repulsive for all values of ?(DMSO). An attempt is made to relate these structural changes to the three most important physical mechanisms that underlie them: the DMSO/water microstructure is strongly dependent on the DMSO volume fraction, DMSO acts as a strong H-bond acceptor, and DMSO is a bad solvent for the protein backbone and a number of relatively polar side groups, but a good solvent for relatively apolar side groups, such as tryptophan. PMID:20731407

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

    PubMed Central

    Girod, Marion; Enjalbert, Quentin; Brunet, Claire; Antoine, Rodolphe; Lemoine, Jrme; 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

  18. Bacteriophage P22 tail protein gene expression.

    PubMed Central

    Adams, M B; Brown, H R; Casjens, S

    1985-01-01

    We have found that mutations which block bacteriophage P22 head assembly at or before the DNA packaging stage (1-, 2-, 3-, 5-, and 8-) cause up to a 20-fold increase in the amount of tail (gene 9) protein made during infection. This correlation seems strong enough to warrant consideration of a control mechanism in which the failure to package DNA per se causes a large increase in the synthesis of tail protein. Our results indicate that one of the repressors required for maintenance of lysogeny, the mnt gene product, may be partially responsible for this phenomenon. Images PMID:3155554

  19. Bacterial genome remodeling through bacteriophage recombination.

    PubMed

    Menouni, Rachid; Hutinet, Geoffrey; Petit, Marie-Agnès; Ansaldi, Mireille

    2015-01-01

    Bacteriophages co-exist and co-evolve with their hosts in natural environments. Virulent phages lyse infected cells through lytic cycles, whereas temperate phages often remain dormant and can undergo lysogenic or lytic cycles. In their lysogenic state, prophages are actually part of the host genome and replicate passively in rhythm with host division. However, prophages are far from being passive residents: they can modify or bring new properties to their host. In this review, we focus on two important phage-encoded recombination mechanisms, i.e. site-specific recombination and homologous recombination, and how they remodel bacterial genomes. PMID:25790500

  20. 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

  1. Bacteriophage biosensors for antibiotic-resistant bacteria.

    PubMed

    Sorokulova, Irina; Olsen, Eric; Vodyanoy, Vitaly

    2014-03-01

    An increasing number of disease-causing bacteria are resistant to one or more anti-bacterial drugs utilized for therapy. Early and speedy detection of these pathogens is therefore very important. Traditional pathogen detection techniques, that include microbiological and biochemical assays are long and labor-intensive, while antibody or DNA-based methods require substantial sample preparation and purification. Biosensors based on bacteriophages have demonstrated remarkable potential to surmount these restrictions and to offer rapid, efficient and sensitive detection technique for antibiotic-resistant bacteria. PMID:24506300

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

    PubMed Central

    Scheie, Paul

    1973-01-01

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

  3. 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

  4. 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…

  5. Effect of lysozyme or antibiotics on fecal zoonotic pathogens in nursery pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lysozyme is a 1,4--N-acetylmuramidase that has antimicrobial properties. The objective of this study was to determine the effect of lysozyme and antibiotics on zoonotic pathogen shedding in feces in nursery pigs housed without and with an indirect disease challenge. Two replicates of 600 pigs eac...

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

    PubMed

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

    2014-10-01

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

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

    ERIC Educational Resources Information Center

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

    2015-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Solovykh, G N; Nemtseva, N V

    1994-01-01

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

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

    ERIC Educational Resources Information Center

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

    2015-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. The primary structure of a novel goose-type lysozyme from rhea egg white.

    PubMed

    Pooart, Jureerut; Torikata, Takao; Araki, Tomohiro

    2004-01-01

    G-type lysozyme is a hydrolytic enzyme sharing a similar tertiary structure with plant chitinase. To discover the relation of function and structure, we analyzed the primary structure of new G-type lysozyme. The complete 185 amino acid residues of lysozyme from rhea egg white were sequenced using the peptides hydrolyzed by trypsin, V8 protease, and cyanogen bromide. Rhea lysozyme had sequence similarity to ostrich, cassowary, goose, and black swan, with 93%, 90%, 83%, and 82%, respectively. The six substituted positions were newly found at positions 3 (Asn), 9 (Ser), 43 (Arg), 114 (Ile), 127 (Met), and 129 (Arg) when compared with ostrich, cassowary, goose, and black swan lysozymes. The amino acid substitutions of rhea lysozyme at subsite B were the same as ostrich and cassowary lysozymes (Ser122 and Met123). This study was also constructed in a phylogenetic tree of G-type lysozyme that can be classified into at least three groups of this enzyme, namely, group 1; rhea, ostrich, and cassowary, group 2; goose, black swan, and chicken, and group 3; Japanese flounder. The amino acid sequences in assembled three alpha-helices found in this enzyme group (Thammasirirak, S., Torikata, T., Takami, K., Murata, K., and Araki, T., Biosci. Biotechnol. Biochem., 66, 147-156 (2002)) were also highly conserved, so that they were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group. PMID:14745179

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

    PubMed

    Valachova, I; Takac, P; Majtan, J

    2014-12-01

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

  14. 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

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

    SciTech Connect

    Flanagan, M.P.

    1987-01-01

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

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

    PubMed

    Revathi, R; Rameshkumar, A; Sivasudha, T

    2016-01-01

    Binding of lysozyme with AgTiO2 nanoparticles was analyzed by using absorption, fluorescence, time resolved and synchronous fluorescence measurements. In the presence of AgTiO2 nanoparticles, the fluorescence intensity of lysozyme was decreased. Static type of binding was confirmed through lifetime and ground state absorption measurements. From the fluorescence quenching data, the binding constant and the number of binding sites were found to be 1.510(4)M(-1) and 1.03, respectively. From the synchronous fluorescence spectroscopic measurements, tryptophan residue in lysozyme was found to have interaction with the nanoparticles. Further, the influence of AgTiO2 nanoparticles on the binding strength of lysozyme with a drug molecule was analyzed through fluorescence quenching methods. The presence of nanoparticles decreases the binding capability of drug with protein. Overall, the observed results will provide basic insights on the utilization of nanoparticles in drug delivery applications. PMID:26210014

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  18. 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 α-helices and β-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.

  19. Renal Lysozyme Levels in Animals Developing “Sterile Pyelonephritis”

    PubMed Central

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

    1971-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1992-02-01

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