Tests of spool models for DNA packaging in phage lambda.

PubMed

Widom, J; Baldwin, R L

1983-12-25

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine. In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map. In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with 32P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found. These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one phage to the next. These results agree with other recent experiments on lambda DNA packaging by Hall & Schellman (1982a,b), and by Haas et al. (1982). A different experiment is also reported. The psoralen derivative aminomethyltrioxalen (AMT) is allowed to intercalate into lambda phage and then the DNA strands are crosslinked by ultraviolet-irradiation after the rapid phase of AMT intercalation is complete. The DNA is subsequently denatured by glyoxal modification and spread for electron microscopy in a cytochrome c film by the formamide method.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda.

PubMed

Chattopadhyay, S; Hung, S C; Stuart, A C; Palmer, A G; Garcia-Mena, J; Das, A; Gottesman, M E

1995-12-19

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively.

Site-specificity of abnormal excision: the mechanism of formation of a specialized transducing bacteriophage lambda plac5.

PubMed Central

Shpakovski, G V; Berlin, Y A

1984-01-01

Molecular mechanism of the specialized transducing bacteriophage lambda plac5 formation has been studied. Phage-bacterial DNA junctions in lambda plac5 DNA are localized and primary structure of regions of the abnormal excisional recombination leading to the phage formation is elucidated; the crossover region proved to be comparable with the central part of attP and attB sites (the core and the adjacent tetranucleotide) in length and degree of homology. Bacterial insert in lambda plac5 DNA is shown to end immediately after Z-Y spacer, the DNA not containing lacY gene segments. The data obtained led to the conclusion of site-specific (homologous) character of abnormal excision upon formation of lambda transducing bacteriophages. Possible mechanisms of the excision are discussed. Images PMID:6091038

Recombination between bacteriophage lambda and plasmid pBR322 in Escherichia coli.

PubMed Central

Pogue-Geile, K L; Dassarma, S; King, S R; Jaskunas, S R

1980-01-01

Recombinant lambda phages were isolated that resulted from recombination between the lambda genome and plasmid pBR322 in Escherichia coli, even though these deoxyribonucleic acids (DNAs) did not share extensive regions of homology. The characterization of these recombinant DNAs by heteroduplex analysis and restriction endonucleases is described. All but one of the recombinants appeared to have resulted from reciprocal recombination between a site on lambda DNA and a site on the plasmid. In general, there were two classes of recombinants. One class appeared to have resulted from recombination at the phage attachment site that probably resulted from lambda integration into secondary attachment sites on the plasmid. Seven different secondary attachment sites on pBR322 were found. The other class resulted from plasmid integration at other sites that were widely scattered on the lambda genome. For this second class of recombinants, more than one site on the plasmid could recombine with lambda DNA. Thus, the recombination did not appear to be site specific with respect to lambda or the plasmid. Possible mechanisms for generating these recombinants are discussed. Images PMID:6247334

Family values in the age of genomics: comparative analyses of temperate bacteriophage HK022.

PubMed

Weisberg, R A; Gottesmann, M E; Hendrix, R W; Little, J W

1999-01-01

HK022 is a temperate coliphage related to phage lambda. Its chromosome has been completely sequenced, and several aspects of its life cycle have been intensively studied. In the overall arrangement, expression, and function of most of its genes, HK022 broadly resembles lambda and other members of the lambda family. Upon closer view, significant differences emerge. The differences reveal alternative strategies used by related phages to cope with similar problems and illuminate previously unknown regulatory and structural motifs. HK022 prophages protect lysogens from superinfection by producing a sequence-specific RNA binding protein that prematurely terminates nascent transcripts of infecting phage. It uses a novel RNA-based mechanism to antiterminate its own early transcription. The HK022 protein shell is strengthened by a complex pattern of covalent subunit interlinking to form a unitary structure that resembles chain-mail armour. Its integrase and repressor proteins are similar to those of lambda, but the differences provide insights into the evolution of biological specificity and the elements needed for construction of a stable genetic switch.

Absorption Kinetics of Phage Lambda on Its Host Under Shear Flow

NASA Astrophysics Data System (ADS)

Yip, C. W.; Wu, X. L.

2000-03-01

Classical blender experiment by Hershey and Chase played a seminal role in illustrating the infectious process of bacteriophage to its host, and showed unequivocally that DNA is responsible for the transmission of heredity. Subsequent works by others have established that interaction between phage particles and bacterial cells is a diffusion-limited process in that, statistically speaking, each collision results in an irreversible infection. However, such a result is hard to reconcile with the fact that the infection appears to be independent of the density of phage receptors on the bacterial cell membrane. Thus, quantitative experiments showing how a phage finds its receptor and how long does it take would be valuable to this paradoxical view. Simple calculations based on Brownian motion of the phage particles show that the interaction time between the receptor and the phage is given by tau=b^2/(5D), where b is the length of the phage and D is its diffusion coefficient. Using a shear flow apparatus we study absorption kinetics of lambda phage on E. Coli (strain YMEL) under different flow conditions, and the results are compared with a simple diffusion model taking into account the hydrodynamic convection and the interaction time tau.

Evaluation of humoral and cellular immune responses against HSV-1 using genetic immunization by filamentous phage particles: a comparative approach to conventional DNA vaccine.

PubMed

Hashemi, Hamidreza; Bamdad, Taravat; Jamali, Abbas; Pouyanfard, Somayeh; Mohammadi, Masoumeh Gorgian

2010-02-01

Phage display is based on expressing peptides as a fusion to one of the phage coat proteins. To date, many vaccine researches have been conducted to display immunogenic peptides or mimotopes of various pathogens and tumors on the surface of filamentous bacteriophages. In recent years as a new approach to application of phages, recombinant bacteriophage lambda particles were used as DNA delivery vehicles to mammalian cells. In this study, recombinant filamentous phage whole particles were used for vaccination of mice. BALB/c mice were inoculated with filamentous phage particles containing expression cassette of Herpes simplex virus 1 (HSV-1) glycoprotein D that has essential roles in the virus attachment and entry. Both humoral and cellular immune responses were measured in the immunized mice and compared to conventional DNA vaccination. A dose-response relationship was observed in both arms of immune responses induced by recombinant filamentous phage inoculation. The results were similar to those from DNA vaccination. Filamentous phages can be considered as suitable alternative candidate vaccines because of easier and more cost-effective production and purification over plasmid DNA or bacteriophage lambda particles. 2009 Elsevier B.V. All rights reserved.

Organization of genes for transcription and translation in the rif region of the Escherichia coli chromosome. [uv radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, M.; Nomura, M.

1979-01-01

The lambda rif/sup d/18 transducing phage is known to carry several genes for components of transcriptional and translational machineries; these genes are clustered in the rif region at 88 min on the Escherichia coli genetic map. They include a set of genes for rRNA's (rrnB), a gene for spacer tRNA, tRNA/sub 2//sup Glu/(tgtB), one of the two genes for EF-TU (tufB), genes for four ribosomal proteins (rplK, A, J, and L), genes for the ..beta.. and ..beta..' subunits of RNA polymerase (rpoB and rpoC), and genes for three tRNA's (tyrU, gluT, and thrT). An additional tRNA gene (subsequently identified asmore » thrU by Landy and his co-workers) and a gene for a protein (protein U) with unknown functions were found to be carried by lambda rif/sup d/18. We analyzed the organization of these genes by using various deletion and hybrid phages derived from lambda rif/sup d/18 and lambda rif/sup d/12, a phage related to lambda rif/sup d/18. The expression of various genes was examined in uv-irradiated cells infected with these transducing phages. Two main conclusions were obtained. First, the four tRNA genes are not cotranscribed with the genes in rrnB, even though these tRNA genes are located close to the distal end of rrnB. Second, the four ribosomal protein genes are organized into two separate transcriptional units; rplK and A are in one unit and rplJ and L are in the second unit.« less

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.

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

PubMed Central

Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

2012-01-01

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop + oriλ+ sequence. PMID:22590552

Use of the microscreen phage-induction assay to assess the genotoxicity of 14 hazardous industrial wastes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Houk, V.S.; DeMarini, D.M.

1988-01-01

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 pg per ml. Comparisons between the ability of these waste samples to induce prophage and their mutagenicity in the Salmonella reverse mutation assay indicate that the phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, themore » Microscreen assay detected as genotoxic five additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed, as are some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.« less

Study of genetic effects of high energy radiations with different ionizing capacities on extracellular phages.

PubMed

Bresler, S E; Kalinin, V L; Kopylova, Y U; Krivisky, A S; Rybchin, V N; Shelegedin, V N

1975-07-01

The inactivating and mutagenic action of high-energy radiations with different ionizing capacities (gamma-rays, protons, alpha-particles and accelerated ions of 12C and 20Ne) was studied by using coliphages lambda11 and SD as subjects. In particular the role of irradiation conditions (broth suspension, pure buffer, dry samples) and of the host functions recA, exrA and polA was investigated. The dose-response curve of induced mutagenesis was studied by measuring the yield of vir mutants in lambda11 and plaque mutants in SD. The following results were obtained. (1) The inactivation kinetics of phages under the action of gamma-rays and protons was first order to a survival of 10(-7). Heavy ions also showed exponential inactivation kinetics to a survival of 10(-4). At higher doses of 20Ne ion bombardment some deviation from one-hit kinetics was observed. For dry samples of phages the dimensions of targets for all types of radiation were approximately proportional to the molecular weights of phage DNA's. For densely ionizing radiation (heavy ions) the inactivating action was 3-5 times weaker than for gamma-rays and protons. (2) Mutagenesis was observed for all types of radiation, but heavy ions were 1-5-2 times less efficient than gamma-rays. For both phages studied the dose-response curve of mutagenesis was non-linear. The dependence on the dose was near to parabolic for lambda11. For SD a plateau or maximum of mutagenesis was observed for the relative number of mutants at a survival of about 10(-4). (3) Host-cell functions recA and exrA were practically indifferent for survival of gamma-irradiated phage lambda11, but indispensable for mutagenesis. Mutation recAI3 abolished induced vir mutations totally and exrA- reduced them significantly. The absence of the function polA had a considerable influence on phage survival, but no effect on vir mutation yield (if compared at the same survival level). (4) In conditions of indirect action of gamma-rays no vir mutations were induced. This is regarded as evidence that the single-strand breaks formed under indirect action conditions cannot serve as pre-mutational damage in DNA.

Antisense RNA: effect of ribosome binding sites, target location, size, and concentration on the translation of specific mRNA molecules.

PubMed

Daugherty, B L; Hotta, K; Kumar, C; Ahn, Y H; Zhu, J D; Pestka, S

1989-01-01

A series of plasmids were constructed to generate RNA complementary to the beta-galactosidase messenger RNA under control of the phage lambda PL promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda PL promoter and/or the lacZ ribosome binding site in reverse orientation. Fragments of lacZ DNA from the 5' and/or the 3' region were used in these constructions. When these anti-mRNA molecules were produced in Escherichia coli 294, maximal inhibition of beta-galactosidase synthesis occurred when a functional ribosome binding site was present near the 5' end of the anti-mRNA and the anti-mRNA synthesized was complementary to the 5' region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5'-coding sequence. Anti-mRNAs producing maximal inhibition of beta-galactosidase synthesis exhibited an anti-lacZ mRNA:normal lacZ mRNA ratio of 100:1 or higher. Those showing lower levels of inhibition exhibited much lower anti-lacZ mRNA:normal lacZ mRNA ratios. A functional ribosome binding site at the 5'-end was found to decrease the decay rate of the anti-lacZ mRNAs. In addition, the incorporation of a transcription terminator just downstream of the antisense segment provided for more efficient inhibition of lacZ mRNA translation due to synthesis of smaller and more abundant anti-lacZ mRNAs. The optimal constructions produced undetectable levels of beta-galactosidase synthesis.

Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology.

PubMed

Tanouchi, Yu; Covert, Markus W

2017-09-19

During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by targeting over 300 unique sites in the host Escherichia coli genome. Moreover, these integration events can have important phenotypic consequences for the host, including changes in cell motility and increased antibiotic resistance. Importantly, the new technologies that we developed to enable this study-sequencing secondary sites using next-generation sequencing and then selecting relevant lysogens using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based selection-are broadly applicable to other phage-bacterium systems. IMPORTANCE Bacteriophages play an important role in bacterial evolution through lysogeny, where the phage genome is integrated into the host chromosome. While phage integration generally occurs at a specific site in the host chromosome, it is also known to occur at other, so-called secondary sites. In this study, we developed a new experimental technology to comprehensively study secondary integration sites and discovered that phage can integrate into over 300 unique sites in the host genome, resulting in significant genetic diversity in bacteria. We further developed an assay to examine the phenotypic consequence of such diverse integration events and found that phage integration can cause changes in evolutionarily relevant traits such as bacterial motility and increases in antibiotic resistance. Importantly, our method is readily applicable to other phage-bacterium systems. Copyright © 2017 Tanouchi and Covert.

On the Internal Structure of Bacteriophage Lambda

PubMed Central

Kaiser, A. D.

1966-01-01

The structure of bacteriophage lambda has been studied by electron microscopy of negatively stained particles. The phage particles will eject their DNA if they are heated or dialyzed against a chelating agent. The ghost particles, so formed, have a channel running down their tails. Since the channel is not visible in normal particles, the channel may be filled with part of the DNA molecule. Up to 30% of the ghosts contain round objects about half the internal diameter of the head. The round objects, called "cores," have the same buoyant density as the coat protein. The core may be a protein spool about which the phage DNA is wound. PMID:5967429

Salt-Dependent DNA-DNA Spacings in Intact Bacteriophage lambda Reflect Relative Importance of DNA Self-Repulsion and Bending Energies

DOE Office of Scientific and Technical Information (OSTI.GOV)

X Qiu; D Rau; V Parsegian

2011-12-31

Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA d spacings in bacteriophage {lambda} with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5 x 10{sup 3} base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in d spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured d spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In themore » commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about 1 kT/bp, an order of magnitude larger than the bending energies.« less

ArgO145, a Stx2a prophage of a bovine O145:H- STEC strain, is closely related to phages of virulent human strains.

PubMed

Krüger, A; Burgán, J; Friedrich, A W; Rossen, J W A; Lucchesi, P M A

2018-06-01

Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both Stx prophages and Stx. In particular, the toxin subtype Stx2a is associated with high virulence and HUS. Here, we report the genome of ArgO145, an inducible Stx2a prophage identified in a bovine O145:H- strain which produced high levels of Shiga toxin and Stx phage particles. The ArgO145 genome shared lambda phage organization, with recombination, regulation, replication, lysis, and head and tail structural gene regions, although some lambda genes encoding regulatory proteins could not be identified. Remarkably, some Stx2a phages of strains isolated from patients in other countries showed high similarity to ArgO145. Copyright © 2018 Elsevier B.V. All rights reserved.

Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1

PubMed Central

Gödeke, Julia; Paul, Kristina; Lassak, Jürgen; Thormann, Kai M

2011-01-01

Shewanella oneidensis MR-1 is capable of forming highly structured surface-attached communities. By DNase I treatment, we demonstrated that extracellular DNA (eDNA) serves as a structural component in all stages of biofilm formation under static and hydrodynamic conditions. We determined whether eDNA is released through cell lysis mediated by the three prophages LambdaSo, MuSo1 and MuSo2 that are harbored in the genome of S. oneidensis MR-1. Mutant analyses and infection studies revealed that all three prophages may individually lead to cell lysis. However, only LambdaSo and MuSo2 form infectious phage particles. Phage release and cell lysis already occur during early stages of static incubation. A mutant devoid of the prophages was significantly less prone to lysis in pure culture. In addition, the phage-less mutant was severely impaired in biofilm formation through all stages of development, and three-dimensional growth occurred independently of eDNA as a structural component. Thus, we suggest that in S. oneidensis MR-1 prophage-mediated lysis results in the release of crucial biofilm-promoting factors, in particular eDNA. PMID:20962878

Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae.

PubMed

Grose, Julianne H; Casjens, Sherwood R

2014-11-01

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships.

Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae

PubMed Central

Grose, Julianne H.; Casjens, Sherwood R.

2014-01-01

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships. PMID:25240328

A kinetic analysis of DNA ejection from tailed phages revealing the prerequisite activation energy.

PubMed

Raspaud, Eric; Forth, Thomas; São-José, Carlos; Tavares, Paulo; de Frutos, Marta

2007-12-01

All tailed bacteriophages follow the same general scheme of infection: they bind to their specific host receptor and then transfer their genome into the bacterium. DNA translocation is thought to be initiated by the strong pressure due to DNA packing inside the capsid. However, the exact mechanism by which each phage controls its DNA ejection remains unknown. Using light scattering, we analyzed the kinetics of in vitro DNA release from phages SPP1 and lambda (Siphoviridae family) and found a simple exponential decay. The ejection characteristic time was studied as a function of the temperature and found to follow an Arrhenius law, allowing us to determine the activation energy that governs DNA ejection. A value of 25-30 kcal/mol is obtained for SPP1 and lambda, comparable to the one measured in vitro for T5 (Siphoviridae) and in vivo for T7 (Podoviridae). This suggests similar mechanisms of DNA ejection control. In all tailed phages, the opening of the connector-tail channel is needed for DNA release and could constitute the limiting step. The common value of the activation energy likely reflects the existence for all phages of an optimum value, ensuring a compromise between efficient DNA delivery and high stability of the virus.

Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

PubMed

Liberek, K; Osipiuk, J; Zylicz, M; Ang, D; Skorko, J; Georgopoulos, C

1990-02-25

The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

A Novel Approach to the Elucidation of the Mechanism of Development of Androgen-Independent Growth of Prostate Cancer

DTIC Science & Technology

2001-01-01

with 1.0 with differential hybridization to the two probes. Phage plaques corresponding ml of RIPA buffer with protease inhibitors (PBS, 1% NP40, 0.5...hybridized to radiolabeled probe prepared from plaque-purified phage . at 10,000 X g for 20 min; supernatants were collected and centrifuged again to...Screening. Lambda phage plaques from the primary unamplified Differential expression screening of cDNA libraries constructed CWR22 library were plated

Phages of Lactobacillus casei/paracasei: response to environmental factors and interaction with collection and commercial strains.

PubMed

Capra, M L; Quiberoni, A; Reinheimer, J

2006-02-01

To investigate the influence of several environmental factors on the viability and cell-adsorption for two Lactobacillus casei/paracasei bacteriophages (PL-1 and J-1). Both phages showed a remarkably high specificity of species, sharing similar host spectra. Two phages and four sensitive strains were used to conform five phage/strain systems. Each showed a particular behaviour (burst size: ranging from 32 to 160 PFU/infective centre; burst time: 120-240 min and latent time: 5-90 min). For both phages, the viability was not significantly affected from pH 4 to 11 (room temperature) and from pH 5 to 10 (37 degrees C). Adsorption rates were not influenced by calcium ions, but decreased after the thermal inactivation of cells. Adsorption rates were high between 0 and 50 degrees C with maximum values at 30 degrees C and pH 6. System PL-1/Lact. paracasei A showed noticeable differences in comparison with the others, being times required to reach 90% of adsorption of 4 h and lower than 45 min, respectively. The data obtained in this work demonstrated that environmental parameters can influence the viability and cell adsorption rates of Lact. casei/paracasei phages. The extent of this influence was phage dependent. This work contributes to the enlargement of the currently scarce knowledge of phages of probiotic bacteria.

Visualization of phage DNA degradation by a type I CRISPR-Cas system at the single-cell level.

PubMed

Guan, Jingwen; Shi, Xu; Burgos, Roberto; Zeng, Lanying

2017-03-01

The CRISPR-Cas system is a widespread prokaryotic defense system which targets and cleaves invasive nucleic acids, such as plasmids or viruses. So far, a great number of studies have focused on the components and mechanisms of this system, however, a direct visualization of CRISPR-Cas degrading invading DNA in real-time has not yet been studied at the single-cell level. In this study, we fluorescently label phage lambda DNA in vivo , and track the labeled DNA over time to characterize DNA degradation at the single-cell level. At the bulk level, the lysogenization frequency of cells harboring CRISPR plasmids decreases significantly compared to cells with a non-CRISPR control. At the single-cell level, host cells with CRISPR activity are unperturbed by phage infection, maintaining normal growth like uninfected cells, where the efficiency of our anti-lambda CRISPR system is around 26%. During the course of time-lapse movies, the average fluorescence of invasive phage DNA in cells with CRISPR activity, decays more rapidly compared to cells without, and phage DNA is fully degraded by around 44 minutes on average. Moreover, the degradation appears to be independent of cell size or the phage DNA ejection site suggesting that Cas proteins are dispersed in sufficient quantities throughout the cell. With the CRISPR-Cas visualization system we developed, we are able to examine and characterize how a CRISPR system degrades invading phage DNA at the single-cell level. This work provides direct evidence and improves the current understanding on how CRISPR breaks down invading DNA.

A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.

PubMed Central

Tabor, S; Richardson, C C

1985-01-01

The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase. Images PMID:3156376

The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI

PubMed Central

Kasarjian, Julie K. A.; Hidaka, Masumi; Horiuchi, Takashi; Iida, Masatake; Ryu, Junichi

2004-01-01

Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT). PMID:15199175

Cloning and study of the pectate lyase gene of Erwinia carotovora

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bukanov, N.O.; Fonshtein, M.Yu.; Evtushenkov, A.N.

1986-04-01

The cloning of the gene of a secretable protein of Erwinia carotovora, pectate lyase, in Escherichia coli was described. Primary cloning was conducted using the phage vector lambda 47.1. In the gene library of E. carotovora obtained, eight phages carrying the gene sought were identified according to the appearance of enzymatic activity of the gene product, pectate lyase, in situ. The BamHI fragment of DNA, common to all these phages, was recloned on the plasmid pUC19. It was shown that the cloned pectate lyase gene is represented on the E. carotovora chromosome in one copy. Methods of production of representativemore » gene libraries on phage vectors from no less than 1 ..mu..g of cloned DNA even for the genomes of eukaryotes have now been developed. Vectors have been created, for example, lambda 47.1, permitting the selection only of hybrid molecules. A number of methods have been developed for the search for a required gene in the library, depending on whether the cloned gene can be expressed or not, and if it can, what properties it will impart to the hybrid clone containing it.« less

Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221.

PubMed

Cohen, P T; Cohen, P

1989-06-15

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.

The Genetic Switch Regulating Activity of Early Promoters of the Temperate Lactococcal Bacteriophage TP901-1

PubMed Central

Madsen, Peter Lynge; Johansen, Annette H.; Hammer, Karin; Brøndsted, Lone

1999-01-01

A functional analysis of open reading frame 4 (ORF4) and ORF5 from the temperate lactococcal phage TP901-1 was performed by mutant and deletion analysis combined with transcriptional studies of the early phage promoters pR and pL. ORF4 (180 amino acids) was identified as a phage repressor necessary for repression of both promoters. Furthermore, the presence of ORF4 confers immunity of the host strain to TP901-1. ORF5 (72 amino acids) was found to be able to inhibit repression of the lytic promoter pL by ORF4. Upon transformation with a plasmid containing both ORF4 and ORF5 and their cognate promoters, clonal variation is observed: in each transformant, either pL is open and pR is closed or vice versa. The repression is still dependent on ORF4, and the presence of ORF5 is needed for the clonal variation. Induction of a repressed pL fusion containing orf4 and orf5 was obtained by addition of mitomycin C, and the induction was also shown to be dependent on the presence of the RecA protein, even though ORF4 does not contain a recognizable autocleavage site. Our results suggest that the relative amounts of the two proteins ORF4 and ORF5 determine the decision between lytic or lysogenic life cycle after phage infection and that a protein complex consisting of ORF4 and ORF5 may constitute a new type of genetic switch in bacteriophages. PMID:10601198

Genes and Structural Proteins of the Phage Syn5 of the Marine Cyanobacteria Synechococcus

DTIC Science & Technology

2005-09-01

typhimurium phage P22, a podoviridae, was shown to possess extensive genomic similarity to coliphage lambda, a siphoviridae (Botstein and Herskowitz...are found among cyanobacteria in the surface waters during the winter. Temperature influences the number of infectious particles produced during lytic...grids and stained with 1% uranyl acetate for 15 minutes, washed three times in double-distilled water , stained in 1% lead citrate for 4 minutes, and

Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

PubMed Central

2009-01-01

Background Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV) DNA levels, which can cause difficulty in the interpretation of viral load. A universal double-stranded DNA control or standard that can be used in all commercial HBV DNA nucleic acid amplification assay kits is urgently needed. By aligning all HBV genotypes (A-H), we found that the surface antigen gene and precore-core gene regions of HBV are the most conserved regions among the different HBV genotypes. We constructed a chimeric fragment by overlapping extension polymerase chain reaction and obtained a 1,349-bp HBVC+S fragment. We then packaged the fragment into lambda phages using a traditional lambda phage cloning procedure. Results The obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits. Conclusions The lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays. PMID:20025781

Oligopeptide M13 Phage Display in Pathogen Research

PubMed Central

Kügler, Jonas; Zantow, Jonas; Meyer, Torsten; Hust, Michael

2013-01-01

Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline. PMID:24136040

Oligopeptide m13 phage display in pathogen research.

PubMed

Kügler, Jonas; Zantow, Jonas; Meyer, Torsten; Hust, Michael

2013-10-16

Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

PubMed Central

Alfano, C; McMacken, R

1988-01-01

The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of lambda DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in lambda DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori lambda DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori lambda, however, later steps in lambda DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of lambda DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands. Images PMID:2847118

Bacteriophage T5 DNA ejection under pressure.

PubMed

Leforestier, A; Brasilès, S; de Frutos, M; Raspaud, E; Letellier, L; Tavares, P; Livolant, F

2008-12-19

The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for lambda and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and lambda, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.

Lambda phage-based vaccine induces antitumor immunity in hepatocellular carcinoma.

PubMed

Iwagami, Yoshifumi; Casulli, Sarah; Nagaoka, Katsuya; Kim, Miran; Carlson, Rolf I; Ogawa, Kosuke; Lebowitz, Michael S; Fuller, Steve; Biswas, Biswajit; Stewart, Solomon; Dong, Xiaoqun; Ghanbari, Hossein; Wands, Jack R

2017-09-01

Hepatocellular carcinoma (HCC) is a difficult to treat tumor with a poor prognosis. Aspartate β-hydroxylase (ASPH) is a highly conserved enzyme overexpressed on the cell surface of both murine and human HCC cells. We evaluated therapeutic effects of nanoparticle lambda (λ) phage vaccine constructs against ASPH expressing murine liver tumors. Mice were immunized before and after subcutaneous implantation of a syngeneic BNL HCC cell line. Antitumor actively was assessed by generation of antigen specific cellular immune responses and the identification of tumor infiltrating lymphocytes. Prophylactic and therapeutic immunization significantly delayed HCC growth and progression. ASPH-antigen specific CD4+ and CD8+ lymphocytes were identified in the spleen of tumor bearing mice and cytotoxicity was directed against ASPH expressing BNL HCC cells. Furthermore, vaccination generated antigen specific Th1 and Th2 cytokine secretion by immune cells. There was widespread necrosis with infiltration of CD3+ and CD8+ T cells in HCC tumors of λ phage vaccinated mice compared to controls. Moreover, further confirmation of anti-tumor effects on ASPH expressing tumor cell growth were obtained in another murine syngeneic vaccine model with pulmonary metastases. These observations suggest that ASPH may serve as a highly antigenic target for immunotherapy.

QUANTITATIVE STUDY OF ENDOLYSIN SYNTHESIS DURING REPRODUCTION OF LAMBDA PHAGES

PubMed Central

Groman, Neal B.; Suzuki, Grace

1963-01-01

Groman, Neal B. (University of Washington, Seattle) and Grace Suzuki. Quantitative study of endolysin synthesis during reproduction of lambda phages. J. Bacteriol. 86:187–194. 1963.—Endolysin is presumed to be a phage-induced enzyme participating in lysis through its destructive action on the host cell wall. A method for assaying endolysin is described, which was utilized in studying endolysin synthesis at 37 and 44 C by induced strains of K-12 (λ), K-12 (λtem), and K-12 (λ112). In all cases, endolysin was detected prior to the appearance of mature, intracellular phage and was detected earlier at 44 C than at 37 C. It was synthesized at a linear rate, as was phage, and both syntheses terminated at the same time. Surprisingly, endolysin also accumulated under conditions in which induced K-12 (λ112) exhibited lysis inhibition. Under these conditions, endolysin concentration per induced cell was 2 to 2.5 times that produced by normally lysing K-12 (λ). Since alterations introduced into the lytic process by temperature, mutation, or both correlate well with the timing and rate of endolysin synthesis, the data tend to support the concept that endolysin determines the kinetics of the process. However, the accumulation of endolysin during lysis inhibition suggests the need for alternative hypotheses. One hypothesis is that although endolysin action is the key to lysis some preliminary steps are required to release the enzyme so that it may contact its substrate in the cell wall. A second hypothesis is that basically the lytic process involves an alteration in the permeability barrier of the cell and that lytic enzymes such as endolysin have evolved as an auxillary but dispensable mechanism to this process. PMID:14058940

Novel DNA packaging recognition in the unusual bacteriophage N15

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feiss, Michael; Geyer, Henriette, E-mail: henriettegeyer@gmail.com; Division of Viral Infections, Robert Koch Institute, Berlin

Phage lambda's cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cos{sup N15}) is closely related to cos{sup λ}, but whereas the cosB{sup N15} subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosB{sup λ}. A bioinformatic study of N15-like phages indicates that cosB{sup N15} also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15more » plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB–λ. The DNA binding domain of TerS-N15 is a dimer. - Highlights: • There are two classes of DNA packaging signals in N15-related phages. • Phage N15's TerS binding site: a critical site and a possible remote accessory site. • Viral DNA recognition signals by the λ-like bacteriophages: the odd case of N15.« less

Stumbling across the Same Phage: Comparative Genomics of Widespread Temperate Phages Infecting the Fish Pathogen Vibrio anguillarum

PubMed Central

Kalatzis, Panos G.; Rørbo, Nanna; Castillo, Daniel; Mauritzen, Jesper Juel; Jørgensen, Jóhanna; Kokkari, Constantina; Zhang, Faxing; Katharios, Pantelis; Middelboe, Mathias

2017-01-01

Nineteen Vibrio anguillarum-specific temperate bacteriophages isolated across Europe and Chile from aquaculture and environmental sites were genome sequenced and analyzed for host range, morphology and life cycle characteristics. The phages were classified as Siphoviridae with genome sizes between 46,006 and 54,201 bp. All 19 phages showed high genetic similarity, and 13 phages were genetically identical. Apart from sporadically distributed single nucleotide polymorphisms (SNPs), genetic diversifications were located in three variable regions (VR1, VR2 and VR3) in six of the phage genomes. Identification of specific genes, such as N6-adenine methyltransferase and lambda like repressor, as well as the presence of a tRNAArg, suggested a both mutualistic and parasitic interaction between phages and hosts. During short term phage exposure experiments, 28% of a V. anguillarum host population was lysogenized by the temperate phages and a genomic analysis of a collection of 31 virulent V. anguillarum showed that the isolated phages were present as prophages in >50% of the strains covering large geographical distances. Further, phage sequences were widely distributed among CRISPR-Cas arrays of publicly available sequenced Vibrios. The observed distribution of these specific temperate Vibriophages across large geographical scales may be explained by efficient dispersal of phages and bacteria in the marine environment combined with a mutualistic interaction between temperate phages and their hosts which selects for co-existence rather than arms race dynamics. PMID:28531104

Membrane fusion during phage lysis.

PubMed

Rajaure, Manoj; Berry, Joel; Kongari, Rohit; Cahill, Jesse; Young, Ry

2015-04-28

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.

Stability, ghost, and strong coupling in nonrelativistic general covariant theory of gravity with {lambda}{ne}1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang Yongqing; Wang Anzhong

2011-05-15

In this paper, we investigate three important issues: stability, ghost, and strong coupling, in the Horava-Melby-Thompson setup of the Horava-Lifshitz theory with {lambda}{ne}1, generalized recently by da Silva. We first develop the general linear scalar perturbations of the Friedmann-Robertson-Walker (FRW) universe with arbitrary spatial curvature and find that an immediate by-product of the setup is that, in all the inflationary models described by a scalar field, the FRW universe is necessarily flat. Applying them to the case of the Minkowski background, we find that it is stable, and, similar to the case {lambda}=1, the spin-0 graviton is eliminated. The vectormore » perturbations vanish identically in the Minkowski background. Thus, similar to general relativity, a free gravitational field in this setup is completely described by a spin-2 massless graviton, even with {lambda}{ne}1. We also study the ghost problem in the FRW background and find explicitly the ghost-free conditions. To study the strong coupling problem, we consider two different kinds of spacetimes, all with the presence of matter: one is cosmological, and the other is static. We find that the coupling becomes strong for a process with energy higher than M{sub pl}|c{sub {psi}|}{sup 5/2} in the flat FRW background and M{sub pl}|c{sub {psi}|}{sup 3} in a static weak gravitational field, where |c{sub {psi}|{identical_to}}|(1-{lambda})/(3{lambda}-1)|{sup 1/2}.« less

Organization and sequence of four flagellin-encoding genes of Edwardsiella icataluri

USDA-ARS?s Scientific Manuscript database

Edwardsiella ictaluri, the cause of enteric septicemia in channel catfish (Ictalurus punctatus), is motile by means of peritrichous flagella. We determined the complete flagellin gene sequences and their organization in E. ictaluri by sequencing genomic segments selected from a lambda-ZAP phage gen...

Burkholderia mallei Cluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages

DTIC Science & Technology

2010-01-01

mallei virAG; Kmr 45 pBHR4-GFP Broad-host-range vector containing gfp from pQBI T7 GFP (Quantum Biotech); Gmr 45 pBHR1-TG pBHR1 containing gfp...Bonanno, J. M. Sauder, S. Pukatzki, S. K. Burley, S. C. Almo, and J. J. Mekalanos. 2009. Type VI secretion apparatus and phage tail-associated protein...14251. 37. Pell, L. G., V. Kanelis, L. W. Donaldson, P. L. Howell, and A. R. Davidson. 2009. The phage lambda major tail protein structure reveals a

Role of C-terminal residues in oligomerization and stability of lambda CII: implications for lysis-lysogeny decision of the phage.

PubMed

Datta, Ajit Bikram; Roy, Siddhartha; Parrack, Pradeep

2005-01-14

A crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein CII, which has several interesting properties. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each. The three-dimensional structure of CII, a homo-tetramer of 97 residue subunits, is unknown. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions. Based on these results, we propose a model for CII structure, in which protein-protein contacts for dimer and tetramer formation are different. The implications of tetrameric organization, essential for CII activity, on the recognition of the direct repeat sequence is discussed.

General transfer matrix formalism to calculate DNA-protein-drug binding in gene regulation: application to OR operator of phage lambda.

PubMed

Teif, Vladimir B

2007-01-01

The transfer matrix methodology is proposed as a systematic tool for the statistical-mechanical description of DNA-protein-drug binding involved in gene regulation. We show that a genetic system of several cis-regulatory modules is calculable using this method, considering explicitly the site-overlapping, competitive, cooperative binding of regulatory proteins, their multilayer assembly and DNA looping. In the methodological section, the matrix models are solved for the basic types of short- and long-range interactions between DNA-bound proteins, drugs and nucleosomes. We apply the matrix method to gene regulation at the O(R) operator of phage lambda. The transfer matrix formalism allowed the description of the lambda-switch at a single-nucleotide resolution, taking into account the effects of a range of inter-protein distances. Our calculations confirm previously established roles of the contact CI-Cro-RNAP interactions. Concerning long-range interactions, we show that while the DNA loop between the O(R) and O(L) operators is important at the lysogenic CI concentrations, the interference between the adjacent promoters P(R) and P(RM) becomes more important at small CI concentrations. A large change in the expression pattern may arise in this regime due to anticooperative interactions between DNA-bound RNA polymerases. The applicability of the matrix method to more complex systems is discussed.

Cloning and Characterization of the P-l Promoter of Bacteriophage Lambda.

ERIC Educational Resources Information Center

Andino, Raul H.; And Others

1986-01-01

Background information, experimental approach, materials needed, procedures used, and typical results obtained are provided for genetic engineering experiments. The course in which these experiments are performed is also described. (JN)

The genome and structural proteome of YuA, a new Pseudomonas aeruginosa phage resembling M6.

PubMed

Ceyssens, Pieter-Jan; Mesyanzhinov, Vadim; Sykilinda, Nina; Briers, Yves; Roucourt, Bart; Lavigne, Rob; Robben, Johan; Domashin, Artem; Miroshnikov, Konstantin; Volckaert, Guido; Hertveldt, Kirsten

2008-02-01

Pseudomonas aeruginosa phage YuA (Siphoviridae) was isolated from a pond near Moscow, Russia. It has an elongated head, encapsulating a circularly permuted genome of 58,663 bp, and a flexible, noncontractile tail, which is terminally and subterminally decorated with short fibers. The YuA genome is neither Mu- nor lambda-like and encodes 78 gene products that cluster in three major regions involved in (i) DNA metabolism and replication, (ii) host interaction, and (iii) phage particle formation and host lysis. At the protein level, YuA displays significant homology with phages M6, phiJL001, 73, B3, DMS3, and D3112. Eighteen YuA proteins were identified as part of the phage particle by mass spectrometry analysis. Five different bacterial promoters were experimentally identified using a promoter trap assay, three of which have a sigma54-specific binding site and regulate transcription in the genome region involved in phage particle formation and host lysis. The dependency of these promoters on the host sigma54 factor was confirmed by analysis of an rpoN mutant strain of P. aeruginosa PAO1. At the DNA level, YuA is 91% identical to the recently (July 2007) annotated phage M6 of the Lindberg typing set. Despite this level of DNA homology throughout the genome, both phages combined have 15 unique genes that do not occur in the other phage. The genome organization of both phages differs substantially from those of the other known Pseudomonas-infecting Siphoviridae, delineating them as a distinct genus within this family.

Analysis of Endonuclease R·EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis

PubMed Central

Helling, Robert B.; Goodman, Howard M.; Boyer, Herbert W.

1974-01-01

By means of agarose-gel electrophoresis, endonuclease R·EcoRI-generated fragments of DNA from various viruses were separated, their molecular weights were determined, and complete or partial fragment maps for lambda, φ80, and hybrid phages were constructed. Images PMID:4372397

Specificity of diffusion channels produced by lambda phage receptor protein of Escherichia coli.

PubMed Central

Luckey, M; Nikaido, H

1980-01-01

The lamB protein, the receptor for phage lambda, was purified from the outer membrane of Escherichia coli K-12 by extraction with Triton X-100 and EDTA, chromatography on DEAE-Sephacel in Triton X-100, exchange of Triton for cholate by gel filtration, and chromatography on Sephacryl S-200 in cholate, NaCl, and EDTA. The purified protein appeared to exist as several oligomeric species. In an equilibrium retention assay with reconstituted vesicles containing phospholipids and lipopolysaccharide, the lamB protein conferred permeability for disaccharides. In a liposome swelling assay designed to measure rates of diffusion, the lamB protein conferred permeability to phospholipid liposomes for a variety of substrates. The rates obtained indicate the permeation facilitated by the lamB protein is specific, discriminating among substrates by both size and configuration. For example, maltose diffused into liposomes 40 times faster than sucrose, about 8 times faster than cellobiose, and about 12 times faster than maltoheptaose. The results suggest that the lamB protein forms a transmembrane channel containing a site (or sites) that loosely interacts with the solutes. Images PMID:6444720

Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.

PubMed Central

Bae, Y S; Kawasaki, I; Ikeda, H; Liu, L F

1988-01-01

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes. Images PMID:2832845

Thermoresistant revertants of an Escherichia coli strain carrying tif-1 and ruv mutations: non-suppressibility of ruv by sfi.

PubMed Central

Otsuji, N; Iyehara-Ogawa, H

1979-01-01

Spontaneous thermoresistant revertants were isolated from Tif1 Ruv- and Tif1 Ruv+ strains of Escherichia coli K-12. They were divided into five groups; backmutants to tif+ and recA structural gene mutants accounted for at least two of these groups. Mutations with an unconditional RecA- phenyotype were detected at a higher frequency in the Tif1 Ruv- strains (65%) than in the Tif1 Ruv+ strains (25%). A third group consisted of revertants exhibiting a RecA- phenotype at low temperature. Revertants with normal recombination ability and UV resistance, but with a thermosensitive defect in propagating lambda bio11 phage, were also isolated (group 4). The alleles responsible for this property were cotransducible with the srl gene, suggesting that they are located at the recA locus. Other revertants, which might carry lex, LEXB, or zab mutations, were UV sensitive and were able to propagate lambda bio11 phage (group 5). The sfi mutation, which suppresses filamentation in the Tif1 and UV-sensitive Lon- strains, does not restore UV resistance of the Ruv- mutant. PMID:374356

[Vitamin para-aminobenzoic acid inhibits development of SOS function in tif-1 mutants of Escherichia coli at nonpermissive temperatures].

PubMed

Vasil'eva, S V; Gorb, T E; Rapoport, I A

1983-12-01

The development of "SOS" inducible functions in lysogenic and non-lysogenic strains of Escherichia coli tif-1 sfiA11 (lambda) at nonpermissive temperature of 42 degrees C was strongly suppressed by para-aminobenzoic acid (PABA). The rate of prophage lambda induction decreased 400 times, as compared to the control level; the efficiency of W-reactivation of UV-irradiated phage lambda decreased 37.5 to 16%. PABA also inhibited to some extent (1.5 times) the process of inducible recombination on the RecF pathway. The processes of spontaneous lambda induction and W-reactivation, as well as spontaneous recombination on RecBC and RecF pathways, were not influenced by PABA. The above data are in accordance with previous studies of PABA action when the manifestation of "SOS" functions was induced by chemical mutagens. The action of PABA has been tentatively interpreted on the basis of negative control of "SOS" repair pathway.

Purification of bacteriophage lambda repressor

PubMed Central

Gao, Ning; Shearwin, Keith; Mack, John; Finzi, Laura; Dunlap, David

2013-01-01

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy “footprinting” assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops. PMID:23831434

Cyborg lectins: novel leguminous lectins with unique specificities.

PubMed

Yamamoto, K; Maruyama, I N; Osawa, T

2000-01-01

Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities.

Divergence and Mosaicism among Virulent Soil Phages of the Burkholderia cepacia Complex‡

PubMed Central

Summer, Elizabeth J.; Gonzalez, Carlos F.; Bomer, Morgan; Carlile, Thomas; Embry, Addie; Kucherka, Amalie M.; Lee, Jonte; Mebane, Leslie; Morrison, William C.; Mark, Louise; King, Maria D.; LiPuma, John J.; Vidaver, Anne K.; Young, Ry

2006-01-01

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the Burkholderia cepacia complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in Photorhabdus luminescens. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages. PMID:16352842

tif-Stimulated deoxyribonucleic acid repair in Escherichia coli K-12.

PubMed Central

Castellazzi, M; Jacques, M; George, J

1980-01-01

Bacterial survival is significantly increased after ultraviolet irradiation in tif sfi cells, provided that the thermosensitive tif mutation has been expressed at 41 degrees C before irradiation. This tif-mediated "reactivation of ultraviolet irradiated bacteria" needs de novo protein synthesis, as is the case for the tif-mediated reactivation of ultraviolet-irradiated phage lambda. However, in striking contrast to the phage reactivation process, this tif-mediated reactivation is no longer associated with mutagenesis. It also requires the presence of the uvrA+ excision function. These results strongly suggest the existence in Escherichia coli K-12 of a repair pathway acting on bacterial deoxyribonucleic acid which is inducible, error free, and uvr dependent. PMID:6451614

Effect of phenolic compounds, ethyl alcohol, and sodium metabisulphite on the lytic activity of phage PL-1 on a Lactobacillus casei S strain.

PubMed

Lee, A; Eschenbruch, R; Waller, J

1985-09-01

The effect of phenolic compounds, ethyl alcohol, and sodium metabisulphite on the lytic activity of virulent bacteriophage PL-1 on a Lactobacillus casei S strain isolated from a lactic acid beverage fermentation was investigated. Catechin, caffeic, and gallic acids, commercially produced red, white, and champagne tannins, ethyl alcohol, and sodium metabisulphite inhibited plaque formation. Catechin, caffeic, and gallic acids were the most effective inhibitors of plaque formation. Commercially supplied oenocyanin was not effective.

Origins of the Xylella fastidiosa prophage-like regions and their impact in genome differentiation.

PubMed

de Mello Varani, Alessandro; Souza, Rangel Celso; Nakaya, Helder I; de Lima, Wanessa Cristina; Paula de Almeida, Luiz Gonzaga; Kitajima, Elliot Watanabe; Chen, Jianchi; Civerolo, Edwin; Vasconcelos, Ana Tereza Ribeiro; Van Sluys, Marie-Anne

2008-01-01

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes.

Origins of the Xylella fastidiosa Prophage-Like Regions and Their Impact in Genome Differentiation

PubMed Central

de Mello Varani, Alessandro; Souza, Rangel Celso; Nakaya, Helder I.; de Lima, Wanessa Cristina; Paula de Almeida, Luiz Gonzaga; Kitajima, Elliot Watanabe; Chen, Jianchi; Civerolo, Edwin; Vasconcelos, Ana Tereza Ribeiro; Van Sluys, Marie-Anne

2008-01-01

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes. PMID:19116666

Genetic characterization of ØVC8 lytic phage for Vibrio cholerae O1.

PubMed

Solís-Sánchez, Alejandro; Hernández-Chiñas, Ulises; Navarro-Ocaña, Armando; De la Mora, Javier; Xicohtencatl-Cortes, Juan; Eslava-Campos, Carlos

2016-03-22

Epidemics and pandemics of cholera, a diarrheal disease, are attributed to Vibrio cholera serogroups O1 and O139. In recent years, specific lytic phages of V. cholera have been proposed to be important factors in the cyclic occurrence of cholera in endemic areas. However, the role and potential participation of lytic phages during long interepidemic periods of cholera in non-endemic regions have not yet been described. The purpose of this study was to isolate and characterize specific lytic phages of V. cholera O1 strains. Sixteen phages were isolated from wastewater samples collected at the Endhó Dam in Hidalgo State, Mexico, concentrated with PEG/NaCl, and purified by density gradient. The lytic activity of the purified phages was tested using different V. cholerae O1 and O139 strains. Phage morphology was visualized by transmission electron microscopy (TEM), and phage genome sequencing was performed using the Genome Analyzer IIx System. Genome assembly and bioinformatics analysis were performed using a set of high-throughput programs. Phage structural proteins were analyzed by mass spectrometry. Sixteen phages with lytic and lysogenic activity were isolated; only phage ØVC8 showed specific lytic activity against V. cholerae O1 strains. TEM images of ØVC8 revealed a phage with a short tail and an isometric head. The ØVC8 genome comprises linear double-stranded DNA of 39,422 bp with 50.8 % G + C. Of the 48 annotated ORFs, 16 exhibit homology with sequences of known function and several conserved domains. Bioinformatics analysis showed multiple conserved domains, including an Ig domain, suggesting that ØVC8 might adhere to different mucus substrates such as the human intestinal epithelium. The results suggest that ØVC8 genome utilize the "single-stranded cohesive ends" packaging strategy of the lambda-like group. The two structural proteins sequenced and analyzed are proteins of known function. ØVC8 is a lytic phage with specific activity against V. cholerae O1 strains and is grouped as a member of the VP2-like phage subfamily. The encoding of an Ig domain by ØVC8 makes this phage a good candidate for use in phage therapy and an alternative tool for monitoring V. cholerae populations.

Promoter/repressor system of Lactobacillus plantarum phage og1e: characterization of the promoters pR49-pR-pL and overproduction of the cro-like protein cng in Escherichia coli.

PubMed

Kakikawa, M; Watanabe, N; Funawatashi, T; Oki, M; Yasukawa, H; Taketo, A; Kodaira, K I

1998-07-30

The Lactobacillus plantarum phage og1e (42259bp) has two repressor-like genes cng and cpg oriented oppositely, accompanied by three potential promoters pR, pL and pR49, and seven operator-like sequences (GATAC-boxes) (Kodaira et al., 1997). In this study, the og1e putative promoters were introduced into the Escherichia coli promoter-detecting plasmid pKK232-8. In E. coli CK111, pR (pKPR1), pL (pKPL1) and pR49 (pKPR49) exhibited distinct CAT activities. When pKPR1 or pKPL1 was coexistent with a compatible plasmid pACYC184 carrying pR-cng (pA4PRCN1), the CAT activity was decreased significantly. On the other hand, cng directed a protein (Cng) of 10.1 kDa in E. coli under the control of T7 promoter. Gel mobility-shift assays demonstrated that Cng binds specifically to a DNA region containing the GATAC-boxes. In addition, primer extension analyses demonstrated that the two sequences pR and pL act as a promoter in L. plantarum as well as in E. coli. These results suggested that the potential promoters pR and pL probably function for the lytic and lysogenic pathways, respectively, and Cng may act as a repressor presumably through the GATAC-boxes as operators.

Genome, Integration, and Transduction of a Novel Temperate Phage of Helicobacter pylori

PubMed Central

Luo, Cheng-Hung; Chiou, Pei-Yu; Yang, Chiou-Ying

2012-01-01

Helicobacter pylori is a common human pathogen that has been identified to be carcinogenic. This study isolated the temperate bacteriophage 1961P from the lysate of a clinical strain of H. pylori isolated in Taiwan. The bacteriophage has an icosahedral head and a short tail, typical of the Podoviridae family. Its double-stranded DNA genome is 26,836 bp long and has 33 open reading frames. Only 9 of the predicted proteins have homologs of known functions, while the remaining 24 are only similar to unknown proteins encoded by Helicobacter prophages and remnants. Analysis of sequences proximal to the phage-host junctions suggests that 1961P may integrate into the host chromosome via a mechanism similar to that of bacteriophage lambda. In addition, 1961P is capable of generalized transduction. To the best of our knowledge, this is the first report of the isolation, characterization, genome analysis, integration, and transduction of a Helicobacter pylori phage. PMID:22696647

Constitutive Expression of a Transcription Termination Factor by a Repressed Prophage: Promoters for Transcribing the Phage HK022 nun Gene

PubMed Central

King, Rodney A.; Madsen, Peter L.; Weisberg, Robert A.

2000-01-01

Lysogens of phage HK022 are resistant to infection by phage λ. Lambda resistance is caused by the action of the HK022 Nun protein, which prematurely terminates early λ transcripts. We report here that transcription of the nun gene initiates at a constitutive prophage promoter, PNun, located just upstream of the protein coding sequence. The 5′ end of the transcript was determined by primer extension analysis of RNA isolated from HK022 lysogens or RNA made in vitro by transcribing a template containing the promoter with purified Escherichia coli RNA polymerase. Inactivation of PNun by mutation greatly reduced Nun activity and Nun antigen in an HK022 lysogen. However, a low level of residual activity was detected, suggesting that a secondary promoter also contributes to nun expression. We found one possible secondary promoter, PNun′, just upstream of PNun. Neither promoter is likely to increase the expression of other phage genes in a lysogen because their transcripts should be terminated downstream of nun. We estimate that HK022 lysogens in stationary phase contain several hundred molecules of Nun per cell and that cells in exponential phase probably contain fewer. PMID:10629193

An Empirically Calibrated Model of Cell Fate Decision Following Viral Infection

NASA Astrophysics Data System (ADS)

Coleman, Seth; Igoshin, Oleg; Golding, Ido

The life cycle of the virus (phage) lambda is an established paradigm for the way genetic networks drive cell fate decisions. But despite decades of interrogation, we are still unable to theoretically predict whether the infection of a given cell will result in cell death or viral dormancy. The poor predictive power of current models reflects the absence of quantitative experimental data describing the regulatory interactions between different lambda genes. To address this gap, we are constructing a theoretical model that captures the known interactions in the lambda network. Model assumptions and parameters are calibrated using new single-cell data from our lab, describing the activity of lambda genes at single-molecule resolution. We began with a mean-field model, aimed at exploring the population averaged gene-expression trajectories under different initial conditions. Next, we will develop a stochastic formulation, to capture the differences between individual cells within the population. The eventual goal is to identify how the post-infection decision is driven by the interplay between network topology, initial conditions, and stochastic effects. The insights gained here will inform our understanding of cell fate choices in more complex cellular systems.

PhD7Faster: predicting clones propagating faster from the Ph.D.-7 phage display peptide library.

PubMed

Ru, Beibei; 't Hoen, Peter A C; Nie, Fulei; Lin, Hao; Guo, Feng-Biao; Huang, Jian

2014-02-01

Phage display can rapidly discover peptides binding to any given target; thus, it has been widely used in basic and applied research. Each round of panning consists of two basic processes: Selection and amplification. However, recent studies have showed that the amplification step would decrease the diversity of phage display libraries due to different propagation capacity of phage clones. This may induce phages with growth advantage rather than specific affinity to appear in the final experimental results. The peptides displayed by such phages are termed as propagation-related target-unrelated peptides (PrTUPs). They would mislead further analysis and research if not removed. In this paper, we describe PhD7Faster, an ensemble predictor based on support vector machine (SVM) for predicting clones with growth advantage from the Ph.D.-7 phage display peptide library. By using reduced dipeptide composition (ReDPC) as features, an accuracy (Acc) of 79.67% and a Matthews correlation coefficient (MCC) of 0.595 were achieved in 5-fold cross-validation. In addition, the SVM-based model was demonstrated to perform better than several representative machine learning algorithms. We anticipate that PhD7Faster can assist biologists to exclude potential PrTUPs and accelerate the finding of specific binders from the popular Ph.D.-7 library. The web server of PhD7Faster can be freely accessed at http://immunet.cn/sarotup/cgi-bin/PhD7Faster.pl.

The First Paenibacillus larvae Bacteriophage Endolysin (PlyPl23) with High Potential to Control American Foulbrood.

PubMed

Oliveira, Ana; Leite, Marta; Kluskens, Leon D; Santos, Sílvio B; Melo, Luís D R; Azeredo, Joana

2015-01-01

Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-positive pathogens. This work describes the characterization of a novel endolysin (PlyPl23) encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restriction to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new endolysin described herein has an N-acetylmuramoyl-L-alanine amidase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0), showing its feasible application in the field. At pH 7.0, a concentration of 0.2 μM of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly, an increase of activity was observed when PlyPl23 was previously incubated in royal jelly. Furthermore, in vivo safety evaluation assays demonstrated that this enzyme is not toxic to the bee larvae. The present work describes for the first time an endolysin encoded in a P. larvae phage that presents high potential to integrate a commercial product to control the problematic American foulbrood.

A new and fast method for preparing high quality lambda DNA suitable for sequencing.

PubMed Central

Manfioletti, G; Schneider, C

1988-01-01

A method is described for the rapid purification of high quality lambda DNA. The method can be used from either liquid or plate lysates and on a small scale or a large scale. It relies on the preadsobtion of all polyanions present in the lysate to an "insoluble" anion-exchange matrix (DEAE or TEAE). Phage particles are then disrupted by combined treatment with EDTA/proteinase K and the resulting DNA is precipitated by the addition of the cationic detergent cetyl (or hexadecyl)-trimethyl ammonium bromide-CTAB ("soluble" anion-exchange matrix). The precipitated CTAB-DNA complex is then exchanged to Na-DNA and ethanol precipitated. The resultant purified DNA is suitable for enzymatic reactions and provides a high quality template for dideoxy-sequence analysis. Images PMID:2966928

Characterization of the relationship between integrase, excisionase and antirepressor activities associated with a superinfecting Shiga toxin encoding bacteriophage

PubMed Central

Fogg, P. C. M.; Rigden, D. J.; Saunders, J. R.; McCarthy, A. J.; Allison, H. E.

2011-01-01

Shigatoxigenic Escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of Shiga toxin-encoding lambdoid bacteriophages. At least some of these Stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host's pathogenic profile. Here, increasing levels of Stx phage Φ24B integrase expression in multiple lysogen cultures are demonstrated along with apparently negligible repression of integrase expression by the cognate CI repressor. The Φ24B int transcription start site and promoter region were identified and found to differ from in silico predictions. The unidirectional activity of this integrase was determined in an in situ, inducible tri-partite reaction. This indicated that Φ24B must encode a novel directionality factor that is controlling excision events during prophage induction. This excisionase was subsequently identified and characterized through complementation experiments. In addition, the previous proposal that a putative antirepressor was responsible for the lack of immunity to superinfection through inactivation of CI has been revisited and a new hypothesis involving the role of this protein in promoting efficient induction of the Φ24B prophage is proposed. PMID:21062824

Unusual Structure of the attB Site of the Site-Specific Recombination System of Lactobacillus delbrueckii Bacteriophage mv4

PubMed Central

Auvray, Frédéric; Coddeville, Michèle; Ordonez, Romy Catoira; Ritzenthaler, Paul

1999-01-01

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3′ end of a tRNASer gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNASer gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model. PMID:10572145

Use of Lambda Phage DNA as a Hybrid Internal Control in a PCR-Enzyme Immunoassay To Detect Chlamydia pneumoniae

PubMed Central

Pham, Dien G.; Madico, Guillermo E.; Quinn, Thomas C.; Enzler, Mark J.; Smith, Thomas F.; Gaydos, Charlotte A.

1998-01-01

An inherent problem in the diagnostic PCR assay is the presence of ill-defined inhibitors of amplification which may cause false-negative results. Addition of an amplifiable fragment of foreign DNA in the PCR to serve as a hybrid internal control (HIC) would allow for a simple way to identify specimens containing inhibitors. Two oligonucleotide hybrid primers were synthesized to contain nucleic acid sequences of the Chlamydia pneumoniae 16S rRNA primers in a position flanking two primers that target the sequences of a 650-bp lambda phage DNA segment. By using the hybrid primers, hybrid DNA comprising a large sequence of lambda phage DNA flanked by short pieces of chlamydia DNA was subsequently generated by PCR, cloned into a plasmid vector, and purified. Plasmids containing the hybrid DNA were diluted and used as a HIC by adding them to each C. pneumoniae PCR test. Consequently, C. pneumoniae primers were able to amplify both chlamydia DNA and the HIC DNA. The production of a 689-bp HIC DNA band on an acrylamide gel indicated that the specimen contained no inhibitors and that internal conditions were compatible with PCR. Subsequently, a biotinylated RNA probe for the HIC was transcribed from a nested sequence of the HIC and was used for its hybridization. Detection of the HIC DNA-RNA hybrid was achieved by enzyme immunoassay (EIA). This PCR-EIA system with a HIC was initially tested with 12 previously PCR-positive and 14 previously PCR-negative specimens. Of the 12 PCR-positive specimens, 11 were reconfirmed as positive; 1 had a negative HIC value, indicating inhibition. Of the 14 previously PCR-negative specimens, 13 were confirmed as true negative; 1 had a negative HIC value, indicating inhibition. The assay was then used with 237 nasopharyngeal specimens from patients with pneumonia. Twenty-one of 237 (8.9%) were positive for C. pneumoniae, and 42 (17.7%) were found to inhibit the PCR. Specimens showing inhibitory activity were diluted 1:10 and were retested. Ten specimens were still inhibitory to the PCR and required further DNA purification. No additional positive samples were detected and 3 nasopharyngeal specimens remained inhibitory to PCR. Coamplification of a HIC DNA can help confirm true-negative PCR results by ruling out the presence of inhibitors of DNA amplification. PMID:9650936

Content and persistence of extracellular DNA in native soils

NASA Astrophysics Data System (ADS)

Blagodatskaya, Evgenia; Blagodatsky, Sergey; Anderson, Traute-Heidi; Kuzyakov, Yakov

2014-05-01

The long-term persistence of soil extracellular DNA is questionable because of high potential activity of nucleases produced by soil microorganisms. By the other hand, the relative persistence of DNA-like biopolymers could be due to their adsorption on clay minerals and humus substances in soil. High-specific and ultra sensitive reagent PicoGreenTM (Molecular Probes) permits the quantitative assessment of microbial dsDNA in diluted soil extracts giving a good tool for tracing the DNA fate in soil. Our goal was to determine intracellular and extracellular DNA content in cambisol (loamy sand) and in chernozem (silty loam) soils and to investigate the possible adsorption and degradation of extracellular DNA in soil. Optimized procedure of mechanical and enzymatic destruction of cell walls was used for direct extraction of microbial DNA with Tris-EDTA buffer (Blagodatskaya et al., 2003). Extracellular dsDNA was determined in distilled water and in Tris-EDTA extracts without enzymatic or mechanical treatments. DNA content was determined after addition of PicoGreen to diluted soil extracts. Degradation of extracellular DNA was traced during 24 h incubation of 2 µg lambda-phage DNA in soil. Possible DNA adsorption to soil matrix was determined by recovery of lambda -phage DNA added to autoclaved soil. Extracellular dsDNA was absent in water extracts of both soils. The content of extracellular dsDNA extracted by Tris-EDTA buffer was 0.46 µg/g in chernozem and 1.59 µg/g in cambisol amounting 0.43 and 2.8% of total dsDNA content in these soils, respectively. 100% and 64.8% of added extracellular lambda -phage dsDNA was found in cambisol and chernozem soils, respectively, in 5 h after application. 39% and 73.5% of added DNA disappeared in cambisol and in chernozem, respectively, during 24 h incubation. Degradation rate of extracellular DNA depended on microbial biomass content, which was 2.5 times higher in chernozem as compared to cambisol. Maximum adsorption of DNA by soils was observed in cambisol and reached 2.7% of added amount. We speculate that probability of gene transfer could be rather high in soils, taking into account possible increase of extracellular DNA content after transient environmental events (i.e. drying - rewetting and freezing - thawing).

Chemical and Molecular Biological Aspects of Alkylhydrazine-Induced Carcinogenesis in Human Cells in Vitro. Revised

DTIC Science & Technology

1984-04-01

weights using the following phage DNA markers : lambda, T2 and T7. The number of alkaline labile sites (breaks due to apurinic sites and phosphotriesters...exhibit cell invasiveness in chick embryo skin, human malignancy specific cell- surface antigenic determinants (19), and produce tumors in pre...they eluted from the column, were identified by co-chromatographed markers , (Chart 2). When randomly proliferating cells were treated with either 1,1-DMH

Breaking Transmission with Vaccines: The Case of Tuberculosis.

PubMed

Gonzalo-Asensio, Jesus; Aguilo, Nacho; Marinova, Dessislava; Martin, Carlos

2017-07-01

Members of the Mycobacterium tuberculosis complex (MTBC) have evolved causing tuberculosis (TB) in different mammalian hosts. MTBC ecotypes have adapted to diverse animal species, with M. bovis being the most common cause of TB in livestock. Cattle-to-human transmission of M. bovis through ingestion of raw milk was common before introduction of the pasteurization process. TB in humans is mainly caused by M. tuberculosis . This bacterium is considered a genetically clonal pathogen that has coevolved with humans due to its ability to manipulate and subvert the immune response. TB is a major public health problem due to airborne person-to-person transmission of M. tuberculosis . The essential yet unanswered question on the natural history of TB is when M. tuberculosis decides to establish latent infection in the host (resambling the lysogenic cycle of lambda phage) or to cause pulmonary disease (comparable to the lytic cycle of lambda phage). In this latter case, M. tuberculosis kills the host with the aim of achieving transmission to new hosts. Combating the TB epidemic requires stopping transmission. M. bovis BCG, the present vaccine against TB, is derived from M. bovis and only protects against disseminated forms of TB. Thus, a priority in TB research is development of new effective vaccines to prevent pulmonary disease. Attenuated vaccines based on M. tuberculosis as MTBVAC are potential candidates that could contribute to break the TB transmission cycle.

Genome of Enterobacteriophage Lula/phi80 and Insights into Its Ability To Spread in the Laboratory Environment

PubMed Central

Rotman, Ella; Kouzminova, Elena; Plunkett, Guy

2012-01-01

The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80's genome layout copies that of lambda, yet homology with other lambdoid phages is mostly limited to the capsid genes. Lula/phi80's DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage's damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80 lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80 SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi80, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion. PMID:23042999

Phage-Encoded Colanic Acid-Degrading Enzyme Permits Lytic Phage Infection of a Capsule-Forming Resistant Mutant Escherichia coli Strain

PubMed Central

Kim, Min Soo; Kim, Young Deuk; Hong, Sung Sik; Park, Kwangseo; Ko, Kwan Soo

2014-01-01

In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria. PMID:25416767

Molecular weights and subunit structure of LamB proteins.

PubMed

Nakae, T; Ishii, J N

1982-01-01

Phage lambda-receptor proteins of Escherichia coli, LamB proteins, form oligomeric aggregates to build transmembrane diffusion pores selective for maltose and maltodextrins. The molecular weights (MW) of functional oligomers as well as dissociated monomers were determined by sedimentation equilibrium analysis in homogeneous non-ionic surfactant and deuterium oxide and in 6 M guanidine-HCl, respectively. The MW of oligomers and monomers appeared as 135 600 and 45 900, respectively. Thus, functional Lamb proteins consisted of three identical subunits.

Fluctuation Pressure Assisted Ejection of DNA From Bacteriophage

NASA Astrophysics Data System (ADS)

Harrison, Michael J.

2011-03-01

The role of thermal pressure fluctuations excited within tightly packaged DNA while it is ejected from protein capsid shells is discussed in a model calculation. At equilibrium before ejection we assume the DNA is folded many times into a bundle of parallel segments that forms an equilibrium conformation at minimum free energy, which presses tightly against capsid walls. Using a canonical ensemble at temperature T we calculate internal pressure fluctuations against a slowly moving or static capsid mantle for an elastic continuum model of the folded DNA bundle. It is found that fluctuating pressures on the capsid from thermal excitation of longitudinal acoustic vibrations in the bundle whose wavelengths are exceeded by the bend persistence length may have root-mean-square values that are several tens of atmospheres for typically small phage dimensions. Comparisons are given with measured data on three mutants of lambda phage with different base pair lengths and total genome ejection pressures.

Ordered shotgun sequencing of a 135 kb Xq25 YAC containing ANT2 and four possible genes, including three confirmed by EST matches.

PubMed Central

Chen, C N; Su, Y; Baybayan, P; Siruno, A; Nagaraja, R; Mazzarella, R; Schlessinger, D; Chen, E

1996-01-01

Ordered shotgun sequencing (OSS) has been successfully carried out with an Xq25 YAC substrate. yWXD703 DNA was subcloned into lambda phage and sequences of insert ends of the lambda subclones were used to generate a map to select a minimum tiling path of clones to be completely sequenced. The sequence of 135 038 nt contains the entire ANT2 cDNA as well as four other candidates suggested by computer-assisted analyses. One of the putative genes is homologous to a gene implicated in Graves' disease and it, ANT2 and two others are confirmed by EST matches. The results suggest that OSS can be applied to YACs in accord with earlier simulations and further indicate that the sequence of the YAC accurately reflects the sequence of uncloned human DNA. PMID:8918809

Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage lambda Q- vector.

PubMed

Oh, Jeong Seok; Cho, Daechul; Park, Tai Hyun

2005-11-01

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage lambda system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage lambda vector with a Q(-) mutation was used to enhance the expression of the lambda system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h(-1) with 1.5 S(0) of the medium supply. The maximum productivity of the total beta-galactosidase was 16.3x10(6) U l(-1) h(-1), which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.

UVB-induced mutagenesis in hairless {lambda}lacZ-transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frijhoff, A.F.W.; Rebel, H.; Mientjes, E.J.

UVB-induced mutagenesis was studied in hairless 40.6 transgenic mice (Muta{trademark}Mouse), which contain the {lambda}gt1OlacZ shuttle vector as a target for mutagenesis. Mice were exposed at the dorsal side to either single doses of 200, 500, 800, or 1000 J/m{sup 2} UVB or to two successive irradiations of either 200 and 800 J/m{sup 2} UVB, with intervals of 1,3, or 5 days, or to 800 and 200 J/m{sup 2} UVB with a 5-day interval. At 23 days after the last exposure, lacZ mutant frequencies (MF) were determined in the epidermis. The lacZ MF increased linearly with increasing dose of UVB. Themore » mutagenic effect of two successive irradiations appeared to be additive. The UV-induced mutation spectrum was dominated by G:C{r_arrow}A:T transitions at dipyrimidine sites. DNA-sequence analysis of spontaneously mutated phages showed a diverse spectrum consisting of insertions, deletions and G:C {r_arrow} A:T transitions at CpG sites. the results indicate that the hairless {lambda}lacZ-transgenic mouse is a suitable in vivo model for studying UVB-induced mutations. 29 refs., 5 tabs.« less

Models of life: epigenetics, diversity and cycles.

PubMed

Sneppen, Kim

2017-04-01

This review emphasizes aspects of biology that can be understood through repeated applications of simple causal rules. The selected topics include perspectives on gene regulation, phage lambda development, epigenetics, microbial ecology, as well as model approaches to diversity and to punctuated equilibrium in evolution. Two outstanding features are repeatedly described. One is the minimal number of rules to sustain specific states of complex systems for a long time. The other is the collapse of such states and the subsequent dynamical cycle of situations that restitute the system to a potentially new metastable state.

Models of life: epigenetics, diversity and cycles

NASA Astrophysics Data System (ADS)

Sneppen, Kim

2017-04-01

This review emphasizes aspects of biology that can be understood through repeated applications of simple causal rules. The selected topics include perspectives on gene regulation, phage lambda development, epigenetics, microbial ecology, as well as model approaches to diversity and to punctuated equilibrium in evolution. Two outstanding features are repeatedly described. One is the minimal number of rules to sustain specific states of complex systems for a long time. The other is the collapse of such states and the subsequent dynamical cycle of situations that restitute the system to a potentially new metastable state.

Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

PubMed

Kügler, Jonas; Wilke, Sonja; Meier, Doris; Tomszak, Florian; Frenzel, André; Schirrmann, Thomas; Dübel, Stefan; Garritsen, Henk; Hock, Björn; Toleikis, Lars; Schütte, Mark; Hust, Michael

2015-02-19

Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

PubMed

Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

2013-01-01

The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

Simulation experiments of the effect of space environment on bacteriophage and DNA thin films

NASA Technical Reports Server (NTRS)

Fekete, A.; Ronto, Gy; Hegedus, M.; Modos, K.; Berces, A.; Kovacs, G.; Lammer, H.; Panitz, C.

2004-01-01

The main goal of PUR experiment (phage and uracil response) is to examine and quantify the effect of specific space conditions on nucleic acid models. To achieve this an improved method was elaborated for the preparation of DNA and bacteriophage thin films. The homogeneity of the films was controlled by UV spectroscopy and microscopy. To provide experimental evidence for the hypothesis that interplanetary transfer of the genetic material is possible, phage T7 and isolated T7 DNA thin films have been exposed to selected space conditions: intense UVC radiation (lambda=254 nm) and high vacuum (10(-4) Pa). The effects of DNA hydration, conformation and packing on UV radiation damage were examined. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.

PubMed

Sun, Xingmin; Mierke, Dale F; Biswas, Tapan; Lee, Sang Yeol; Landy, Arthur; Radman-Livaja, Marta

2006-11-17

The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.

Suppressor Analysis of the Fusogenic Lambda Spanins.

PubMed

Cahill, Jesse; Rajaure, Manoj; Holt, Ashley; Moreland, Russell; O'Leary, Chandler; Kulkarni, Aneesha; Sloan, Jordan; Young, Ry

2017-07-15

The final step of lysis in phage λ infections of Escherichia coli is mediated by the spanins Rz and Rz1. These proteins form a complex that bridges the cell envelope and that has been proposed to cause fusion of the inner and outer membranes. Accordingly, mutations that block spanin function are found within coiled-coil domains and the proline-rich region, motifs essential in other fusion systems. To gain insight into spanin function, pseudorevertant alleles that restored plaque formation for lysis-defective mutants of Rz and Rz1 were selected. Most second-site suppressors clustered within a coiled-coil domain of Rz near the outer leaflet of the cytoplasmic membrane and were not allele specific. Suppressors largely encoded polar insertions within the hydrophobic core of the coiled-coil interface. Such suppressor changes resulted in decreased proteolytic stability of the Rz double mutants in vivo Unlike the wild type, in which lysis occurs while the cells retain a rod shape, revertant alleles with second-site suppressor mutations supported lysis events that were preceded by spherical cell formation. This suggests that destabilization of the membrane-proximal coiled coil restores function for defective spanin alleles by increasing the conformational freedom of the complex at the cost of its normal, all-or-nothing functionality. IMPORTANCE Caudovirales encode cell envelope-spanning proteins called spanins, which are thought to fuse the inner and outer membranes during phage lysis. Recent genetic analysis identified the functional domains of the lambda spanins, which are similar to class I viral fusion proteins. While the pre- and postfusion structures of model fusion systems have been well characterized, the intermediate structure(s) formed during the fusion reaction remains elusive. Genetic analysis would be expected to identify functional connections between intermediates. Since most membrane fusion systems are not genetically tractable, only few such investigations have been reported. Here, we report a suppressor analysis of lambda spanin function. To our knowledge this is the first suppression analysis of a class I-like complex and also the first such analysis of a prokaryote membrane fusion system. Copyright © 2017 American Society for Microbiology.

Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes.

PubMed

Park, Jong-Uk; Jo, Jae-Hyung; Kim, Young-Ji; Chung, So-Sun; Lee, Jin-Ho; Lee, Hyune Hwan

2008-04-01

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the lambdaOL1 and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes.. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the OL1 from the lambdaPL promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one lambdaOL1, and CJ1OX2, which has two successive lambdaOL1, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

PubMed

Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

2017-11-01

CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4.

PubMed

Lin, H; Rao, V B; Black, L W

1999-06-04

Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites. In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro. The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly internalizes terminase regions within the portal in the packasome complex. Both similarities and differences are seen in comparison to analogous sites which have been identified in phages T3 and lambda. Copyright 1999 Academic Press.

Survival of prototype strains of somatic coliphage families in environmental waters and when exposed to UV low-pressure monochromatic radiation or heat.

PubMed

Lee, Hee Suk; Sobsey, Mark D

2011-06-01

The potential use of specific somatic coliphage taxonomic groups as viral indicators based on their persistence and prevalence in water was investigated. Representative type strains of the 4 major somatic coliphage taxonomic groups were seeded into reagent water and an ambient surface water source of drinking water and the survival of the added phages was measured over 90 days at temperatures of 23-25 and 4 °C. Microviridae (type strain PhiX174), Siphoviridae (type strain Lambda), and Myoviridae (type strain T4) viruses were the most persistent in water at the temperatures tested. The Microviridae (type strain PhiX174) and the Siphoviridae (type strain Lambda) were the most resistant viruses to UV radiation and the Myoviridae (type strain T4) and the Microviridae (type strain PhiX174) were the most resistant viruses to heat. Based on their greater persistence in water over time and their relative resistance to heat and/or UV radiation, the Myoviridae (type strain T4), the Microviridae (type strain PhiX174), and the Siphoviridae (type strain Lambda) were the preferred candidate somatic coliphages as fecal indicator viruses in water, with the Microviridae (type strain PhiX174) the most resistant to these conditions overall. Copyright © 2011 Elsevier Ltd. All rights reserved.

Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach.

PubMed

Lehmann, Andreas; Wixted, Josephine H F; Shapovalov, Maxim V; Roder, Heinrich; Dunbrack, Roland L; Robinson, Matthew K

2015-01-01

Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

Binding of transcription termination protein nun to nascent RNA and template DNA.

PubMed

Watnick, R S; Gottesman, M E

1999-12-17

The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation. RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA. To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts. When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template. Nun may arrest transcription by anchoring RNAP to DNA.

New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method

PubMed Central

Kasarjian, Julie K. A.; Iida, Masatake; Ryu, Junichi

2003-01-01

The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test. To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I). Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10–1–10–2). When multiple recognition sites were present, greater reductions in EOT values were observed. Once established in the cell, the plasmids were subjected to modification (EOT = 1.0). We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells. Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively. Eco1158I, an isoschizomer of EcoBI, was also found in this study. PMID:12595571

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts.

PubMed

Hirozane-Kishikawa, Tomoko; Shiraki, Toshiyuki; Waki, Kazunori; Nakamura, Mari; Arakawa, Takahiro; Kawai, Jun; Fagiolini, Michela; Hensch, Takao K; Hayashizaki, Yoshihide; Carninci, Piero

2003-09-01

The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.

Highly repressible expression system for cloning genes that specify potentially toxic proteins.

PubMed Central

O'Connor, C D; Timmis, K N

1987-01-01

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational. Images PMID:2443481

Domestication of Lambda Phage Genes into a Putative Third Type of Replicative Helicase Matchmaker

PubMed Central

Brézellec, Pierre; Petit, Marie-Agnès; Pasek, Sophie; Vallet-Gely, Isabelle; Possoz, Christophe

2017-01-01

Abstract At the onset of the initiation of chromosome replication, bacterial replicative helicases are recruited and loaded on the DnaA-oriC nucleoprotein platform, assisted by proteins like DnaC/DnaI or DciA. Two orders of bacteria appear, however, to lack either of these factors, raising the question of the essentiality of these factors in bacteria. Through a phylogenomic approach, we identified a pair of genes that could have substituted for dciA. The two domesticated genes are specific of the dnaC/dnaI- and dciA-lacking organisms and apparently domesticated from lambdoid phage genes. They derive from λO and λP and were renamed dopC and dopE, respectively. DopE is expected to bring the replicative helicase to the bacterial origin of replication, while DopC might assist DopE in this function. The confirmation of the implication of DopCE in the handling of the replicative helicase at the onset of replication in these organisms would generalize to all bacteria and therefore to all living organisms the need for specific factors dedicated to this function. PMID:28854626

Analysis of noise in quorum sensing.

PubMed

Cox, Chris D; Peterson, Gregory D; Allen, Michael S; Lancaster, Joseph M; McCollum, James M; Austin, Derek; Yan, Ling; Sayler, Gary S; Simpson, Michael L

2003-01-01

Noise may play a pivotal role in gene circuit functionality, as demonstrated for the genetic switch in the bacterial phage lambda. Like the lambda switch, bacterial quorum sensing (QS) systems operate within a population and contain a bistable switching element, making it likely that noise plays a functional role in QS circuit operation. Therefore, a detailed analysis of the noise behavior of QS systems is needed. We have developed a set of tools generally applicable to the analysis of gene circuits, with an emphasis on investigations in the frequency domain (FD), that we apply here to the QS system in the marine bacterium Vibrio fischeri. We demonstrate that a tight coupling between exact stochastic simulation and FD analysis provides insights into the structure/function relationships in the QS circuit. Furthermore, we argue that a noise analysis is incomplete without consideration of the power spectral densities (PSDs) of the important molecular output signals. As an example we consider reversible reactions in the QS circuit, and show through analysis and exact stochastic simulation that these circuits make significant and dynamic modifications to the noise spectra. In particular, we demonstrate a "whitening" effect, which occurs as the noise is processed through these reversible reactions.

Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richardson, C.C.

1993-12-31

This project focuses on the DNA polymerase (gene 5 protein) of phage T7 for use in DNA sequence analysis. Gene 5 protein interacts with accessory proteins to acquire properties essential for DNA replication. One goal is to understand these interactions in order to modify the proteins for use in DNA sequencing. E. coli thioredoxin, binds to gene 5 protein and clamps it to a primer-template. They have analyzed the binding of gene 5 protein-thioredoxin to primer-templates and have defined the optimal conditions to form an extremely stable complex with a dNTP in the polymerase catalytic site. The spatial proximity ofmore » these components has been determined using fluorescence emission anisotropy. The T7 DNA binding protein, the gene 2.5 protein, interacts with gene 5 protein and gene 4 protein to increase processivity and primer synthesis, respectively. Mutant gene 2.5 proteins have been isolated that do not interact with T7 DNA polymerase and can not support T7 growth. The nucleotide binding site of the T7 helicase has been identified and mutations affecting the site provide information on how the hydrolysis of NTPs fuel its unidirectional translocation. The sequence, GTC, has been shown to be necessary and sufficient for recognition by the T7 primase. The T7 gene 5.5 protein interacts with the E. coli nucleoid protein, H-NS, and also overcomes the phage {lambda} rex restriction system.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maaloum, M.; Muller, P.; Beker, A-F.

Almost two decades ago, measurements of force versus extension on isolated double-stranded DNA molecules revealed a force plateau. This unusual stretching phenomenon in DNA suggests that the long molecules may be extended from the usual B form into a new conformation. Different models have been proposed to describe the nature of DNA in its stretched form, S-DNA. Using atomic force microscopy combined with a molecular combing method, we identified the structure of {lambda}-phage DNA for different stretching values. We provide strong evidence for the existence of a first-order transition between B form and S form. Beyond a certain extension ofmore » the natural length, DNA molecules adopt a new double-helix conformation characterized by a diameter of 1.2 nm and a helical pitch of18 nm.« less

A Single Molecule Study of Two Bacteriophage Epigenetic Switches

NASA Astrophysics Data System (ADS)

Wang, Haowei

Epigenetic switches allow organisms to evolve into different states by activating/repressing different sets of genes without mutations of the underlying DNA sequence. The study of epigenetic switches is very important to understand the mechanism of human development, the origin of cancer, mental illness and fundamental processes such as gene regulation. The coliphage lambda epigenetic switch, which allows switching from lysogeny to lysis, has been studied for more than 50 years as a paradigm, and has recently received renewed attention. Atomic force microscopy (AFM) was used here to show that the lambda repressor oligomerizes on DNA, primarily as a dodecamer, to secure a DNA loop, which is the basis of the lambda switch. This study also provides support for the idea that specifically bound repressor stabilizes adjacent, non-specifically bound repressor molecules, which confers robustness to the switch. 186 is a member of a different coliphage family. One of the major differences between the two coliphage families is that lambda phages can be induced to switch from the lysogenic to the lytic state by UV radiation, but most coliphages of P2 family, to which 186 belongs, cannot. Interaction between coliphage 186 repressor and DNA is characterized by AFM and tethered particle motion (TPM). To expedite analysis of the AFM data, MatLab codes were written to automate the laborious, manual tracing procedures. The programs automatically recognize DNA segments and protein particles in an image, in order to measure the DNA length and position of bound particles as well as their height, diameter and volume. Application of these algorithms greatly improved the efficiency of AFM analysis. It was showed that 186 CI dimers form heptameric wheels, which induce DNA wrapping and different kinds of DNA looping producing various conformations of nucleoprotein complexes. Information about the dynamics of DNA wrapping and looping on 186 CI particles was also obtained by TPM.

Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli

NASA Astrophysics Data System (ADS)

Küpper, Hans; Keller, Walter; Kurz, Christina; Forss, Sonja; Schaller, Heinz

1981-02-01

Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322. A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus. The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage λ PL promoter. In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay.

Pulse Detecting Genetic Circuit – A New Design Approach

PubMed Central

Inniss, Mara; Iba, Hitoshi; Way, Jeffrey C.

2016-01-01

A robust cellular counter could enable synthetic biologists to design complex circuits with diverse behaviors. The existing synthetic-biological counters, responsive to the beginning of the pulse, are sensitive to the pulse duration. Here we present a pulse detecting circuit that responds only at the falling edge of a pulse–analogous to negative edge triggered electric circuits. As biological events do not follow precise timing, use of such a pulse detector would enable the design of robust asynchronous counters which can count the completion of events. This transcription-based pulse detecting circuit depends on the interaction of two co-expressed lambdoid phage-derived proteins: the first is unstable and inhibits the regulatory activity of the second, stable protein. At the end of the pulse the unstable inhibitor protein disappears from the cell and the second protein triggers the recording of the event completion. Using stochastic simulation we showed that the proposed design can detect the completion of the pulse irrespective to the pulse duration. In our simulation we also showed that fusing the pulse detector with a phage lambda memory element we can construct a counter which can be extended to count larger numbers. The proposed design principle is a new control mechanism for synthetic biology which can be integrated in different circuits for identifying the completion of an event. PMID:27907045

Pulse Detecting Genetic Circuit - A New Design Approach.

PubMed

Noman, Nasimul; Inniss, Mara; Iba, Hitoshi; Way, Jeffrey C

2016-01-01

A robust cellular counter could enable synthetic biologists to design complex circuits with diverse behaviors. The existing synthetic-biological counters, responsive to the beginning of the pulse, are sensitive to the pulse duration. Here we present a pulse detecting circuit that responds only at the falling edge of a pulse-analogous to negative edge triggered electric circuits. As biological events do not follow precise timing, use of such a pulse detector would enable the design of robust asynchronous counters which can count the completion of events. This transcription-based pulse detecting circuit depends on the interaction of two co-expressed lambdoid phage-derived proteins: the first is unstable and inhibits the regulatory activity of the second, stable protein. At the end of the pulse the unstable inhibitor protein disappears from the cell and the second protein triggers the recording of the event completion. Using stochastic simulation we showed that the proposed design can detect the completion of the pulse irrespective to the pulse duration. In our simulation we also showed that fusing the pulse detector with a phage lambda memory element we can construct a counter which can be extended to count larger numbers. The proposed design principle is a new control mechanism for synthetic biology which can be integrated in different circuits for identifying the completion of an event.

Protein chainmail variants in dsDNA viruses

PubMed Central

Zhou, Z. Hong; Chiou, Joshua

2017-01-01

First discovered in bacteriophage HK97, biological chainmail is a highly stable system formed by concatenated protein rings. Each subunit of the ring contains the HK97-like fold, which is characterized by its submarine-like shape with a 5-stranded β sheet in the axial (A) domain, spine helix in the peripheral (P) domain, and an extended (E) loop. HK97 capsid consists of covalently-linked copies of just one HK97-like fold protein and represents the most effective strategy to form highly stable chainmail needed for dsDNA genome encapsidation. Recently, near-atomic resolution structures enabled by cryo electron microscopy (cryoEM) have revealed a range of other, more complex variants of this strategy for constructing dsDNA viruses. The first strategy, exemplified by P22-like phages, is the attachment of an insertional (I) domain to the core 5-stranded β sheet of the HK97-like fold. The atomic models of the Bordetella phage BPP-1 showcases an alternative topology of the classic HK97 topology of the HK97-like fold, as well as the second strategy for constructing stable capsids, where an auxiliary jellyroll protein dimer serves to cement the non-covalent chainmail formed by capsid protein subunits. The third strategy, found in lambda-like phages, uses auxiliary protein trimers to stabilize the underlying non-covalent chainmail near the 3-fold axis. Herpesviruses represent highly complex viruses that use a combination of these strategies, resulting in four-level hierarchical organization including a non-covalent chainmail formed by the HK97-like fold domain found in the floor region. A thorough understanding of these structures should help unlock the enigma of the emergence and evolution of dsDNA viruses and inform bioengineering efforts based on these viruses. PMID:29177192

Stable micron-scale holes are a general feature of canonical holins

PubMed Central

Savva, Christos G.; Dewey, Jill S.; Moussa, Samir H.; To, Kam H.; Holzenburg, Andreas; Young, Ry

2014-01-01

Summary At a programmed time in phage infection cycles, canonical holins suddenly trigger to cause lethal damage to the cytoplasmic membrane, resulting in the cessation of respiration and the non-specific release of pre-folded, fully active endolysins to the periplasm. For the paradigm holin S105 of lambda, triggering is correlated with the formation of micron-scale membrane holes, visible as interruptions in the bilayer in cryo-electron microscopic images and tomographic reconstructions. Here we report that the size distribution of the holes is stable for long periods after triggering. Moreover, early triggering caused by an early lysis allele of S105 formed approximately the same number of holes, but the lesions were significantly smaller. In contrast, early triggering prematurely induced by energy poisons resulted in many fewer visible holes, consistent with previous sizing studies. Importantly, the unrelated canonical holins P2 Y and T4 T were found to cause the formation of holes of approximately the same size and number as for lambda. In contrast, no such lesions were visible after triggering of the pinholin S2168. These results generalize the hole formation phenomenon for canonical holins. A model is presented suggesting the unprecedentedly large size of these holes is related to the timing mechanism. PMID:24164554

Stable micron-scale holes are a general feature of canonical holins.

PubMed

Savva, Christos G; Dewey, Jill S; Moussa, Samir H; To, Kam H; Holzenburg, Andreas; Young, Ry

2014-01-01

At a programmed time in phage infection cycles, canonical holins suddenly trigger to cause lethal damage to the cytoplasmic membrane, resulting in the cessation of respiration and the non-specific release of pre-folded, fully active endolysins to the periplasm. For the paradigm holin S105 of lambda, triggering is correlated with the formation of micron-scale membrane holes, visible as interruptions in the bilayer in cryo-electron microscopic images and tomographic reconstructions. Here we report that the size distribution of the holes is stable for long periods after triggering. Moreover, early triggering caused by an early lysis allele of S105 formed approximately the same number of holes, but the lesions were significantly smaller. In contrast, early triggering prematurely induced by energy poisons resulted in many fewer visible holes, consistent with previous sizing studies. Importantly, the unrelated canonical holins P2 Y and T4 T were found to cause the formation of holes of approximately the same size and number as for lambda. In contrast, no such lesions were visible after triggering of the pinholin S(21) 68. These results generalize the hole formation phenomenon for canonical holins. A model is presented suggesting the unprecedentedly large size of these holes is related to the timing mechanism. © 2013 John Wiley & Sons Ltd.

Induction of lambda prophage by 213 nm laser radiation: a quantitative comparison with 193 nm excimer radiation using image analysis.

PubMed

Matchette, L S; Grossman, L W; Hahn, D W; Cooney, C

1996-03-01

We compared the DNA damage produced by radiation from two UV laser wavelengths, 213 nm and 193 nm, with that produced by noncoherent 254 nm radiation. Following irradiation of Escherichia coli BR339, a bacteriophage lambda lysogen containing the lacZ gene, pro-phage induction was measured by assaying for beta-galactosidase. Because of the limited penetration by UV laser wavelengths an agar overlay of the lysogen was used as the irradiation target. Irradiation of 254 nm was performed in buffer suspension followed by transfer of 5 microL spots onto assay plants. Computer image analysis was used to monitor the rate of product formation, observed as an increase in optical density of the irradiated zones on assay plates. We found that the rate of product formation was a more reproducible unit of comparison than the optical density present at the end of the reaction. Although the rate of product formation was not linearly related to enzyme concentration, the data could be fit to a simple logarithmic function. Using this method, we concluded that the DNA damaging ability of 213 nm radiation was 10 times more efficient than 193 nm radiation and about 100 times less efficient than 254 nm noncoherent radiation.

Assignment of the human type I IMP dehydrogenase gene (IMPDH1) to chromosome 7q31.3-q32

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, Jing Jin; Kaiser-Rogers, K.; Rao, K.

Two phage {lambda} clones that contain 5{prime} portion of the human type I inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) gene were isolated. Both polymerase chain reaction analysis of a panel of human-mouse and human-hamster cell somatic hybrids using primers specific for the type I IMPDH gene and fluorescence in situ hybridization with metaphase human chromosome using type I IMPDH genomic DNA as probes indicate that the type I IMPDH gene (symbol IMPDH1) is located on chromosome 7. Sequential GTG-banding was performed to assign the band location of the type I IMPDH gene to chromosome 7q31.3-q32. 16 refs., 1 fig., 1more » tab.« less

tif-1 mutation alters polynucleotide recognition by the recA protein of Escherichia coli.

PubMed Central

McEntee, K; Weinstock, G M

1981-01-01

The requirements for polynucleotide-dependent hydrolysis of ATP and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (recA+ protein) and the tif-1 mutant form [tif(recA) protein] of the recA gene product. The recA+ and tif(recA) proteins catalyze both reactions in the presence of long single-stranded DNAs or certain deoxyhomopolymers. However, short oligonucleotides [(dT)12, (dA)14] stimulate neither the protease nor the ATPase activities of the recA+ protein. In contrast, these short oligonucleotides activate tif(recA) protein to cleave lambda repressor without stimulating its ATPase activity. Moreover, both the ATPase and protease activities of the tif(recA) protein are stimulated by poly(rU) and poly(rC) whereas the recA+ protein does not respond to these ribopolymers. We have purified the recA protein from a strain in which the tif mutation is intragenically suppressed. This mutant protein (recA629) is inactive in the presence of (dT)12, (dA)14, poly(rU), and poly(rC) for lambda repressor cleavage and ATP hydrolysis. These results argue that the tif-1 mutation (or mutations) alters the DNA binding site of the recA protein. We suggest that in vivo the tif(recA) protein is activated for cleaving repressors of SOS genes by complex formation with short single-stranded regions or gaps that normally occur near the growing fork of replicating chromosomes and are too short for activating the recA+ enzyme. This mechanism can account for the expression of SOS functions in the absence of DNA damage in tif mutant strains. Images PMID:7031642

Determination of the termination efficiency of the transcription terminator using different fluorescent profiles in green fluorescent protein mutants.

PubMed

Nojima, Takahiko; Lin, Angela C; Fujii, Teruo; Endo, Isao

2005-12-01

An approach in determining the intrinsic termination efficiency (%T) of transcription termination using green fluorescent protein (GFP) mutants was developed. This approach utilizes a cassette vector in which the tested terminator is introduced between two GFP mutant genes: an ultraviolet-optimized mutant (GFPuv: F99S, M153T, V163A) and a blue-shifted mutant (BFP: F64L, S65T, T145F). The ratio of the fluorescence intensity of BFP to GFPuv after transcription and translation represents the termination efficiency of the terminator. E. coli ribosomal RNA operon T1 terminator, phage lambda terminator site R2, E. coli tryptophane attenuater were introduced into the vector, and their transcriptional efficiencies were estimated as 89, 79, and 24%, respectively, showing good agreement with published data.

Occurrence of small Hsd plasmids in Salmonella typhi, Shigella boydii, and Escherichia coli.

PubMed Central

Yoshida, Y; Mise, K

1986-01-01

The natural occurrence of small Hsd (host specificity for DNA) plasmids was demonstrated in restriction endonuclease-producing strains of Salmonella typhi, Shigella boydii, and Escherichia coli. The five Hsd plasmids isolated were between 5.0 and 12.2 kilobases long. The copy number of all the Hsd plasmids was high (more than 10 copies per cell). Introduction of these small plasmids into E. coli strain 0 drastically lowered the efficiency of plating of the lambda.0 phages (the efficiency of plating was less than 5 X 10(-5) PFU-1). High restriction endonuclease activities were detected in the Hsd plasmid-positive strains because of the elevated copy numbers of the hsdR+ gene. The advantages of using E. coli strains containing the small Hsd plasmids for purification of type II restriction endonucleases are discussed. Images PMID:3003023

Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

PubMed Central

Hayes, Sidney; Erker, Craig; Horbay, Monique A.; Marciniuk, Kristen; Wang, Wen; Hayes, Connie

2013-01-01

The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step. PMID:23389467

Cloning structural genes for Treponema pallidum immunogens and characterisation of recombinant treponemal surface protein, P2 (P2 star).

PubMed Central

Peterson, K M; Baseman, J B; Alderete, J F

1987-01-01

A genomic library consisting of partially digested 10 to 20 kilobase pair fragments of Treponema pallidum deoxyribonucleic acid (DNA) was constructed using bacteriophage lambda EMBL-3 as the vector. Positive clones expressing T pallidum antigens were detected with sera from experimentally infected rabbits. Treponemal proteins ranging in molecular weight from 37,000 daltons to 120,000 daltons were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting of phage lysate proteins. One recombinant phage was examined further and contained an insert encoding a prominent treponemal 37,000 dalton protein. The recombinant protein was not recognised by antiserum directed against a fibronectin binding treponemal adhesion that contained the same electrophoretic mobility. Neither did antibody to the recombinant 37,000 dalton protein react with any treponemal proteins purified by fibronectin affinity chromatography. The recombinant protein in Escherichia coli lysates was labelled P2 (P2 star) to differentiate it from the comigrating adhesin protein called P2. Native P2 protein was present on T pallidum surfaces as shown by radioimmunoprecipitation assays with extrinsically labelled organisms. A cross reactive molecule like P2 was not synthesised by the avirulent spirochaete, T phagedenis biotype Reiter, which indicated that P2 is a protein specific to virulent T pallidum organisms. Finally, only sera of patients with primary syphilis possessed appreciable concentrations of antibody to recombinant P2 protein. Images PMID:3315959

Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli.

PubMed Central

Rule, G S; Pratt, E A; Chin, C C; Wold, F; Ho, C

1985-01-01

Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH. Images PMID:3882663

On the equivalence of some spectral sequences for Serre fibrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onishchenko, Aleksandr Yu; Popelenskii, Fedor Yu

2011-04-30

Several different constructions of a spectral sequence for a Serre fibration {pi}:E{yields}B over a compact simply connected manifold B are considered in this paper. Namely, we consider the spectral sequence for the minimal model ({Lambda}Vx{Lambda}W,d) of the fibration, along with the spectral sequences arising from the Cech filtration in the complexes C*(U,A*{sub PL}({pi}{sup -1}(U))) and C*(U,S*({pi}{sup -1}(U))), where U=(U) is a covering of the base B. It is known that all these spectral sequences have the same terms E{sub 2}*{sup ,}*=H*(X)xH{sup *}(F) and converge to the cohomology of the total space E. A new natural isomorphism of these spectral sequencesmore » is constructed in every term E{sub r} with r{>=}2. It is also proved that in the case of a smooth locally trivial fibration these spectral sequences are isomorphic to the spectral sequences of the complex of smooth forms {Omega}*(E) and of the Cech-de Rham complex. It is therefore established that all these constructions give the same spectral sequence, starting from the E{sub 2} term. Bibliography: 9 titles.« less

On the equivalence of some spectral sequences for Serre fibrations

NASA Astrophysics Data System (ADS)

Onishchenko, Aleksandr Yu; Popelenskii, Fedor Yu

2011-04-01

Several different constructions of a spectral sequence for a Serre fibration \\pi\\colon E \\to B over a compact simply connected manifold B are considered in this paper. Namely, we consider the spectral sequence for the minimal model (\\Lambda V\\otimes \\Lambda W,d) of the fibration, along with the spectral sequences arising from the Čech filtration in the complexes \\check{C}^*(\\mathscr{U}, A_{PL}^*(\\pi^{-1}(U))) and \\check{C}^*(\\mathscr{U}, S^*(\\pi^{-1}(U))), where \\mathscr{U}=\\{U\\} is a covering of the base B. It is known that all these spectral sequences have the same terms E_2^{*,*}=H^*(X)\\otimes H^*(F) and converge to the cohomology of the total space E. A new natural isomorphism of these spectral sequences is constructed in every term E_r with r\\ge2. It is also proved that in the case of a smooth locally trivial fibration these spectral sequences are isomorphic to the spectral sequences of the complex of smooth forms \\Omega^*(E) and of the Čech-de Rham complex. It is therefore established that all these constructions give the same spectral sequence, starting from the E_2 term. Bibliography: 9 titles.

Escherichia coli mutants impaired in maltodextrin transport.

PubMed

Wandersman, C; Schwartz, M; Ferenci, T

1979-10-01

Wild-type Escherichia coli K-12 was found to grow equally well on maltose and on maltodextrins containing up to seven glucose residues. Three classes of mutants unable to grow on maltodextrins, but still able to grow on maltose, were investigated in detail. The first class, already known, was composed of phage lambda-resistant mutants, which lack the outer membrane protein coded by gene lamB. These mutants grow on maltose and maltotriose but not at all on maltotetraose and longer maltodextrins which cannot cross the outer membrane. A second class of mutants were affected in malE, the structural gene of the periplasmic maltose binding protein. The maltose binding proteins isolated from the new mutants were altered in their substrate binding properties, but not in a way that could account for the mutant phenotypes. Rather, the results of growth experiments and transport studies suggest that these malE mutants are impaired in their ability to transport maltodextrins across the outer membrane. This implies that the maltose binding protein (in wild-type strains) cooperates with the lambda receptor in permeation through the outer membrane. The last class of mutants described in this paper were affected in malG, or perhaps in an as yet undetected gene close to malG. They were defective in the transfer of maltodextrins from the periplasmic space to the cytoplasm but only slightly affected in the transport of maltose.

T7 RNA polymerase-driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis.

PubMed

Juhas, Mario; Ajioka, James W

2017-11-01

The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome. In this system, T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. We showed that T7 RNA polymerase significantly increases efficiency of cell lysis and transfer of the plasmid and bacterial artificial chromosome-encoded DNA from the lysed E. coli into B. subtilis. The T7 RNA polymerase-driven inducible cell lysis system is suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Determination of transcriptional units and gene products from the ftsA region of Escherichia coli.

PubMed Central

Lutkenhaus, J F; Wu, H C

1980-01-01

Lambda transducing phage gamma 16-2 carries the genes envA, ftsZ, ftsA, ddl, and murC and directs the synthesis of six unique proteins in ultraviolet-irradiated cells. Various derivatives of gamma 16-2 carrying smaller segments of the bacterial deoxyribonucleic acid have also been analyzed for their capacity to direct protein synthesis in ultraviolet-irradiated cells. These results, in combination with genetic results, have allowed the gene product of each of these genes to be assigned. In addition, an unidentified gene was located counterclockwise to murC between murC and murF. Analysis of the direction of transcription indicates that murC, ddl, ftsA, and ftsZ are transcribed clockwise on the Escherichia coli genetic map, and envA is transcribed counterclockwise. In addition, it is shown that each of the genes envA, ftsZ, and ftsA can be expressed independently. Images PMID:6447690

Long-range correlations and charge transport properties of DNA sequences

NASA Astrophysics Data System (ADS)

Liu, Xiao-liang; Ren, Yi; Xie, Qiong-tao; Deng, Chao-sheng; Xu, Hui

2010-04-01

By using Hurst's analysis and transfer approach, the rescaled range functions and Hurst exponents of human chromosome 22 and enterobacteria phage lambda DNA sequences are investigated and the transmission coefficients, Landauer resistances and Lyapunov coefficients of finite segments based on above genomic DNA sequences are calculated. In a comparison with quasiperiodic and random artificial DNA sequences, we find that λ-DNA exhibits anticorrelation behavior characterized by a Hurst exponent 0.5

Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse.

PubMed Central

Kominami, R; Mishima, Y; Urano, Y; Sakai, M; Muramatsu, M

1982-01-01

A Eco RI 6.6 kb DNA fragment containing the 3'-end of 28S ribosomal RNA gene of the mouse was detected by Southern blot hybridization, and cloned in a lambda-phage vector. The site of transcription termination and the processed 3'-end of 28S RNA were determined on the cloned fragment and the surrounding nucleotide sequence determined. The 3'-terminal nucleotides of mouse 28S RNA are similar to those of yeast, Drosophila and Xenopus although the homology was lost drastically beyond the 3'-end of 28S RNA. 45S precursor RNA terminated at 30 nucleotides downstream from the 3'-end of 28S RNA gene. A structure of a dyad symmetry with a loop was found immediately prior to the termination site of 45S RNA. The rDNA termination site thus shares some common features with termination sites recognized by other RNA polymerases. Images PMID:6281727

Single-Molecule Denaturation Mapping of DNA in Nanofluidic Channels

NASA Astrophysics Data System (ADS)

Reisner, Walter; Larsen, Niels; Silahtaroglu, Asli; Kristensen, Anders; Tommerup, Niels; Tegenfeldt, Jonas O.; Flyvbjerg, Henrik

2010-03-01

Nanochannel based DNA stretching can serve as a platform for a new optical mapping technique based on measuring the pattern of partial melting along the extended molecules. We partially melt DNA extended in nanofluidic channels via a combination of local heating and added chemical denaturants. The melted molecules, imaged via a standard fluorescence videomicroscopy setup, exhibit a nonuniform fluorescence profile corresponding to a series of local dips and peaks in the intensity trace along the stretched molecule. We show that this barcode is consistent with the presence of locally melted regions along the molecule and can be explained by calculations of sequence-dependent melting probability. Specifically, we obtain experimental melting profiles for T4, T7, lambda-phage and bacterial artificial chromosome DNA (from human chromosome 12) and compare these profiles to theory. In addition, we demonstrate that the BAC melting profile can be used to align the BAC to its correct position on chromosome 12.

Biophysics and bioinformatics of transcription regulation in bacteria and bacteriophages

NASA Astrophysics Data System (ADS)

Djordjevic, Marko

2005-11-01

Due to rapid accumulation of biological data, bioinformatics has become a very important branch of biological research. In this thesis, we develop novel bioinformatic approaches and aid design of biological experiments by using ideas and methods from statistical physics. Identification of transcription factor binding sites within the regulatory segments of genomic DNA is an important step towards understanding of the regulatory circuits that control expression of genes. We propose a novel, biophysics based algorithm, for the supervised detection of transcription factor (TF) binding sites. The method classifies potential binding sites by explicitly estimating the sequence-specific binding energy and the chemical potential of a given TF. In contrast with the widely used information theory based weight matrix method, our approach correctly incorporates saturation in the transcription factor/DNA binding probability. This results in a significant reduction in the number of expected false positives, and in the explicit appearance---and determination---of a binding threshold. The new method was used to identify likely genomic binding sites for the Escherichia coli TFs, and to examine the relationship between TF binding specificity and degree of pleiotropy (number of regulatory targets). We next address how parameters of protein-DNA interactions can be obtained from data on protein binding to random oligos under controlled conditions (SELEX experiment data). We show that 'robust' generation of an appropriate data set is achieved by a suitable modification of the standard SELEX procedure, and propose a novel bioinformatic algorithm for analysis of such data. Finally, we use quantitative data analysis, bioinformatic methods and kinetic modeling to analyze gene expression strategies of bacterial viruses. We study bacteriophage Xp10 that infects rice pathogen Xanthomonas oryzae. Xp10 is an unusual bacteriophage, which has morphology and genome organization that most closely resembles temperate phages, such as lambda. It, however, encodes its own T7-like RNA polymerase (characteristic of virulent phages), whose role in gene expression was unclear. Our analysis resulted in quantitative understanding of the role of both host and phage RNA polymerase, and in the identification of the previously unknown promoter sequence for Xp10 RNA polymerase. More generally, an increasing number of phage genomes are being sequenced every year, and we expect that methods of quantitative data analysis that we introduced will provide an efficient way to study gene expression strategies of novel bacterial viruses.

[Construction of the new Escherichia coli K-12 wild-type strain with improved growth characteristics for application in metabolic engineering].

PubMed

Biriukova, I V; Krylov, A A; Kiseleva, E M; Minaeva, N I; Mashko, S V

2010-03-01

MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The "polar effect" of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val(S) phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph(wt) from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying lambda attL-Cm(R)-lambda attR and transduced with phage P1 into MG1655; later, the Cm(R) marker was removed with the use of lambda Xis/Int recombinase. Parallel to this procedure, a spontaneous Val(R) mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of Val (50 microg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val(R) strain had been generated in the region of the identified E. coli K-12 ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val(R) phenotype in the obtained strain, was transferred to MG1655-rph(wt) using cotransduction, by analogy to the transfer of rph(wt). Evaluation of rates of aerobically growing cells (microm, hour(-1)) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655, MG1655-rph(wt), and MG1655-(rph(wt), ilvG-15), respectively.

Analysis of autoimmune bone marrow by antibody-phage display: somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes.

PubMed

Seal, S N; Hoet, R M; Raats, J M; Radic, M Z

2000-09-01

To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.

Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages

PubMed Central

Datta, Simanti; Costantino, Nina; Zhou, Xiaomei; Court, Donald L.

2008-01-01

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (λ) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria. PMID:18230724

Molecular cloning and characterization of Hymenolepis diminuta alpha-tubulin gene.

PubMed

Mohajer-Maghari, Behrokh; Amini-Bavil-Olyaee, Samad; Webb, Rodney A; Coe, Imogen R

2007-02-01

To isolate a full-length alpha-tubulin cDNA from an eucestode, Hymenolepis diminuta, a lambda phage cDNA library was constructed. The alpha-tubulin gene was cloned, sequenced and characterized. The H. diminuta alpha-tubulin consisted of 450 amino acids. This protein contained putative sites for all posttranslational modifications as detyrosination/tyrosination at the carboxyl-terminal of protien, phosphorylation at residues R79 and K336, glycylation/glutamylation at residue G445 and acetylation at residue K40. Comparisons of H. diminuta alpha-tubulin with all full-length alpha-tubulin proteins revealed that H. diminuta alpha-tubulin possesses 10 distinctive residues, which are not found in any other alpha-tubulins. Phylogenetic analysis showed that H. diminuta alpha-tubulin has grouped in a separated branch adjacent eucestode and trematodes branch with 92% bootstrap value (1000 replicates). In conclusion, this is the first report of H. diminuta cDNA library construction, cloning and characterization of H. diminuta alpha-tubulin gene.

Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification.

PubMed

Carnes, Aaron E; Hodgson, Clague P; Luke, Jeremy M; Vincent, Justin M; Williams, James A

2009-10-15

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non-ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid-liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts.

The effects of temperature and magnetic flux on electron transport through a four-channel DNA model

NASA Astrophysics Data System (ADS)

Lee, Sunhee; Hedin, Eric; Joe, Yong

2010-03-01

The temperature dependence of the conductivity of lambda phage DNA has been measured by Tran et al [1] experimentally, where the conductivity displayed strong (weak) temperature dependence above (below) a threshold temperature. In order to understand the temperature effects of electron transport theoretically, we study a two-dimensional and four-channel DNA model using a tight-binding (TB) Hamiltonian. The thermal effects within a TB model are incorporated into the hopping integral and the relative twist angle from its equilibrium value between base-pairs. Since these thermal structural fluctuations localize the electronic wave functions in DNA, we examine a temperature-dependent localization length, a temperature-driven transmission, and current-voltage characteristics in this system. In addition, we incorporate magnetic field effects into the analysis of the transmission through DNA in order to modulate the quantum interference between the electron paths that comprise the 4-channel structure. [1] P. Tran, B. Alavi, and G. Gruner, PRL 85, 1564 (2000).

A novel cell autolysis system for cost-competitive downstream processing.

PubMed

Hajnal, Ivan; Chen, Xiangbin; Chen, Guo-Qiang

2016-11-01

The industrial production of low value-added biological products poses significant challenges due to cost pressures. In recent years, it has been argued that synthetic biology approaches will lead to breakthroughs that eliminate price bottlenecks for the production of a wide range of biological products including bioplastics and biofuels. One significant bottleneck lies in the necessity to break the tough cell walls of microbes in order to release intracellular products. We here report the implementation of the first synthetic biology standard part based on the lambda phage SRRz genes and a synthetic ribosome binding site (RBS) that works in Escherichia coli and Halomonas campaniensis, which enables the producer strains to induce lysis after the addition of small amounts (1-5 %) of solvents or to spontaneously lyse during the stresses of downstream processing, and thus has the potential to eliminate the mechanical cell disruption step as both an efficiency bottleneck and a significant capex barrier when implementing downstream bioprocesses.

A homolog of Escherichia coli RecA in mitochondria of the cellular slime mold Dictyostelium discoideum.

PubMed

Hasegawa, Yasuna; Wakabayashi, Masayuki; Nakamura, Shogo; Kodaira, Ken-ichi; Shinohara, Hiroaki; Yasukawa, Hiro

2004-05-04

The cellular slime mold Dictyostelium discoideum expresses a gene encoding a 452-amino-acid polypeptide that is 47% identical to Escherichia coli RecA. A recA-deficient E. coli, JE6651, was transformed by pYSN1, which was designed to express the truncated form of the D. discoideum gene, and used in suppression assays. The viability of the transformant, JE6651(pYSN1), increased following UV irradiation or mitomycin C treatment. Phage lambda (red(-) gam(-)), which required RecA activity for DNA packaging, formed plaques on a lawn of JE6651(pYSN1). These results indicate that the gene product has a DNA recombination activity. Fluorescence of D. discoideum protein fused with GFP was detected in mitochondria. The gene disruption mutant was hypersensitive to UV-light (254nm), mitomycin C and H(2)O(2), indicating that D. discoideum recA is important for survival following exposure to DNA damaging agents.

[Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

PubMed

Chen, Xiao-hong; Chen, Zhi; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan; Yao, Hang-ping

2005-03-01

To construct a cDNA library from human liver tissue of cirrhosis. The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

Assignment of the gene encoding the 5-HT{sub 1E} serotonin receptor (S31) (locus HTR1E) to human chromosome 6q14-q15

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levy, F.O.; Tasken, K.; Solberg, R.

1994-08-01

The human gene for the 5-HT{sub 1E} serotonin receptor was recently cloned, but no chromosomal assignment has yet been given to this gene (locus HTR1E). In this work, we demonstrate by two independent polymerase chain reactions on a panel of human-hamster somatic cell hybrid genomic DNA that the 5-HT{sub 1E} serotonin receptor gene is localized on human chromosome 6. Furthermore, by means of in situ hybridization to human metaphase chromosomes, using the cloned 5-HT{sub 1E} receptor gene (phage clone {lambda}-S31) as a probe, we demonstrate that this gene is localized to the q14-q15 region on chromosome 6. Screening of genomicmore » DNA from 15 unrelated Caucasian individuals, using as a probe the open reading frame of the cloned 5-HT{sub 1E} receptor gene, did not reveal any restriction fragment length polymorphisms with the enzymes BamHI, BanII, BglII, EcoRI, HincII, HindIII, HinfI, MspI, PstI, and PvuII. Since the 5-HT{sub 1E} receptor is found mainly in the cerebral cortex and abnormal function of the serotonergic system has been implicated in a variety of neurologic and psychiatric diseases, the precise chromosomal assignment of the 5-HT{sub 1E} receptor gene is the crucial first step toward the evaluation of this locus as a candidate for mutations in such syndromes. 28 refs., 2 figs., 2 tabs.« less

Packaging of a unit-length viral genome: the role of nucleotides and the gpD decoration protein in stable nucleocapsid assembly in bacteriophage lambda.

PubMed

Yang, Qin; Maluf, Nasib Karl; Catalano, Carlos Enrique

2008-11-28

The developmental pathways for a variety of eukaryotic and prokaryotic double-stranded DNA viruses include packaging of viral DNA into a preformed procapsid structure, catalyzed by terminase enzymes and fueled by ATP hydrolysis. In most instances, a capsid expansion process accompanies DNA packaging, which significantly increases the volume of the capsid to accommodate the full-length viral genome. "Decoration" proteins add to the surface of the expanded capsid lattice, and the terminase motors tightly package DNA, generating up to approximately 20 atm of internal capsid pressure. Herein we describe biochemical studies on genome packaging using bacteriophage lambda as a model system. Kinetic analysis suggests that the packaging motor possesses at least four ATPase catalytic sites that act cooperatively to effect DNA translocation, and that the motor is highly processive. While not required for DNA translocation into the capsid, the phage lambda capsid decoration protein gpD is essential for the packaging of the penultimate 8-10 kb (15-20%) of the viral genome; virtually no DNA is packaged in the absence of gpD when large DNA substrates are used, most likely due to a loss of capsid structural integrity. Finally, we show that ATP hydrolysis is required to retain the genome in a packaged state subsequent to condensation within the capsid. Presumably, the packaging motor continues to "idle" at the genome end and to maintain a positive pressure towards the packaged state. Surprisingly, ADP, guanosine triphosphate, and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) similarly stabilize the packaged viral genome despite the fact that they fail to support genome packaging. In contrast, the poorly hydrolyzed ATP analog ATP-gammaS only partially stabilizes the nucleocapsid, and a DNA is released in "quantized" steps. We interpret the ensemble of data to indicate that (i) the viral procapsid possesses a degree of plasticity that is required to accommodate the packaging of large DNA substrates; (ii) the gpD decoration protein is required to stabilize the fully expanded capsid; and (iii) nucleotides regulate high-affinity DNA binding interactions that are required to maintain DNA in the packaged state.

Five-Body Cluster Structure of the Double-{Lambda} Hypernucleus {sub {Lambda}{Lambda}}{sup 11}Be

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hiyama, E.; Kamimura, M.; Yamamoto, Y.

2010-05-28

Energy levels of the double {Lambda} hypernucleus, {sub {Lambda}{Lambda}}{sup 11}Be are calculated within the framework of a {alpha}{alpha}n{Lambda}{Lambda} five-body model. Interactions between constituent particles are determined so as to reproduce reasonably the observed low-energy properties of the {alpha}{alpha}, {alpha}{alpha}n nuclei and the existing data for {Lambda}-binding energies of the {alpha}{Lambda}, {alpha}{alpha}{Lambda}, {alpha}n{Lambda}, and {alpha}{alpha}n{Lambda} systems. An effective {Lambda}{Lambda} interaction is constructed so as to reproduce, within the {alpha}{Lambda}{Lambda} three-body model, the B{sub {Lambda}{Lambda}}of {sub {Lambda}{Lambda}}{sup 6}He, which was extracted from the emulsion experiment, the NAGARA event. With no adjustable parameters for the {alpha}{alpha}n{Lambda}{Lambda} system, B{sub {Lambda}{Lambda}}of the ground and boundmore » excited states of {sub {Lambda}{Lambda}}{sup 11}Be are calculated with the Gaussian expansion method. The Hida event, recently observed at KEK-E373 experiment, is interpreted as an observation of the ground state of the {sub {Lambda}{Lambda}}{sup 11}Be.« less

Purification and DNA-binding properties of the cro-type regulatory repressor protein cng encoded by the Lactobacillus plantarum phage phi g1e.

PubMed

Kakikawa, M; Ohkubo, S; Sakate, T; Sayama, M; Taketo, A; Kodaira, K

2000-05-16

The putative repressor protein Cng (10kDa on an SDS gel) for the lytic pathway of Lactobacillus plantarum phage φg1e was purified using the Escherichia coli Pt7 system, and its DNA-binding ability for the seven operator-like sequences, the GATAC-boxes (Gb1 to Gb7), was investigated in vitro. In gel-shift assays, Cng selectively bound to the DNA fragments containing the GATAC-box(es). In addition, DNase I footprinting analysis with supercoiled DNA demonstrated that Cng can specifically cover about a 25bp region centered around each of the GATAC-boxes, although two boxes, Gb4 and Gb6, were only partially protected. Moreover, protein crosslinking experiments using glutaraldehyde suggested that Cng most likely functions as a dimer. On the other hand, the binding ability of Cpg for the GATAC-boxes in supercoiled DNA was also examined under the same conditions as in Cng; unlike Cng, Cpg covered Gb4 and Gb6 completely sufficiently as well as the other five boxes. Thus, the present and previous [Kakikawa et al., Gene 215 (1998) 371-379; 242 (2000) 155-166] results indicate a possibility that the two proteins Cng and Cpg selectively bind to the GATAC-boxes that act as operators, and can decide between the lytic or lysogenic pathways through repression of the promoter activity of P(R) as well as P(L).

Study of {Lambda}-{Lambda} oscillation in quantum coherent {Lambda}{Lambda} by using J/{psi}{yields}{Lambda}{Lambda} decay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang Xianwei; Department of Physics, Henan Normal University, Xinxiang 453007; Li Haibo

2010-03-01

We discuss the possibility of searching for the {Lambda}-{Lambda} oscillations for coherent {Lambda}{Lambda} production in the J/{psi}{yields}{Lambda}{Lambda} decay process. The sensitivity of measurement of {Lambda}-{Lambda} oscillation in the external field at BES-III experiment is considered. These considerations indicate an alternative way to probe the {Delta}B=2 amplitude in addition to neutron oscillation experiments. Both coherent and time-dependent information can be used to extract the {Lambda}-{Lambda} oscillation parameter. With one year's luminosity at BES-III, we can set an upper limit of {delta}m{sub {Lambda}{Lambda}<}10{sup -15} MeV at 90% confidence level, corresponding to about 10{sup -6} s of {Lambda}-{Lambda} oscillation time.

Phage Wrapping with Cationic Polymers Eliminates Non-specific Binding between M13 Phage and High pI Target Proteins

PubMed Central

Lamboy, Jorge A.; Arter, Jessica A.; Knopp, Kristeene A.; Der, Denise; Overstreet, Cathie M.; Palermo, Edmund; Urakami, Hiromitsu; Yu, Ting-Bin; Tezgel, Ozgul; Tew, Gregory; Guan, Zhibin; Kuroda, Kenichi; Weiss, Gregory A.

2011-01-01

M13 phage have provided scaffolds for nanostructure synthesis based upon self-assembled inorganic and hard materials interacting with phage-displayed peptides. Additionally, phage display has been used to identify binders to plastic, TiO2, and other surfaces. However, synthesis of phage-based materials through the hybridization of soft materials with the phage surface remains unexplored. Here, we present an efficient “phage wrapping” strategy for the facile synthesis of phage coated with soluble, cationic polymers. Polymers bearing high positive charge densities demonstrated the most effective phage wrapping, as shown by assays for blocking non-specific binding of the anionic phage coat to a high pI target protein. The results establish the functional group requirements for hybridizing phage with soft materials, and solve a major problem in phage display – non-specific binding by the phage to high pI target proteins. PMID:19856910

Phage wrapping with cationic polymers eliminates nonspecific binding between M13 phage and high pI target proteins.

PubMed

Lamboy, Jorge A; Arter, Jessica A; Knopp, Kristeene A; Der, Denise; Overstreet, Cathie M; Palermo, Edmund F; Urakami, Hiromitsu; Yu, Ting-Bin; Tezgel, Ozgul; Tew, Gregory N; Guan, Zhibin; Kuroda, Kenichi; Weiss, Gregory A

2009-11-18

M13 phage have provided scaffolds for nanostructure synthesis based upon self-assembled inorganic and hard materials interacting with phage-displayed peptides. Additionally, phage display has been used to identify binders to plastic, TiO(2), and other surfaces. However, synthesis of phage-based materials through the hybridization of soft materials with the phage surface remains unexplored. Here, we present an efficient "phage wrapping" strategy for the facile synthesis of phage coated with soluble, cationic polymers. Polymers bearing high positive charge densities demonstrated the most effective phage wrapping, as shown by assays for blocking nonspecific binding of the anionic phage coat to a high pI target protein. The results establish the functional group requirements for hybridizing phage with soft materials and solve a major problem in phage display-nonspecific binding by the phage to high pI target proteins.

Basic Phage Mathematics.

PubMed

Abedon, Stephen T; Katsaounis, Tena I

2018-01-01

Basic mathematical descriptions are useful in phage ecology, applied phage ecology such as in the course of phage therapy, and also toward keeping track of expected phage-bacterial interactions as seen during laboratory manipulation of phages. The most basic mathematical descriptor of phages is their titer, that is, their concentration within stocks, experimental vessels, or other environments. Various phenomena can serve to modify phage titers, and indeed phage titers can vary as a function of how they are measured. An important aspect of how changes in titers can occur results from phage interactions with bacteria. These changes tend to vary in degree as a function of bacterial densities within environments, and particularly densities of those bacteria that are susceptible to or at least adsorbable by a given phage type. Using simple mathematical models one can describe phage-bacterial interactions that give rise particularly to phage adsorption events. With elaboration one can consider changes in both phage and bacterial densities as a function of both time and these interactions. In addition, phages along with their impact on bacteria can be considered as spatially constrained processes. In this chapter we consider the simpler of these concepts, providing in particular detailed verbal explanations toward facile mathematical insight. The primary goal is to stimulate a more informed use and manipulation of phages and phage populations within the laboratory as well as toward more effective phage application outside of the laboratory, such as during phage therapy. More generally, numerous issues and approaches to the quantification of phages are considered along with the quantification of individual, ecological, and applied properties of phages.

A novel dual vector coexpressing PhiX174 lysis E gene and staphylococcal nuclease A gene on the basis of lambda promoter pR and pL, respectively.

PubMed

Fu, Lixia; Lu, Chengping

2013-06-01

Bacterial ghost is a novel vaccine platform, and its safe and efficient production depends largely upon a suitable and functional vector. In this study, a series of temperature-inducible plasmids, carrying Phix174 lysis gene E and/or staphylococcal nuclease A (SNA) gene, were constructed and evaluated in Escherichia coli. The results showed that the direct product of SNA (pBV220-SNA) could degrade the plasmid and genomic DNA of E. coli while the fusion product of gene E and partial Cro gene (pKF396M-2) lost the ability to lyse the host strain. The insertion of enhancer T7g10 elements and Shine-Dalgarno box (ESD) between them (pKF396M-3) could resume the function of gene E. Using plasmid pKF396M-4 with gene E and SNA, respectively, under the immediate control of promoter pR and pL, the remnant plasmids and genomic DNA of E. coli were eliminated, and the rates of inactivation increased by two orders of magnitude over that obtained with the exclusive use of E-mediated lysis plasmid. By substituting these two genes with customized multiple cloning sites sequences, the plasmid could be modified to a dual expression vector (pKF396M-5).

Analyses of Short-Term Antagonistic Evolution of Pseudomonas aeruginosa Strain PAO1 and Phage KPP22 (Myoviridae Family, PB1-Like Virus Genus).

PubMed

Uchiyama, Jumpei; Suzuki, Masato; Nishifuji, Koji; Kato, Shin-Ichiro; Miyata, Reina; Nasukawa, Tadahiro; Yamaguchi, Kotoe; Takemura-Uchiyama, Iyo; Ujihara, Takako; Shimakura, Hidekatsu; Murakami, Hironobu; Okamoto, Noriaki; Sakaguchi, Yoshihiko; Shibayama, Keigo; Sakaguchi, Masahiro; Matsuzaki, Shigenobu

2016-08-01

Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy. Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

[Isolation and characterization of siphovirus phages infecting bovine Streptococcus agalactiae].

PubMed

Bai, Qinqin; Yang, Yongchun; Lu, Chengping

2016-02-04

To isolate and identify Streptococcus agalactiae phages and screen candidate phages to control infection caused by bovine S. agalactiae. We used two methods for isolation of S. agalactiae phages, namely (1) isolation of phages from milk and environmental samples, and (2) isolation of phages via induction of lysogens with Mitomycin C. Double-layer agar culture method was used to purify phages. Then the newly obtained phages, with S. agalactiae phage JX01 isolated from mastitis milk, were comparatively analyzed in the following aspects: morphology of phages by transmission electron microscopy, host range of phages to 55 S. agalactiae strains and other Streptococcus strains, phages DNA using EcoR I, Xba I, Pst I and Sal I, the optical multiplicity of infection, absorption curve and one step growth curve, and the stability of phages at different storage conditions. The comparative analysis of the 3 novel phages LYGO9, HZ04 and pA11 (induced from S. agalctiae bovine clinical isolate HAJL2011070601) with JX01 showed that the 4 phages were classified as the member of Siphovirdae family. EcoR I, Sal I, Xba I and Pst I separately digested the 4 phages DNA provided 4, 3, 3 and 2 profiles, respectively. This suggested that they were different strains. All the 4 phages specifically infected bovine S. agalactiae isolates. LYGO9, pA11, JX01 and HZ04 could lyse 12, 13, 20 and 23 of 42 tested bovine S. agalctiae isolates, respectively. This clearly indicated that these 4 phages are closely related. The 3 new phages which specifically lyse bovine S. agalactiae isolates are siphovirus phages. Phage LYGO9 was shown having a short latent period and a larger burst size.

Finding a needle in the virus metagenome haystack--micro-metagenome analysis captures a snapshot of the diversity of a bacteriophage armoire.

PubMed

Ray, Jessica; Dondrup, Michael; Modha, Sejal; Steen, Ida Helene; Sandaa, Ruth-Anne; Clokie, Martha

2012-01-01

Viruses are ubiquitous in the oceans and critical components of marine microbial communities, regulating nutrient transfer to higher trophic levels or to the dissolved organic pool through lysis of host cells. Hydrothermal vent systems are oases of biological activity in the deep oceans, for which knowledge of biodiversity and its impact on global ocean biogeochemical cycling is still in its infancy. In order to gain biological insight into viral communities present in hydrothermal vent systems, we developed a method based on deep-sequencing of pulsed field gel electrophoretic bands representing key viral fractions present in seawater within and surrounding a hydrothermal plume derived from Loki's Castle vent field at the Arctic Mid-Ocean Ridge. The reduction in virus community complexity afforded by this novel approach enabled the near-complete reconstruction of a lambda-like phage genome from the virus fraction of the plume. Phylogenetic examination of distinct gene regions in this lambdoid phage genome unveiled diversity at loci encoding superinfection exclusion- and integrase-like proteins. This suggests the importance of fine-tuning lyosgenic conversion as a viral survival strategy, and provides insights into the nature of host-virus and virus-virus interactions, within hydrothermal plumes. By reducing the complexity of the viral community through targeted sequencing of prominent dsDNA viral fractions, this method has selectively mimicked virus dominance approaching that hitherto achieved only through culturing, thus enabling bioinformatic analysis to locate a lambdoid viral "needle" within the greater viral community "haystack". Such targeted analyses have great potential for accelerating the extraction of biological knowledge from diverse and poorly understood environmental viral communities.

Expert Opinion on Three Phage Therapy Related Topics: Bacterial Phage Resistance, Phage Training and Prophages in Bacterial Production Strains.

PubMed

Rohde, Christine; Resch, Grégory; Pirnay, Jean-Paul; Blasdel, Bob G; Debarbieux, Laurent; Gelman, Daniel; Górski, Andrzej; Hazan, Ronen; Huys, Isabelle; Kakabadze, Elene; Łobocka, Małgorzata; Maestri, Alice; Almeida, Gabriel Magno de Freitas; Makalatia, Khatuna; Malik, Danish J; Mašlaňová, Ivana; Merabishvili, Maia; Pantucek, Roman; Rose, Thomas; Štveráková, Dana; Van Raemdonck, Hilde; Verbeken, Gilbert; Chanishvili, Nina

2018-04-05

Phage therapy is increasingly put forward as a "new" potential tool in the fight against antibiotic resistant infections. During the "Centennial Celebration of Bacteriophage Research" conference in Tbilisi, Georgia on 26-29 June 2017, an international group of phage researchers committed to elaborate an expert opinion on three contentious phage therapy related issues that are hampering clinical progress in the field of phage therapy. This paper explores and discusses bacterial phage resistance, phage training and the presence of prophages in bacterial production strains while reviewing relevant research findings and experiences. Our purpose is to inform phage therapy stakeholders such as policy makers, officials of the competent authorities for medicines, phage researchers and phage producers, and members of the pharmaceutical industry. This brief also points out potential avenues for future phage therapy research and development as it specifically addresses those overarching questions that currently call for attention whenever phages go into purification processes for application.

Expert Opinion on Three Phage Therapy Related Topics: Bacterial Phage Resistance, Phage Training and Prophages in Bacterial Production Strains

PubMed Central

Rohde, Christine; Resch, Grégory; Blasdel, Bob G.; Gelman, Daniel; Górski, Andrzej; Hazan, Ronen; Huys, Isabelle; Kakabadze, Elene; Łobocka, Małgorzata; Maestri, Alice; Makalatia, Khatuna; Malik, Danish J.; Mašlaňová, Ivana; Merabishvili, Maia; Rose, Thomas; Štveráková, Dana; Van Raemdonck, Hilde; Verbeken, Gilbert; Chanishvili, Nina

2018-01-01

Phage therapy is increasingly put forward as a “new” potential tool in the fight against antibiotic resistant infections. During the “Centennial Celebration of Bacteriophage Research” conference in Tbilisi, Georgia on 26–29 June 2017, an international group of phage researchers committed to elaborate an expert opinion on three contentious phage therapy related issues that are hampering clinical progress in the field of phage therapy. This paper explores and discusses bacterial phage resistance, phage training and the presence of prophages in bacterial production strains while reviewing relevant research findings and experiences. Our purpose is to inform phage therapy stakeholders such as policy makers, officials of the competent authorities for medicines, phage researchers and phage producers, and members of the pharmaceutical industry. This brief also points out potential avenues for future phage therapy research and development as it specifically addresses those overarching questions that currently call for attention whenever phages go into purification processes for application. PMID:29621199

Shell-model predictions for Lambda Lambda hypernuclei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gal, A.; Millener, D.

2011-06-02

It is shown how the recent shell-model determination of {Lambda}N spin-dependent interaction terms in {Lambda} hypernuclei allows for a reliable deduction of {Lambda}{Lambda} separation energies in {Lambda}{Lambda} hypernuclei across the nuclear p shell. Comparison is made with the available data, highlighting {sub {Lambda}{Lambda}}{sup 11}Be and {sub {Lambda}{Lambda}}{sup 12}Be which have been suggested as possible candidates for the KEK-E373 HIDA event.

Characterization of Two Virulent Phages of Lactobacillus plantarum

PubMed Central

Briggiler Marcó, Mariángeles; Garneau, Josiane E.; Tremblay, Denise; Quiberoni, Andrea

2012-01-01

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria. PMID:23042172

Toward Understanding Phage:Host Interactions in the Rumen; Complete Genome Sequences of Lytic Phages Infecting Rumen Bacteria

PubMed Central

Gilbert, Rosalind A.; Kelly, William J.; Altermann, Eric; Leahy, Sinead C.; Minchin, Catherine; Ouwerkerk, Diane; Klieve, Athol V.

2017-01-01

The rumen is known to harbor dense populations of bacteriophages (phages) predicted to be capable of infecting a diverse range of rumen bacteria. While bacterial genome sequencing projects are revealing the presence of phages which can integrate their DNA into the genome of their host to form stable, lysogenic associations, little is known of the genetics of phages which utilize lytic replication. These phages infect and replicate within the host, culminating in host lysis, and the release of progeny phage particles. While lytic phages for rumen bacteria have been previously isolated, their genomes have remained largely uncharacterized. Here we report the first complete genome sequences of lytic phage isolates specifically infecting three genera of rumen bacteria: Bacteroides, Ruminococcus, and Streptococcus. All phages were classified within the viral order Caudovirales and include two phage morphotypes, representative of the Siphoviridae and Podoviridae families. The phage genomes displayed modular organization and conserved viral genes were identified which enabled further classification and determination of closest phage relatives. Co-examination of bacterial host genomes led to the identification of several genes responsible for modulating phage:host interactions, including CRISPR/Cas elements and restriction-modification phage defense systems. These findings provide new genetic information and insights into how lytic phages may interact with bacteria of the rumen microbiome. PMID:29259581

Advance in phage display technology for bioanalysis.

PubMed

Tan, Yuyu; Tian, Tian; Liu, Wenli; Zhu, Zhi; J Yang, Chaoyong

2016-06-01

Phage display technology has emerged as a powerful tool for target gene expression and target-specific ligand selection. It is widely used to screen peptides, proteins and antibodies with the advantages of simplicity, high efficiency and low cost. A variety of targets, including ions, small molecules, inorganic materials, natural and biological polymers, nanostructures, cells, bacteria, and even tissues, have been demonstrated to generate specific binding ligands by phage display. Phages and target-specific ligands screened by phage display have been widely used as affinity reagents in therapeutics, diagnostics and biosensors. In this review, comparisons of different types of phage display systems are first presented. Particularly, microfluidic-based phage display, which enables screening with high throughput, high efficiency and integration, is highlighted. More importantly, we emphasize the advances in biosensors based on phages or phage-derived probes, including nonlytic phages, lytic phages, peptides or proteins screened by phage display, phage assemblies and phage-nanomaterial complexes. However, more efficient and higher throughput phage display methods are still needed to meet an explosion in demand for bioanalysis. Furthermore, screening of cyclic peptides and functional peptides will be the hotspot in bioanalysis. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineered phages for electronics.

PubMed

Cui, Yue

2016-11-15

Phages are traditionally widely studied in biology and chemistry. In recent years, engineered phages have attracted significant attentions for functionalization or construction of electronic devices, due to their specific binding, catalytic, nucleating or electronic properties. To apply the engineered phages in electronics, these are a number of interesting questions: how to engineer phages for electronics? How are the engineered phages characterized? How to assemble materials with engineered phages? How are the engineered phages micro or nanopatterned? What are the strategies to construct electronics devices with engineered phages? This review will highlight the early attempts to address these questions and explore the fundamental and practical aspects of engineered phages in electronics, including the approaches for selection or expression of specific peptides on phage coat proteins, characterization of engineered phages in electronics, assembly of electronic materials, patterning of engineered phages, and construction of electronic devices. It provides the methodologies and opens up ex-cit-ing op-por-tu-ni-ties for the development of a variety of new electronic materials and devices based on engineered phages for future applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Measurement of the forward-backward asymmetry of $$\\Lambda$$ and $$\\bar{\\Lambda}$$ production in $$p \\bar{p}$$ collisions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abazov, Victor Mukhamedovich

Here, we studymore » $$\\Lambda$$ and $$\\bar{\\Lambda}$$ production asymmetries in $$p \\bar{p} \\rightarrow \\Lambda (\\bar{\\Lambda}) X$$, $$p \\bar{p} \\rightarrow J/\\psi \\Lambda (\\bar{\\Lambda}) X$$, and $$p \\bar{p} \\rightarrow \\mu^\\pm \\Lambda (\\bar{\\Lambda}) X$$ events recorded by the D0 detector at the Fermilab Tevatron collider at $$\\sqrt{s} = 1.96$$ TeV. We find an excess of $$\\Lambda$$'s ($$\\bar{\\Lambda}$$'s) produced in the proton (antiproton) direction. This forward-backward asymmetry is measured as a function of rapidity. We confirm that the $$\\bar{\\Lambda}/\\Lambda$$ production ratio, measured by several experiments with various targets and a wide range of energies, is a universal function of "rapidity loss", i.e., the rapidity difference of the beam proton and the lambda.« less

Measurement of the forward-backward asymmetry of $$\\Lambda$$ and $$\\bar{\\Lambda}$$ production in $$p \\bar{p}$$ collisions

DOE PAGES

Abazov, Victor Mukhamedovich

2016-02-09

Here, we studymore » $$\\Lambda$$ and $$\\bar{\\Lambda}$$ production asymmetries in $$p \\bar{p} \\rightarrow \\Lambda (\\bar{\\Lambda}) X$$, $$p \\bar{p} \\rightarrow J/\\psi \\Lambda (\\bar{\\Lambda}) X$$, and $$p \\bar{p} \\rightarrow \\mu^\\pm \\Lambda (\\bar{\\Lambda}) X$$ events recorded by the D0 detector at the Fermilab Tevatron collider at $$\\sqrt{s} = 1.96$$ TeV. We find an excess of $$\\Lambda$$'s ($$\\bar{\\Lambda}$$'s) produced in the proton (antiproton) direction. This forward-backward asymmetry is measured as a function of rapidity. We confirm that the $$\\bar{\\Lambda}/\\Lambda$$ production ratio, measured by several experiments with various targets and a wide range of energies, is a universal function of "rapidity loss", i.e., the rapidity difference of the beam proton and the lambda.« less

Infective and inactivated filamentous phage as carriers for immunogenic peptides.

PubMed

Samoylova, Tatiana I; Norris, Mandy D; Samoylov, Alexandre M; Cochran, Anna M; Wolfe, Karen G; Petrenko, Valery A; Cox, Nancy R

2012-07-01

The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines. Copyright © 2012 Elsevier B.V. All rights reserved.

Measurements of J/{psi} and {psi}(2S) decays into {lambda}{lambda}{pi}{sup 0} and {lambda}{lambda}{eta}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ablikim, M.; Bai, J. Z.; Cai, X.

2007-11-01

Using 58x10{sup 6} J/{psi} and 14x10{sup 6} {psi}(2S) events collected by the BESII detector at the BEPC, branching fractions or upper limits for the decays J/{psi} and {psi}(2S){yields}{lambda}{lambda}{pi}{sup 0} and {lambda}{lambda}{eta} are measured. For the isospin violating decays, the upper limits are determined to be B(J/{psi}{yields}{lambda}{lambda}{pi}{sup 0})<6.4x10{sup -5} and B[{psi}(2S){yields}{lambda}{lambda}{pi}{sup 0}]<4.9x10{sup -5} at the 90% confidence level. The isospin conserving process J/{psi}{yields}{lambda}{lambda}{eta} is observed for the first time, and its branching fraction is measured to be B(J/{psi}{yields}{lambda}{lambda}{eta})=(2.62{+-}0.60{+-}0.44)x10{sup -4}, where the first error is statistical and the second one is systematic. No {lambda}{lambda}{eta} signal is observed in {psi}(2S) decays, and B[{psi}(2S){yields}{lambda}{lambda}{eta}]<1.2x10{supmore » -4} is set at the 90% confidence level. Branching fractions of J/{psi} decays into {sigma}{sup +}{pi}{sup -}{lambda} and {sigma}{sup -}{pi}{sup +}{lambda} are also reported, and the sum of these branching fractions is determined to be B(J/{psi}{yields}{sigma}{sup +}{pi}{sup -}{lambda}+c.c.)=(1.52{+-}0.08{+-}0.16)x10{sup -3}.« less

Measurement of the branching fraction $${\\mathcal{B}}(\\Lambda^0_b\\rightarrow \\Lambda^+_c\\pi^-\\pi^+\\pi^-)$$ at CDF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aaltonen, T.; /Helsinki Inst. of Phys.; Alvarez Gonzalez, B.

We report an analysis of the {Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}{sup +}{pi}{sup -}{pi}{sup +}{pi}{sup -} decay in a data sample collected by the CDF II detector at the Fermilab Tevatron corresponding to 2.4 fb{sup -1} of integrated luminosity. We reconstruct the currently largest samples of the decay modes {Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}(2595){sup +}{pi}{sup -} (with {Lambda}{sub c}(2595){sup +} {yields} {Lambda}{sub c}{sup +}{pi}{sup +}{pi}{sup -}), {Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}(2625){sup +}{pi}{sup -} (with {Lambda}{sub c}(2625){sup +} {yields} {Lambda}{sub c}{sup +}{pi}{sup +}{pi}{sup -}), {Lambda}{sub b}{sup 0} {yields} {Sigma}{sub c}(2455){sup ++}{pi}{sup -}{pi}{sup -} (with {Sigma}{sub c}(2455){sup ++} {yields} {Lambda}{submore » c}{sup +}{pi}{sup +}), and {Lambda}{sub b}{sup 0} {yields} {Sigma}{sub c}(2455)0{pi}{sup +}{pi}{sup -} (with {Sigma}{sub c}(2455)0 {yields} {Lambda}{sub c}{sup +}{pi}{sup -}) and measure the branching fractions relative to the {Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}{sup +}{pi}{sup -} branching fraction. We measure the ratio {Beta}({Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}{sup +}{pi}{sup -}{pi}{sup +}{pi}{sup -})/ {Beta}({Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}{sup +}{pi}{sup -})=3.04 {+-} 0.33(stat){sub -0.55}{sup +0.70}(syst) which is used to derive {Beta}({Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}{sup +}{pi}{sup -}{pi}{sup +}{pi}{sup -})=(26.8{sub -11.2}{sup +11.9}) x 10{sup -3}.« less

Genomic, proteomic and bioinformatic analysis of two temperate phages in Roseobacter clade bacteria isolated from the deep-sea water.

PubMed

Tang, Kai; Lin, Dan; Zheng, Qiang; Liu, Keshao; Yang, Yujie; Han, Yu; Jiao, Nianzhi

2017-06-27

Marine phages are spectacularly diverse in nature. Dozens of roseophages infecting members of Roseobacter clade bacteria were isolated and characterized, exhibiting a very high degree of genetic diversity. In the present study, the induction of two temperate bacteriophages, namely, vB_ThpS-P1 and vB_PeaS-P1, was performed in Roseobacter clade bacteria isolated from the deep-sea water, Thiobacimonas profunda JLT2016 and Pelagibaca abyssi JLT2014, respectively. Two novel phages in morphological, genomic and proteomic features were presented, and their phylogeny and evolutionary relationships were explored by bioinformatic analysis. Electron microscopy showed that the morphology of the two phages were similar to that of siphoviruses. Genome sequencing indicated that the two phages were similar in size, organization, and content, thereby suggesting that these shared a common ancestor. Despite the presence of Mu-like phage head genes, the phages are more closely related to Rhodobacter phage RC1 than Mu phages in terms of gene content and sequence similarity. Based on comparative genomic and phylogenetic analysis, we propose a Mu-like head phage group to allow for the inclusion of Mu-like phages and two newly phages. The sequences of the Mu-like head phage group were widespread, occurring in each investigated metagenomes. Furthermore, the horizontal exchange of genetic material within the Mu-like head phage group might have involved a gene that was associated with phage phenotypic characteristics. This study is the first report on the complete genome sequences of temperate phages that infect deep-sea roseobacters, belonging to the Mu-like head phage group. The Mu-like head phage group might represent a small but ubiquitous fraction of marine viral diversity.

Molecular Basis for Lytic Bacteriophage Resistance in Enterococci.

PubMed

Duerkop, Breck A; Huo, Wenwen; Bhardwaj, Pooja; Palmer, Kelli L; Hooper, Lora V

2016-08-30

The human intestine harbors diverse communities of bacteria and bacteriophages. Given the specificity of phages for their bacterial hosts, there is growing interest in using phage therapies to combat the rising incidence of multidrug-resistant bacterial infections. A significant barrier to such therapies is the rapid development of phage-resistant bacteria, highlighting the need to understand how bacteria acquire phage resistance in vivo Here we identify novel lytic phages in municipal raw sewage that kill Enterococcus faecalis, a Gram-positive opportunistic pathogen that resides in the human intestine. We show that phage infection of E. faecalis requires a predicted integral membrane protein that we have named PIPEF (for phage infection protein from E. faecalis). We find that PIPEF is conserved in E. faecalis and harbors a 160-amino-acid hypervariable region that determines phage tropism for distinct enterococcal strains. Finally, we use a gnotobiotic mouse model of in vivo phage predation to show that the sewage phages temporarily reduce E. faecalis colonization of the intestine but that E. faecalis acquires phage resistance through mutations in PIPEF Our findings define the molecular basis for an evolutionary arms race between E. faecalis and the lytic phages that prey on them. They also suggest approaches for engineering E. faecalis phages that have altered host specificity and that can subvert phage resistance in the host bacteria. Bacteriophage therapy has received renewed attention as a potential solution to the rise in antibiotic-resistant bacterial infections. However, bacteria can acquire phage resistance, posing a major barrier to phage therapy. To overcome this problem, it is necessary to understand phage resistance mechanisms in bacteria. We have unraveled one such resistance mechanism in Enterococcus faecalis, a Gram-positive natural resident of the human intestine that has acquired antibiotic resistance and can cause opportunistic infections. We have identified a cell wall protein hypervariable region that specifies phage tropism in E. faecalis Using a gnotobiotic mouse model of in vivo phage predation, we show that E. faecalis acquires phage resistance through mutations in this cell wall protein. Our findings define the molecular basis for lytic phage resistance in E. faecalis They also suggest opportunities for engineering E. faecalis phages that circumvent the problem of bacterial phage resistance. Copyright © 2016 Duerkop et al.

Study of B{yields}{lambda}{sub c}{lambda}{sub c} and B{yields}{lambda}{sub c}{lambda}{sub c}K

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, H.-Y.; Hsiao, Y.-K.; Chua, C.-K.

2009-06-01

We study the doubly charmful two-body and three-body baryonic B decays B{yields}{lambda}{sub c}{sup +}{lambda}{sub c}{sup -} and B{yields}{lambda}{sub c}{sup +}{lambda}{sub c}{sup -}K. As pointed out before, a naive estimate of the branching ratio O(10{sup -8}) for the latter decay is too small by 3 to 4 orders of magnitude compared to experiment. Previously, it has been shown that a large enhancement for the {lambda}{sub c}{sup +}{lambda}{sub c}{sup -}K production can occur due to a charmoniumlike resonance (e.g. X(4630) discovered by Belle) with a mass near the {lambda}{sub c}{lambda}{sub c} threshold. Motivated by the BABAR's observation of a resonance in themore » {lambda}{sub c}K system with a mass of order 2930 MeV, we study in this work the contribution to B{yields}{lambda}{sub c}{sup +}{lambda}{sub c}{sup -}K from the intermediate state {xi}{sub c}(2980) which is postulated to be a first positive-parity excited D-wave charmed baryon state. Assuming that a soft qq quark pair is produced through the {sigma} and {pi} meson exchanges in the configuration for B{yields}{xi}{sub c}(2980){lambda}{sub c} and {lambda}{sub c}{lambda}{sub c}, it is found that branching ratios of B{yields}{lambda}{sub c}{sup +}{lambda}{sub c}{sup -}K and B{yields}{lambda}{sub c}{sup +}{lambda}{sub c}{sup -} are of order 3.5x10{sup -4} and 5x10{sup -5}, respectively, in agreement with experiment except that the prediction for the {lambda}{sub c}{lambda}{sub c}K{sup -} is slightly smaller. In conjunction with our previous analysis, we conclude that the enormously large rate of B{yields}{lambda}{sub c}{sup +}{lambda}{sub c}{sup -}K arises from the resonances {xi}{sub c}(2980) and X(4630)« less

Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

PubMed Central

Rousseau, Geneviève M.; Capra, María L.; Quiberoni, Andrea; Tremblay, Denise M.; Labrie, Simon J.

2015-01-01

Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts. PMID:26475105

Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

PubMed Central

Gillis, Annika; Mahillon, Jacques

2014-01-01

Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here. PMID:25010767

Measurement of the $$\\Lambda_b$$ cross section and the $$_{\\bar{\\Lambda}_b}$$ to $$\\Lambda_b$$ ratio with $$J/\\Psi \\Lambda$$ decays in $pp$ collisions at $$\\sqrt{s}=7$$ TeV

DOE PAGES

Chatrchyan, Serguei; et al.

2013-07-16

The Lambda(b) differential production cross section and the cross section ratio anti-Lambda(b)/Lambda(b) are measured as functions of transverse momentum pt(Lambda(b)) and rapidity abs(y(Lambda(b))) in pp collisions at sqrt(s) = 7 TeV using data collected by the CMS experiment at the LHC. The measurements are based on Lambda(b) decays reconstructed in the exclusive final state J/Psi Lambda, with the subsequent decays J/Psi to an opposite-sign muon pair and Lambda to proton pion, using a data sample corresponding to an integrated luminosity of 1.9 inverse femtobarns. The product of the cross section times the branching ratio for Lambda(b) to J/Psi Lambda versusmore » pt(Lambda(b)) falls faster than that of b mesons. The measured value of the cross section times the branching ratio for pt(Lambda(b)) > 10 GeV and abs(y(Lambda(b))) < 2.0 is 1.06 +/- 0.06 +/- 0.12 nb, and the integrated cross section ratio for anti-Lambda(b)/Lambda(b) is 1.02 +/- 0.07 +/- 0.09, where the uncertainties are statistical and systematic, respectively.« less

The Use of a Selenium-Peptide to Specifically Inactivate Yersinia pestis

DTIC Science & Technology

2005-10-01

Control 2 (F1 Antibody+Anti-F1 Antibody) 2ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab 1ug/ mlF1 Ab+1011 Phage+Anti-F1 Antibody .5ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab...25ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .125ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .0625ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .03125ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab...0156ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .0078ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .0039ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .00195ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab

Landscape Phage: Evolution from Phage Display to Nanobiotechnology.

PubMed

Petrenko, Valery A

2018-06-07

The development of phage engineering technology has led to the construction of a novel type of phage display library-a collection of nanofiber materials with diverse molecular landscapes accommodated on the surface of phage particles. These new nanomaterials, called the "landscape phage", serve as a huge resource of diagnostic/detection probes and versatile construction materials for the preparation of phage-functionalized biosensors and phage-targeted nanomedicines. Landscape-phage-derived probes interact with biological threat agents and generate detectable signals as a part of robust and inexpensive molecular recognition interfaces introduced in mobile detection devices. The use of landscape-phage-based interfaces may greatly improve the sensitivity, selectivity, robustness, and longevity of these devices. In another area of bioengineering, landscape-phage technology has facilitated the development and testing of targeted nanomedicines. The development of high-throughput phage selection methods resulted in the discovery of a variety of cancer cell-associated phages and phage proteins demonstrating natural proficiency to self-assemble into various drug- and gene-targeting nanovehicles. The application of this new "phage-programmed-nanomedicines" concept led to the development of a number of cancer cell-targeting nanomedicine platforms, which demonstrated anticancer efficacy in both in vitro and in vivo experiments. This review was prepared to attract the attention of chemical scientists and bioengineers seeking to develop functionalized nanomaterials and use them in different areas of bioscience, medicine, and engineering.

Bypassing bacterial infection in phage display by sequencing DNA released from phage particles.

PubMed

Villequey, Camille; Kong, Xu-Dong; Heinis, Christian

2017-11-01

Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type. A potential strategy for bypassing the bacterial infection step is to directly sequence DNA extracted from phage particles after a single round of phage panning using high-throughput sequencing. In this work, we have quantified the fraction of phage clones that can be identified by directly sequencing DNA from phage particles. The results show that the DNA of essentially all of the phage particles can be 'decoded', and that the sequence coverage for mutants equals that of amplified DNA extracted from cells infected with wild-type phage. This procedure is particularly attractive for selections with phage that have a compromised infection capacity, and it may allow phage display to be performed with particles that are not infective at all. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

PubMed Central

Forsman, Päivi; Alatossava, Tapani

1991-01-01

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages. Images PMID:16348513

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species

PubMed Central

Szymczak, Paula; Neves, Ana Rute; Kot, Witold; Hansen, Lars H.; Lametsch, René; Neve, Horst; Franz, Charles M. A. P.

2016-01-01

ABSTRACT Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed. IMPORTANCE Streptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations. PMID:28039135

Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships.

PubMed

Pope, Welkin H; Mavrich, Travis N; Garlena, Rebecca A; Guerrero-Bustamante, Carlos A; Jacobs-Sera, Deborah; Montgomery, Matthew T; Russell, Daniel A; Warner, Marcie H; Hatfull, Graham F

2017-08-15

The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are "singletons" with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable. IMPORTANCE Despite the numerical dominance of bacteriophages in the biosphere, there is a dearth of complete genomic sequences. Current genomic information reveals that phages are highly diverse genomically and have mosaic architectures formed by extensive horizontal genetic exchange. Comparative analysis of 79 phages of Gordonia shows them to not only be highly diverse, but to present a spectrum of relatedness. Most are distantly related to phages of the phylogenetically proximal host Mycobacterium smegmatis , although one group of Gordonia phages is more closely related to mycobacteriophages than to the other Gordonia phages. Phage genome sequence space remains largely unexplored, but further isolation and genomic comparison of phages targeted at related groups of hosts promise to reveal pathways of bacteriophage evolution. Copyright © 2017 Pope et al.

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species.

PubMed

Szymczak, Paula; Janzen, Thomas; Neves, Ana Rute; Kot, Witold; Hansen, Lars H; Lametsch, René; Neve, Horst; Franz, Charles M A P; Vogensen, Finn K

2017-03-01

Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos - or pac -type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos - or pac -type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos - or pac -type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis , extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed. IMPORTANCE Streptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations. Copyright © 2017 Szymczak et al.

Receptor Diversity and Host Interaction of Bacteriophages Infecting Salmonella enterica Serovar Typhimurium

PubMed Central

Kim, Hyeryen; Choi, Younho; Heu, Sunggi; Ryu, Sangryeol

2012-01-01

Background Salmonella enterica subspecies enterica serovar Typhimurium is a Gram-negative pathogen causing salmonellosis. Salmonella Typhimurium-targeting bacteriophages have been proposed as an alternative biocontrol agent to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated. Methodology/Principal Findings Twenty-five Salmonella phages were isolated and their receptors were identified by screening a Tn5 random mutant library of S. Typhimurium SL1344. Among them, three types of receptors were identified flagella (11 phages), vitamin B12 uptake outer membrane protein, BtuB (7 phages) and lipopolysaccharide-related O-antigen (7 phages). TEM observation revealed that the phages using flagella (group F) or BtuB (group B) as a receptor belong to Siphoviridae family, and the phages using O-antigen of LPS as a receptor (group L) belong to Podoviridae family. Interestingly, while some of group F phages (F-I) target FliC host receptor, others (F-II) target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain. Conclusions/Significance In this report, three receptor groups of 25 newly isolated S. Typhimurium-targeting phages were determined. Among them, two subgroups of group F phages interact with their host receptors in different manner. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for Salmonella phages and will inform development of optimal phage therapy for protection against Salmonella. PMID:22927964

Investigation of interaction between magnetic silica particles and lambda phage DNA fragment.

PubMed

Smerkova, Kristyna; Dostalova, Simona; Vaculovicova, Marketa; Kynicky, Jindrich; Trnkova, Libuse; Kralik, Miroslav; Adam, Vojtech; Hubalek, Jaromir; Provaznik, Ivo; Kizek, Rene

2013-12-01

Nucleic acids belong to the most important molecules and therefore the understanding of their properties, function and behavior is crucial. Even though a range of analytical and biochemical methods have been developed for this purpose, one common step is essential for all of them - isolation of the nucleic acid from the from complex sample matrix. The use of magnetic particles for the separation of nucleic acids has many advantages over other isolation methods. In this study, an isolation procedure for extraction of DNA was optimized. Each step of the isolation process including washing, immobilization and elution was optimized and therefore the efficiency was increased from 1.7% to 28.7% and the total time was shortened from 75 to 30min comparing to the previously described method. Quantification of the particular parameter influence was performed by square-wave voltammetry using hanging drop mercury electrode. Further, we compared the optimized method with standard chloroform extraction and applied on isolation of DNA from Staphylococcus aureus and Escherichia coli. Copyright © 2013 Elsevier B.V. All rights reserved.

Low-power microwave-mediated heating for microchip-based PCR.

PubMed

Marchiarullo, Daniel J; Sklavounos, Angelique H; Oh, Kyudam; Poe, Brian L; Barker, N Scott; Landers, James P

2013-09-07

Microwave energy has been used to rapidly heat food and drinks for decades, in addition to assisting other chemical reactions. However, only recently has microwave energy been applied in microfluidic systems to heat solution in reaction chambers, in particular, the polymerase chain reaction (PCR). One of the difficulties in developing microwave-mediated heating on a microchip is the construction of the appropriate architecture for delivery of the energy to specific micro-areas on the microchip. This work employs commercially-available microwave components commonly used in the wireless communications industry to generate a microwave signal, and a microstrip transmission line to deliver the energy to a 1 μL reaction chamber fabricated in plastic microdevices. A model was developed to create transmission lines that would optimally transmit energy to the reaction chamber at a given frequency, minimizing energy usage while focusing microwave delivery to the target chamber. Two different temperature control methods were demonstrated, varying microwave power or frequency. This system was used to amplify a fragment of the lambda-phage genome, thereby demonstrating its potential for integration into a portable PCR system.

Sequence and structural characterization of great salt lake bacteriophage CW02, a member of the T7-like supergroup.

PubMed

Shen, Peter S; Domek, Matthew J; Sanz-García, Eduardo; Makaju, Aman; Taylor, Ryan M; Hoggan, Ryan; Culumber, Michele D; Oberg, Craig J; Breakwell, Donald P; Prince, John T; Belnap, David M

2012-08-01

Halophage CW02 infects a Salinivibrio costicola-like bacterium, SA50, isolated from the Great Salt Lake. Following isolation, cultivation, and purification, CW02 was characterized by DNA sequencing, mass spectrometry, and electron microscopy. A conserved module of structural genes places CW02 in the T7 supergroup, members of which are found in diverse aquatic environments, including marine and freshwater ecosystems. CW02 has morphological similarities to viruses of the Podoviridae family. The structure of CW02, solved by cryogenic electron microscopy and three-dimensional reconstruction, enabled the fitting of a portion of the bacteriophage HK97 capsid protein into CW02 capsid density, thereby providing additional evidence that capsid proteins of tailed double-stranded DNA phages have a conserved fold. The CW02 capsid consists of bacteriophage lambda gpD-like densities that likely contribute to particle stability. Turret-like densities were found on icosahedral vertices and may represent a unique adaptation similar to what has been seen in other extremophilic viruses that infect archaea, such as Sulfolobus turreted icosahedral virus and halophage SH1.

Therapeutic and prophylactic applications of bacteriophage components in modern medicine.

PubMed

Adhya, Sankar; Merril, Carl R; Biswas, Biswajit

2014-01-01

As the interactions of phage with mammalian innate and adaptive immune systems are better delineated and with our ability to recognize and eliminate toxins and other potentially harmful phage gene products, the potential of phage therapies is now being realized. Early efforts to use phage therapeutically were hampered by inadequate phage purification and limited knowledge of phage-bacterial and phage-human relations. However, although use of phage as an antibacterial therapy in countries that require controlled clinical studies has been hampered by the high costs of patient trials, their use as vaccines and the use of phage components such as lysolytic enzymes or lysozymes has progressed to the point of commercial applications. Recent studies concerning the intimate associations between mammalian hosts and bacterial and phage microbiomes should hasten this progress.

Current insights into phage biodiversity and biogeography.

PubMed

Thurber, Rebecca Vega

2009-10-01

Phages exert tremendous ecological and evolutionary forces directly on their bacterial hosts. Phage induced cell lysis also indirectly contributes to organic and inorganic nutrient recycling. Phage abundance, diversity, and distribution are therefore important parameters in ecosystem function. The assumption that phage consortia are ubiquitous and homogenous across habitats (everything is everywhere) is currently being re-evaluated. New studies on phage biogeography have found that some phages are globally distributed while others are unique and perhaps endemic to specific environments. Furthermore, advances in technology have allowed scientists to conduct experiments aimed at analyzing phage consortia over temporal scales, and surprisingly have found reoccurring patterns. This review discusses currents in the field of phage ecology with particular focus on efforts to characterize phage diversity and biogeography across various spatial and temporal scales.

FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

PubMed Central

Krueger, A. P.; Scribner, E. J.; Brown, B. B.

1946-01-01

1. The reaction between an antistaphlycoccal phage and the homologous bacterium has been studied, applying the following experimental technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining [phage]. (b) Sampling was done at short intervals; i.e., every 0.1 hour. (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of < 95 per cent of free phage. 2. Using these technics, the reaction was followed: (a) with pH maintained at 6.10 and temperature at 28°C. to slow the process; (b) with pH maintained at 7.2 and temperature at 36°C. 3. In addition separate experiments were performed on the sorption of phage by bacteria at 30°, 23°, and 0°C. 4. At pH 6.10 and 28°C. the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant. (b) When 90 per cent of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques; i.e., phage is formed intracellularly, but is retained within cellular confines. (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque). (d) With the concentrations of phage and bacteria used in the experiment carried out at pH 6.1 and 28°C. there are two further increments in [phage]act. before massive lysis begins. (e) During terminal lysis there are sharp rises in the curves for [total phage]plaq., [extracellular phage]act., and [extracellular phage]plaq.. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being of sufficient size to be removed by the super-cel filters. 5. At pH 7.2 and 36°C. the steps in the phage production curve as determined by activity assay and plaque count are much less prominent than those observed at pH 6.1 and 28°C. However, the plateaus described by Ellis and Delbrück (10) for B. coli and coli phage can be detected also in the present case if frequent samples are taken. 6. The sorption experiments show a significant rise in the rate of phage uptake with increase in temperature, again supporting the view that the reaction involves more than a purely physical adsorption. 7. Delbrück's objections to: (a) the use of the activity assay for determining [total phage] in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. 8. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in solution or phage bound to living susceptible cells, under the conditions of the experiments reported here and in earlier work. 9. The titration values obtained in the experiments designed to exhibit intracellular phage precursor are not the result of artifacts as Delbrück has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out. PMID:19873475

Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy

PubMed Central

Khan Mirzaei, Mohammadali; Nilsson, Anders S.

2015-01-01

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection. PMID:25761060

[The expression of interferon-lambda1 in CHO cell].

PubMed

Yuan, Wu-Mei; Ma, Fen-Lian; Zhang, Qian; Zheng, Wen-Zhi; Zheng, Li-Shu

2013-06-01

To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells. Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1. Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity. Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.

Energy of the ground and 2{sup +} excited states of {sub {lambda}}{sub {lambda}}{sup 10}Be: A partial ten-body model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shoeb, Mohammad; Sonika

2009-08-15

The energies of the ground and excited 2{sup +} states of {sub {lambda}}{sub {lambda}}{sup 10}Be have been calculated variationally in the Monte Carlo framework. The hypernucleus is treated as a partial ten-body problem in the {lambda}{lambda}+{alpha}{alpha} model where nucleonic degrees of freedom of {alpha}'s are taken into consideration ignoring the antisymmetrization between two {alpha}'s. The central two-body {lambda}N and {lambda}{lambda} and the three-body dispersive and two-pion exchange {lambda}NN forces, constrained by the {lambda}p scattering data and the observed ground state energies of {sub {lambda}}{sup 5}He and {sub {lambda}}{sub {lambda}}{sup 6}He, are employed. The product-type trial wave function predicts binding energymore » for the ground state considerably less than for the event reported by Danysz et al.; however, it is consistent with the value deduced assuming a {gamma} ray of 3.04 MeV must have escaped undetected in the decay of the product {sub {lambda}}{sup 9}Be* {yields} {sub {lambda}}{sup 9}Be+{gamma} of the emulsion event {sub {lambda}}{sub {lambda}}{sup 10}Be{yields} {pi}{sup -}+p+{sub {lambda}}{sup 9}Be* and for the excited 2{sup +} state closer to the value measured in the Demachi-Yanagi event. The hypernucleus {sub {lambda}}{sub {lambda}}{sup 10}Be has an oblate shape in the excited state. These results are consistent with the earlier four-body {alpha} cluster model approach where {alpha}'s are assumed to be structureless entities.« less

Diversity and geographical distribution of Flavobacterium psychrophilum isolates and their phages: patterns of susceptibility to phage infection and phage host range.

PubMed

Castillo, Daniel; Christiansen, Rói Hammershaimb; Espejo, Romilio; Middelboe, Mathias

2014-05-01

Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that "enhanced infection" is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.

In vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with Escherichia coli.

PubMed

Weiss, Marietta; Denou, Emmanuel; Bruttin, Anne; Serra-Moreno, Ruth; Dillmann, Marie-Lise; Brüssow, Harald

2009-10-10

The gut transit of T4 phages was studied in axenic mice mono-colonized with the non-pathogenic Escherichia coli strain K-12. Thirty minutes, 1 and 2 h after phage feeding, T4 phage had reached the jejunum, ileum and cecum, respectively. Phage was found in the lumen and was also associated with the mucosa. One day later no phage was detected in the feces. Compared to germ-free control animals, oral T4 phage led to a 300-fold higher fecal phage titer in mice mono-colonized with E. coli strain WG-5. The in vivo T4 phage replication was transient and reached peak fecal titers about 8 h after oral phage application followed by a rapid titer decrease over two days. Similar data were obtained in mice colonized with E. coli strain Nissle. In contrast, orally applied T7 phage experienced a massive and sustained in vivo replication in mice mono-colonized with E. coli strain WG-5 irrespective whether phage or E. coli host was applied first. T7 phage replication occurred mainly in the large intestine. High titers of T7 phage and high E. coli cell counts coexisted in the feces. The observation of only 20% T7 phage-resistant fecal E. coli colonies suggests a refuge model where phage-sensitive E. coli cells are physically or physiologically protected from phage infection in the gut. The difference between T7 and T4 with respect to gut replication might partly reflect their distinct in vitro capacity to replicate on slowly growing cells.

Whey powders are a rich source and excellent storage matrix for dairy bacteriophages.

PubMed

Wagner, Natalia; Brinks, Erik; Samtlebe, Meike; Hinrichs, Jörg; Atamer, Zeynep; Kot, Witold; Franz, Charles M A P; Neve, Horst; Heller, Knut J

2017-01-16

Thirteen whey powders and 5 whey powder formulations were screened for the presence of dairy bacteriophages using a representative set of 8 acid-producing Lactococcus lactis and 5 Streptococcus thermophilus, and 8 flavour-producing Leuconostoc pseudomesenteroides and Leuconostoc mesenteroides strains. Lytic L. lactis phages were detected in all samples, while S. thermophilus and Leuconostoc phages were present in 50% or 40% of the samples, respectively. Maximal phage titers were 6×10 7 plaque-forming units (pfu)/g of whey powder for L. lactis phages, 1×10 7 pfu/g for Leuconostoc phages and 1×10 5 pfu/g for S. thermophilus phages. In total, 55 phages were isolated and characterized. Thirty one of the 33 lactococcal phages tested belonged to the wide-spread 936 phage group. In the course of this study, a PCR detection method for Leuconostoc phages (Ali et al., 2013) was adapted to new phage isolates. Furthermore, a remarkably high stability of phages in whey powder samples was documented during a long-term storage period of 4 years. Copyright © 2016 Elsevier B.V. All rights reserved.

In vivo growth rates are poorly correlated with phage therapy success in a mouse infection model.

PubMed

Bull, J J; Otto, G; Molineux, I J

2012-02-01

Two classes of phages yield profoundly different levels of recovery in mice experimentally infected with an Escherichia coli O18:K1:H7 strain. Phages requiring the K1 capsule for infection (K1-dep) rescue virtually all infected mice, whereas phages not requiring the capsule (K1-ind) rescue modest numbers (∼30%). To rescue infected mice, K1-ind phages require at least a 10(6)-fold-higher inoculum than K1-dep phages. Yet their in vivo growth dynamics are only modestly inferior to those of K1-dep phages, and competition between the two phage types in the same mouse reveals only a slight growth advantage for the K1-dep phage. The in vivo growth rate seems unlikely to be the primary determinant of phage therapy success. An alternative explanation is that the success of K1-dep phages is due substantially to their proteomic composition. They encode an enzyme that degrades the K1 capsule, which has been shown in other work to be sufficient to cure infection in the complete absence of phages.

Filamentous Phage: Structure and Biology.

PubMed

Rakonjac, Jasna; Russel, Marjorie; Khanum, Sofia; Brooke, Sam J; Rajič, Marina

2017-01-01

Ff filamentous phage (fd, M13 and f1) of Escherichia coli have been the workhorse of phage display technology for the past 30 years. Dominance of Ff over other bacteriophage in display technology stems from the titres that are about 100-fold higher than any other known phage, efficacious transformation ensuring large library size and superior stability of the virion at high temperatures, detergents and pH extremes, allowing broad range of biopanning conditions in screening phage display libraries. Due to the excellent understanding of infection and assembly requirements, Ff phage have also been at the core of phage-assisted continual protein evolution strategies (PACE). This chapter will give an overview of the Ff filamentous phage structure and biology, emphasizing those properties of the Ff phage life cycle and virion that are pertinent to phage display applications.

Characterisation of a novel enterobacteria phage, CAjan, isolated from rat faeces.

PubMed

Carstens, Alexander B; Kot, Witold; Lametsch, Rene; Neve, Horst; Hansen, Lars H

2016-08-01

In this study, we describe the isolation and characterisation of the novel enterobacteria phage CAjan. This phage belongs to the order Caudovirales and the family Siphoviridae. The phage possesses a linear, double-stranded DNA genome consisting of 59,670 bp with a G+C content of 44.7 % and 91 predicted open reading frames (ORFs). Putative functions were assigned to 39 of the ORFs (37.4 %). The phage structural genes were furthermore functionally characterised by LC MS/MS. CAjan, together with Escherichia phage Seurat and Escherichia phage slur01, represent a novel and genetically distinct clade of Siphoviridae phages that could be considered to constitute a new phage genus. Despite limited sequence similarity, the phages in this group share a number of other common features, including genome structure and the presence of queuosine biosynthesis genes.

Bacteriophage Ecology in a Commercial Cucumber Fermentation

PubMed Central

Pérez-Díaz, I. M.; Hayes, J. S.; Breidt, F.

2012-01-01

To reduce high-salt waste from cucumber fermentations, low-salt fermentations are under development. These fermentations may require the use of starter cultures to ensure normal fermentations. Because potential phage infection can cause starter culture failure, it is important to understand phage ecology in the fermentations. This study investigated the phage ecology in a commercial cucumber fermentation. Brine samples taken from a fermentation tank over a 90-day period were plated onto deMan-Rogosa-Sharpe agar plates. A total of 576 lactic acid bacterial isolates were randomly selected to serve as potential hosts for phage isolation. Filtered brine served as a phage source. Fifty-seven independent phage isolates were obtained, indicating that 10% of the bacterial isolates were sensitive to phage attack. Phage hosts include Lactobacillus brevis (67% of all hosts), Lactobacillus plantarum (21%), Weissella paramesenteroides, Weissella cibaria, and Pediococcus ethanolidurans. Nearly 50% of phages were isolated on day 14, and the majority of them attacked L. brevis. Some phages had a broad host range and were capable of infecting multiple hosts in two genera. Other phages were species specific or strain specific. About 30% of phage isolates produced turbid pinpoint plaques or only caused reduced cell growth on the bacterial lawns. Six phages with distinct host ranges were characterized. The data from this study showed that abundant and diverse phages were present in the commercial cucumber fermentation, which could cause significant mortality to the lactic acid bacteria population. Therefore, a phage control strategy may be needed in low-salt cucumber fermentations. PMID:23023756

Bacteriophage of Haemophilus influenzae III. Morphology, DNA Homology, and Immunity Properties of HP1c1, S2, and the Defective Bacteriophage from Strain Rd

PubMed Central

Boling, Maxon E.; Allison, David P.; Setlow, Jane K.

1973-01-01

The phages HP1c1 and S2 and a defective phage of Haemophilus influenzae have been compared. The morphology of the phages and the mol wt of their DNAs are similar, although the defective phage appears to have a different tail plate region. Electron microscope observation indicates that the defective phage does not attach to the cell surface, and its DNA appears to lack cohesive ends. The homology of the DNAs of the phages has been measured by hydridization. DNA from the defective phage shows little or no homology with the other phage DNAs. HP1c1 and S2 DNAs show a high level of homology. Each of these phages can form plaques on lawns of the lysogen of the other phage but at reduced plating efficiencies, suggesting that the two phages have related but not identical immunity systems. Images PMID:4540713

Efficacy of bacteriophage therapy in a model of Burkholderia cenocepacia pulmonary infection

PubMed Central

Carmody, Lisa A.; Gill, Jason J.; Summer, Elizabeth J.; Sajjan, Uma S.; Gonzalez, Carlos F.; Young, Ryland F.; LiPuma, John J.

2009-01-01

The therapeutic potential of bacteriophage (phage) in a mouse model of acute B. cenocepacia pulmonary infection was assessed. Phage were administered by either intranasal (i.n.) inhalation or intraperitoneal (i.p.) injection. Bacterial density, macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-α (TNFα) levels were significantly reduced in lungs of mice treated with i.p. phage. No significant differences in lung bacterial density or MIP-2 levels were found between untreated mice and mice treated with i.n. phage, i.p. UV-inactivated phage, or i.p. λ phage controls. Mock-infected mice treated with phage showed no significant increase in lung MIP-2 or TNFα levels compared to mock-infected / mock-treated mice. We have demonstrated the efficacy of phage therapy in an acute B. cenocepacia lung infection model. Systemic administration of phage was more effective than inhalational administration, suggesting that circulating phage have better access to bacteria in lung compared to topical phage. PMID:20001604

Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries

PubMed Central

Noppe, Wim; Plieva, Fatima; Galaev, Igor Yu; Pottel, Hans; Deckmyn, Hans; Mattiasson, Bo

2009-01-01

Background Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library. Results Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages. Conclusion Chromato-panning allows combining several steps of the panning procedure resulting in 4–8 fold decrease of total time needed for phage selection. PMID:19292898

Characterization of Natronobacterium magadii phage phi Ch1, a unique archaeal phage containing DNA and RNA.

PubMed

Witte, A; Baranyi, U; Klein, R; Sulzner, M; Luo, C; Wanner, G; Krüger, D H; Lubitz, W

1997-02-01

A novel archaeal bacteriophage, phi Ch1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage-cured strain N. magadii(L13) was used to demonstrate infectivity of phage phi Ch1. The turbid-plaque morphology and the fact that N. magadii cells isolated from plaques were able to produce phage indicated that phi Ch1 is a temperate phage. The phage morphology resembles other members of Myoviridae-infecting Halobacterium species. In solution below 2M NaCl, the phage lost its morphological stability and infectivity. One- and two-dimensional SDS-PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points. Southern blot analysis of chromosomal DNA of a lysogenic N. magadii strain showed that phi Ch1 exists as a chromosomally integrated prophage. The phage particles contain both double-stranded, linear DNA (approx. 55 kbp) as well as several RNA species (80-700 nucleotides). Hybridization of labelled RNA fragments to total DNA from N. magadii and phi Ch1 showed that the virion-associated RNA is host encoded. Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation. Phage phi Ch1 is the first virus described for the genus natronobacterium, and the first phage containing DNA and RNA in mature phage particles.

Citrulline-modified phage display: a novel high-throughput discovery approach for the identification of citrulline-containing ligands.

PubMed

Somers, Klaartje; Stinissen, Piet; Somers, Veerle

2011-06-01

Phage display is a high-throughput technology used to identify ligands for a given target. A drawback of the approach is the absence of PTMs in phage-displayed peptides. The applicability of phage display could be broadened considerably by the implementation of PTMs in this system. The aim of this study was to investigate the possible application of citrullination, a PTM of an arginine into a citrulline amino acid, in filamentous (M13) and lytic (T7) phage display. After in vitro citrullination of T7 and M13 phages, citrullination was confirmed and the infectivity of both citrullinated and non-citrullinated phage was compared by titer determination. We demonstrated the successful in vitro citrullination of T7 and M13 phage-displayed peptides. This in vitro modification did not affect the viability or infectivity of the T7 virions, a necessary prerequisite for the implementation of this approach in T7 phage display. For M13 phage, however, the infecting phage titer decreased five-fold upon citrullination, limiting the use of this modification in M13 phage display. In conclusion, in vitro citrullination can be applied in T7 phage display giving rise to a high-throughput and sensitive approach to identify citrulline-containing ligands by the use of the strengths of phage display technology. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

On The Influence Of Vector Design On Antibody Phage Display

PubMed Central

Soltes, Glenn; Hust, Michael; Ng, Kitty K.Y.; Bansal, Aasthaa; Field, Johnathan; Stewart, Donald I.H.; Dübel, Stefan; Cha, Sanghoon; Wiersma, Erik J

2007-01-01

Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other. Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids. Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection. PMID:16996161

On the influence of vector design on antibody phage display.

PubMed

Soltes, Glenn; Hust, Michael; Ng, Kitty K Y; Bansal, Aasthaa; Field, Johnathan; Stewart, Donald I H; Dübel, Stefan; Cha, Sanghoon; Wiersma, Erik J

2007-01-20

Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other. Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids. Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection.

Beauty Baryons at CDF and DO

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kryemadhi, Abaz

The results from Tevatron in the baryonic sector are presented. The lifetime of {lambda}b {yields} J/{psi}{lambda}, the observation of hadronic decay of {lambda}b {yields} {lambda}c{pi}, the semileptonic decays of {lambda}b {yields} {lambda}c{mu}{nu}, the hadronization of the b-baryons, and the {lambda}b decays to {lambda}b {yields} p{pi} and {lambda}b {yields} pK are discussed. These measurements paint a nice picture of our understanding of the beauty baryons.

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

PubMed

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

2013-11-14

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

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

PubMed Central

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

2013-01-01

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

Improved measurement of the form factors in the decay lambda+c-->lambda + nue.

PubMed

Hinson, J W; Huang, G S; Lee, J; Miller, D H; Pavlunin, V; Rangarajan, R; Sanghi, B; Shibata, E I; Shipsey, I P J; Cronin-Hennessy, D; Park, C S; Park, W; Thayer, J B; Thorndike, E H; Coan, T E; Gao, Y S; Liu, F; Stroynowski, R; Artuso, M; Boulahouache, C; Blusk, S; Dambasuren, E; Dorjkhaidav, O; Mountain, R; Muramatsu, H; Nandakumar, R; Skwarnicki, T; Stone, S; Wang, J C; Csorna, S E; Danko, I; Bonvicini, G; Cinabro, D; Dubrovin, M; McGee, S; Bornheim, A; Lipeles, E; Pappas, S P; Shapiro, A; Sun, W M; Weinstein, A J; Briere, R A; Chen, G P; Ferguson, T; Tatishvili, G; Vogel, H; Watkins, M E; Adam, N E; Alexander, J P; Berkelman, K; Boisvert, V; Cassel, D G; Duboscq, J E; Ecklund, K M; Ehrlich, R; Galik, R S; Gibbons, L; Gittelman, B; Gray, S W; Hartill, D L; Heltsley, B K; Hsu, L; Jones, C D; Kandaswamy, J; Kreinick, D L; Magerkurth, A; Mahlke-Krüger, H; Meyer, T O; Mistry, N B; Patterson, J R; Peterson, D; Pivarski, J; Richichi, S J; Riley, D; Sadoff, A J; Schwarthoff, H; Shepherd, M R; Thayer, J G; Urner, D; Wilksen, T; Warburton, A; Weinberger, M; Athar, S B; Avery, P; Breva-Newell, L; Potlia, V; Stoeck, H; Yelton, J; Benslama, K; Cawlfield, C; Eisenstein, B I; Gollin, G D; Karliner, I; Lowrey, N; Plager, C; Sedlack, C; Selen, M; Thaler, J J; Williams, J; Edwards, K W; Besson, D; Anderson, S; Frolov, V V; Gong, D T; Kubota, Y; Li, S Z; Poling, R; Smith, A; Stepaniak, C J; Urheim, J; Metreveli, Z; Seth, K K; Tomaradze, A; Zweber, P; Ahmed, S; Alam, M S; Ernst, J; Jian, L; Saleem, M; Wappler, F; Arms, K; Eckhart, E; Gan, K K; Gwon, C; Honscheid, K; Kagan, H; Kass, R; Pedlar, T K; von Toerne, E; Severini, H; Skubic, P; Dytman, S A; Mueller, J A; Nam, S; Savinov, V

2005-05-20

Using the CLEO detector at the Cornell Electron Storage Ring, we have studied the distribution of kinematic variables in the decay lambda(+)(c)lambda--> e(+)nu(e). By performing a four-dimensional maximum likelihood fit, we determine the form factor ratio, R= f(2)/f(1) = -0.31 +/- 0.05(stat) +/- 0.04(syst), the pole mass, M(pole) = [2.21 +/- 0.08(stat) +/- 0.14(syst)] GeV/c(2), and the decay asymmetry parameter of the lambda(+)(c), alpha (lambda(c)) = -0.86 +/-0.03(stat) +/- 0.02(syst), for q(2) = 0.67 (GeV/c(2))(2). We compare the angular distributions of the lambda(+)(c) and lambda(-)(c) and find no evidence for CP violation: A(lambda(c)) = (alpha(lambda(c)) + alpha (lambda(c)))/(alpha(lambda(c))-alpha(lambda(c))) = 0.00 +/- 0.03(stat) +/- 0.01(syst) +/- 0.02, where the third error is from the uncertainty in the world average of the CP-violating parameter, A(lambda), for ppi(-).

Marine phages as excellent tracers for reactive colloidal transport in porous media

NASA Astrophysics Data System (ADS)

Ghanem, Nawras; Chatzinotas, Antonis; Harms, Hauke; Wick, Lukas Y.

2016-04-01

Question: Here we evaluate marine phages as specific markers of hydrological flow and reactive transport of colloidal particles in the Earth's critical zone (CZ). Marine phages and their bacterial hosts are naturally absent in the CZ, and can be detected with extremely high sensitivity. In the framework of the DFG Collaborative Research Center AquaDiva, we asked the following questions: (1) Are marine phages useful specific markers of hydrological flow and reactive transport in porous media? and (2) Which phage properties are relevant drivers for the transport of marine phages in porous media? Methods: Seven marine phages from different families (as well two commonly used terrestrial phages) were selected based on their morphology, size and physico-chemical surface properties (surface charge and hydrophobicity). Phage properties were assessed by electron microscopy, dynamic light scattering and water contact angle analysis (CA). Sand-filled laboratory percolation columns were used to study transport. The breakthrough curves of the phages were analyzed using the clean bed filtration theory and the XDLVO theory of colloid stability, respectively. Phages were quantified by a modified high- throughput plaque assay and a culture-independent particle counting method approach. Results: Our data show that most marine tested phages exhibited highly variable transport rates and deposition efficiency, yet generally high colloidal stability and viability. We find that size, morphology and hydrophobicity are key factors shaping the transport efficiency of phages. Differing deposition efficiencies of the phages were also supported by calculated XDLVO interaction energy profile. Conclusion: Marine phages have a high potential for the use as sensitive tracers in terrestrial habitats with their surface properties playing a crucial role for their transport. Marine phages however, exhibit differences in their deposition efficiency depending on their morphology, hydrophobicity and availability.

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

PubMed

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2015-02-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Observation of B{sup 0}{yields}{lambda}{lambda}K{sup 0} and B{sup 0}{yields}{lambda}{lambda}K*{sup 0} at Belle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Y.-W.; Wang, M.-Z.; Chao, Y.

2009-03-01

We study the charmless decays B{yields}{lambda}{lambda}h, where h stands for {pi}{sup +}, K{sup +}, K{sup 0},K*{sup +}, or K*{sup 0}, using a 605 fb{sup -1} data sample collected at the {upsilon}(4S) resonance with the Belle detector at the KEKB asymmetric energy e{sup +}e{sup -} collider. We observe B{sup 0}{yields}{lambda}{lambda}K{sup 0} and B{sup 0}{yields}{lambda}{lambda}K*{sup 0} with branching fractions of (4.76{sub -0.68}{sup +0.84}(stat){+-}0.61(syst))x10{sup -6} and (2.46{sub -0.72}{sup +0.87}{+-}0.34)x10{sup -6}, respectively. The significances of these signals in the threshold-mass enhanced mass region, M{sub {lambda}}{sub {lambda}}<2.85 GeV/c{sup 2}, are 12.4{sigma} and 9.3{sigma}, respectively. We also update the branching fraction B(B{sup +}{yields}{lambda}{lambda}K{sup +})=(3.38{sub -0.36}{sup +0.41}{+-}0.41)x10{supmore » -6} with better accuracy, and report the following measurement or 90% confidence level upper limit in the threshold-mass-enhanced region: B(B{sup +}{yields}{lambda}{lambda}K*{sup +})=(2.19{sub -0.88}{sup +1.13}{+-}0.33)x10{sup -6} with 3.7{sigma} significance; B(B{sup +}{yields}{lambda}{lambda}{pi}{sup +})<0.94x10{sup -6}. A related search for B{sup 0}{yields}{lambda}{lambda}D{sup 0} yields a branching fraction B(B{sup 0}{yields}{lambda}{lambda}D{sup 0})=(1.05{sub -0.44}{sup +0.57}{+-}0.14)x10{sup -5}. This may be compared with the large, {approx}10{sup -4}, branching fraction observed for B{sup 0}{yields}ppD{sup 0}. The M{sub {lambda}}{sub {lambda}} enhancements near threshold and related angular distributions for the observed modes are also reported.« less

Phage Therapy Approaches to Reducing Pathogen Persistence and Transmission in Animal Production Environments: Opportunities and Challenges.

PubMed

Colavecchio, Anna; Goodridge, Lawrence D

2017-06-01

The era of genomics has allowed for characterization of phages for use as antimicrobials to treat animal infections with a level of precision never before realized. As more research in phage therapy has been conducted, several advantages of phage therapy have been realized, including the ubiquitous nature, specificity, prevalence in the biosphere, and low inherent toxicity of phages, which makes them a safe and sustainable technology for control of animal diseases. These unique qualities of phages have led to several opportunities with respect to emerging trends in infectious disease treatment. However, the opportunities are tempered by several challenges to the successful implementation of phage therapy, such as the fact that an individual phage can only infect one or a few bacterial strains, meaning that large numbers of different phages will likely be needed to treat infections caused by multiple species of bacteria. In addition, phages are only effective if enough of them can reach the site of bacterial colonization, but clearance by the immune system upon introduction to the animal is a reality that must be overcome. Finally, bacterial resistance to the phages may develop, resulting in treatment failure. Even a successful phage infection and lysis of its host has consequences, because large amounts of endotoxin are released upon lysis of Gram-negative bacteria, which can lead to local and systemic complications. Overcoming these challenges will require careful design and development of phage cocktails, including comprehensive characterization of phage host range and assessment of immunological risks associated with phage treatment.

Twelve previously unknown phage genera are ubiquitous in global oceans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh B

Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in unknowns dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four wellknown viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria.more » Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage host systems for experimental hypothesis testing.« less

Somatostatin displayed on filamentous phage as a receptor-specific agonist

PubMed Central

Rousch, Mat; Lutgerink, Jan T; Coote, James; de Bruïne, Adriaan; Arends, Jan-Willem; Hoogenboom, Hennie R

1998-01-01

In search of methods to identify bio-active ligands specific for G protein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional screening method. First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti-somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO-K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries. Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor-specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling. The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio-active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand. PMID:9776337

Characterization of novel Staphylococcus aureus lytic phage and defining their combinatorial virulence using the OmniLog® system

PubMed Central

Estrella, Luis A.; Quinones, Javier; Henry, Matthew; Hannah, Ryan M.; Pope, Robert K.; Hamilton, Theron; Teneza-mora, Nimfa; Hall, Eric; Biswajit, Biswas

2016-01-01

ABSTRACT Skin and soft tissue infections (SSTI) caused by methicillin resistant Staphylococcus aureus (MRSA) are difficult to treat. Bacteriophage (phage) represent a potential alternate treatment for antibiotic resistant bacterial infections. In this study, 7 novel phage with broad lytic activity for S. aureus were isolated and identified. Screening of a diverse collection of 170 clinical isolates by efficiency of plating (EOP) assays shows that the novel phage are virulent and effectively prevent growth of 70–91% of MRSA and methicillin sensitive S. aureus (MSSA) isolates. Phage K, which was previously identified as having lytic activity on S. aureus was tested on the S. aureus collection and shown to prevent growth of 82% of the isolates. These novel phage group were examined by electron microscopy, the results of which indicate that the phage belong to the Myoviridae family of viruses. The novel phage group requires β-N-acetyl glucosamine (GlcNac) moieties on cell wall teichoic acids for infection. The phage were distinct from, but closely related to, phage K as characterized by restriction endonuclease analysis. Furthermore, growth rate analysis via OmniLog® microplate assay indicates that a combination of phage K, with phage SA0420ᶲ1, SA0456ᶲ1 or SA0482ᶲ1 have a synergistic phage-mediated lytic effect on MRSA and suppress formation of phage resistance. These results indicate that a broad spectrum lytic phage mixture can suppress the emergence of resistant bacterial populations and hence have great potential for combating S. aureus wound infections. PMID:27738555

Twelve previously unknown phage genera are ubiquitous in global oceans.

PubMed

Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh; Corrier, Kristen; Riemann, Lasse; Verberkmoes, Nathan C; Sullivan, Matthew B

2013-07-30

Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing.

Safety analysis of a Russian phage cocktail: From MetaGenomic analysis to oral application in healthy human subjects

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCallin, Shawna, E-mail: semccallin@yahoo.com; Alam Sarker, Shafiqul, E-mail: sasarker@icddrb.org; Barretto, Caroline, E-mail: Caroline.Barretto@rdls.nestle.com

Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb,more » including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure. - Highlights: • We analyzed the composition of a commercial Russian phage cocktail. • The cocktail consists of at least 10 different phage genera. • No undesired genes were detected. • No adverse effects were seen upon oral application in a small human clinical trial.« less

Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage.

PubMed

Brockmann, Eeva-Christine; Lamminmäki, Urpo; Saviranta, Petri

2005-06-20

Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.

A simple and rapid method to isolate purer M13 phage by isoelectric precipitation.

PubMed

Dong, Dexian; Sutaria, Sanjana; Hwangbo, Je Yeol; Chen, P

2013-09-01

M13 virus (phage) has been extensively used in phage display technology and nanomaterial templating. Our research aimed to use M13 phage to template sulfur nanoparticles for making lithium ion batteries. Traditional methods for harvesting M13 phage from Escherichia coli employ polyethylene glycol (PEG)-based precipitation, and the yield is usually measured by plaque counting. With this method, PEG residue is present in the M13 phage pellet and is difficult to eliminate. To resolve this issue, a method based on isoelectric precipitation was introduced and tested. The isoelectric method resulted in the production of purer phage with a higher yield, compared to the traditional PEG-based method. There is no significant variation in infectivity of the phage prepared using isoelectric precipitation, and the dynamic light scattering data indirectly prove that the phage structure is not damaged by pH adjustment. To maximize phage production, a dry-weight yield curve of M13 phage for various culture times was produced. The yield curve is proportional to the growth curve of E. coli. On a 200-mL culture scale, 0.2 g L(-1) M13 phage (dry-weight) was produced by the isoelectric precipitation method.

Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein.

PubMed

Osipiuk, J; Georgopoulos, C; Zylicz, M

1993-03-05

It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site. The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site. The first step of this disassembly reaction is the binding of DnaK to the lambda P protein. In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex. We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE. Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex. Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone. Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK. However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein. These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

Lytic and inhibition responses to bacteriophages among marine bacteria, with special reference to the origin of phage-host systems

NASA Astrophysics Data System (ADS)

Moebus, K.

1983-12-01

The results of phage-host cross-reaction tests reported by Moebus & Nattkemper (1981) were re-examined using serially diluted bacteriophage suspensions to elicit the actual type of reaction between the bacteria and phage lysates tested. More than 1450 phage-host systems were studied at 25 °C incubation temperature. Among the nearly 300 phage strains used, 29 were identified as temperate ones. In about 65 % of the phage-host systems bacteriophage propagation was indicated by plaque formation. The remaining systems were characterized by the “inhibition” reaction of bacteria to phage lysates indicated by homogenously reduced bacterial growth within the test area without production of progeny phages. Since crude phage lysates had to be used, it remains obscure whether agents other than infective phage particles (defective ones or bacteriocins) caused this reaction. Among 269 systems of the inhibition type which were also tested at 5° and 15 °C, 54 were observed to propagate phages at one of or both the lower temperatures. Plaques produced at 15 °C with several phage-host systems were found to yield only few progeny phages which generally could not be propagated to produce high-titer phage stocks. With one system temperature-sensitive phage mutants were isolated. The probability of inhibition reactions occurring was found to be higher with phage-host systems isolated east of the Azores than with systems derived from the western Atlantic. With systems from the last mentioned area the proportion of inhibition versus lytic responses of bacteria to phages was observed to increase with the distance between the stations where both parts of the systems were derived. The latter findings are discussed in view of the assumption that bacterial and bacteriophage populations undergo genetic changes while being transported from west to east.

EFFECT OF SODIUM CHLORIDE ON STAPHYLOCOCCUS-PHAGE RELATIONSHIPS

PubMed Central

West, B.; Kelly, Florene C.; Shields, Doris A.

1963-01-01

West, B. (University of Oklahoma Medical Center, Oklahoma City), Florene C. Kelly, and Doris A. Shields. Effect of sodium chloride on staphylococcus-phage relationships. J. Bacteriol. 86:773–780. 1963.—Phage patterns of 21 phage-propagating strains of staphylococci on medium with high NaCl content appeared to be an expression of the staphylococcal cells, as well as of the salt tolerance of the phages. Serological group A phages, previously found to be NaCl-tolerant in the free state, were capable of lysing susceptible staphylococci on 3, 7.5, and 10% NaCl Trypticase Soy Agar. None of the other phages tested was active when the medium contained 7.5 and 10% NaCl. Increasing the NaCl content of the medium rarely resulted in nonspecific reactions; rather the effect was, generally, a narrowing of the phage spectrum of the cells, with persistence in the phage pattern of the phage, or phages, which were propagated on the cells being tested. Although NaCl tolerance of the phages was the chief limiting factor of phage activity in the presence of 7.5 and 10% NaCl, reactions on salt medium also depended on the degree of susceptibility of cells to phage on routine typing medium and to certain other unexplained factors. In some instances, under the influence of increased NaCl, significant lysis at 1000 RTD was replaced by thinning of growth (inhibition), with or without the presence of plaques. Conversely, certain phage-cell combinations, which gave inhibition at 1000 RTD on standard medium produced some degree of lysis when the NaCl concentration was increased. Studies of phage 81 and its propagating strain showed that replication of phage occurred in 10% NaCl medium, although adsorption diminished as salt concentration was increased, and the time required to reach maximal lytic activity was delayed. PMID:14066474

Phage-Phagocyte Interactions and Their Implications for Phage Application as Therapeutics

PubMed Central

Jończyk-Matysiak, Ewa; Weber-Dąbrowska, Beata; Owczarek, Barbara; Międzybrodzki, Ryszard; Łusiak-Szelachowska, Marzanna; Łodej, Norbert; Górski, Andrzej

2017-01-01

Phagocytes are the main component of innate immunity. They remove pathogens and particles from organisms using their bactericidal tools in the form of both reactive oxygen species and degrading enzymes—contained in granules—that are potentially toxic proteins. Therefore, it is important to investigate the possible interactions between phages and immune cells and avoid any phage side effects on them. Recent progress in knowledge concerning the influence of phages on phagocytes is also important as such interactions may shape the immune response. In this review we have summarized the current knowledge on phage interactions with phagocytes described so far and their potential implications for phage therapy. The data suggesting that phage do not downregulate important phagocyte functions are especially relevant for the concept of phage therapy. PMID:28613272

Phage-Phagocyte Interactions and Their Implications for Phage Application as Therapeutics.

PubMed

Jończyk-Matysiak, Ewa; Weber-Dąbrowska, Beata; Owczarek, Barbara; Międzybrodzki, Ryszard; Łusiak-Szelachowska, Marzanna; Łodej, Norbert; Górski, Andrzej

2017-06-14

Phagocytes are the main component of innate immunity. They remove pathogens and particles from organisms using their bactericidal tools in the form of both reactive oxygen species and degrading enzymes-contained in granules-that are potentially toxic proteins. Therefore, it is important to investigate the possible interactions between phages and immune cells and avoid any phage side effects on them. Recent progress in knowledge concerning the influence of phages on phagocytes is also important as such interactions may shape the immune response. In this review we have summarized the current knowledge on phage interactions with phagocytes described so far and their potential implications for phage therapy . The data suggesting that phage do not downregulate important phagocyte functions are especially relevant for the concept of phage therapy.

Free Shiga toxin 1-encoding bacteriophages are less prevalent than Shiga toxin 2 phages in extraintestinal environments.

PubMed

Grau-Leal, Ferran; Quirós, Pablo; Martínez-Castillo, Alexandre; Muniesa, Maite

2015-11-01

Stx bacteriophages are involved in the pathogenicity of Stx-producing Escherichia coli. Induction of the Stx phage lytic cycle increases Stx expression and releases Stx phages that reach extracellular environments. Stx phage family comprises different phages that harbour any stx subtype. Stx2 is closely related with severe disease and therefore previous studies focused on free Stx2 phages in extraintestinal environments. To provide similar information regarding Stx1 phages, we evaluate free Stx1 phages in 357 samples of human and animal wastewater, faeces, river water, soil, sludge and food. Our method, based on quantification of stx1 in the DNA from the viral fraction, was validated using electron microscopy counting of phages and infectivity. The overall prevalence of Stx1 phages was very low: 7.6% of positive samples and values below 3 × 10(3) GC (gene copies) ml(-1) . These results contrast starkly with the abundance of Stx2 phages in the samples (68.4%). This environmental scarcity of free Stx1 phages is attributed to their lower rates of induction and the fact that Stx1 does not require phage induction to be expressed because it possesses an independent promoter. The implications of the low prevalence of free Stx1 phages for the emergence of new pathogenic strains in the environment are discussed. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine.

PubMed

Cao, Binrui; Yang, Mingying; Mao, Chuanbin

2016-06-21

Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (∼900 nm long and ∼8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic-inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic differentiation; (4) discovered that phage could induce angiogenesis and osteogenesis for MSC-based vascularized bone regeneration; (5) identified novel breast cancer cell-targeting and MSC-targeting peptides and used them to significantly improve the efficiency of targeted cancer therapy and MSC-based gene delivery, respectively; (6) employed engineered phage as a probe to achieve ultrasensitive detection of biomarkers from serum of human patients for disease diagnosis; and (7) constructed centimeter-scale 3D multilayered phage assemblies with the potential application as scaffolds for bone regeneration and functional device fabrication. Our findings demonstrated that phage is indeed a very powerful supramacromolecule suitable for not only developing novel nanostructures and biomaterials but also advancing important fields in biomedicine, including molecular targeting, cancer diagnosis and treatment, drug and gene delivery, stem cell fate direction, and tissue regeneration. Our successes in exploiting phage in chemistry, materials, and medicine suggest that phage itself is nontoxic at the cell level and can be safely used for detecting biomarkers in vitro. Moreover, although we have demonstrated successful in vivo tissue regeneration induced by phage, we believe future studies are needed to evaluate the in vivo biodistribution and potential risks of the phage-based biomaterials.

Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine

PubMed Central

Cao, Binrui; Yang, Mingying; Mao, Chuanbin

2016-01-01

CONSPECTUS Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (~900 nm long and ~8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic–inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic differentiation; (4) discovered that phage could induce angiogenesis and osteogenesis for MSC-based vascularized bone regeneration; (5) identified novel breast cancer cell-targeting and MSC-targeting peptides and used them to significantly improve the efficiency of targeted cancer therapy and MSC-based gene delivery, respectively; (6) employed engineered phage as a probe to achieve ultrasensitive detection of biomarkers from serum of human patients for disease diagnosis; and (7) constructed centimeter-scale 3D multilayered phage assemblies with the potential application as scaffolds for bone regeneration and functional device fabrication. Our findings demonstrated that phage is indeed a very powerful supramacromolecule suitable for not only developing novel nanostructures and biomaterials but also advancing important fields in biomedicine, including molecular targeting, cancer diagnosis and treatment, drug and gene delivery, stem cell fate direction, and tissue regeneration. Our successes in exploiting phage in chemistry, materials, and medicine suggest that phage itself is nontoxic at the cell level and can be safely used for detecting biomarkers in vitro. Moreover, although we have demonstrated successful in vivo tissue regeneration induced by phage, we believe future studies are needed to evaluate the in vivo biodistribution and potential risks of the phage-based biomaterials. PMID:27153341

Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

PubMed Central

Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia; Breitbart, Mya; Edwards, Robert A.

2015-01-01

Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set of publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. We propose adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution. PMID:26005436

Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

DOE PAGES

Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia; ...

2015-05-08

Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set ofmore » publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. By adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.« less

Phage survival: the biodegradability of M13 phage display library in vitro.

PubMed

Tóthová, L'ubomíra; Bábíčková, Janka; Celec, Peter

2012-01-01

Administration of bacteriophages is used for phage therapy modulation of gut microbiome or for in vivo phage display. The aim of the study was to analyze the survival of M13 phage in different body fluids and tissues in vitro. The survival of M13 phage was measured in vitro in human blood, saliva, urine, artificial gastric juice (AGJ), and mouse homogenates of stomach, jejunum, and colon after defined time points (5, 15, or 45 Min). The plates were inspected after overnight incubation and the plaques were counted. No phage was recovered after 5 Min of incubation with AGJ. In urine, the phage survival was decreased by 44% after 5 Min of incubation (P = 0.004). In saliva, the recovered titer was decreased by 33% and 88% (P < 0.05) after 15 and 45 Min, respectively. Phage coincubation with jejunum homogenate led to significant decrease of phage titer by 72% (P < 0.01) after 15 Min and by 99% (P < 0.001) after 45 Min. Decreased survival of M13 phage depending on time of incubation was proved under several in vitro conditions, with low pH in the AGJ having the most detrimental effect on phage survival. Phage pharmacokinetics described in vitro might have applications for the use of bacteriophages in vivo. © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia

Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set ofmore » publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. By adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.« less

Characterizing RecA-Independent Induction of Shiga toxin2-Encoding Phages by EDTA Treatment

PubMed Central

Imamovic, Lejla; Muniesa, Maite

2012-01-01

Background The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. Methodology/Principal Findings The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg2+. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction. Conclusions/Significance Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains. PMID:22393404

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

PubMed Central

Vongkamjan, Kitiya; Switt, Andrea Moreno; den Bakker, Henk C.; Fortes, Esther D.

2012-01-01

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 104 PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools. PMID:23042180

Genomes and Characterization of Phages Bcep22 and BcepIL02, Founders of a Novel Phage Type in Burkholderia cenocepacia▿†

PubMed Central

Gill, Jason J.; Summer, Elizabeth J.; Russell, William K.; Cologna, Stephanie M.; Carlile, Thomas M.; Fuller, Alicia C.; Kitsopoulos, Kate; Mebane, Leslie M.; Parkinson, Brandi N.; Sullivan, David; Carmody, Lisa A.; Gonzalez, Carlos F.; LiPuma, John J.; Young, Ry

2011-01-01

Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages. PMID:21804006

Genome Sequences of 12 Cluster AN Arthrobacter Phages

PubMed Central

Lee-Soety, Julia Y.; Bhatt, Shantanu; Adair, Tamarah L.; Bonilla, J. Alfred; Klyczek, Karen K.; Harrison, Melinda; Garlena, Rebecca A.; Bowman, Charles A.; Russell, Daniel A.; Jacobs-Sera, Deborah

2017-01-01

ABSTRACT Twelve siphoviral phages isolated using Arthrobacter sp. strain ATCC 21022 were sequenced. The phages all have relatively small genomes, ranging from 15,319 to 15,556 bp. All 12 phages are closely related to previously described cluster AN Arthrobacter phages. PMID:29122859

Novel Phage Group Infecting Lactobacillus delbrueckii subsp. lactis, as Revealed by Genomic and Proteomic Analysis of Bacteriophage Ldl1

PubMed Central

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio

2014-01-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ± 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species. PMID:25501478

Emerging methods to study bacteriophage infection at the single-cell level.

PubMed

Dang, Vinh T; Sullivan, Matthew B

2014-01-01

Bacteria and their viruses (phages) are abundant across diverse ecosystems and their interactions influence global biogeochemical cycles and incidence of disease. Problematically, both classical and metagenomic methods insufficiently assess the host specificity of phages and phage-host infection dynamics in nature. Here we review emerging methods to study phage-host interaction and infection dynamics with a focus on those that offer resolution at the single-cell level. These methods leverage ever-increasing sequence data to identify virus signals from single-cell amplified genome datasets or to produce primers/probes to target particular phage-bacteria pairs (digital PCR and phageFISH), even in complex communities. All three methods enable study of phage infection of uncultured bacteria from environmental samples, while the latter also discriminates between phage-host interaction outcomes (e.g., lytic, chronic, lysogenic) in model systems. Together these techniques enable quantitative, spatiotemporal studies of phage-bacteria interactions from environmental samples of any ecosystem, which will help elucidate and predict the ecological and evolutionary impacts of specific phage-host pairings in nature.

[Peptide phage display in biotechnology and biomedicine].

PubMed

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

Inhaled phage therapy: a promising and challenging approach to treat bacterial respiratory infections.

PubMed

Bodier-Montagutelli, Elsa; Morello, Eric; L'Hostis, Guillaume; Guillon, Antoine; Dalloneau, Emilie; Respaud, Renaud; Pallaoro, Nikita; Blois, Hélène; Vecellio, Laurent; Gabard, Jérôme; Heuzé-Vourc'h, Nathalie

2017-08-01

Bacterial respiratory tract infections (RTIs) are increasingly difficult to treat due to evolving antibiotic resistance. In this context, bacteriophages (or phages) are part of the foreseen alternatives or combination therapies. Delivering phages through the airways seems more relevant to accumulate these natural antibacterial viruses in proximity to their bacterial host, within the infectious site. Areas covered: This review addresses the potential of phage therapy to treat RTIs and discusses preclinical and clinical results of phages administration in this context. Recent phage formulation and aerosolization attempts are also reviewed, raising technical challenges to achieve efficient pulmonary deposition via inhalation. Expert opinion: Overall, the inhalation of phages as antibacterial treatment seems both clinically relevant and technically feasible. Several crucial points still need to be investigated, such as phage product pharmacokinetics and immunogenicity. Furthermore, given phage-specific features, appropriate regulatory and manufacturing guidelines will need to be defined. Finally, randomized controlled clinical trials should be carried out to establish phage therapy's clinical positioning in the antimicrobial arsenal against RTIs.

Safety analysis of a Russian phage cocktail: from metagenomic analysis to oral application in healthy human subjects.

PubMed

McCallin, Shawna; Alam Sarker, Shafiqul; Barretto, Caroline; Sultana, Shamima; Berger, Bernard; Huq, Sayeda; Krause, Lutz; Bibiloni, Rodrigo; Schmitt, Bertrand; Reuteler, Gloria; Brüssow, Harald

2013-09-01

Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb, including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure. Copyright © 2013 Elsevier Inc. All rights reserved.

Diversity of phage infection types and associated terminology: the problem with 'Lytic or lysogenic'.

PubMed

Hobbs, Zack; Abedon, Stephen T

2016-04-01

Bacteriophages, or phages, are viruses of members of domain Bacteria. These viruses play numerous roles in shaping the diversity of microbial communities, with impact differing depending on what infection strategies specific phages employ. From an applied perspective, these especially are communities containing undesired or pathogenic bacteria that can be modified through phage-mediated bacterial biocontrol, that is, through phage therapy. Here we seek to categorize phages in terms of their infection strategies as well as review or suggest more descriptive, accurate or distinguishing terminology. Categories can be differentiated in terms of (1) whether or not virion release occurs (productive infections versus lysogeny, pseudolysogeny and/or the phage carrier state), (2) the means of virion release (lytic versus chronic release) and (3) the degree to which phages are genetically equipped to display lysogenic cycles (temperate versus non-temperate phages). We address in particular the use or overuse of what can be a somewhat equivocal phrase, 'Lytic or lysogenic', especially when employed as a means of distinguishing among phages types. We suggest that the implied dichotomy is inconsistent with both modern as well as historical understanding of phage biology. We consider, therefore, less ambiguous terminology for distinguishing between 'Lytic' versus 'Lysogenic' phage types. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships

PubMed Central

Pope, Welkin H.; Mavrich, Travis N.; Garlena, Rebecca A.; Guerrero-Bustamante, Carlos A.; Jacobs-Sera, Deborah; Montgomery, Matthew T.; Russell, Daniel A.; Warner, Marcie H.

2017-01-01

ABSTRACT The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are “singletons” with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable. PMID:28811342

The scope of phage display for membrane proteins.

PubMed

Vithayathil, Rosemarie; Hooy, Richard M; Cocco, Melanie J; Weiss, Gregory A

2011-12-09

Numerous examples of phage display applied to soluble proteins demonstrate the power of the technique for protein engineering, affinity reagent discovery and structure-function studies. Recent reports have expanded phage display to include membrane proteins (MPs). The scope and limitations of MP display remain undefined. Therefore, we report data from the phage display of representative types of membrane-associated proteins including plasma, nuclear, peripheral, single and multipass. The peripheral MP neuromodulin displays robustly with packaging by conventional M13-KO7 helper phage. The monotopic MP Nogo-66 can also display on the phage surface, if packaged by the modified M13-KO7(+) helper phage. The modified phage coat of KO7(+) can better mimic the zwitterionic character of the plasma membrane. Four examples of putatively α-helical, integral MPs failed to express as fusions to an anchoring phage coat protein and therefore did not display on the phage surface. However, the β-barrel MPs ShuA (Shigella heme uptake A) and MOMP (major outer membrane protein), which pass through the membrane 22 and 16 times, respectively, can display surprisingly well on the surfaces of both conventional and KO7(+) phages. The results provide a guide for protein engineering and large-scale mutagenesis enabled by the phage display of MPs. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bacteriophages of wastewater foaming-associated filamentous Gordonia reduce host levels in raw activated sludge.

PubMed

Liu, Mei; Gill, Jason J; Young, Ry; Summer, Elizabeth J

2015-09-09

Filamentous bacteria are a normal and necessary component of the activated sludge wastewater treatment process, but the overgrowth of filamentous bacteria results in foaming and bulking associated disruptions. Bacteriophages, or phages, were investigated for their potential to reduce the titer of foaming bacteria in a mixed-microbial activated sludge matrix. Foaming-associated filamentous bacteria were isolated from activated sludge of a commercial wastewater treatment plan and identified as Gordonia species by 16S rDNA sequencing. Four representative phages were isolated that target G. malaquae and two un-named Gordonia species isolates. Electron microscopy revealed the phages to be siphophages with long tails. Three of the phages--GordTnk2, Gmala1, and GordDuk1--had very similar ~76 kb genomes, with >93% DNA identity. These genomes shared limited synteny with Rhodococcus equi phage ReqiDocB7 and Gordonia phage GTE7. In contrast, the genome of phage Gsput1 was smaller (43 kb) and was not similar enough to any known phage to be placed within an established phage type. Application of these four phages at MOIs of 5-15 significantly reduced Gordonia host levels in a wastewater sludge model by approximately 10-fold as compared to non-phage treated reactors. Phage control was observed for nine days after treatment.

A highly specific phage defense system is a conserved feature of the Vibrio cholerae mobilome

PubMed Central

O’Hara, Brendan J.

2017-01-01

Vibrio cholerae-specific bacteriophages are common features of the microbial community during cholera infection in humans. Phages impose strong selective pressure that favors the expansion of phage-resistant strains over their vulnerable counterparts. The mechanisms allowing virulent V. cholerae strains to defend against the ubiquitous threat of predatory phages have not been established. Here, we show that V. cholerae PLEs (phage-inducible chromosomal island-like elements) are widespread genomic islands dedicated to phage defense. Analysis of V. cholerae isolates spanning a 60-year collection period identified five unique PLEs. Remarkably, we found that all PLEs (regardless of geographic or temporal origin) respond to infection by a myovirus called ICP1, the most prominent V. cholerae phage found in cholera patient stool samples from Bangladesh. We found that PLE activity reduces phage genome replication and accelerates cell lysis following ICP1 infection, killing infected host cells and preventing the production of progeny phage. PLEs are mobilized by ICP1 infection and can spread to neighboring cells such that protection from phage predation can be horizontally acquired. Our results reveal that PLEs are a persistent feature of the V. cholerae mobilome that are adapted to providing protection from a single predatory phage and advance our understanding of how phages influence pathogen evolution. PMID:28594826

A highly specific phage defense system is a conserved feature of the Vibrio cholerae mobilome.

PubMed

O'Hara, Brendan J; Barth, Zachary K; McKitterick, Amelia C; Seed, Kimberley D

2017-06-01

Vibrio cholerae-specific bacteriophages are common features of the microbial community during cholera infection in humans. Phages impose strong selective pressure that favors the expansion of phage-resistant strains over their vulnerable counterparts. The mechanisms allowing virulent V. cholerae strains to defend against the ubiquitous threat of predatory phages have not been established. Here, we show that V. cholerae PLEs (phage-inducible chromosomal island-like elements) are widespread genomic islands dedicated to phage defense. Analysis of V. cholerae isolates spanning a 60-year collection period identified five unique PLEs. Remarkably, we found that all PLEs (regardless of geographic or temporal origin) respond to infection by a myovirus called ICP1, the most prominent V. cholerae phage found in cholera patient stool samples from Bangladesh. We found that PLE activity reduces phage genome replication and accelerates cell lysis following ICP1 infection, killing infected host cells and preventing the production of progeny phage. PLEs are mobilized by ICP1 infection and can spread to neighboring cells such that protection from phage predation can be horizontally acquired. Our results reveal that PLEs are a persistent feature of the V. cholerae mobilome that are adapted to providing protection from a single predatory phage and advance our understanding of how phages influence pathogen evolution.

High stability of Stx2 phage in food and under food-processing conditions.

PubMed

Rode, Tone Mari; Axelsson, Lars; Granum, Per Einar; Heir, Even; Holck, Askild; L'abée-Lund, Trine M

2011-08-01

Bacteriophages (phages) carrying Shiga toxin genes constitute a major virulence attribute in enterohemorrhagic Escherichia coli (EHEC). Several EHEC outbreaks have been linked to food. The survival of such strains in different foods has received much attention, while the fate of the mobile Shiga toxin-converting phages (Stx phages) has been less studied. We have investigated the stability of an Stx phage in several food products and examined how storage, food processing, and disinfection influence the infectivity of phage particles. The study involved a recombinant Stx phage (Δstx::cat) of an E. coli O103:H25 strain from a Norwegian outbreak in 2006. Temperature, matrix, and time were factors of major importance for the stability of phage particles. Phages stored at cooling temperatures (4°C) showed a dramatic reduction in stability compared to those stored at room temperature. The importance of the matrix was evident at higher temperatures (60°C). Phages in ground beef were below the detection level when heated to 60°C for more than 10 min, while phages in broth exposed to the same heating conditions showed a 5-log-higher stability. The phages tolerated desiccation poorly but were infective for a substantial period of time in solutions. Under moist conditions, they also had a high ability to tolerate exposure to several disinfectants. In a dry-fermented sausage model, phages were shown to infect E. coli in situ. The results show that Stx phage particles can maintain their infectivity in foods and under food-processing conditions.

Analysis of whole genome sequencing for the Escherichia coli O157:H7 typing phages.

PubMed

Cowley, Lauren A; Beckett, Stephen J; Chase-Topping, Margo; Perry, Neil; Dallman, Tim J; Gally, David L; Jenkins, Claire

2015-04-08

Shiga toxin producing Escherichia coli O157 can cause severe bloody diarrhea and haemolytic uraemic syndrome. Phage typing of E. coli O157 facilitates public health surveillance and outbreak investigations, certain phage types are more likely to occupy specific niches and are associated with specific age groups and disease severity. The aim of this study was to analyse the genome sequences of 16 (fourteen T4 and two T7) E. coli O157 typing phages and to determine the genes responsible for the subtle differences in phage type profiles. The typing phages were sequenced using paired-end Illumina sequencing at The Genome Analysis Centre and the Animal Health and Veterinary Laboratories Agency and bioinformatics programs including Velvet, Brig and Easyfig were used to analyse them. A two-way Euclidian cluster analysis highlighted the associations between groups of phage types and typing phages. The analysis showed that the T7 typing phages (9 and 10) differed by only three genes and that the T4 typing phages formed three distinct groups of similar genomic sequences: Group 1 (1, 8, 11, 12 and 15, 16), Group 2 (3, 6, 7 and 13) and Group 3 (2, 4, 5 and 14). The E. coli O157 phage typing scheme exhibited a significantly modular network linked to the genetic similarity of each group showing that these groups are specialised to infect a subset of phage types. Sequencing the typing phage has enabled us to identify the variable genes within each group and to determine how this corresponds to changes in phage type.

Comparative genomics of 9 novel Paenibacillus larvae bacteriophages

PubMed Central

Stamereilers, Casey; LeBlanc, Lucy; Yost, Diane; Amy, Penny S.; Tsourkas, Philippos K.

2016-01-01

ABSTRACT American Foulbrood Disease, caused by the bacterium Paenibacillus larvae, is one of the most destructive diseases of the honeybee, Apis mellifera. Our group recently published the sequences of 9 new phages with the ability to infect and lyse P. larvae. Here, we characterize the genomes of these P. larvae phages, compare them to each other and to other sequenced P. larvae phages, and putatively identify protein function. The phage genomes are 38–45 kb in size and contain 68–86 genes, most of which appear to be unique to P. larvae phages. We classify P. larvae phages into 2 main clusters and one singleton based on nucleotide sequence identity. Three of the new phages show sequence similarity to other sequenced P. larvae phages, while the remaining 6 do not. We identified functions for roughly half of the P. larvae phage proteins, including structural, assembly, host lysis, DNA replication/metabolism, regulatory, and host-related functions. Structural and assembly proteins are highly conserved among our phages and are located at the start of the genome. DNA replication/metabolism, regulatory, and host-related proteins are located in the middle and end of the genome, and are not conserved, with many of these genes found in some of our phages but not others. All nine phages code for a conserved N-acetylmuramoyl-L-alanine amidase. Comparative analysis showed the phages use the “cohesive ends with 3′ overhang” DNA packaging strategy. This work is the first in-depth study of P. larvae phage genomics, and serves as a marker for future work in this area. PMID:27738559

Cytotoxic Tumor-Targeting Peptides From In Vivo Phage Display.

PubMed

Northup, Jessica R Newton; Deutscher, Susan L

2016-01-01

We previously utilized an in vivo peptide phage display selection technique, which included the use of detergent elution of phage from excised tumor, to obtain tumor-targeting phage with the ability to extravasate the vasculature and bind directly to prostate tumor tissue. It is hypothesized that this same in vivo phage selection technique can be used to functionally select for molecules that not only bind to cancer cells but also kill them. Here we analyzed two different in vivo phage display selected phage clones, G1 and H5, retrieved from PC-3 human prostate carcinoma xenografted tumors. First, cell de-attachment as an endpoint criterion for apoptosis and cell cycle was examined. After 2.5 hours incubation with G1 phage, PC-3 cell attachment was reduced by 23.8% and the percent of cell population in M phase reduced by 32.1%. In comparison, PC-3 cells incubated with H5 phage had a reduction of 25.0% cell attachment and 33.6% of cell population in M phase. These changes in combination with elevated caspase activation within cells in M phase, and no significant changes to G1/G0 or S phase cell populations suggest that the cytotoxic phages are targeting actively dividing PC-3 cells. Microscopic studies were also performed to further analyze the nature of cytotoxicity of these two phage clones. It was found that G1 phage induced and co- localized with tubulin based projections within apoptotic cells, while H5 phage did not. These phage may form the foundation for a new class of targeted prostate cancer therapeutic agents.

High Stability of Stx2 Phage in Food and under Food-Processing Conditions ▿

PubMed Central

Rode, Tone Mari; Axelsson, Lars; Granum, Per Einar; Heir, Even; Holck, Askild; L'Abée-Lund, Trine M.

2011-01-01

Bacteriophages (phages) carrying Shiga toxin genes constitute a major virulence attribute in enterohemorrhagic Escherichia coli (EHEC). Several EHEC outbreaks have been linked to food. The survival of such strains in different foods has received much attention, while the fate of the mobile Shiga toxin-converting phages (Stx phages) has been less studied. We have investigated the stability of an Stx phage in several food products and examined how storage, food processing, and disinfection influence the infectivity of phage particles. The study involved a recombinant Stx phage (Δstx::cat) of an E. coli O103:H25 strain from a Norwegian outbreak in 2006. Temperature, matrix, and time were factors of major importance for the stability of phage particles. Phages stored at cooling temperatures (4°C) showed a dramatic reduction in stability compared to those stored at room temperature. The importance of the matrix was evident at higher temperatures (60°C). Phages in ground beef were below the detection level when heated to 60°C for more than 10 min, while phages in broth exposed to the same heating conditions showed a 5-log-higher stability. The phages tolerated desiccation poorly but were infective for a substantial period of time in solutions. Under moist conditions, they also had a high ability to tolerate exposure to several disinfectants. In a dry-fermented sausage model, phages were shown to infect E. coli in situ. The results show that Stx phage particles can maintain their infectivity in foods and under food-processing conditions. PMID:21685156

Humoral immune responses against gonadotropin releasing hormone elicited by immunization with phage-peptide constructs obtained via phage display.

PubMed

Samoylov, Alexandre; Cochran, Anna; Schemera, Bettina; Kutzler, Michelle; Donovan, Caitlin; Petrenko, Valery; Bartol, Frank; Samoylova, Tatiana

2015-12-20

Phage display is based on genetic engineering of phage coat proteins resulting in fusion peptides displayed on the surface of phage particles. The technology is widely used for generation of phages with novel characteristics for numerous applications in biomedicine and far beyond. The focus of this study was on development of phage-peptide constructs that stimulate production of antibodies against gonadotropin releasing hormone (GnRH). Phage-peptide constructs that elicit production of neutralizing GnRH antibodies can be used for anti-fertility and anti-cancer applications. Phage-GnRH constructs were generated via selection from a phage display library using several types of GnRH antibodies as selection targets. Such phage constructs were characterized for sequence similarities to GnRH peptide and frequency of their occurrence in the selection rounds. Five of the constructs with suitable characteristics were tested in mice as a single dose 5×10(11) virions (vir) vaccine and were found to be able to stimulate production of GnRH-specific antibodies, but not to suppress testosterone (indirect indicator of GnRH antibody neutralizing properties). Next, one of the constructs was tested at a higher dose of 2×10(12) vir per mouse in combination with a poly(lactide-co-glycolide) (PLGA)-based adjuvant. This resulted in multifold increase in GnRH antibody production and significant reduction of serum testosterone, indicating that antibodies produced in response to the phage-GnRH immunization possess neutralizing properties. To achieve optimal immune responses for desired applications, phage-GnRH constructs can be modified with respect to flanking sequences of GnRH-like peptides displayed on phage. Anticipated therapeutic effects also might be attained using optimized phage doses, a combination of several constructs in a single treatment, or application of adjuvants and advanced phage delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus.

PubMed

Zago, Miriam; Scaltriti, Erika; Fornasari, Maria Emanuela; Rivetti, Claudio; Grolli, Stefano; Giraffa, Giorgio; Ramoni, Roberto; Carminati, Domenico

2012-01-01

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥6log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions. Copyright © 2011 Elsevier B.V. All rights reserved.

Tables of Transition Probabilities and Branching Ratios for Electric Dipole Transitions Between Arbitrary Levels of Hydrogen-Like Atoms

NASA Technical Reports Server (NTRS)

Omidvar, K.

1980-01-01

Branching ratios in hydrogen-like atoms due to electric-dipole transitions are tabulated for the initial principal and angular momentum quantum number n, lambda, and final principal and angular momentum quantum numbers n, lambda. In table 1, transition probabilities are given for transitions n, lambda, yields n, where sums have been made with respect to lambda. In this table, 2 or = n' or = 10, o or = lambda' or = n'-1, and 1 or = n or = n'-1. In addition, averages with respect to lambda' and sums with respect to n, and lifetimes are given. In table 2, branching ratios are given for transitions n' lambda' yields ni, where sums have been made with respect to lambda. In these tables, 2 or = n' or = 10, 0 or = lambda', n'-1, and 1 or = n or = n'-1. Averages with respect to lambda' are also given. In table 3, branching ratios are given for transitions n' lambda' yields in lambda, where 1 or = n or = 5, 0 or = lambda or = n-1, n n' or = 15, and 0 or = lambda' or = n(s), where n(s), is the smaller of the two numbers n'-1 and 6. Averages with respect to lambda' are given.

Measurement of the Λ b cross section and the Λ ¯ b to Λ b ratio with J / ψ Λ decays in pp collisions at s = 7 TeV

DOE PAGES

Chatrchyan, S.; Khachatryan, V.; Sirunyan, A. M.; ...

2012-05-31

The Lambda(b) differential production cross section and the cross section ratio anti-Lambda(b)/Lambda(b) are measured as functions of transverse momentum pt(Lambda(b)) and rapidity abs(y(Lambda(b))) in pp collisions at sqrt(s) = 7 TeV using data collected by the CMS experiment at the LHC. The measurements are based on Lambda(b) decays reconstructed in the exclusive final state J/Psi Lambda, with the subsequent decays J/Psi to an opposite-sign muon pair and Lambda to proton pion, using a data sample corresponding to an integrated luminosity of 1.9 inverse femtobarns. The product of the cross section times the branching ratio for Lambda(b) to J/Psi Lambda versusmore » pt(Lambda(b)) falls faster than that of b mesons. The measured value of the cross section times the branching ratio for pt(Lambda(b)) > 10 GeV and abs(y(Lambda(b))) < 2.0 is 1.06 +/- 0.06 +/- 0.12 nb, and the integrated cross section ratio for anti-Lambda(b)/Lambda(b) is 1.02 +/- 0.07 +/- 0.09, where the uncertainties are statistical and systematic, respectively.« less

{lambda}{sub b}{yields}p, {lambda} transition form factors from QCD light-cone sum rules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Yuming; Lue Caidian; Shen Yuelong

2009-10-01

Light-cone sum rules for the {lambda}{sub b}{yields}p, {lambda} transition form factors are derived from the correlation functions expanded by the twist of the distribution amplitudes of the {lambda}{sub b} baryon. In terms of the {lambda}{sub b} three-quark distribution amplitude models constrained by the QCD theory, we calculate the form factors at small momentum transfers and compare the results with those estimated in the conventional light-cone sum rules (LCSR) and perturbative QCD approaches. Our results indicate that the two different versions of sum rules can lead to the consistent numbers of form factors responsible for {lambda}{sub b}{yields}p transition. The {lambda}{sub b}{yields}{lambda}more » transition form factors from LCSR with the asymptotic {lambda} baryon distribution amplitudes are found to be almost 1 order larger than those obtained in the {lambda}{sub b}-baryon LCSR, implying that the preasymptotic corrections to the baryonic distribution amplitudes are of great importance. Moreover, the SU(3) symmetry breaking effects between the form factors f{sub 1}{sup {lambda}{sub b}}{sup {yields}}{sup p} and f{sub 1}{sup {lambda}{sub b}}{sup {yields}}{sup {lambda}} are computed as 28{sub -8}{sup +14}% in the framework of {lambda}{sub b}-baryon LCSR.« less

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

First observation and measurement of the resonant structure of the lambda_b->lambda_c pi-pi+pi- decay mode

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azzurri, P.; Barria, P.; Ciocci, M.A.

The authors present the first observation of the {Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}{sup +}{pi}{sup -}{pi}{sup +}{pi}{sup -} decay using data from an integrated luminosity of approximately 2.4 fb{sup -1} of p{bar p} collisions at {radical}s = 1.96 TeV, collected with the CDF II detector at the Fermilab Tevatron. They also present the first observation of the resonant decays {Lambda}{sub b}{sup 0} {yields} {Sigma}{sub c}(2455){sup 0} {pi}{sup +}{pi}{sup -} {yields} {Lambda}{sub c}{sup +}{pi}{sup -}{pi}{sup +}{pi}{sup -}, {Lambda}{sub b}{sup 0} {yields} {Sigma}{sub c}(2455){sup ++}{pi}{sup -}{pi}{sup -} {yields} {Lambda}{sub c}{sup +}{pi}{sup -}{pi}{sup +}{pi}{sup -}, {Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}(2595){sup +}{pi}{sup -}more » {yields} {Lambda}{sub c}{sup +}{pi}{sup -}{pi}{sup +}{pi}{sup -} and {Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}(2625){sup +}{pi}{sup -} {yields} {Lambda}{sub c}{sup +}{pi}{sup -}{pi}{sup +}{pi}{sup -}, and measure their relative branching ratios.« less

Spin correlations in the {Lambda}{Lambda} and {Lambda}{Lambda}-bar systems generated in relativistic heavy-ion collisions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyuboshitz, V. L.; Lyuboshitz, V. V., E-mail: Valery.Lyuboshitz@jinr.r

2010-05-15

Spin correlations for the {Lambda}{Lambda} and {Lambda}{Lambda}-bar pairs, generated in relativistic heavy-ion collisions, and related angular correlations at the joint registration of hadronic decays of two hyperons, in which space parity is not conserved, are analyzed. The correlation tensor components can be derived from the double angular distribution of products of two decays by the method of 'moments'. The properties of the 'trace' of the correlation tensor (a sum of three diagonal components), determining the relative fractions of the triplet states and singlet state of respective pairs, are discussed. Spin correlations for two identical particles ({Lambda}{Lambda}) and two nonidentical particlesmore » ({Lambda}{Lambda}-bar) are considered from the viewpoint of the conventional model of one-particle sources. In the framework of this model, correlations vanish at sufficiently large relative momenta. However, under these conditions, in the case of two nonidentical particles ({Lambda}{Lambda}-bar) a noticeable role is played by two-particle annihilation (two-quark, two-gluon) sources, which lead to the difference of the correlation tensor from zero. In particular, such a situation may arise when the system passes through the 'mixed phase.'« less

Therapeutic use of chimeric bacteriophage (phage) lysins in staphylococcal endophthalmitis

USDA-ARS?s Scientific Manuscript database

Purpose: Phage endolysins are peptidoglycan hydrolases that are produced at the end of the phage lytic cycle to digest the host bacterial cell wall, facilitating the release of mature phage progeny. The aim of this study is to determine the antimicrobial activity of chimeric phage lysins against cli...

Phage display for generating peptide reagents.

PubMed

Brigati, Jennifer R; Samoylova, Tatiana I; Jayanna, Prashanth K; Petrenko, Valery A

2008-02-01

This unit presents detailed protocols for selection and propagation of landscape phages, which are fusions of filamentous phage fd (or its close relatives M13 and f1) and foreign DNA that result in chimeric phage virions with foreign peptides (8 to 9 amino acids long) covering the entire surface of the phage particles. These landscape phages bind specifically to mammalian and bacterial cells, spores, or discrete molecular targets. (c) 2008 by John Wiley & Sons, Inc.

Thioredoxin is required for filamentous phage assembly.

PubMed Central

Russel, M; Model, P

1985-01-01

Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly. Images PMID:3881756

Effectiveness of the lactococcal abortive infection systems AbiA, AbiE, AbiF and AbiG against P335 type phages.

PubMed

Tangney, Mark; Fitzgerald, Gerald F

2002-04-23

Four lactococcal abortive infection mechanisms were introduced into strains which were sensitive hosts for P335 type phages and plaque assay experiments performed to assess their effect on five lactococcal bacteriophages from this family. Results indicate that AbiA inhibits all five P335 phages tested, while AbiG affects phiP335 itself and phiQ30 but not the other P335 species phages. AbiA was shown to retard phage Q30 DNA replication as previously reported for other phages. It was also demonstrated that AbiG, previously shown to act at a point after DNA replication in the cases of c2 type and 936 type phages, acts at the level of, or prior to phage Q30 DNA replication. AbiE and AbiF had no effect on the P335 type phages examined.

Novel chitosan film embedded with liposome-encapsulated phage for biocontrol of Escherichia coli O157:H7 in beef.

PubMed

Cui, Haiying; Yuan, Lu; Lin, Lin

2017-12-01

In recent years, phages used for the reduction of pathogenic bacteria have fostered many attentions, but they are liable to lost bioactivity in food due to the presence of acidic compounds, enzymes and evaporite materials. To improve the stability of phages, a chitosan edible film containing liposome-encapsulated phage was engineered in the present study. The characteristics of liposome-encapsulated phage and the chitosan film containing liposome-encapsulated phage were investigated. The encapsulation efficiency of phages in liposome reached 57.66±0.12%. Besides, the desirable physical properties of chitosan film were obtained. The chitosan film embedded with liposome-encapsulated phage exhibited high antibacterial activity against Escherichia coli O157:H7, without the impact on the sensory properties of beef. Hence, chitosan film containing liposome-encapsulated phage could be a promising antibacterial packaging for beef preservation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Polar flagella rotation in Vibrio parahaemolyticus confers resistance to bacteriophage infection

PubMed Central

Zhang, Hui; Li, Lu; Zhao, Zhe; Peng, Daxin; Zhou, Xiaohui

2016-01-01

Bacteriophage has been recognized as a novel approach to treat bacterial infectious diseases. However, phage resistance may reduce the efficacy of phage therapy. Here, we described a mechanism of bacterial resistance to phage infections. In Gram-negative enteric pathogen Vibrio parahaemolyticus, we found that polar flagella can reduce the phage infectivity. Deletion of polar flagella, but not the lateral flagella, can dramatically promote the adsorption of phage to the bacteria and enhances the phage infectivity to V. parahaemolyticus, indicating that polar flagella play an inhibitory role in the phage infection. Notably, it is the rotation, not the physical presence, of polar flagella that inhibits the phage infection of V. parahaemolyticus. Strikingly, phage dramatically reduces the virulence of V. parahaemolyticus only when polar flagella were absent both in vitro and in vivo. These results indicated that polar flagella rotation is a previously unidentified mechanism that confers bacteriophage resistance. PMID:27189325

Phage display—A powerful technique for immunotherapy

PubMed Central

Bazan, Justyna; Całkosiński, Ireneusz; Gamian, Andrzej

2012-01-01

One of the most effective molecular diversity techniques is phage display. This technology is based on a direct linkage between phage phenotype and its encapsulated genotype, which leads to presentation of molecule libraries on the phage surface. Phage display is utilized in studying protein-ligand interactions, receptor binding sites and in improving or modifying the affinity of proteins for their binding partners. Generating monoclonal antibodies and improving their affinity, cloning antibodies from unstable hybridoma cells and identifying epitopes, mimotopes and functional or accessible sites from antigens are also important advantages of this technology. Techniques originating from phage display have been applied to transfusion medicine, neurological disorders, mapping vascular addresses and tissue homing of peptides. Phages have been applicable to immunization therapies, which may lead to development of new tools used for treating autoimmune and cancer diseases. This review describes the phage display technology and presents the recent advancements in therapeutic applications of phage display. PMID:22906939

Bacteriophage Procurement for Therapeutic Purposes

PubMed Central

Weber-Dąbrowska, Beata; Jończyk-Matysiak, Ewa; Żaczek, Maciej; Łobocka, Małgorzata; Łusiak-Szelachowska, Marzanna; Górski, Andrzej

2016-01-01

Bacteriophages (phages), discovered 100 years ago, are able to infect and destroy only bacterial cells. In the current crisis of antibiotic efficacy, phage therapy is considered as a supplementary or even alternative therapeutic approach. Evolution of multidrug-resistant and pandrug-resistant bacterial strains poses a real threat, so it is extremely important to have the possibility to isolate new phages for therapeutic purposes. Our phage laboratory and therapy center has extensive experience with phage isolation, characterization, and therapeutic application. In this article we present current progress in bacteriophages isolation and use for therapeutic purposes, our experience in this field and its practical implications for phage therapy. We attempt to summarize the state of the art: properties of phages, the methods for their isolation, criteria of phage selection for therapeutic purposes and limitations of their use. Perspectives for the use of genetically engineered phages to specifically target bacterial virulence-associated genes are also briefly presented. PMID:27570518

Development of Engineered Bacteriophages for Escherichia coli Detection and High-Throughput Antibiotic Resistance Determination.

PubMed

Chen, Juhong; Alcaine, Samuel D; Jackson, Angelyca A; Rotello, Vincent M; Nugen, Sam R

2017-04-28

T7 bacteriophages (phages) have been genetically engineered to carry the lacZ operon, enabling the overexpression of beta-galactosidase (β-gal) during phage infection and allowing for the enhanced colorimetric detection of Escherichia coli (E. coli). Following the phage infection of E. coli, the enzymatic activity of the released β-gal was monitored using a colorimetric substrate. Compared with a control T7 phage, our T7 lacZ phage generated significantly higher levels of β-gal expression following phage infection, enabling a lower limit of detection for E. coli cells. Using this engineered T7 lacZ phage, we were able to detect E. coli cells at 10 CFU·mL -1 within 7 h. Furthermore, we demonstrated the potential for phage-based sensing of bacteria antibiotic resistance profiling using our T7 lacZ phage, and subsequent β-gal expression to detect antibiotic resistant profile of E. coli strains.

The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808

PubMed Central

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-01-01

The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed. PMID:24400001

Phage therapy dosing: The problem(s) with multiplicity of infection (MOI).

PubMed

Abedon, Stephen T

2016-01-01

The concept of bacteriophage multiplicity of infection (MOI) - ratios of phages to bacteria - historically has been less easily applied than many phage workers would prefer or, perhaps, may be aware. Here, toward clarification of the concept, I discuss multiplicity of infection in terms of semantics, history, mathematics, pharmacology, and actual practice. For phage therapy and other biocontrol purposes it is desirable, especially, not to solely employ MOI to describe what phage quantities have been applied during dosing. Why? Bacterial densities can change between bacterial challenge and phage application, may not be easily determined immediately prior to phage dosing, and/or target bacterial populations may not be homogeneous with regard to phage access and thereby inconsistent in terms of what MOI individual bacteria experience. Toward experiment reproducibility and as practiced generally for antibacterial application, phage dosing instead should be described in terms of concentrations of formulations (phage titers) as well as volumes applied and, in many cases, absolute numbers of phages delivered. Such an approach typically will be far more desirable from a pharmacological perspective than solely indicating ratios of agents to bacteria. This essay was adapted, with permission, from an appendix of the 2011 monograph, Bacteriophages and Biofilms , Nova Science Publishers.

The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808.

PubMed

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-12-24

The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed.

Stochasticity in the Expression of LamB and its Affect on λ phage Infection

NASA Astrophysics Data System (ADS)

Chapman, Emily; Wu, Xiao-Lun

2006-03-01

λ phage binds to E. Coli's lamB protein and injects its DNA into the cell. The phage quickly replicates and after a latent period the bacteria bursts, emitting mature phages. We developed a mathematical model based on the known physical events that occur when a λ phage infects an E.Coli cell. The results of these models predict that the bacteria and phage populations become extinct unless the parameters of the model are very finely tuned, which is untrue in the nature. The lamB protein is part of the maltose regulon and can be repressed to minimal levels when grown in the absence of inducer. Therefore, a cell that is not expressing any lamB protein at that moment is resistant against phage infection. We studied the dynamic relationship between λ phage and E. Coli when the concentration of phage greatly outnumbers the concentration of bacteria. We study how the stochasticity of the expression of lamB affects the percentage of cells that the λ phage infects. We show that even in the case when the maltose regulon is fully induced a percentage of cells continue to persist against phage infection.

Phage Therapy: Eco-Physiological Pharmacology

PubMed Central

Abedon, Stephen T.

2014-01-01

Bacterial virus use as antibacterial agents, in the guise of what is commonly known as phage therapy, is an inherently physiological, ecological, and also pharmacological process. Physiologically we can consider metabolic properties of phage infections of bacteria and variation in those properties as a function of preexisting bacterial states. In addition, there are patient responses to pathogenesis, patient responses to phage infections of pathogens, and also patient responses to phage virions alone. Ecologically, we can consider phage propagation, densities, distribution (within bodies), impact on body-associated microbiota (as ecological communities), and modification of the functioning of body “ecosystems” more generally. These ecological and physiological components in many ways represent different perspectives on otherwise equivalent phenomena. Comparable to drugs, one also can view phages during phage therapy in pharmacological terms. The relatively unique status of phages within the context of phage therapy as essentially replicating antimicrobials can therefore result in a confluence of perspectives, many of which can be useful towards gaining a better mechanistic appreciation of phage therapy, as I consider here. Pharmacology more generally may be viewed as a discipline that lies at an interface between organism-associated phenomena, as considered by physiology, and environmental interactions as considered by ecology. PMID:25031881

In silico analysis of AHJD-like viruses, Staphylococcus aureus phages S24-1 and S13′, and study of phage S24-1 adsorption

PubMed Central

Uchiyama, Jumpei; Takemura-Uchiyama, Iyo; Kato, Shin-ichiro; Sato, Miho; Ujihara, Takako; Matsui, Hidehito; Hanaki, Hideaki; Daibata, Masanori; Matsuzaki, Shigenobu

2014-01-01

Staphylococcus aureus is a clinically important bacterium that is commensal in both humans and animals. Bacteriophage (phage) attachment to the host bacterial surface is an important process during phage infection, which involves interactions between phage receptor-binding proteins and host receptor molecules. However, little information is available on the receptor-binding protein of S. aureus phages. S. aureus virulent phages S24-1 and S13′ (family Podoviridae, genus AHJD-like viruses) were isolated from sewage. In the present study, we investigated the receptor-binding protein of AHJD-like viruses using phage S24-1. First, based on a comparative genomic analysis of phages S24-1 and S13′, open reading frame 16 (ORF16) of phage S24-1 was speculated to be the receptor-binding protein, which possibly determines the host range. Second, we demonstrated that this was the receptor-binding protein of phage S24-1. Third, our study suggested that wall teichoic acids in the cell walls of S. aureus are the main receptor molecules for ORF16 and phage S24-1. Finally, the C-terminal region of ORF16 may be essential for binding to S. aureus. These results strongly suggest that ORF16 of phage S24-1 and its homologs may be the receptor-binding proteins of AHJD-like viruses. PMID:24591378

Adapter-directed display: a modular design for shuttling display on phage surfaces.

PubMed

Wang, Kevin Caili; Wang, Xinwei; Zhong, Pingyu; Luo, Peter Peizhi

2010-02-05

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems. Copyright 2009 Elsevier Ltd. All rights reserved.

Application of a phage in decontaminating Vibrio parahaemolyticus in oysters.

PubMed

Zhang, Hui; Yang, Zhenquan; Zhou, Yan; Bao, Hongduo; Wang, Ran; Li, Tingwu; Pang, Maoda; Sun, Lichang; Zhou, Xiaohui

2018-06-20

Vibrio parahaemolyticus is a major pathogen that is mainly associated with seafood and is a global concern of food safety. With high prevalence of contamination in food, efficient strategy is needed to decontaminate those contaminated foods and control the emergence of vibriosis. In the present study, a V. parahaemolyticus-specific phage vB_VpaS_OMN (designated as phage OMN) was isolated from oyster. Phage OMN had good pH (5-9) and temperature tolerance (<50 °C). Phage OMN exhibited broad host range against isolates of V. parahaemolyticus (20/31). After treatment with phage OMN in the liquid condition for 7 h, the number of V. parahaemolyticus was reduced significantly compared to control treatment. When phage OMN was applied to oyster samples for 48 and 72 h, 90% and 99%, respectively, of V. parahaemolyticus was inactivated on Oyster meat surface. Sequence analysis showed that phage OMN had a 42.202 bp genome and revealed about 59.04% homology with Cronobacter phage vB_CsaP_Ss1. Only 10 CDSs can be predicted based on the GenBank database, while 42% of the CDSs were unique to OMN and had no known function, indicating that phage OMN is a new lytic phage. Fully understanding of the function for the phage genes and the properties of the phage is important for the development of strategies to control V. parahaemolyticus contamination in oysters and disease in aquaculture. Copyright © 2018 Elsevier B.V. All rights reserved.

Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity.

PubMed

Murphy, James; Klumpp, Jochen; Mahony, Jennifer; O'Connell-Motherway, Mary; Nauta, Arjen; van Sinderen, Douwe

2014-10-01

So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return. Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages. SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.

Metagenomic Analysis of Dairy Bacteriophages: Extraction Method and Pilot Study on Whey Samples Derived from Using Undefined and Defined Mesophilic Starter Cultures

PubMed Central

Muhammed, Musemma K.; Kot, Witold; Neve, Horst; Mahony, Jennifer; Castro-Mejía, Josué L.; Krych, Lukasz; Hansen, Lars H.; Nielsen, Dennis S.; Sørensen, Søren J.; Heller, Knut J.; van Sinderen, Douwe

2017-01-01

ABSTRACT Despite being potentially highly useful for characterizing the biodiversity of phages, metagenomic studies are currently not available for dairy bacteriophages, partly due to the lack of a standard procedure for phage extraction. We optimized an extraction method that allows the removal of the bulk protein from whey and milk samples with losses of less than 50% of spiked phages. The protocol was applied to extract phages from whey in order to test the notion that members of Lactococcus lactis 936 (now Sk1virus), P335, c2 (now C2virus) and Leuconostoc phage groups are the most frequently encountered in the dairy environment. The relative abundance and diversity of phages in eight and four whey mixtures from dairies using undefined mesophilic mixed-strain cultures containing Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc species (i.e., DL starter cultures) and defined cultures, respectively, were assessed. Results obtained from transmission electron microscopy and high-throughput sequence analyses revealed the dominance of Lc. lactis 936 phages (order Caudovirales, family Siphoviridae) in dairies using undefined DL starter cultures and Lc. lactis c2 phages (order Caudovirales, family Siphoviridae) in dairies using defined cultures. The 936 and Leuconostoc phages demonstrated limited diversity. Possible coinduction of temperate P335 prophages and satellite phages in one of the whey mixtures was also observed. IMPORTANCE The method optimized in this study could provide an important basis for understanding the dynamics of the phage community (abundance, development, diversity, evolution, etc.) in dairies with different sizes, locations, and production strategies. It may also enable the discovery of previously unknown phages, which is crucial for the development of rapid molecular biology-based methods for phage burden surveillance systems. The dominance of only a few phage groups in the dairy environment signifies the depth of knowledge gained over the past decades, which served as the basis for designing current phage control strategies. The presence of a correlation between phages and the type of starter cultures being used in dairies might help to improve the selection and/or design of suitable, custom, and cost-efficient phage control strategies. PMID:28754704

UTILIZING SYNTHETIC UV SPECTRA TO EXPLORE THE PHYSICAL BASIS FOR THE CLASSIFICATION OF LAMBDA BOÖTIS STARS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Kwang-Ping; Johnson, Dustin M.; Tarbell, Erik S.

2016-04-15

Lambda Boo-type stars are a group of late B to early F-type Population I dwarfs that show mild to extreme deficiencies of iron-peak elements (up to 2 dex), but their C, N, O, and S abundances are near solar. This intriguing stellar class has recently regained the spotlight because of the directly imaged planets around a confirmed Lambda Boo star, HR 8799, and a suggested Lambda Boo star, Beta Pictoris. The discovery of a giant asteroid belt around Vega, another possible Lambda Boo star, also suggests hidden planets. The possible link between Lambda Boo stars and planet-bearing stars motivates usmore » to study Lambda Boo stars systematically. Since the peculiar nature of the prototype Lambda Boötis was first noticed in 1943, Lambda Boo candidates published in the literature have been selected using widely different criteria. In order to determine the origin of Lambda Boo stars’ unique abundance pattern and to better discriminate between theories explaining the Lambda Boo phenomenon, a consistent working definition of Lambda Boo stars is needed. We have re-evaluated all published Lambda Boo candidates and their available ultraviolet and visible spectra. In this paper, using observed and synthetic spectra, we explore the physical basis for the classification of Lambda Boo stars, and develop quantitative criteria that discriminate metal-poor stars from bona fide Lambda Boo stars. Based on these stricter Lambda Boo classification criteria, we conclude that neither Beta Pictoris nor Vega should be classified as Lambda Boo stars.« less

Bivariate- distribution for transition matrix elements in Breit-Wigner to Gaussian domains of interacting particle systems.

PubMed

Kota, V K B; Chavda, N D; Sahu, R

2006-04-01

Interacting many-particle systems with a mean-field one-body part plus a chaos generating random two-body interaction having strength lambda exhibit Poisson to Gaussian orthogonal ensemble and Breit-Wigner (BW) to Gaussian transitions in level fluctuations and strength functions with transition points marked by lambda = lambda c and lambda = lambda F, respectively; lambda F > lambda c. For these systems a theory for the matrix elements of one-body transition operators is available, as valid in the Gaussian domain, with lambda > lambda F, in terms of orbital occupation numbers, level densities, and an integral involving a bivariate Gaussian in the initial and final energies. Here we show that, using a bivariate-t distribution, the theory extends below from the Gaussian regime to the BW regime up to lambda = lambda c. This is well tested in numerical calculations for 6 spinless fermions in 12 single-particle states.

A new helper phage for improved monovalent display of Fab molecules.

PubMed

Beaber, John W; Tam, Eric M; Lao, Llewelyn S; Rondon, Isaac J

2012-02-28

Phage display technology is a powerful tool for the identification of novel antibodies for drug discovery. Phage display libraries have been constructed with massive diversity, but their use may be hindered by limited antibody display levels when rescued with the M13KO7 helper phage. Variants of M13KO7 have been constructed previously that increase the levels of display of rescued phage, but all produce phage that display multiple copies of the antibody fragment on their surface and have reduced titer and infectivity. In this study, we describe a new helper phage, XP5, which increased the display level of Fab molecules more than two-fold compared to phage rescued with M13KO7. XP5 uses a combination of ribosome binding site spacing alterations and rare codon clusters to reduce the expression of pIII from the helper phage. This reduction in pIII expression leads to an increase in the incorporation of pIII-Fab fusions during phage rescue. The rescued phage displayed a single copy of the Fab molecule, preventing any avidity effects during the selection process. This also suggests that the percentage of the population of phage displaying a Fab molecule is increased when rescued with XP5. Additionally, the phage titers and infectivity are comparable to libraries rescued with M13KO7. After two rounds of panning we observed a nearly 5-fold increase in the number of antigen binding Fab molecules compared to panning conducted with the same library rescued with M13KO7. The nature of the mutations in XP5 makes it a universal substitute for M13KO7 in pIII-based phage display, compatible with most phagemids and bacterial strains. Copyright © 2011 Elsevier B.V. All rights reserved.

Sequence Analysis of Leuconostoc mesenteroides Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation▿

PubMed Central

Lu, Z.; Altermann, E.; Breidt, F.; Kozyavkin, S.

2010-01-01

Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB). Leuconostoc mesenteroides initiates fermentation. As fermentation proceeds, L. mesenteroides dies off and other LAB complete the fermentation. Phages infecting L. mesenteroides may significantly influence the die-off of L. mesenteroides. However, no L. mesenteroides phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of L. mesenteroides phage Φ1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G+C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules. In silico analyses showed that Φ1-A4 is a unique lytic phage with a large-scale genome inversion (∼30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that Φ1-A4 clusters with several Lactococcus phages. To our knowledge, Φ1-A4 is the first genetically characterized L. mesenteroides phage. PMID:20118355

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage.

PubMed

Ploss, Martin; Kuhn, Andreas

2011-09-26

Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

PubMed Central

2011-01-01

Background Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies. PMID:21943062

An Insight into Phage Diversity at Environmental Habitats using Comparative Metagenomics Approach.

PubMed

Parmar, Krupa; Dafale, Nishant; Pal, Rajesh; Tikariha, Hitesh; Purohit, Hemant

2018-02-01

Bacteriophages play significant role in driving microbial diversity; however, little is known about the diversity of phages in different ecosystems. A dynamic predator-prey mechanism called "kill the winner" suggests the elimination of most active bacterial populations through phages. Thus, interaction between phage and host has an effect on the composition of microbial communities in ecosystems. In this study, secondary phage metagenome data from aquatic habitats: wastewater treatment plant (WWTP), fresh, marine, and hot water spring habitat were analyzed using MG-RAST and STAMP tools to explore the diversity of the viruses. Differential relative abundance of phage families-Siphoviridae (34%) and Myoviridae (26%) in WWTP, Myoviridae (30%) and Podoviridae (23%) in fresh water, and Myoviridae (41%) and Podoviridae (8%) in marine-was found to be a discriminating factor among four habitats while Rudiviridae (9%), Globuloviridae (8%), and Lipothrixviridae (1%) were exclusively observed in hot water spring. Subsequently, at genera level, Bpp-1-like virus, Chlorovirus, and T4-like virus were found abundant in WWTP, fresh, and marine habitat, respectively. PCA analysis revealed completely disparate composition of phage in hot water spring from other three ecosystems. Similar analysis of relative abundance of functional features corroborated observations from taxa analysis. Functional features corresponding to phage packaging machinery, replication, integration and excision, and gene transfer discriminated among four habitats. The comparative metagenomics approach exhibited genetically distinct phage communities among four habitats. Results revealed that selective distribution of phage communities would help in understanding the role of phages in food chains, nutrient cycling, and microbial ecology. Study of specific phages would also help in controlling environmental pathogens including MDR bacterial populations using phage therapy approach by selective mining and isolation of phages against specific pathogens persisting in a given environment.

Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae.

PubMed

Kęsik-Szeloch, Agata; Drulis-Kawa, Zuzanna; Weber-Dąbrowska, Beata; Kassner, Jerzy; Majkowska-Skrobek, Grażyna; Augustyniak, Daria; Lusiak-Szelachowska, Marzanna; Zaczek, Maciej; Górski, Andrzej; Kropinski, Andrew M

2013-03-28

Members of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies. Electron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages. Of the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.

Anti-CDR3 Therapy for B-Cell Malignancies

DTIC Science & Technology

2013-10-01

1-5 in the report). The "Tomlinson" human antibody phage library will be used to pan for antibodies that bind these target CDR3s and not the parent...selection of phage to a test antigen. Successful expression will lead directly to the selection of CDR3-specific antibody-encoding phage : from which we... phage that bind to the purified candidate antibody and not to irrelevant antibodies. Phage are from single chain Fv library. Sequence phage that

Host exopolysaccharide quantity and composition impact Erwinia amylovora bacteriophage pathogenesis.

PubMed

Roach, Dwayne R; Sjaarda, David R; Castle, Alan J; Svircev, Antonet M

2013-05-01

Erwinia amylovora bacteriophages (phages) belonging to the Myoviridae and Podoviridae families demonstrated a preference for either high-exopolysaccharide-producing (HEP) or low-exopolysaccharide-producing (LEP) bacterial hosts when grown on artificial medium without or with sugar supplementation. Myoviridae phages produced clear plaques on LEP hosts and turbid plaques on HEP hosts. The reverse preference was demonstrated by most Podoviridae phages, where clear plaques were seen on HEP hosts. Efficiency of plating (EOP) was determined by comparing phage growth on the original isolation host to the that on the LEP or HEP host. Nine of 10 Myoviridae phages showed highest EOPs on LEP hosts, and 8 of 11 Podoviridae phages had highest EOPs on HEP hosts. Increasing the production of EPS on sugar-supplemented medium or decreasing production by knocking out the synthesis of amylovoran or levan, the two EPSs produced by E. amylovora, indicated that these components play crucial roles in phage infection. Amylovoran was virtually essential for proliferation of most Podoviridae phages when phage population growth was compared to the wild type. Decreased levan production resulted in a significant reduction of progeny from phages in the Myoviridae family. Thus, Podoviridae phages are adapted to hosts that produce high levels of exopolysaccharides and are dependent on host-produced amylovoran for pathogenesis. Myoviridae phages are adapted to hosts that produce lower levels of exopolysaccharides and host-produced levan.

Host Exopolysaccharide Quantity and Composition Impact Erwinia amylovora Bacteriophage Pathogenesis

PubMed Central

Roach, Dwayne R.; Sjaarda, David R.; Svircev, Antonet M.

2013-01-01

Erwinia amylovora bacteriophages (phages) belonging to the Myoviridae and Podoviridae families demonstrated a preference for either high-exopolysaccharide-producing (HEP) or low-exopolysaccharide-producing (LEP) bacterial hosts when grown on artificial medium without or with sugar supplementation. Myoviridae phages produced clear plaques on LEP hosts and turbid plaques on HEP hosts. The reverse preference was demonstrated by most Podoviridae phages, where clear plaques were seen on HEP hosts. Efficiency of plating (EOP) was determined by comparing phage growth on the original isolation host to the that on the LEP or HEP host. Nine of 10 Myoviridae phages showed highest EOPs on LEP hosts, and 8 of 11 Podoviridae phages had highest EOPs on HEP hosts. Increasing the production of EPS on sugar-supplemented medium or decreasing production by knocking out the synthesis of amylovoran or levan, the two EPSs produced by E. amylovora, indicated that these components play crucial roles in phage infection. Amylovoran was virtually essential for proliferation of most Podoviridae phages when phage population growth was compared to the wild type. Decreased levan production resulted in a significant reduction of progeny from phages in the Myoviridae family. Thus, Podoviridae phages are adapted to hosts that produce high levels of exopolysaccharides and are dependent on host-produced amylovoran for pathogenesis. Myoviridae phages are adapted to hosts that produce lower levels of exopolysaccharides and host-produced levan. PMID:23503310

Bacteriophages of wastewater foaming-associated filamentous Gordonia reduce host levels in raw activated sludge

PubMed Central

Liu, Mei; Gill, Jason J.; Young, Ry; Summer, Elizabeth J.

2015-01-01

Filamentous bacteria are a normal and necessary component of the activated sludge wastewater treatment process, but the overgrowth of filamentous bacteria results in foaming and bulking associated disruptions. Bacteriophages, or phages, were investigated for their potential to reduce the titer of foaming bacteria in a mixed-microbial activated sludge matrix. Foaming-associated filamentous bacteria were isolated from activated sludge of a commercial wastewater treatment plan and identified as Gordonia species by 16S rDNA sequencing. Four representative phages were isolated that target G. malaquae and two un-named Gordonia species isolates. Electron microscopy revealed the phages to be siphophages with long tails. Three of the phages - GordTnk2, Gmala1, and GordDuk1 - had very similar ~76 kb genomes, with >93% DNA identity. These genomes shared limited synteny with Rhodococcus equi phage ReqiDocB7 and Gordonia phage GTE7. In contrast, the genome of phage Gsput1 was smaller (43 kb) and was not similar enough to any known phage to be placed within an established phage type. Application of these four phages at MOIs of 5–15 significantly reduced Gordonia host levels in a wastewater sludge model by approximately 10-fold as compared to non-phage treated reactors. Phage control was observed for nine days after treatment. PMID:26349678

Identification of chondrocyte-binding peptides by phage display.

PubMed

Cheung, Crystal S F; Lui, Julian C; Baron, Jeffrey

2013-07-01

As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders. Copyright © 2013 Orthopaedic Research Society.

Engineering M13 for phage display.

PubMed

Sidhu, S S

2001-09-01

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.

Therapeutic and Prophylactic Applications of Bacteriophage Components in Modern Medicine

PubMed Central

Adhya, Sankar; Merril, Carl R.; Biswas, Biswajit

2014-01-01

As the interactions of phage with mammalian innate and adaptive immune systems are better delineated and with our ability to recognize and eliminate toxins and other potentially harmful phage gene products, the potential of phage therapies is now being realized. Early efforts to use phage therapeutically were hampered by inadequate phage purification and limited knowledge of phage–bacterial and phage–human relations. However, although use of phage as an antibacterial therapy in countries that require controlled clinical studies has been hampered by the high costs of patient trials, their use as vaccines and the use of phage components such as lysolytic enzymes or lysozymes has progressed to the point of commercial applications. Recent studies concerning the intimate associations between mammalian hosts and bacterial and phage microbiomes should hasten this progress. PMID:24384811

Bacteriophages in food fermentations: new frontiers in a continuous arms race.

PubMed

Samson, Julie E; Moineau, Sylvain

2013-01-01

Phage contamination represents an important risk to any process requiring bacterial growth, particularly in the biotechnology and food industries. The presence of unwanted phages may lead to manufacturing delays, lower quality product, or, in the worst cases, total production loss. Thus, constant phage monitoring and stringent application of the appropriate control measures are indispensable. In fact, a systematic preventive approach to phage contamination [phage analysis and critical control points (PACCP)] should be put in place. In this review, sources of phage contamination and novel phage detection methods are described, with an emphasis on bacterial viruses that infect lactic acid bacteria used in food fermentations. Recent discoveries related to antiphage systems that are changing our views on phage-host interactions are highlighted. Finally, future directions are also discussed.

Isolation and characterization of Yersinia-specific bacteriophages from pig stools in Finland.

PubMed

Salem, M; Virtanen, S; Korkeala, H; Skurnik, M

2015-03-01

Bacteriophages infect bacteria, and they are present everywhere in the world including the intestinal tracts of animals. Yersiniosis is a common foodborne infection caused by Yersinia enterocolitica and Yersinia pseudotuberculosis. As these bacteria are frequently isolated from pigs, we wanted to know whether Yersinia-specific bacteriophages are also present in the pig stools and, if so, whether there is a positive or negative association between the prevalence of the Yersinia phages and the pathogenic Yersinia in the stool samples. Altogether 793 pig stool samples collected between November 2010 and March 2012 from 14 Finnish pig farms were screened for the presence of bacteriophages able to infect Y. enterocolitica serotype O:3, O:5,27 or O:9 strains, or Y. pseudotuberculosis serotype O:1a, O:1b or O:3 strains. Yersinia phages were isolated from 90 samples from eight farms. Yersinia enterocolitica O:3 was infected by 59 phages, 28 phages infected serotypes O:3 and O:5,27, and eight phages infected serotypes O:3, O:5,27 and O:9, and Y. pseudotuberculosis O:1a by eight phages. Many phages originating from pigs in the same farm were identical based on their restriction enzyme digestion patterns; 20 clearly different phages were selected for further characterization. Host ranges of these phages were tested with 94 Yersinia strains. Six of the phages infected eight strains, 13 phages infected three strains, and one phage infected only one strain, indicating that the phages had a relatively narrow host range. There was a clear association between the presence of the host bacteria and specific phages in the stools. The isolated bacteriophages may have potential as biocontrol agents for yersiniosis in both humans and pigs in future, and as alternatives or in addition to antibiotics. To our knowledge, this is the first reported isolation of Yersinia-specific phages from pig stool samples. © 2014 The Society for Applied Microbiology.

Purification of phage display-modified bacteriophage T4 by affinity chromatography

PubMed Central

2011-01-01

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development. PMID:21627821

Envisaging bacteria as phage targets

PubMed Central

Abedon, Stephen T.

2011-01-01

It can be difficult to appreciate just how small bacteria and phages are or how large, in comparison, the volumes that they occupy. A single milliliter, for example, can represent to a phage what would be, with proper scaling, an “ocean” to you and me. Here I illustrate, using more easily visualized macroscopic examples, the difficulties that a phage, as a randomly diffusing particle, can have in locating bacteria to infect. I conclude by restating the truism that the rate of phage adsorption to a given target bacterium is a function of phage density, that is, titer, in combination with the degree of bacterial susceptibility to adsorption by an encountering phage. PMID:23616932

PhiSiGns: an online tool to identify signature genes in phages and design PCR primers for examining phage diversity.

PubMed

Dwivedi, Bhakti; Schmieder, Robert; Goldsmith, Dawn B; Edwards, Robert A; Breitbart, Mya

2012-03-04

Phages (viruses that infect bacteria) have gained significant attention because of their abundance, diversity and important ecological roles. However, the lack of a universal gene shared by all phages presents a challenge for phage identification and characterization, especially in environmental samples where it is difficult to culture phage-host systems. Homologous conserved genes (or "signature genes") present in groups of closely-related phages can be used to explore phage diversity and define evolutionary relationships amongst these phages. Bioinformatic approaches are needed to identify candidate signature genes and design PCR primers to amplify those genes from environmental samples; however, there is currently no existing computational tool that biologists can use for this purpose. Here we present PhiSiGns, a web-based and standalone application that performs a pairwise comparison of each gene present in user-selected phage genomes, identifies signature genes, generates alignments of these genes, and designs potential PCR primer pairs. PhiSiGns is available at (http://www.phantome.org/phisigns/; http://phisigns.sourceforge.net/) with a link to the source code. Here we describe the specifications of PhiSiGns and demonstrate its application with a case study. PhiSiGns provides phage biologists with a user-friendly tool to identify signature genes and design PCR primers to amplify related genes from uncultured phages in environmental samples. This bioinformatics tool will facilitate the development of novel signature genes for use as molecular markers in studies of phage diversity, phylogeny, and evolution.

PhiSiGns: an online tool to identify signature genes in phages and design PCR primers for examining phage diversity

PubMed Central

2012-01-01

Background Phages (viruses that infect bacteria) have gained significant attention because of their abundance, diversity and important ecological roles. However, the lack of a universal gene shared by all phages presents a challenge for phage identification and characterization, especially in environmental samples where it is difficult to culture phage-host systems. Homologous conserved genes (or "signature genes") present in groups of closely-related phages can be used to explore phage diversity and define evolutionary relationships amongst these phages. Bioinformatic approaches are needed to identify candidate signature genes and design PCR primers to amplify those genes from environmental samples; however, there is currently no existing computational tool that biologists can use for this purpose. Results Here we present PhiSiGns, a web-based and standalone application that performs a pairwise comparison of each gene present in user-selected phage genomes, identifies signature genes, generates alignments of these genes, and designs potential PCR primer pairs. PhiSiGns is available at (http://www.phantome.org/phisigns/; http://phisigns.sourceforge.net/) with a link to the source code. Here we describe the specifications of PhiSiGns and demonstrate its application with a case study. Conclusions PhiSiGns provides phage biologists with a user-friendly tool to identify signature genes and design PCR primers to amplify related genes from uncultured phages in environmental samples. This bioinformatics tool will facilitate the development of novel signature genes for use as molecular markers in studies of phage diversity, phylogeny, and evolution. PMID:22385976

Milk Contamination and Resistance to Processing Conditions Determine the Fate of Lactococcus lactis Bacteriophages in Dairies

PubMed Central

Madera, Carmen; Monjardín, Cristina; Suárez, Juan E.

2004-01-01

Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72°C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent. PMID:15574937

Influence of RNase E deficiency on the production of stx2-bearing phages and Shiga toxin in an RNase E-inducible strain of enterohaemorrhagic Escherichia coli (EHEC) O157:H7.

PubMed

Thuraisamy, Thujitha; Lodato, Patricia B

2018-05-01

In enterohaemorrhagic Escherichia coli (EHEC), stx1 or stx2 genes encode Shiga toxin (Stx1 or Stx2, respectively) and are carried by prophages. The production and release of both stx phages and toxin occur upon initiation of the phage lytic cycle. Phages can further disseminate stx genes by infecting naïve bacteria in the intestine. Here, the effect of RNase E deficiency on these two virulence traits was investigated. Cultures of the EHEC strains TEA028-rne containing low versus normal RNase E levels or the parental strain (TEA028) were treated with mitomycin C (MMC) to induce the phage lytic cycle. Phages and Stx2 titres were quantified by the double-agar assay and the receptor ELISA technique, respectively. RNase E deficiency in MMC-treated cells significantly reduced the yield of infectious stx2 phages. Delayed cell lysis and the appearance of encapsidated phage DNA copies suggest a slow onset of the lytic cycle. However, these observations do not entirely explain the decrease of phage yields. stx1 phages were not detected under normal or deficient RNase E levels. After an initial delay, high levels of toxin were finally produced in MMC-treated cultures. RNase E scarcity reduces stx2 phage production but not toxin. Normal concentrations of RNase E are likely required for correct phage morphogenesis. Our future work will address the mechanism of RNase E action on phage morphogenesis.

Correlation Measurement of Lambda-anti-Lambda, Lambda-Lambda and anti-Lambda-anti-Lambda with the ATLAS detector at s=7 TeV

NASA Astrophysics Data System (ADS)

Cheng, Hok-Chuen

This thesis summaries the measurements of correlations between Lambda 0Lambda0, Lambda0Lambda 0, and Lambda0Lambda 0 hyperon pairs produced inclusively at the LHC, which are useful for a better understanding of the quark-antiquark pair production and jet fragmentation and hadronization processes. The analysis is based on hyperon pairs selected using the muon and minimum bias data samples collected at the ATLAS experiment from proton-proton collisions at a center-of-mass energy of 7 TeV in 2010. Excess Lambda0Lambda 0 are observed near the production threshold and are identified to be originated from the parton system in the string model in the MC sample, decaying either directly or through heavy strange resonances such as Sigma0 and Sigma*(1385). Dynamical correlations have been explored through a correlation function defined as the ratio of two-particle to single-particle densities. Positive correlation is observed for Lambda0Lambda0 and anticorrelation is observed for Lambda0Lambda 0 and Lambda0Lambda 0 for Q in [0,2] GeV. The structure replicates similar correlations in pp, pp, and pppp events in PYTHIA generator as predicted by the Lund string fragmentation model. Parameters of the "popcorn" mechanism implemented in the PYTHIA generator are tuned and are found to have little impact on the structure observed. The spin composition of the sample is extracted using a data-driven reference sample built by event mixing. Appropriate corrections have been made to the kinematic distributions in the reference sample by kinematic weighting to make sure that the detector effects are well modeled. A modified Pearson's chi2 test statistics is calculated for the costheta* distribution to determine the best-fitted A-value for data. The results are consistent with zero for both like-type and unlike-type hyperon pairs in Q ∈ [0,10] GeV and Q ∈ [1,10] GeV respectively. The data statistics in the range of Q ∈ [0, 1] GeV is currently too low for the estimation of the emitter size for Fermi-Dirac correlation.

Phage as a template to grow bone mineral nanocrystals.

PubMed

Cao, Binrui; Xu, Hong; Mao, Chuanbin

2014-01-01

Phage display is a biotechnique that fuses functional peptides on the outer surface of filamentous phage by inserting DNA encoding the peptides into the genes of its coat proteins. The resultant peptide-displayed phage particles have been widely used as biotemplates for the synthesis of functional hybrid nanomaterials. Here, we describe the bioengineering of M13 filamentous phage to surface-display bone mineral (hydroxyapatite (HAP))-nucleating peptides derived from dentin matrix protein-1 and using the engineered phage as a biotemplate to grow HAP nanocrystals.

Phage Therapy: Beyond Antibacterial Action.

PubMed

Górski, Andrzej; Jończyk-Matysiak, Ewa; Międzybrodzki, Ryszard; Weber-Dąbrowska, Beata; Łusiak-Szelachowska, Marzanna; Bagińska, Natalia; Borysowski, Jan; Łobocka, Małgorzata B; Węgrzyn, Alicja; Węgrzyn, Grzegorz

2018-01-01

Until recently, phages were considered as mere "bacteria eaters" with potential for use in combating antimicrobial resistance. The real value of phage therapy assessed according to the standards of evidence-based medicine awaits confirmation by clinical trials. However, the progress in research on phage biology has shed more light on the significance of phages. Accumulating data indicate that phages may also interact with eukaryotic cells. How such interactions could be translated into advances in medicine (especially novel means of therapy) is discussed herein.

Development of a High Throughput Assay for Indirectly Measuring Phage Growth Using the OmniLog (trademark) System

DTIC Science & Technology

2012-09-01

and phage Giraffe exhibits a similar morphology (Fig. 1A). Phage BA39 (Fig. 1E) appears to belong to the Myoviridae family (icosahedral head and...see later) but this assignment is tentative. Monitoring the kinetics of bacterial growth using OmniLogTM system upon infection with Giraffe phage...spores with and without infection by Giraffe phage are shown in Figure 2A and B respectively. The growth of 7702 without phage infection followed a

Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

NASA Astrophysics Data System (ADS)

Essler, Markus; Ruoslahti, Erkki

2002-02-01

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

Recombinant phage probes for Listeria monocytogenes

NASA Astrophysics Data System (ADS)

Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

2007-10-01

Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

The Human Gut Phage Community and Its Implications for Health and Disease.

PubMed

Manrique, Pilar; Dills, Michael; Young, Mark J

2017-06-08

In this review, we assess our current understanding of the role of bacteriophages infecting the human gut bacterial community in health and disease. In general, bacteriophages contribute to the structure of their microbial communities by driving host and viral diversification, bacterial evolution, and by expanding the functional diversity of ecosystems. Gut bacteriophages are an ensemble of unique and shared phages in individuals, which encompass temperate phages found predominately as prophage in gut bacteria (prophage reservoir) and lytic phages. In healthy individuals, only a small fraction of the prophage reservoir is activated and found as extracellular phages. Phage community dysbiosis is characterized by a shift in the activated prophage community or an increase of lytic phages, and has been correlated with disease, suggesting that a proper balance between lysis and lysogeny is needed to maintain health. Consequently, the concept of microbial dysbiosis might be extended to the phage component of the microbiome as well. Understanding the dynamics and mechanisms to restore balance after dysbiosis is an active area of research. The use of phage transplants to re-establish health suggests that phages can be used as disease treatment. Such advances represent milestones in our understanding of gut phages in human health and should fuel research on their role in health and disease.

Ciprofloxacin and Trimethoprim Cause Phage Induction and Virulence Modulation in Staphylococcus aureus

PubMed Central

Goerke, Christiane; Köller, Johanna; Wolz, Christiane

2006-01-01

In Staphylococcus aureus strains of human origin, phages which integrate into the chromosomal gene coding for β-hemolysin (hlb) are widely distributed. Most of them encode accessory virulence determinants such as staphylokinase (sak) or enterotoxins. Here, we analyzed the effects of ciprofloxacin and trimethoprim on phage induction and expression of phage-encoded virulence factors by using isolates from patients with cystic fibrosis for which the induction of hlb-converting phages was demonstrated in vivo (C. Goerke, S. Matias y Papenberg, S. Dasbach, K. Dietz, R. Ziebach, B. C. Kahl, and C. Wolz, J. Infect. Dis. 189:724-734, 2004) as well as a φ13 lysogen of phage-cured strain 8325-4. Treatment of lysogens with subinhibitory concentrations of either antibiotic resulted in (i) delysogenization of strains resembling the isolates picked up after chronic lung infection and (ii) replication of phages in the bacterial host in a dose-dependent manner. Ciprofloxacin treatment resulted in enhanced recA transcription, indicating involvement of the SOS response in phage mobilization. Induction of φ13 was linked to elevated expression of the phage-encoded virulence gene sak, chiefly due to the activation of latent phage promoters. In summary, we could show the induction of hlb-converting phages and a subsequent virulence modulation of the host bacterium by ciprofloxacin and trimethoprim. PMID:16377683

Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp.

PubMed Central

Hendrix, Roger W.; Dedrick, Rebekah; Mitchell, Kaitlin; Ko, Ching-Chung; Russell, Daniel; Bell, Emma; Gregory, Matthew; Bibb, Maureen J.; Pethick, Florence; Jacobs-Sera, Deborah; Herron, Paul; Buttner, Mark J.; Hatfull, Graham F.

2013-01-01

The genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, ϕHau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage ϕHau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species. PMID:23995638

A bioinformatic analysis of ribonucleotide reductase genes in phage genomes and metagenomes

PubMed Central

2013-01-01

Background Ribonucleotide reductase (RNR), the enzyme responsible for the formation of deoxyribonucleotides from ribonucleotides, is found in all domains of life and many viral genomes. RNRs are also amongst the most abundant genes identified in environmental metagenomes. This study focused on understanding the distribution, diversity, and evolution of RNRs in phages (viruses that infect bacteria). Hidden Markov Model profiles were used to analyze the proteins encoded by 685 completely sequenced double-stranded DNA phages and 22 environmental viral metagenomes to identify RNR homologs in cultured phages and uncultured viral communities, respectively. Results RNRs were identified in 128 phage genomes, nearly tripling the number of phages known to encode RNRs. Class I RNR was the most common RNR class observed in phages (70%), followed by class II (29%) and class III (28%). Twenty-eight percent of the phages contained genes belonging to multiple RNR classes. RNR class distribution varied according to phage type, isolation environment, and the host’s ability to utilize oxygen. The majority of the phages containing RNRs are Myoviridae (65%), followed by Siphoviridae (30%) and Podoviridae (3%). The phylogeny and genomic organization of phage and host RNRs reveal several distinct evolutionary scenarios involving horizontal gene transfer, co-evolution, and differential selection pressure. Several putative split RNR genes interrupted by self-splicing introns or inteins were identified, providing further evidence for the role of frequent genetic exchange. Finally, viral metagenomic data indicate that RNRs are prevalent and highly dynamic in uncultured viral communities, necessitating future research to determine the environmental conditions under which RNRs provide a selective advantage. Conclusions This comprehensive study describes the distribution, diversity, and evolution of RNRs in phage genomes and environmental viral metagenomes. The distinct distributions of specific RNR classes amongst phages, combined with the various evolutionary scenarios predicted from RNR phylogenies suggest multiple inheritance sources and different selective forces for RNRs in phages. This study significantly improves our understanding of phage RNRs, providing insight into the diversity and evolution of this important auxiliary metabolic gene as well as the evolution of phages in response to their bacterial hosts and environments. PMID:23391036

First genome sequences of Achromobacter phages reveal new members of the N4 family.

PubMed

Wittmann, Johannes; Dreiseikelmann, Brigitte; Rohde, Manfred; Meier-Kolthoff, Jan P; Bunk, Boyke; Rohde, Christine

2014-01-27

Multi-resistant Achromobacter xylosoxidans has been recognized as an emerging pathogen causing nosocomially acquired infections during the last years. Phages as natural opponents could be an alternative to fight such infections. Bacteriophages against this opportunistic pathogen were isolated in a recent study. This study shows a molecular analysis of two podoviruses and reveals first insights into the genomic structure of Achromobacter phages so far. Growth curve experiments and adsorption kinetics were performed for both phages. Adsorption and propagation in cells were visualized by electron microscopy. Both phage genomes were sequenced with the PacBio RS II system based on single molecule, real-time (SMRT) technology and annotated with several bioinformatic tools. To further elucidate the evolutionary relationships between the phage genomes, a phylogenomic analysis was conducted using the genome Blast Distance Phylogeny approach (GBDP). In this study, we present the first detailed analysis of genome sequences of two Achromobacter phages so far. Phages JWAlpha and JWDelta were isolated from two different waste water treatment plants in Germany. Both phages belong to the Podoviridae and contain linear, double-stranded DNA with a length of 72329 bp and 73659 bp, respectively. 92 and 89 putative open reading frames were identified for JWAlpha and JWDelta, respectively, by bioinformatic analysis with several tools. The genomes have nearly the same organization and could be divided into different clusters for transcription, replication, host interaction, head and tail structure and lysis. Detailed annotation via protein comparisons with BLASTP revealed strong similarities to N4-like phages. Analysis of the genomes of Achromobacter phages JWAlpha and JWDelta and comparisons of different gene clusters with other phages revealed that they might be strongly related to other N4-like phages, especially of the Escherichia group. Although all these phages show a highly conserved genomic structure and partially strong similarities at the amino acid level, some differences could be identified. Those differences, e.g. the existence of specific genes for replication or host interaction in some N4-like phages, seem to be interesting targets for further examination of function and specific mechanisms, which might enlighten the mechanism of phage establishment in the host cell after infection.

Engineering filamentous phage carriers to improve focusing of antibody responses against peptides.

PubMed

van Houten, Nienke E; Henry, Kevin A; Smith, George P; Scott, Jamie K

2010-03-02

The filamentous bacteriophage are highly immunogenic particles that can be used as carrier proteins for peptides and presumably other haptens and antigens. Our previous work demonstrated that the antibody response was better focused against a synthetic peptide if it was conjugated to phage as compared to the classical carrier, ovalbumin. We speculated that this was due, in part, to the relatively low surface complexity of the phage. Here, we further investigate the phage as an immunogenic carrier, and the effect reducing its surface complexity has on the antibody response against peptides that are either displayed as recombinant fusions to the phage coat or are chemically conjugated to it. Immunodominant regions of the minor coat protein, pIII, were removed from the phage surface by excising its N1 and N2 domains (Delta3 phage variant), whereas immunodominant epitopes of the major coat protein, pVIII, were altered by reducing the charge of its surface-exposed N-terminal residues (Delta8 phage variant). Immunization of mice revealed that the Delta3 variant was less immunogenic than wild-type (WT) phage, whereas the Delta8 variant was more immunogenic. The immunogenicity of two different peptides was tested in the context of the WT and Delta3 phage in two different forms: (i) as recombinant peptides fused to pVIII, and (ii) as synthetic peptides conjugated to the phage surface. One peptide (MD10) in its recombinant form produced a stronger anti-peptide antibody response fused to the WT carrier compared to the Delta3 phage carrier, and did not elicit a detectable anti-peptide response in its synthetic form conjugated to either phage carrier. This trend was reversed for a different peptide (4E10(L)), which did not produce a detectable anti-peptide antibody response as a recombinant fusion; yet, as a chemical conjugate to Delta3 phage, but not WT phage, it elicited a highly focused anti-peptide antibody response that exceeded the anti-carrier response by approximately 65-fold. The results suggest that focusing of the antibody response against synthetic peptides can be improved by decreasing the antigenic complexity of the phage surface. Copyright 2010 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akaishi, Yoshinori; College of Science and Technology, Nihon University, Funabashi 274-8501; Myint, Khin Swe

The overbinding problem of {sub {lambda}}{sup 5}He is solved by introducing a concept of coherent {lambda}-{sigma} coupling which is equivalent to a {lambda}NN three-body force. This three-body force is coherently enhanced in the 0{sup +} states of {sub {lambda}}{sup 4}H and {sub {lambda}}{sup 4}He. The 0{sup +}-1{sup +} splitting in these hypernuclei is mainly due to coherent {lambda}-{sigma} coupling and partly due to the {lambda}N spin-spin interaction. A {lambda}NN three-body potential is derived from the coupled-channel treatment. The origin of the repulsive and attractive nature of the three-body force is discussed. Coherent {lambda}-{sigma} coupling becomes more important in neutron-rich hypernucleimore » and especially in neutron-star matter at high densities. The possible existence of ''hyperheavy hydrogen'', {sub {lambda}}{sup 6}H, is suggested.« less

Comparative Persistence of Subgroups of F-Specific RNA Phages in River Water

PubMed Central

Yang, Yongheng

2013-01-01

F-specific (F+) RNA phages are widely used as indicators for the presence of fecal contamination and/or enteric viruses in water, and identifying subgroups of F+ RNA phages provides an approach for microbial source tracking. Different survival characteristics of the F+ RNA phage subgroups result in a misinterpretation of their original proportion in water, thus giving misleading information when they are used for microbial source tracking. This study investigated the comparative persistence of subgroups of F+ RNA phages in river water under different conditions. Results suggested that temperature and pH are the major factors affecting the persistence of F+ RNA phages in river water, and organic substances promote phage survival. The comparative persistence patterns of subgroups of F+ RNA phages varied and may bias extrapolation of their initial proportions in surface water. Thus, the characteristics of water should be taken into consideration and the results should be carefully interpreted when F+ RNA phages are used for microbial source tracking. PMID:23686274

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in {approx}300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressedmore » for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.« less

Metagenomic Analysis of Dairy Bacteriophages: Extraction Method and Pilot Study on Whey Samples Derived from Using Undefined and Defined Mesophilic Starter Cultures.

PubMed

Muhammed, Musemma K; Kot, Witold; Neve, Horst; Mahony, Jennifer; Castro-Mejía, Josué L; Krych, Lukasz; Hansen, Lars H; Nielsen, Dennis S; Sørensen, Søren J; Heller, Knut J; van Sinderen, Douwe; Vogensen, Finn K

2017-10-01

Despite being potentially highly useful for characterizing the biodiversity of phages, metagenomic studies are currently not available for dairy bacteriophages, partly due to the lack of a standard procedure for phage extraction. We optimized an extraction method that allows the removal of the bulk protein from whey and milk samples with losses of less than 50% of spiked phages. The protocol was applied to extract phages from whey in order to test the notion that members of Lactococcus lactis 936 (now Sk1virus ), P335, c2 (now C2virus ) and Leuconostoc phage groups are the most frequently encountered in the dairy environment. The relative abundance and diversity of phages in eight and four whey mixtures from dairies using undefined mesophilic mixed-strain cultures containing Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc species (i.e., DL starter cultures) and defined cultures, respectively, were assessed. Results obtained from transmission electron microscopy and high-throughput sequence analyses revealed the dominance of Lc. lactis 936 phages (order Caudovirales , family Siphoviridae ) in dairies using undefined DL starter cultures and Lc. lactis c2 phages (order Caudovirales , family Siphoviridae ) in dairies using defined cultures. The 936 and Leuconostoc phages demonstrated limited diversity. Possible coinduction of temperate P335 prophages and satellite phages in one of the whey mixtures was also observed. IMPORTANCE The method optimized in this study could provide an important basis for understanding the dynamics of the phage community (abundance, development, diversity, evolution, etc.) in dairies with different sizes, locations, and production strategies. It may also enable the discovery of previously unknown phages, which is crucial for the development of rapid molecular biology-based methods for phage burden surveillance systems. The dominance of only a few phage groups in the dairy environment signifies the depth of knowledge gained over the past decades, which served as the basis for designing current phage control strategies. The presence of a correlation between phages and the type of starter cultures being used in dairies might help to improve the selection and/or design of suitable, custom, and cost-efficient phage control strategies. Copyright © 2017 American Society for Microbiology.

Dispersal and survival of Flavobacterium psychrophilum phages in vivo in rainbow trout and in vitro under laboratory conditions: implications for their use in phage therapy.

PubMed

Madsen, Lone; Bertelsen, Sif K; Dalsgaard, Inger; Middelboe, Mathias

2013-08-01

Attention has been drawn to phage therapy as an alternative approach for controlling pathogenic bacteria such as Flavobacterium psychrophilum in salmonid aquaculture, which can give rise to high mortalities, especially in rainbow trout fry. Recently, phages have been isolated with a broad host range and a strong lytic potential against pathogenic F. psychrophilum under experimental conditions. However, little is known about the fate of phages at environmental conditions. Here, we quantified the dispersal and fate of F. psychrophilum phages and hosts in rainbow trout fry after intraperitoneal injection. Both phages and bacteria were isolated from the fish organs for up to 10 days after injection, and coinjection with both bacteria and phages resulted in a longer persistence of the phage in the fish organs, than when the fish had been injected with the phages only. The occurrence of both phage and bacterium was most prevalent in the kidney and spleen, with only minor occurrence in the brain. The experiment showed that injected phages were rapidly spread in the internal organs of the fish, also in the absence of bacteria. Parallel examination of the regulation of bacteriophage infectivity in controlled laboratory experiments at various environmental conditions showed that pH had only minor effects on long-term (3 months) phage infectivity within a pH range of 4.5 to 7.5, whereas phage infectivity was immediately lost at pH 3. In the absence of host cells, phage infectivity decreased by a factor of 10,000 over 55 days in untreated pond water, while the sterilization and removal of particles caused a 100-fold increase in phage survival relative to the control. In addition, F. psychrophilum-specific phages maintained their infectivity for ∼2 months in glycerol at -80°C, whereas infectivity decreased by a factor 10 when kept in a buffer at 20°C. Only a very small degradation in infectivity was seen when bacteriophages were added and dried on fish feed pellets. Together, these results indicate that application of bacteriophages represents a promising approach for the control of F. psychrophilum infections in trout and suggest fish feed as a potential delivery method.

Comparative studies with tox plus and tox minus corynebacteriophages.

PubMed

Holmes, R K; Barksdale, L

1970-06-01

The characteristics of nine inducible temperate corynebacteriophages designated alpha(tox+), beta(tox+), P(tox+), gamma(tox-), pi(tox+), K(tox-), rho(tox-), L(tox+), and delta(tox+) have been compared. Virion morphology and ability to recombine genetically with the well-studied phage beta(tox+) have been correlated with other properties of the phages, and the distribution of the genetic marker tox+ among related and relatively unrelated corynebacteriophages has been analyzed. The immunity specificity, host range, and plaque morphology of each phage were determined. The phages can be separated into five groups with different immunity specificities. Each type of host range previously recognized in mutants of phage beta(tox+) was present in one or more of the phages included in the present study, and the phages were found to produce plaques of several different morphological types. Representative phages with each of the five types of immunity specificity were further characterized with respect to virion morphology, ability to recombine with phage beta(tox+), latent period, average burst size, and neutralization by homologous and heterologous antiphage sera. All of these phages have polyhedral heads and long slender tails, but two distinct morphological types were distinguished by the sizes and proportions of the components of the virions. Only phages of the same morphological type as beta(tox+) were capable of genetic recombination with beta(tox+), but morphological similarity between phages was not sufficient to insure interfertility. The phages which recombined with beta(tox+) resembled one another in plaque morphology, latent period, and average burst size, whereas phages which failed to recombine with beta(tox+) differed in these characteristics. The phages capable of genetic recombination with beta(tox+) were found to differ from each other in immunity specificity, host range, neutralization by antiphage sera, and toxinogenicity. Thus, these latter characteristics are of limited value in establishing the extent of relatedness between corynebacteriophages. The genetic marker tox+ was not consistently correlated with any other property of the corynebacteriophages analyzed in this study. The most striking finding regarding the distribution of the tox+ marker is its presence both in beta(tox+) and delta(tox+), phages which fail to recombine genetically and which differ in virion morphology. The presence of the tox+ marker in genetically unrelated corynebacteriophages poses many questions concerning the origin(s) of tox+ and the evolution of the phage-host interactions which determine the ability of corynebacteria to synthesize diphtherial toxin.

Comparative Studies with tox+ and tox− Corynebacteriophages 1

PubMed Central

Holmes, Randall K.; Barksdale, Lane

1970-01-01

The characteristics of nine inducible temperate corynebacteriophages designated αtox+, βtox+, Ptox+, γtox−, πtox+, Ktox−, ρtox−, Ltox+, and δtox+ have been compared. Virion morphology and ability to recombine genetically with the well-studied phage βtox+ have been correlated with other properties of the phages, and the distribution of the genetic marker tox+ among related and relatively unrelated corynebacteriophages has been analyzed. The immunity specificity, host range, and plaque morphology of each phage were determined. The phages can be separated into five groups with different immunity specificities. Each type of host range previously recognized in mutants of phage βtox+ was present in one or more of the phages included in the present study, and the phages were found to produce plaques of several different morphological types. Representative phages with each of the five types of immunity specificity were further characterized with respect to virion morphology, ability to recombine with phage βtox+, latent period, average burst size, and neutralization by homologous and heterologous antiphage sera. All of these phages have polyhedral heads and long slender tails, but two distinct morphological types were distinguished by the sizes and proportions of the components of the virions. Only phages of the same morphological type as βtox+ were capable of genetic recombination with βtox+, but morphological similarity between phages was not sufficient to insure interfertility. The phages which recombined with βtox+ resembled one another in plaque morphology, latent period, and average burst size, whereas phages which failed to recombine with βtox+ differed in these characteristics. The phages capable of genetic recombination with βtox+ were found to differ from each other in immunity specificity, host range, neutralization by antiphage sera, and toxinogenicity. Thus, these latter characteristics are of limited value in establishing the extent of relatedness between corynebacteriophages. The genetic marker tox+ was not consistently correlated with any other property of the corynebacteriophages analyzed in this study. The most striking finding regarding the distribution of the tox+ marker is its presence both in βtox+ and δtox+, phages which fail to recombine genetically and which differ in virion morphology. The presence of the tox+ marker in genetically unrelated corynebacteriophages poses many questions concerning the origin(s) of tox+ and the evolution of the phage-host interactions which determine the ability of corynebacteria to synthesize diphtherial toxin. Images PMID:4193835

Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

PubMed

Osipiuk, J; Zylicz, M

1991-01-01

Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

Measurement of sigma Lambda b0/sigma B0 x B(Lambda b0-->Lambda c+pi-)/B(B0-->D+pi-) in pp collisions at square root s=1.96 TeV.

PubMed

Abulencia, A; Adelman, J; Affolder, T; Akimoto, T; Albrow, M G; Ambrose, D; Amerio, S; Amidei, D; Anastassov, A; Anikeev, K; Annovi, A; Antos, J; Aoki, M; Apollinari, G; Arguin, J-F; Arisawa, T; Artikov, A; Ashmanskas, W; Attal, A; Azfar, F; Azzi-Bacchetta, P; Azzurri, P; Bacchetta, N; Badgett, W; Barbaro-Galtieri, A; Barnes, V E; Barnett, B A; Baroiant, S; Bartsch, V; Bauer, G; Bedeschi, F; Behari, S; Belforte, S; Bellettini, G; Bellinger, J; Belloni, A; Benjamin, D; Beretvas, A; Beringer, J; Berry, T; Bhatti, A; Binkley, M; Bisello, D; Blair, R E; Blocker, C; Blumenfeld, B; Bocci, A; Bodek, A; Boisvert, V; Bolla, G; Bolshov, A; Bortoletto, D; Boudreau, J; Boveia, A; Brau, B; Brigliadori, L; Bromberg, C; Brubaker, E; Budagov, J; Budd, H S; Budd, S; Budroni, S; Burkett, K; Busetto, G; Bussey, P; Byrum, K L; Cabrera, S; Campanelli, M; Campbell, M; Canelli, F; Canepa, A; Carillo, S; Carlsmith, D; Carosi, R; Casarsa, M; Castro, A; Catastini, P; Cauz, D; Cavalli-Sforza, M; Cerri, A; Cerrito, L; Chang, S H; Chen, Y C; Chertok, M; Chiarelli, G; Chlachidze, G; Chlebana, F; Cho, I; Cho, K; Chokheli, D; Chou, J P; Choudalakis, G; Chuang, S H; Chung, K; Chung, W H; Chung, Y S; Ciljak, M; Ciobanu, C I; Ciocci, M A; Clark, A; Clark, D; Coca, M; Compostella, G; Convery, M E; Conway, J; Cooper, B; Copic, K; Cordelli, M; Cortiana, G; Crescioli, F; Cuenca Almenar, C; Cuevas, J; Culbertson, R; Cully, J C; Cyr, D; DaRonco, S; D'Auria, S; Davies, T; D'Onofrio, M; Dagenhart, D; de Barbaro, P; De Cecco, S; Deisher, A; De Lentdecker, G; Dell'Orso, M; Delli Paoli, F; Demortier, L; Deng, J; Deninno, M; De Pedis, D; Derwent, P F; Di Giovanni, G P; Dionisi, C; Di Ruzza, B; Dittmann, J R; DiTuro, P; Dörr, C; Donati, S; Donega, M; Dong, P; Donini, J; Dorigo, T; Dube, S; Efron, J; Erbacher, R; Errede, D; Errede, S; Eusebi, R; Fang, H C; Farrington, S; Fedorko, I; Fedorko, W T; Feild, R G; Feindt, M; Fernandez, J P; Field, R; Flanagan, G; Foland, A; Forrester, S; Foster, G W; Franklin, M; Freeman, J C; Furic, I; Gallinaro, M; Galyardt, J; Garcia, J E; Garberson, F; Garfinkel, A F; Gay, C; Gerberich, H; Gerdes, D; Giagu, S; Giannetti, P; Gibson, A; Gibson, K; Gimmell, J L; Ginsburg, C; Giokaris, N; Giordani, M; Giromini, P; Giunta, M; Giurgiu, G; Glagolev, V; Glenzinski, D; Gold, M; Goldschmidt, N; Goldstein, J; Gomez, G; Gomez-Ceballos, G; Goncharov, M; González, O; Gorelov, I; Goshaw, A T; Goulianos, K; Gresele, A; Griffiths, M; Grinstein, S; Grosso-Pilcher, C; Group, R C; Grundler, U; Guimaraes da Costa, J; Gunay-Unalan, Z; Haber, C; Hahn, K; Hahn, S R; Halkiadakis, E; Hamilton, A; Han, B-Y; Han, J Y; Handler, R; Happacher, F; Hara, K; Hare, M; Harper, S; Harr, R F; Harris, R M; Hartz, M; Hatakeyama, K; Hauser, J; Heijboer, A; Heinemann, B; Heinrich, J; Henderson, C; Herndon, M; Heuser, J; Hidas, D; Hill, C S; Hirschbuehl, D; Hocker, A; Holloway, A; Hou, S; Houlden, M; Hsu, S-C; Huffman, B T; Hughes, R E; Husemann, U; Huston, J; Incandela, J; Introzzi, G; Iori, M; Ishizawa, Y; Ivanov, A; Iyutin, B; James, E; Jang, D; Jayatilaka, B; Jeans, D; Jensen, H; Jeon, E J; Jindariani, S; Jones, M; Joo, K K; Jun, S Y; Jung, J E; Junk, T R; Kamon, T; Karchin, P E; Kato, Y; Kemp, Y; Kephart, R; Kerzel, U; Khotilovich, V; Kilminster, B; Kim, D H; Kim, H S; Kim, J E; Kim, M J; Kim, S B; Kim, S H; Kim, Y K; Kimura, N; Kirsch, L; Klimenko, S; Klute, M; Knuteson, B; Ko, B R; Kondo, K; Kong, D J; Konigsberg, J; Korytov, A; Kotwal, A V; Kovalev, A; Kraan, A C; Kraus, J; Kravchenko, I; Kreps, M; Kroll, J; Krumnack, N; Kruse, M; Krutelyov, V; Kubo, T; Kuhlmann, S E; Kuhr, T; Kusakabe, Y; Kwang, S; Laasanen, A T; Lai, S; Lami, S; Lammel, S; Lancaster, M; Lander, R L; Lannon, K; Lath, A; Latino, G; Lazzizzera, I; Le, Y; LeCompte, T; Lee, J; Lee, J; Lee, Y J; Lee, S W; Lefèvre, R; Leonardo, N; Leone, S; Levy, S; Lewis, J D; Lin, C; Lin, C S; Lindgren, M; Lipeles, E; Lister, A; Litvintsev, D O; Liu, T; Lockyer, N S; Loginov, A; Loreti, M; Loverre, P; Lu, R-S; Lucchesi, D; Lujan, P; Lukens, P; Lungu, G; Lyons, L; Lys, J; Lysak, R; Lytken, E; Mack, P; MacQueen, D; Madrak, R; Maeshima, K; Makhoul, K; Maki, T; Maksimovic, P; Malde, S; Manca, G; Margaroli, F; Marginean, R; Marino, C; Marino, C P; Martin, A; Martin, M; Martin, V; Martínez, M; Maruyama, T; Mastrandrea, P; Masubuchi, T; Matsunaga, H; Mattson, M E; Mazini, R; Mazzanti, P; McFarland, K S; McIntyre, P; McNulty, R; Mehta, A; Mehtala, P; Menzemer, S; Menzione, A; Merkel, P; Mesropian, C; Messina, A; Miao, T; Miladinovic, N; Miles, J; Miller, R; Mills, C; Milnik, M; Mitra, A; Mitselmakher, G; Miyamoto, A; Moed, S; Moggi, N; Mohr, B; Moore, R; Morello, M; Movilla Fernandez, P; Mülmenstädt, J; Mukherjee, A; Muller, Th; Mumford, R; Murat, P; Nachtman, J; Nagano, A; Naganoma, J; Nakano, I; Napier, A; Necula, V; Neu, C; Neubauer, M S; Nielsen, J; Nigmanov, T; Nodulman, L; Norniella, O; Nurse, E; Oh, S H; Oh, Y D; Oksuzian, I; Okusawa, T; Oldeman, R; Orava, R; Osterberg, K; Pagliarone, C; Palencia, E; Papadimitriou, V; Paramonov, A A; Parks, B; Pashapour, S; Patrick, J; Pauletta, G; Paulini, M; Paus, C; Pellett, D E; Penzo, A; Phillips, T J; Piacentino, G; Piedra, J; Pinera, L; Pitts, K; Plager, C; Pondrom, L; Portell, X; Poukhov, O; Pounder, N; Prakoshyn, F; Pronko, A; Proudfoot, J; Ptohos, F; Punzi, G; Pursley, J; Rademacker, J; Rahaman, A; Ranjan, N; Rappoccio, S; Reisert, B; Rekovic, V; Renton, P; Rescigno, M; Richter, S; Rimondi, F; Ristori, L; Robson, A; Rodrigo, T; Rogers, E; Rolli, S; Roser, R; Rossi, M; Rossin, R; Ruiz, A; Russ, J; Rusu, V; Saarikko, H; Sabik, S; Safonov, A; Sakumoto, W K; Salamanna, G; Saltó, O; Saltzberg, D; Sánchez, C; Santi, L; Sarkar, S; Sartori, L; Sato, K; Savard, P; Savoy-Navarro, A; Scheidle, T; Schlabach, P; Schmidt, E E; Schmidt, M P; Schmitt, M; Schwarz, T; Scodellaro, L; Scott, A L; Scribano, A; Scuri, F; Sedov, A; Seidel, S; Seiya, Y; Semenov, A; Sexton-Kennedy, L; Sfyrla, A; Shapiro, M D; Shears, T; Shepard, P F; Sherman, D; Shimojima, M; Shochet, M; Shon, Y; Shreyber, I; Sidoti, A; Sinervo, P; Sisakyan, A; Sjolin, J; Slaughter, A J; Slaunwhite, J; Sliwa, K; Smith, J R; Snider, F D; Snihur, R; Soderberg, M; Soha, A; Somalwar, S; Sorin, V; Spalding, J; Spinella, F; Spreitzer, T; Squillacioti, P; Stanitzki, M; Staveris-Polykalas, A; St Denis, R; Stelzer, B; Stelzer-Chilton, O; Stentz, D; Strologas, J; Stuart, D; Suh, J S; Sukhanov, A; Sun, H; Suzuki, T; Taffard, A; Takashima, R; Takeuchi, Y; Takikawa, K; Tanaka, M; Tanaka, R; Tecchio, M; Teng, P K; Terashi, K; Thom, J; Thompson, A S; Thomson, E; Tipton, P; Tiwari, V; Tkaczyk, S; Toback, D; Tokar, S; Tollefson, K; Tomura, T; Tonelli, D; Torre, S; Torretta, D; Tourneur, S; Trischuk, W; Tseng, J; Tsuchiya, R; Tsuno, S; Turini, N; Ukegawa, F; Unverhau, T; Uozumi, S; Usynin, D; Vallecorsa, S; van Remortel, N; Varganov, A; Vataga, E; Vázquez, F; Velev, G; Veramendi, G; Veszpremi, V; Vidal, R; Vila, I; Vilar, R; Vine, T; Vollrath, I; Volobouev, I; Volpi, G; Würthwein, F; Wagner, P; Wagner, R G; Wagner, R L; Wagner, J; Wagner, W; Wallny, R; Wang, S M; Warburton, A; Waschke, S; Waters, D; Wester, W C; Whitehouse, B; Whiteson, D; Wicklund, A B; Wicklund, E; Williams, G; Williams, H H; Wilson, P; Winer, B L; Wittich, P; Wolbers, S; Wolfe, C; Wright, T; Wu, X; Wynne, S M; Yagil, A; Yamamoto, K; Yamaoka, J; Yamashita, T; Yang, C; Yang, U K; Yang, Y C; Yao, W M; Yeh, G P; Yoh, J; Yorita, K; Yoshida, T; Yu, G B; Yu, I; Yu, S S; Yun, J C; Zanello, L; Zanetti, A; Zaw, I; Zhang, X; Zhou, J; Zucchelli, S

2007-03-23

We present the first observation of the baryon decay Lambda b0-->Lambda c+pi- followed by Lambda c+-->pK-pi+ in 106 pb-1 pp collisions at square root s=1.96 TeV in the CDF experiment. In order to reduce systematic error, the measured rate for Lambda b0 decay is normalized to the kinematically similar meson decay B0-->D+pi- followed by D+-->pi+K-pi+. We report the ratio of production cross sections (sigma) times the ratio of branching fractions (B) for the momentum region integrated above pT>6 GeV/c and pseudorapidity range |eta|<1.3: sigma(pp-->Lambda b0X)/sigma(pp-->B0X)xB(Lambda b0-->Lambda c+pi-)/B(B0-->D+pi-)=0.82+/-0.08(stat)+/-0.11(syst)+/-0.22[B(Lambda c+-->pK-pi+)].

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

Real-time analysis of dual-display phage immobilization and autoantibody screening using quartz crystal microbalance with dissipation monitoring.

PubMed

Rajaram, Kaushik; Losada-Pérez, Patricia; Vermeeren, Veronique; Hosseinkhani, Baharak; Wagner, Patrick; Somers, Veerle; Michiels, Luc

2015-01-01

Over the last three decades, phage display technology has been used for the display of target-specific biomarkers, peptides, antibodies, etc. Phage display-based assays are mostly limited to the phage ELISA, which is notorious for its high background signal and laborious methodology. These problems have been recently overcome by designing a dual-display phage with two different end functionalities, namely, streptavidin (STV)-binding protein at one end and a rheumatoid arthritis-specific autoantigenic target at the other end. Using this dual-display phage, a much higher sensitivity in screening specificities of autoantibodies in complex serum sample has been detected compared to single-display phage system on phage ELISA. Herein, we aimed to develop a novel, rapid, and sensitive dual-display phage to detect autoantibodies presence in serum samples using quartz crystal microbalance with dissipation monitoring as a sensing platform. The vertical functionalization of the phage over the STV-modified surfaces resulted in clear frequency and dissipation shifts revealing a well-defined viscoelastic signature. Screening for autoantibodies using antihuman IgG-modified surfaces and the dual-display phage with STV magnetic bead complexes allowed to isolate the target entities from complex mixtures and to achieve a large response as compared to negative control samples. This novel dual-display strategy can be a potential alternative to the time consuming phage ELISA protocols for the qualitative analysis of serum autoantibodies and can be taken as a departure point to ultimately achieve a point of care diagnostic system.

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin

PubMed Central

Liu, Jared; Yan, Riceley; Zhong, Qiao; Ngo, Sam; Bangayan, Nathanael J; Nguyen, Lin; Lui, Timothy; Liu, Minghsun; Erfe, Marie C; Craft, Noah; Tomida, Shuta; Li, Huiying

2015-01-01

The viral population, including bacteriophages, is an important component of the human microbiota, yet is poorly understood. We aim to determine whether bacteriophages modulate the composition of the bacterial populations, thus potentially playing a role in health or disease. We investigated the diversity and host interactions of the bacteriophages of Propionibacterium acnes, a major human skin commensal implicated in acne pathogenesis. By sequencing 48 P. acnes phages isolated from acne patients and healthy individuals and by analyzing the P. acnes phage populations in healthy skin metagenomes, we revealed that P. acnes phage populations in the skin microbial community are often dominated by one strain. We also found phage strains shared among both related and unrelated individuals, suggesting that a pool of common phages exists in the human population and that transmission of phages may occur between individuals. To better understand the bacterium–phage interactions in the skin microbiota, we determined the outcomes of 74 genetically defined Propionibacterium strains challenged by 15 sequenced phages. Depending on the Propionibacterium lineage, phage infection can result in lysis, pseudolysogeny, or resistance. In type II P. acnes strains, we found that encoding matching clustered regularly interspaced short palindromic repeat spacers is insufficient to confer phage resistance. Overall, our findings suggest that the prey–predator relationship between bacteria and phages may have a role in modulating the composition of the microbiota. Our study also suggests that the microbiome structure of an individual may be an important factor in the design of phage-based therapy. PMID:25848871

Phage Biodiversity in Artisanal Cheese Wheys Reflects the Complexity of the Fermentation Process

PubMed Central

Mahony, Jennifer; Moscarelli, Angelo; Kelleher, Philip; Lugli, Gabriele A.; Ventura, Marco; Settanni, Luca; van Sinderen, Douwe

2017-01-01

Dairy fermentations constitute a perfect “breeding ground” for bacteriophages infecting starter cultures, particularly strains of Lactococcus lactis. In modern fermentations, these phages typically belong to one of three groups, i.e., the 936, P335, and c2 phage groups. Traditional production methods present fewer chemical and physical barriers to phage proliferation compared to modern production systems, while the starter cultures used are typically complex, variable, and undefined. In the current study, a variety of cheese whey, animal-derived rennet, and vat swab samples from artisanal cheeses produced in Sicily were analysed for the presence of lactococcal phages to assess phage diversity in such environments. The complete genomes of 18 representative phage isolates were sequenced, allowing the identification of 10 lactococcal 949 group phages, six P087 group phages, and two members of the 936 group phages. The genetic diversity of these isolates was examined using phylogenetic analysis as well as a focused analysis of the receptor binding proteins, which dictate specific interactions with the host-encoded receptor. Thermal treatments at 63 °C and 83 °C indicate that the 949 phages are particularly sensitive to thermal treatments, followed by the P087 and 936 isolates, which were shown to be much less sensitive to such treatments. This difference may explain the relatively low frequency of isolation of the so-called “rare” 949 and P087 group phages in modern fermentations. PMID:28300778

Characterization and adsorption of Lactobacillus virulent phage P1.

PubMed

Chen, X; Xi, Y; Zhang, H; Wang, Z; Fan, M; Liu, Y; Wu, W

2016-09-01

Bacteriophage infection of lactic acid bacteria is considered an important problem worldwide in the food fermentation industry, as it may produce low quality or unsafe foods, cause fermentation failure, and result in economic losses. To increase current knowledge on the properties of Lactobacillus virulent phages, we evaluated the effect of divalent cations, temperature, pH, and chloramphenicol on the adsorption ability of Lactobacillus virulent phage P1. Phage P1 was isolated from the abnormal fermentation liquid of Lactobacillus plantarum IMAU10120. The results showed that this phage belonged to the Siphoviridae family. The latent period of this phage was 45min, and the burst time was 90min. Burst size was 132.88±2.37 phage counts expressed per milliliter per infective center. This phage showed good tolerance at different temperatures, but incubation at 50°C only affected its adsorption. Adsorption rate reached a maximum value between 30 and 42°C. A high adsorption value of phage infectivity was obtained from pH 6 to 8. Moreover, calcium ions promoted and increased the adsorption capacity of phage P1, but magnesium ions had negative effects. Chloramphenicol had no effect on phage adsorption. This study increased current knowledge on the characterization and biological aspects of Lactobacillus virulent phages, and may provide some basic information that can be used to design successful antiphage strategies in the food industry. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin.

PubMed

Liu, Jared; Yan, Riceley; Zhong, Qiao; Ngo, Sam; Bangayan, Nathanael J; Nguyen, Lin; Lui, Timothy; Liu, Minghsun; Erfe, Marie C; Craft, Noah; Tomida, Shuta; Li, Huiying

2015-09-01

The viral population, including bacteriophages, is an important component of the human microbiota, yet is poorly understood. We aim to determine whether bacteriophages modulate the composition of the bacterial populations, thus potentially playing a role in health or disease. We investigated the diversity and host interactions of the bacteriophages of Propionibacterium acnes, a major human skin commensal implicated in acne pathogenesis. By sequencing 48 P. acnes phages isolated from acne patients and healthy individuals and by analyzing the P. acnes phage populations in healthy skin metagenomes, we revealed that P. acnes phage populations in the skin microbial community are often dominated by one strain. We also found phage strains shared among both related and unrelated individuals, suggesting that a pool of common phages exists in the human population and that transmission of phages may occur between individuals. To better understand the bacterium-phage interactions in the skin microbiota, we determined the outcomes of 74 genetically defined Propionibacterium strains challenged by 15 sequenced phages. Depending on the Propionibacterium lineage, phage infection can result in lysis, pseudolysogeny, or resistance. In type II P. acnes strains, we found that encoding matching clustered regularly interspaced short palindromic repeat spacers is insufficient to confer phage resistance. Overall, our findings suggest that the prey-predator relationship between bacteria and phages may have a role in modulating the composition of the microbiota. Our study also suggests that the microbiome structure of an individual may be an important factor in the design of phage-based therapy.

Antibody Production in Response to Staphylococcal MS-1 Phage Cocktail in Patients Undergoing Phage Therapy.

PubMed

Żaczek, Maciej; Łusiak-Szelachowska, Marzanna; Jończyk-Matysiak, Ewa; Weber-Dąbrowska, Beata; Międzybrodzki, Ryszard; Owczarek, Barbara; Kopciuch, Agnieszka; Fortuna, Wojciech; Rogóż, Paweł; Górski, Andrzej

2016-01-01

In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties toward applied phages (K rate). Among 20 examined patients receiving the MS-1 phage cocktail orally and/or locally, the majority did not show a noticeably higher level of antiphage antibodies in their sera during phage administration. Even in those individual cases with an increased immune response, mostly by induction of IgG and IgM, the presence of antiphage antibodies did not translate into unsatisfactory clinical results of phage therapy. On the other hand, a negative outcome of the treatment occurred in some patients who showed relatively weak production of antiphage antibodies before and during treatment. This may imply that possible induction of antiphage antibodies is not an obstacle to the implementation of phage therapy and support our assumption that the outcome of the phage treatment does not primarily depend on the appearance of antiphage antibodies in sera of patients during therapy. These conclusions are in line with our previous findings. The confirmation of this thesis is of great interest as regards the efficacy of phage therapy in humans.

Bioengineering bacteriophages to enhance the sensitivity of phage amplification-based paper fluidic detection of bacteria.

PubMed

Alcaine, S D; Law, K; Ho, S; Kinchla, A J; Sela, D A; Nugen, S R

2016-08-15

Bacteriophage (phage) amplification is an attractive method for the detection of bacteria due to a narrow phage-host specificity, short amplification times, and the phages' ability to differentiate between viable and non-viable bacterial cells. The next step in phage-based bacteria detection is leveraging bioengineered phages to create low-cost, rapid, and easy-to-use detection platforms such as lateral flow assays. Our work establishes the proof-of-concept for the use of bioengineered T7 phage strains to increase the sensitivity of phage amplification-based lateral flow assays. We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 10(3)CFU/mL of Escherichia coli in broth after 7h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100CFU/100mL in inoculated river water after 9h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of an RNase III Binding Site in Transcription Termination at λ nutL by HK022 Nun Protein

PubMed Central

Washburn, Robert S.; Court, Donald L.; Gottesman, Max E.

2006-01-01

The phage HK022 Nun protein excludes phage λ by binding nascent λ pL and pR transcripts at nutL and nutR, respectively, and inducing transcription termination just downstream of these sites. Termination is more efficient at nutL than at nutR. One difference between nutL and nutR is the presence of RNase III processing sites (rIII) located immediately promoter distal to λ nutL. We found that deletion of rIII dramatically reduced Nun transcription arrest in vitro but had little effect on termination in vivo. However, consistent with the in vitro results, overexpression of a transcript carrying nutL and rIII efficiently titrated Nun, allowing λ to grow on a strain that expressed Nun, whereas a transcript carrying only nutL or nutL-rIII with nucleotides 97 to 141 deleted was ineffective. Rnc70, an RNase III mutant that binds but does not cleave rIII, also prevented Nun-mediated λ exclusion. We propose that rIII enhances the on-rate of Nun at nutL, stimulating Nun-mediated arrest in vitro. We have shown that a specific element in rIII, i.e., box C (G89GUGUGUG), strongly enhances arrest on rIII+ templates. Nun-rIII interactions do not stimulate Nun termination in vivo, presumably because formation of the Nun-nutL complex is normally not rate-limiting in the cell. In contrast to Nun, N is not occluded by Rnc70 and is not efficiently titrated by a nutL-rIII transcript. PMID:16980485

The genomes and comparative genomics of Lactobacillus delbrueckii phages.

PubMed

Riipinen, Katja-Anneli; Forsman, Päivi; Alatossava, Tapani

2011-07-01

Lactobacillus delbrueckii phages are a great source of genetic diversity. Here, the genome sequences of Lb. delbrueckii phages LL-Ku, c5 and JCL1032 were analyzed in detail, and the genetic diversity of Lb. delbrueckii phages belonging to different taxonomic groups was explored. The lytic isometric group b phages LL-Ku (31,080 bp) and c5 (31,841 bp) showed a minimum nucleotide sequence identity of 90% over about three-fourths of their genomes. The genomic locations of their lysis modules were unique, and the genomes featured several putative overlapping transcription units of genes. LL-Ku and c5 virions displayed peptidoglycan hydrolytic activity associated with a ~36-kDa protein similar in size to the endolysin. Unexpectedly, the 49,433-bp genome of the prolate phage JCL1032 (temperate, group c) revealed a conserved gene order within its structural genes. Lb. delbrueckii phages representing groups a (a phage LL-H), b and c possessed only limited protein sequence homology. Genomic comparison of LL-Ku and c5 suggested that diversification of Lb. delbrueckii phages is mainly due to insertions, deletions and recombination. For the first time, the complete genome sequences of group b and c Lb. delbrueckii phages are reported.

Use of phages to control Campylobacter spp.

PubMed

Janež, Nika; Loc-Carrillo, Catherine

2013-10-01

The use of phages to control pathogenic bacteria has been investigated since they were first discovered in the beginning of the 1900s. Over the last century we have slowly gained an in-depth understanding of phage biology including which phage properties are desirable when considering phage as biocontrol agents and which phage characteristics to potentially avoid. Campylobacter infections are amongst the most frequently encountered foodborne bacterial infections around the world. Handling and consumption of raw or undercooked poultry products have been determined to be the main route of transmission. The ability to use phages to target these bacteria has been studied for more than a decade and although we have made progress towards deciphering how best to use phages to control Campylobacter associated with poultry production, there is still much work to be done. This review outlines methods to improve the isolation of these elusive phages, as well as methods to identify desirable characteristics needed for a successful outcome. It also highlights the body of research undertaken so far and what criteria to consider when doing in-vivo studies, especially because some in-vitro studies have not been found to translate into to phage efficacy in-vivo. © 2013. Published by Elsevier B.V. All rights reserved.

Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages

PubMed Central

Adair, Tamarah L.; Afram, Patricia; Allen, Katherine G.; Archambault, Megan L.; Aziz, Rahat M.; Bagnasco, Filippa G.; Ball, Sarah L.; Barrett, Natalie A.; Benjamin, Robert C.; Blasi, Christopher J.; Borst, Katherine; Braun, Mary A.; Broomell, Haley; Brown, Conner B.; Brynell, Zachary S.; Bue, Ashley B.; Burke, Sydney O.; Casazza, William; Cautela, Julia A.; Chen, Kevin; Chimalakonda, Nitish S.; Chudoff, Dylan; Connor, Jade A.; Cross, Trevor S.; Curtis, Kyra N.; Dahlke, Jessica A.; Deaton, Bethany M.; Degroote, Sarah J.; DeNigris, Danielle M.; DeRuff, Katherine C.; Dolan, Milan; Dunbar, David; Egan, Marisa S.; Evans, Daniel R.; Fahnestock, Abby K.; Farooq, Amal; Finn, Garrett; Fratus, Christopher R.; Gaffney, Bobby L.; Garlena, Rebecca A.; Garrigan, Kelly E.; Gibbon, Bryan C.; Goedde, Michael A.; Guerrero Bustamante, Carlos A.; Harrison, Melinda; Hartwell, Megan C.; Heckman, Emily L.; Huang, Jennifer; Hughes, Lee E.; Hyduchak, Kathryn M.; Jacob, Aswathi E.; Kaku, Machika; Karstens, Allen W.; Kenna, Margaret A.; Khetarpal, Susheel; King, Rodney A.; Kobokovich, Amanda L.; Kolev, Hannah; Konde, Sai A.; Kriese, Elizabeth; Lamey, Morgan E.; Lantz, Carter N.; Lapin, Jonathan S.; Lawson, Temiloluwa O.; Lee, In Young; Lee, Scott M.; Lee-Soety, Julia Y.; Lehmann, Emily M.; London, Shawn C.; Lopez, A. Javier; Lynch, Kelly C.; Mageeney, Catherine M.; Martynyuk, Tetyana; Mathew, Kevin J.; Mavrich, Travis N.; McDaniel, Christopher M.; McDonald, Hannah; McManus, C. Joel; Medrano, Jessica E.; Mele, Francis E.; Menninger, Jennifer E.; Miller, Sierra N.; Minick, Josephine E.; Nabua, Courtney T.; Napoli, Caroline K.; Nkangabwa, Martha; Oates, Elizabeth A.; Ott, Cassandra T.; Pellerino, Sarah K.; Pinamont, William J.; Pirnie, Ross T.; Pizzorno, Marie C.; Plautz, Emilee J.; Pope, Welkin H.; Pruett, Katelyn M.; Rickstrew, Gabbi; Rimple, Patrick A.; Rinehart, Claire A.; Robinson, Kayla M.; Rose, Victoria A.; Russell, Daniel A.; Schick, Amelia M.; Schlossman, Julia; Schneider, Victoria M.; Sells, Chloe A.; Sieker, Jeremy W.; Silva, Morgan P.; Silvi, Marissa M.; Simon, Stephanie E.; Staples, Amanda K.; Steed, Isabelle L.; Stowe, Emily L.; Stueven, Noah A.; Swartz, Porter T.; Sweet, Emma A.; Sweetman, Abigail T.; Tender, Corrina; Terry, Katrina; Thomas, Chrystal; Thomas, Daniel S.; Thompson, Allison R.; Vanderveen, Lorianna; Varma, Rohan; Vaught, Hannah L.; Vo, Quynh D.; Vonberg, Zachary T.; Ware, Vassie C.; Warrad, Yasmene M.; Wathen, Kaitlyn E.; Weinstein, Jonathan L.; Wyper, Jacqueline F.; Yankauskas, Jakob R.; Zhang, Christine

2017-01-01

The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single Arthrobacter sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45–68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these Arthrobacter phages are primarily lytic, and only the singleton Galaxy is likely temperate. PMID:28715480

Capture and detection of T7 bacteriophages on a nanostructured interface.

PubMed

Han, Jin-Hee; Wang, Min S; Das, Jayanti; Sudheendra, L; Vonasek, Erica; Nitin, Nitin; Kennedy, Ian M

2014-04-09

A highly ordered array of T7 bacteriophages was created by the electrophoretic capture of phages onto a nanostructured array with wells that accommodated the phages. Electrophoresis of bacteriophages was achieved by applying a positive potential on an indium tin oxide electrode at the bottom of the nanowells. Nanoscale arrays of phages with different surface densities were obtained by changing the electric field applied to the bottom of the nanowells. The applied voltage was shown to be the critical factor in generating a well-ordered phage array. The number of wells occupied by a phage, and hence the concentration of phages in a sample solution, could be quantified by using a DNA intercalating dye that rapidly stains the T7 phage. The fluorescence signal was enhanced by the intrinsic photonic effect made available by the geometry of the platform. It was shown that the quantification of phages on the array was 6 orders of magnitude better than could be obtained with a fluorescent plate reader. The device opens up the possibility that phages can be detected directly without enrichment or culturing, and by detecting phages that specifically infect bacteria of interest, rapid pathogen detection becomes possible.

Capture and Detection of T7 Bacteriophages on a Nanostructured Interface

PubMed Central

2015-01-01

A highly ordered array of T7 bacteriophages was created by the electrophoretic capture of phages onto a nanostructured array with wells that accommodated the phages. Electrophoresis of bacteriophages was achieved by applying a positive potential on an indium tin oxide electrode at the bottom of the nanowells. Nanoscale arrays of phages with different surface densities were obtained by changing the electric field applied to the bottom of the nanowells. The applied voltage was shown to be the critical factor in generating a well-ordered phage array. The number of wells occupied by a phage, and hence the concentration of phages in a sample solution, could be quantified by using a DNA intercalating dye that rapidly stains the T7 phage. The fluorescence signal was enhanced by the intrinsic photonic effect made available by the geometry of the platform. It was shown that the quantification of phages on the array was 6 orders of magnitude better than could be obtained with a fluorescent plate reader. The device opens up the possibility that phages can be detected directly without enrichment or culturing, and by detecting phages that specifically infect bacteria of interest, rapid pathogen detection becomes possible. PMID:24650205

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.

Review: Efficiency of Physical and Chemical Treatments on the Inactivation of Dairy Bacteriophages

PubMed Central

Guglielmotti, Daniela M.; Mercanti, Diego J.; Reinheimer, Jorge A.; Quiberoni, Andrea del L.

2011-01-01

Bacteriophages can cause great economic losses due to fermentation failure in dairy plants. Hence, physical and chemical treatments of raw material and/or equipment are mandatory to maintain phage levels as low as possible. Regarding thermal treatments used to kill pathogenic bacteria or achieve longer shelf-life of dairy products, neither low temperature long time nor high temperature short time pasteurization were able to inactivate most lactic acid bacteria (LAB) phages. Even though most phages did not survive 90°C for 2 min, there were some that resisted 90°C for more than 15 min (conditions suggested by the International Dairy Federation, for complete phage destruction). Among biocides tested, ethanol showed variable effectiveness in phage inactivation, since only phages infecting dairy cocci and Lactobacillus helveticus were reasonably inactivated by this alcohol, whereas isopropanol was in all cases highly ineffective. In turn, peracetic acid has consistently proved to be very fast and efficient to inactivate dairy phages, whereas efficiency of sodium hypochlorite was variable, even among different phages infecting the same LAB species. Both alkaline chloride foam and ethoxylated non-ylphenol with phosphoric acid were remarkably efficient, trait probably related to their highly alkaline or acidic pH values in solution, respectively. Photocatalysis using UV light and TiO2 has been recently reported as a feasible option to industrially inactivate phages infecting diverse LAB species. Processes involving high pressure were barely used for phage inactivation, but until now most studied phages revealed high resistance to these treatments. To conclude, and given the great phage diversity found on dairies, it is always advisable to combine different anti-phage treatments (biocides, heat, high pressure, photocatalysis), rather than using them separately at extreme conditions. PMID:22275912

In vitro management of hospital Pseudomonas aeruginosa biofilm using indigenous T7-like lytic phage.

PubMed

Ahiwale, Sangeeta; Tamboli, Nilofer; Thorat, Kiran; Kulkarni, Rajendra; Ackermann, Hans; Kapadnis, Balasaheb

2011-02-01

Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1-HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.

Incorporating Phage Therapy into WPI Dip Coatings for Applications on Fresh Whole and Cut Fruit and Vegetable Surfaces.

PubMed

Vonasek, Erica L; Choi, Angela H; Sanchez, Juan; Nitin, Nitin

2018-06-15

There is a significant unmet need to develop antimicrobial solutions to reduce the risk of contamination in fresh produce. Bacteriophages have been proposed as a potential approach for controlling foodborne pathogens. This study evaluated the combination of edible dip coatings with T7 bacteriophages on whole and cut produce. The evaluation includes an assessment of phage loading, phage storage stability, antimicrobial activity, and phage stability during simulated gastric digestion on sliced cucumbers, sliced apples, and whole cherry tomatoes. In this evaluation, phages coated on fresh produce using edible whey protein isolate (WPI) were compared with phages coated from an aqueous suspension (control coating). The results demonstrated that WPI coatings load more phages than the control and enhanced phage stability during cold storage (4 °C) for cut apples and whole cherry tomatoes. Phage stability decreased by 1 to 3 log(PFU) in a simulated gastric environment. Phage antimicrobial activity against Escherichia coli BL21 decreased 2 to 4 log(CFU) of bacteria on cut apples and whole cherry tomatoes, while no significant bacterial reduction was observed for sliced cucumbers. Overall, the results show that WPI dip coating provides phage loading, stability, and antimicrobial activity to produce surfaces compared to the control coating, and thus may be considered an effective approach for extending phage therapy on fresh produce. The practical application is to prevent bacterial cross contamination of fresh produce by using a combination of edible coating with bacteriophages. The results demonstrate enhanced loading and stability of phages on fresh produce when used in combination with an edible coating. © 2018 Institute of Food Technologists®.

Three novel Pseudomonas phages isolated from composting provide insights into the evolution and diversity of tailed phages.

PubMed

Amgarten, Deyvid; Martins, Layla Farage; Lombardi, Karen Cristina; Antunes, Luciana Principal; de Souza, Ana Paula Silva; Nicastro, Gianlucca Gonçalves; Kitajima, Elliott Watanabe; Quaggio, Ronaldo Bento; Upton, Chris; Setubal, João Carlos; da Silva, Aline Maria

2017-05-04

Among viruses, bacteriophages are a group of special interest due to their capacity of infecting bacteria that are important for biotechnology and human health. Composting is a microbial-driven process in which complex organic matter is converted into humus-like substances. In thermophilic composting, the degradation activity is carried out primarily by bacteria and little is known about the presence and role of bacteriophages in this process. Using Pseudomonas aeruginosa as host, we isolated three new phages from a composting operation at the Sao Paulo Zoo Park (Brazil). One of the isolated phages is similar to Pseudomonas phage Ab18 and belongs to the Siphoviridae YuA-like viral genus. The other two isolated phages are similar to each other and present genomes sharing low similarity with phage genomes in public databases; we therefore hypothesize that they belong to a new genus in the Podoviridae family. Detailed genomic descriptions and comparisons of the three phages are presented, as well as two new clusters of phage genomes in the Viral Orthologous Clusters database of large DNA viruses. We found sequences encoding homing endonucleases that disrupt a putative ribonucleotide reductase gene and an RNA polymerase subunit 2 gene in two of the phages. These findings provide insights about the evolution of two-subunits RNA polymerases and the possible role of homing endonucleases in this process. Infection tests on 30 different strains of bacteria reveal a narrow host range for the three phages, restricted to P. aeruginosa PA14 and three other P. aeruginosa clinical isolates. Biofilm dissolution assays suggest that these phages could be promising antimicrobial agents against P. aeruginosa PA14 infections. Analyses on composting metagenomic and metatranscriptomic data indicate association between abundance variations in both phage and host populations in the environment. The results about the newly discovered and described phages contribute to the understanding of tailed bacteriophage diversity, evolution, and role in the complex composting environment.

Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases

PubMed Central

De Paepe, Marianne; Hutinet, Geoffrey; Son, Olivier; Amarir-Bouhram, Jihane; Schbath, Sophie; Petit, Marie-Agnès

2014-01-01

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself. PMID:24603854

Target-specific copper hybrid T7 phage particles.

PubMed

Dasa, Siva Sai Krishna; Jin, Qiaoling; Chen, Chin-Tu; Chen, Liaohai

2012-12-18

Target-specific nanoparticles have attracted significant attention recently, and have greatly impacted life and physical sciences as new agents for imaging, diagnosis, and therapy, as well as building blocks for the assembly of novel complex materials. While most of these particles are synthesized by chemical conjugation of an affinity reagent to polymer or inorganic nanoparticles, we are promoting the use of phage particles as a carrier to host organic or inorganic functional components, as well as to display the affinity reagent on the phage surface, taking advantage of the fact that some phages host well-established vectors for protein expression. An affinity reagent can be structured in a desired geometry on the surface of phage particles, and more importantly, the number of the affinity reagent molecules per phage particle can be precisely controlled. We previously have reported the use of the T7 phage capsid as a template for synthesizing target-specific metal nanoparticles. In this study herein, we reported the synthesis of nanoparticles using an intact T7 phage as a scaffold from which to extend 415 copies of a peptide that contains a hexahistidine (6His) motif for capture of copper ions and staging the conversion of copper ions to copper metal, and a cyclic Arginine-Glycine-Aspartic Acid (RGD4C) motif for targeting integrin and cancer cells. We demonstrated that the recombinant phage could load copper ions under low bulk copper concentrations without interfering with its target specificity. Further reduction of copper ions to copper metal rendered a very stable copper hybrid T7 phage, which prevents the detachment of copper from phage particles and maintains the phage structural integrity even under harsh conditions. Cancer cells (MCF-7) can selectively uptake copper hybrid T7 phage particles through ligand-mediated transmembrane transportation, whereas normal control cells (MCF-12F) uptake 1000-fold less. We further demonstrated that copper hybrid T7 phage could be endocytosed by cancer cells in culture.

Comparative genomic and morphological analyses of Listeria phages isolated from farm environments.

PubMed

Denes, Thomas; Vongkamjan, Kitiya; Ackermann, Hans-Wolfgang; Moreno Switt, Andrea I; Wiedmann, Martin; den Bakker, Henk C

2014-08-01

The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large genome (~135-kb) myoviruses belonging to the genus “Twort-like viruses,” three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (~66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (~56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence.

PubMed

Xie, Yicheng; Wahab, Laith; Gill, Jason J

2018-04-12

Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence

PubMed Central

Xie, Yicheng; Wahab, Laith

2018-01-01

Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format. PMID:29649135

Stable carbon isotope fractionation during the biodegradation of lambda-cyhalothrin.

PubMed

Shen, Xiaoli; Xu, Zemin; Zhang, Xichang; Yang, Fangxing

2015-11-01

In this study, the microbial degradation of lambda-cyhalothrin in soil was investigated using compound-specific stable isotope analysis. The results revealed that lambda-cyhalothrin was biodegraded in soil under laboratory conditions. The half-lives of lambda-cyhalothrin were determined to be 49 and 161 days in non-sterile and sterile soils spiked with 2mg/kg lambda-cyhalothrin and 84 and 154 days in non-sterile and sterile soils spiked with 10mg/kg lambda-cyhalothrin, respectively. The biodegradation of lambda-cyhalothrin resulted in carbon isotope fractionation, which shifted from -29.0‰ to -26.5‰ in soil spiked with 2mg/kg lambda-cyhalothrin, and to -27.5‰ with 10mg/kg lambda-cyhalothrin. A relationship was established between the stable carbon isotope fraction and the residual concentrations of lambda-cyhalothrin by the Rayleigh equation in which the carbon isotope enrichment factor ε of the microbial degradation of lambda-cyhalothrin in the soil was calculated as -2.53‰. This study provides an approach to quantitatively evaluate the biodegradation of lambda-cyhalothrin in soil in field studies. Copyright © 2015 Elsevier B.V. All rights reserved.

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Bacteriophage as models for virus removal from Pacific oysters (Crassostrea gigas) during re-laying.

PubMed Central

Humphrey, T. J.; Martin, K.

1993-01-01

A study was undertaken to examine the feasibility of using naturally-occurring bacteriophages to assess the impact of re-laying on levels of viral contamination in Crassostrea gigas, the Pacific oyster. Two phages were chosen. One, male-specific (F+), was enumerated using Salmonella typhimurium. The other, a somatic phage, was detected using an, as yet, uncharacterized Escherichia coli. Investigations, using a variety of re-laying sites, demonstrated that numbers of F+ phage in oyster tissue declined more rapidly than those of somatic phage. For example, in oysters placed in commercially-used sea water ponds, F+ phage reached undetectable levels within 2-3 weeks, whereas somatic phage could still be detected 5 weeks after re-laying. The studies suggest that F+ phage may not be a suitable indicator for virus removal and that somatic phage may be better suited to this role. PMID:8405159

Construction of a filamentous phage display peptide library.

PubMed

Fagerlund, Annette; Myrset, Astrid Hilde; Kulseth, Mari Ann

2014-01-01

The concept of phage display is based on insertion of random oligonucleotides at an appropriate location within a structural gene of a bacteriophage. The resulting phage will constitute a library of random peptides displayed on the surface of the bacteriophages, with the encoding genotype packaged within each phage particle. Using a phagemid/helper phage system, the random peptides are interspersed between wild-type coat proteins. Libraries of phage-expressed peptides may be used to search for novel peptide ligands to target proteins. The success of finding a peptide with a desired property in a given library is highly dependent on the diversity and quality of the library. The protocols in this chapter describe the construction of a high-diversity library of phagemid vector encoding fusions of the phage coat protein pVIII with random peptides, from which a phage library displaying random peptides can be prepared.

Pf16 and phiPMW: Expanding the realm of Pseudomonas putida bacteriophages

PubMed Central

Krylov, Victor N.; Shaburova, Olga V.; McGrath, John W.; Allen, Christopher C. R.; Quinn, John P.; Kulakov, Leonid A.

2017-01-01

We present the analysis of two novel Pseudomonas putida phages, pf16 and phiPMW. Pf16 represents a peripherally related T4-like phage, and is the first of its kind infecting a Pseudomonad, with evidence suggesting cyanophage origins. Extensive divergence has resulted in pf16 occupying a newly defined clade designated as the pf16-related phages, lying at the interface of the Schizo T-Evens and Exo T-Evens. Recombination with an ancestor of the P. putida phage AF is likely responsible for the tropism of this phage. phiPMW represents a completely novel Pseudomonas phage with a genome containing substantial genetic novelty through its many hypothetical proteins. Evidence suggests that this phage has been extensively shaped through gene transfer events and vertical evolution. Phylogenetics shows that this phage has an evolutionary history involving FelixO1-related viruses but is in itself highly distinct from this group. PMID:28877269

Dynamics of a family of transcendental meromorphic functions having rational Schwarzian derivative

NASA Astrophysics Data System (ADS)

Sajid, M.; Kapoor, G. P.

2007-02-01

In the present paper, a class of critically finite transcendental meromorphic functions having rational Schwarzian derivative is introduced and the dynamics of functions in one parameter family is investigated. It is found that there exist two parameter values [lambda]*=[phi](0)>0 and , where and is the real root of [phi]'(x)=0, such that the Fatou sets of f[lambda](z) for [lambda]=[lambda]* and [lambda]=[lambda]** contain parabolic domains. A computationally useful characterization of the Julia set of the function f[lambda](z) as the complement of the basin of attraction of an attracting real fixed point of f[lambda](z) is established and applied for the generation of the images of the Julia sets of f[lambda](z). Further, it is observed that the Julia set of explodes to whole complex plane for [lambda]>[lambda]**. Finally, our results found in the present paper are compared with the recent results on dynamics of one parameter families [lambda]tanz, [R.L. Devaney, L. Keen, Dynamics of meromorphic maps: Maps with polynomial Schwarzian derivative, Ann. Sci. Ecole Norm. Sup. 22 (4) (1989) 55-79; L. Keen, J. Kotus, Dynamics of the family [lambda]tan(z), Conform. Geom. Dynam. 1 (1997) 28-57; G.M. Stallard, The Hausdorff dimension of Julia sets of meromorphic functions, J. London Math. Soc. 49 (1994) 281-295] and , [lambda]>0 [G.P. Kapoor, M. Guru Prem Prasad, Dynamics of : The Julia set and bifurcation, Ergodic Theory Dynam. Systems 18 (1998) 1363-1383].

Evolved Populations of Shigella flexneri Phage Sf6 Acquire Large Deletions, Altered Genomic Architecture, and Faster Life Cycles.

PubMed

Dover, John A; Burmeister, Alita R; Molineux, Ian J; Parent, Kristin N

2016-09-19

Genomic architecture is the framework within which genes and regulatory elements evolve and where specific constructs may constrain or potentiate particular adaptations. One such construct is evident in phages that use a headful packaging strategy that results in progeny phage heads packaged with DNA until full rather than encapsidating a simple unit-length genome. Here, we investigate the evolution of the headful packaging phage Sf6 in response to barriers that impede efficient phage adsorption to the host cell. Ten replicate populations evolved faster Sf6 life cycles by parallel mutations found in a phage lysis gene and/or by large, 1.2- to 4.0-kb deletions that remove a mobile genetic IS911 element present in the ancestral phage genome. The fastest life cycles were found in phages that acquired both mutations. No mutations were found in genes encoding phage structural proteins, which were a priori expected from the experimental design that imposed a challenge for phage adsorption by using a Shigella flexneri host lacking receptors preferred by Sf6. We used DNA sequencing, molecular approaches, and physiological experiments on 82 clonal isolates taken from all 10 populations to reveal the genetic basis of the faster Sf6 life cycle. The majority of our isolates acquired deletions in the phage genome. Our results suggest that deletions are adaptive and can influence the duration of the phage life cycle while acting in conjunction with other lysis time-determining point mutations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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

2015-11-03

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.

Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10.

PubMed

Bari, S M Nayeemul; Walker, Forrest C; Cater, Katie; Aslan, Barbaros; Hatoum-Aslan, Asma

2017-12-15

Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multisubunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. On the basis of this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.

Development of phoH as a Novel Signature Gene for Assessing Marine Phage Diversity▿

PubMed Central

Goldsmith, Dawn B.; Crosti, Giuseppe; Dwivedi, Bhakti; McDaniel, Lauren D.; Varsani, Arvind; Suttle, Curtis A.; Weinbauer, Markus G.; Sandaa, Ruth-Anne; Breitbart, Mya

2011-01-01

Phages play a key role in the marine environment by regulating the transfer of energy between trophic levels and influencing global carbon and nutrient cycles. The diversity of marine phage communities remains difficult to characterize because of the lack of a signature gene common to all phages. Recent studies have demonstrated the presence of host-derived auxiliary metabolic genes in phage genomes, such as those belonging to the Pho regulon, which regulates phosphate uptake and metabolism under low-phosphate conditions. Among the completely sequenced phage genomes in GenBank, this study identified Pho regulon genes in nearly 40% of the marine phage genomes, while only 4% of nonmarine phage genomes contained these genes. While several Pho regulon genes were identified, phoH was the most prevalent, appearing in 42 out of 602 completely sequenced phage genomes. Phylogenetic analysis demonstrated that phage phoH sequences formed a cluster distinct from those of their bacterial hosts. PCR primers designed to amplify a region of the phoH gene were used to determine the diversity of phage phoH sequences throughout a depth profile in the Sargasso Sea and at six locations worldwide. phoH was present at all sites examined, and a high diversity of phoH sequences was recovered. Most phoH sequences belonged to clusters without any cultured representatives. Each depth and geographic location had a distinct phoH composition, although most phoH clusters were recovered from multiple sites. Overall, phoH is an effective signature gene for examining phage diversity in the marine environment. PMID:21926220

Efficacy and Safety of a Bovine-Associated Staphylococcus aureus Phage Cocktail in a Murine Model of Mastitis.

PubMed

Breyne, Koen; Honaker, Ryan W; Hobbs, Zachary; Richter, Manuela; Żaczek, Maciej; Spangler, Taylor; Steenbrugge, Jonas; Lu, Rebecca; Kinkhabwala, Anika; Marchon, Bruno; Meyer, Evelyne; Mokres, Lucia

2017-01-01

Overuse of antibiotics is a major problem in the treatment of bovine mastitis, and antibiotic treatment is frequently non-curative, thus alternative treatments are necessary. The primary aim of this study was to evaluate the efficacy of a purified phage cocktail for treatment of bovine Staphylococcus aureus mastitis in a well-defined mouse model. Candidate phages were selected based on their in vitro performance and subsequently processed into an optimally composed phage cocktail. The highest scoring phages were further tested for efficacy and resistance suppression in broth and raw milk, with and without supplemental IgG. As these in vitro results displayed significant decreases in CFU, the cocktail was purified for testing in vivo . Lactating mice were intramammarily inoculated with S. aureus N305 (ATCC 29740), a clinical bovine mastitis isolate commonly used for experimental infection of dairy cows. The phage cocktail was applied via the same route 4 h post-inoculation. Treated mammary glands were graded for gross pathological appearance and excised for bacterial and phage load quantification as well as histopathology. Observation of gross macroscopic and histopathological changes and CFU quantification demonstrated that the phage cocktail treatment significantly improved mastitis pathology and decreased bacterial counts. Phage PFU quantification indicated that the tested phage cocktail treatment was able to maintain high intramammary phage titers without spreading systemically. The in vivo results complement the in vitro data and support our concept of phage therapy as an innovative alternative or supplementation therapy to antibiotics for the treatment of bovine mastitis.

Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

PubMed Central

Tucker, Kimberly P; Parsons, Rachel; Symonds, Erin M; Breitbart, Mya

2011-01-01

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem. PMID:21124487

Dynamics of success and failure in phage and antibiotic therapy in experimental infections.

PubMed

Bull, J J; Levin, Bruce R; DeRouin, Terry; Walker, Nina; Bloch, Craig A

2002-11-26

In 1982 Smith and Huggins showed that bacteriophages could be at least as effective as antibiotics in preventing mortality from experimental infections with a capsulated E. coli (K1) in mice. Phages that required the K1 capsule for infection were more effective than phages that did not require this capsule, but the efficacies of phages and antibiotics in preventing mortality both declined with time between infection and treatment, becoming virtually ineffective within 16 hours. We develop quantitative microbiological procedures that (1) explore the in vivo processes responsible for the efficacy of phage and antibiotic treatment protocols in experimental infections (the Resistance Competition Assay, or RCA), and (2) survey the therapeutic potential of phages in vitro (the Phage Replication Assay or PRA). We illustrate the application and utility of these methods in a repetition of Smith and Huggins' experiments, using the E. coli K1 mouse thigh infection model, and applying treatments of phages or streptomycin. 1) The Smith and Huggins phage and antibiotic therapy results are quantitatively and qualitatively robust. (2) Our RCA values reflect the microbiological efficacies of the different phages and of streptomycin in preventing mortality, and reflect the decline in their efficacy with a delay in treatment. These results show specifically that bacteria become refractory to treatment over the term of infection. (3) The K1-specific and non-specific phages had similar replication rates on bacteria grown in broth (based on the PRA), but the K1-specific phage had markedly greater replication rates in mouse serum.

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice.

PubMed

Filippov, Andrey A; Sergueev, Kirill V; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T; Mueller, Allen J; Fernandez-Prada, Carmen M; Nikolich, Mikeljon P

2011-01-01

Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

PubMed Central

Filippov, Andrey A.; Sergueev, Kirill V.; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T.; Mueller, Allen J.; Fernandez-Prada, Carmen M.; Nikolich, Mikeljon P.

2011-01-01

Background Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis. PMID:21980477

Filamentous phages of Ralstonia solanacearum: double-edged swords for pathogenic bacteria.

PubMed

Yamada, Takashi

2013-01-01

Some phages from genus Inovirus use host or bacteriophage-encoded site-specific integrases or recombinases establish a prophage state. During integration or excision, a superinfective form can be produced. The three states (free, prophage, and superinfective) of such phages exert different effects on host bacterial phenotypes. In Ralstonia solanacearum, the causative agent of bacterial wilt disease of crops, the bacterial virulence can be positively or negatively affected by filamentous phages, depending on their state. The presence or absence of a repressor gene in the phage genome may be responsible for the host phenotypic differences (virulent or avirulent) caused by phage infection. This strategy of virulence control may be widespread among filamentous phages that infect pathogenic bacteria of plants.

Modeling the interactions between pathogenic bacteria, bacteriophage and immune response

NASA Astrophysics Data System (ADS)

Leung, Chung Yin (Joey); Weitz, Joshua S.

The prevalence of antibiotic-resistant strains of pathogenic bacteria has led to renewed interest in the use of bacteriophage (phage), or virus that infects bacteria, as a therapeutic agent against bacterial infections. However, little is known about the theoretical mechanism by which phage therapy may work. In particular, interactions between the bacteria, the phage and the host immune response crucially influences the outcome of the therapy. Few models of phage therapy have incorporated all these three components, and existing models suffer from unrealistic assumptions such as unbounded growth of the immune response. We propose a model of phage therapy with an emphasis on nonlinear feedback arising from interactions with bacteria and the immune response. Our model shows a synergistic effect between the phage and the immune response which underlies a possible mechanism for phage to catalyze the elimination of bacteria even when neither the immune response nor phage could do so alone. We study the significance of this effect for different parameters of infection and immune response, and discuss its implications for phage therapy.

Phage-Mediated Gene Therapy.

PubMed

Hosseinidoust, Zeinab

2017-01-01

Bacteriophages (bacterial viruses) have long been under investigation as vectors for gene therapy. Similar to other viral vectors, the phage coat proteins have evolved over millions of years to protect the viral genome from degradation post injection, offering protection for the valuable therapeutic sequence. However, what sets phage apart from other viral gene delivery vectors is their safety for human use and the relative ease by which foreign molecules can be expressed on the phage outer surface, enabling highly targeted gene delivery. The latter property also makes phage a popular choice for gene therapy target discovery through directed evolution. Although promising, phage-mediated gene therapy faces several outstanding challenges, the most notable being lower gene delivery efficiency compared to animal viruses, vector stability, and nondesirable immune stimulation. This review presents a critical review of promises and challenges of employing phage as gene delivery vehicles as well as an introduction to the concept of phage-based microbiome therapy as the new frontier and perhaps the most promising application of phage-based gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A comparative study of different strategies for removal of endotoxins from bacteriophage preparations.

PubMed

Van Belleghem, Jonas D; Merabishvili, Maya; Vergauwen, Bjorn; Lavigne, Rob; Vaneechoutte, Mario

2017-01-01

Bacterial endotoxins have high immunogenicity. Phage biology studies as well as therapeutic phage applications necessitate highly purified phage particles. In this study, we compared combinations of seven different endotoxin removal strategies and validated their endotoxin removal efficacy for five different phages (i.e. four Pseudomonas aeruginosa phages and one Staphylococcus aureus phage). These purification strategies included Endotrap HD column purification and/or CsCl density centrifugation in combination with Endotrap purification, followed by organic solvent (1-octanol), detergent (Triton X-100), enzymatic inactivation of the endotoxin using alkaline phosphatase and CIM monolytic anion exchange chromatography. We show that CsCl density purification of the P. aeruginosa phages, at an initial concentration of 10 12 -10 13 pfu/ml, led to the strongest reduction of endotoxins, with an endotoxin removal efficacy of up to 99%, whereas additional purification methods did not result in a complete removal of endotoxins from the phage preparations and only yielded an additional endotoxin removal efficacy of 23 to 99%, sometimes accompanied with strong losses in phage titer. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterizing Phage Genomes for Therapeutic Applications

PubMed Central

Philipson, Casandra W.; Voegtly, Logan J.; Lueder, Matthew R.; Long, Kyle A.; Rice, Gregory K.; Frey, Kenneth G.; Biswas, Biswajit; Cer, Regina Z.; Hamilton, Theron; Bishop-Lilly, Kimberly A.

2018-01-01

Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA) for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates. PMID:29642590

Transcription attenuation is the major mechanism by which the leu operon of Salmonella typhimurium is controlled.

PubMed

Searles, L L; Wessler, S R; Calvo, J M

1983-01-25

Three mutations, each causing constitutive expression of the Salmonella typhimurium leu operon, were cloned into phage vector lambda gt4 on EcoRI DNA fragments carrying all of that operon except for part of the promoter-distal last gene. Sequence analysis of DNA from these phage demonstrated that each contains a single base change in the leu attenuator. Transcription of mutant DNA in vitro resulted in transcription beyond the usual site of termination. The level of beta-IPM dehydrogenase, the leuB enzyme, was elevated 40-fold in a strain carrying one of these mutations, and starvation of this strain for leucine had little effect on the amount of activity expressed. Using a strain with a wild-type promoter-leader region of the leu operon, the rates of synthesis and degradation of leu leader RNA and readthrough RNA (leu mRNA) were measured by DNA-RNA hybridizations with specific DNA probes. The rate of synthesis of the leu leader was about the same in cells grown with excess or with limiting leucine. On the other hand, the rate of synthesis of leu mRNA was 12-fold higher for cells grown in limiting leucine as opposed to excess leucine. The rate of degradation of these RNA species was the same under both conditions of growth. Thus, the variation in expression of the leu operon observed for cells grown in minimal medium is, for the most part, not caused by control over the frequency of initiation or by the differential stability of these RNA species. Rather, the variation is a direct result of the frequency of transcription termination at an attenuator site. These results taken together suggest that transcription attenuation is the major mechanism by which leucine regulates expression of the leu operon of S. typhimurium for cells growing in a minimal medium.

Jovian Chromophore Characteristics from Multispectral HST Images

NASA Technical Reports Server (NTRS)

Strycker, Paul D.; Chanover, Nancy J.; Simon-Miller, Amy A.; Banfield, Don; Gierasch, Peter J.

2011-01-01

The chromophores responsible for coloring the jovian atmosphere are embedded within Jupiter's vertical aerosol structure. Sunlight propagates through this vertical distribution of aerosol particles, whose colors are defined by omega-bar (sub 0)(lambda), and we remotely observe the culmination of the radiative transfer as I/F(lambda). In this study, we employed a radiative transfer code to retrieve omega-bar (sub 0)(lambda) for particles in Jupiter's tropospheric haze at seven wavelengths in the near-UV and visible regimes. The data consisted of images of the 2008 passage of Oval BA to the south of the Great Red Spot obtained by the Wide Field Planetary Camera 2 on-board the Hubble Space Telescope. We present derived particle colors for locations that were selected from 14 weather regions, which spanned a large range of observed colors. All omega-bar (sub 0)(lambda) curves were absorbing in the blue, and omega-bar (sub 0)(lambda) increased monotonically to approximately unity as wavelength increased. We found accurate fits to all omega-bar (sub 0)(lambda) curves using an empirically derived functional form: omega-bar (sub 0)(lambda) = 1 A exp(-B lambda). The best-fit parameters for the mean omega-bar (sub 0)(lambda) curve were A = 25.4 and B = 0.0149 for lambda in units of nm. We performed a principal component analysis (PCA) on our omega-bar (sub 0)(lambda) results and found that one or two independent chromophores were sufficient to produce the variations in omega-bar (sub 0)(lambda). A PCA of I/F(lambda) for the same jovian locations resulted in principal components (PCs) with roughly the same variances as the omega-bar (sub 0)(lambda) PCA, but they did not result in a one-to-one mapping of PC amplitudes between the omega-bar (sub 0)(lambda) PCA and I/F(lambda) PCA. We suggest that statistical analyses performed on I/ F(lambda) image cubes have limited applicability to the characterization of chromophores in the jovian atmosphere due to the sensitivity of 1/ F(lambda) to horizontal variations in the vertical aerosol distribution.

Mechanisms of Hyperbilirubinemia During Peginterferon Lambda-1a Therapy for Chronic Hepatitis C Infection: A Retrospective Investigation.

PubMed

Zwirtes, Ricardo; Narasimhan, Premkumar; Wind-Rotolo, Megan M; Xu, Dong; Hruska, Matthew W; Kishnani, Narendra; Colston, Elizabeth M; Srinivasan, Subasree

2016-11-01

The phase 2b EMERGE study compared the efficacy/safety of peginterferon lambda-1a (Lambda) and peginterferon alfa-2a (Alfa), both with ribavirin (RBV), for treatment of chronic hepatitis C virus (HCV) infection. A key safety finding was a higher frequency of hyperbilirubinemia with Lambda/RBV versus Alfa/RBV. To characterize mechanisms of hyperbilirubinemia associated with Lambda/RBV, we conducted a retrospective analysis of safety data from the HCV genotype 1 and genotype 4 cohort of the EMERGE study. Subjects were randomized to once-weekly Lambda (120/180/240 μg) or Alfa (180 μg), with daily RBV, for 48 weeks. Early-onset Lambda/RBV-related hyperbilirubinemia events (6-12 weeks) resulted mostly from RBV-induced hemolysis evidenced by sustained reticulocytosis and a predominantly unconjugated pattern of hyperbilirubinemia. The higher hyperbilirubinemia frequency with Lambda/RBV versus Alfa/RBV was attributed to bone marrow suppression known to occur with Alfa but not Lambda. Late-onset (>12 weeks) Lambda/RBV-related hyperbilirubinemia events occurred most frequently with higher Lambda doses and were associated with increased levels of hepatic transaminase and direct bilirubin fractions compared with early events. This dual pattern of hyperbilirubinemia observed while on Lambda/RBV treatment is thought to be caused by exaggerated RBV-induced hemolysis in early-onset events compared with possible direct Lambda-induced hepatocellular toxicity in late-onset events.

Rearrangement and expression of the human {Psi}C{lambda}6 gene segment results in a surface Ig receptor with a truncated light chain constant region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stiernholm, N.B.J.; Verkoczy, L.K.; Berinstein, N.L.

1995-05-01

The constant region of the human Ig{lambda} locus consists of seven tandemly organized J-C gene segments. Although it has been established that the J-C{lambda}1, J-C{lambda}2, J-C{lambda}3, and J-C{lambda}7 gene segments are functional, and code for the four distinct Ig{lambda} isotypes found in human serum, the J-C{lambda}4, J-C{lambda}5, and J-C{lambda}6 gene segments are generally considered to be pseudogenes. Although one example of a functional J-C{lambda}6 gene segment has been documented, in the majority of cases, J-C{lambda}6 is rendered nonfunctional by virtue of a single duplication of four nucleotides, creating a premature translational arrest. We show here that rearrangements to the J-C{lambda}6more » gene segment do occur, and that such a rearrangement encodes an Ig{lambda} protein that lacks the terminal end of the constant region. We also show that this truncated protein is expressed on the surface with the IgH chain, creating an unusual surface Ig (sIg) receptor (sIg{triangle}CL). Cells that express this receptor on the surface do so at significantly reduced levels compared with clonally related variants, which express sIg receptors with conventional Ig{lambda} L chains. However, the effects of sIg cross-linking on tyrosine phosphorylation and surface expression of the CD25 and CD71 Ags are similar in cells that express conventional sIg receptors and in those that express sIg{triangle}CL receptors, suggesting that the latter could possibly function as an Ag receptor. 35 refs., 7 figs.« less

Existence and non-uniqueness of similarity solutions of a boundary-layer problem

NASA Technical Reports Server (NTRS)

Hussaini, M. Y.; Lakin, W. D.

1986-01-01

A Blasius boundary value problem with inhomogeneous lower boundary conditions f(0) = 0 and f'(0) = - lambda with lambda strictly positive was considered. The Crocco variable formulation of this problem has a key term which changes sign in the interval of interest. It is shown that solutions of the boundary value problem do not exist for values of lambda larger than a positive critical value lambda. The existence of solutions is proven for 0 lambda lambda by considering an equivalent initial value problem. It is found however that for 0 lambda lambda, solutions of the boundary value problem are nonunique. Physically, this nonuniqueness is related to multiple values of the skin friction.

Existence and non-uniqueness of similarity solutions of a boundary layer problem

NASA Technical Reports Server (NTRS)

Hussaini, M. Y.; Lakin, W. D.

1984-01-01

A Blasius boundary value problem with inhomogeneous lower boundary conditions f(0) = 0 and f'(0) = - lambda with lambda strictly positive was considered. The Crocco variable formulation of this problem has a key term which changes sign in the interval of interest. It is shown that solutions of the boundary value problem do not exist for values of lambda larger than a positive critical value lambda. The existence of solutions is proven for 0 lambda lambda by considering an equivalent initial value problem. It is found however that for 0 lambda lambda, solutions of the boundary value problem are nonunique. Physically, this nonuniqueness is related to multiple values of the skin friction.

Measurement of the Lambda b0 lifetime in Lambda b0-->J/psi Lambda 0 in pp collisions at square root s=1.96 TeV.

PubMed

Abulencia, A; Adelman, J; Affolder, T; Akimoto, T; Albrow, M G; Ambrose, D; Amerio, S; Amidei, D; Anastassov, A; Anikeev, K; Annovi, A; Antos, J; Aoki, M; Apollinari, G; Arguin, J-F; Arisawa, T; Artikov, A; Ashmanskas, W; Attal, A; Azfar, F; Azzi-Bacchetta, P; Azzurri, P; Bacchetta, N; Badgett, W; Barbaro-Galtieri, A; Barnes, V E; Barnett, B A; Baroiant, S; Bartsch, V; Bauer, G; Bedeschi, F; Behari, S; Belforte, S; Bellettini, G; Bellinger, J; Belloni, A; Benjamin, D; Beretvas, A; Beringer, J; Berry, T; Bhatti, A; Binkley, M; Bisello, D; Blair, R E; Blocker, C; Blumenfeld, B; Bocci, A; Bodek, A; Boisvert, V; Bolla, G; Bolshov, A; Bortoletto, D; Boudreau, J; Boveia, A; Brau, B; Brigliadori, L; Bromberg, C; Brubaker, E; Budagov, J; Budd, H S; Budd, S; Budroni, S; Burkett, K; Busetto, G; Bussey, P; Byrum, K L; Cabrera, S; Campanelli, M; Campbell, M; Canelli, F; Canepa, A; Carrillo, S; Carlsmith, D; Carosi, R; Carron, S; Casarsa, M; Castro, A; Catastini, P; Cauz, D; Cavalli-Sforza, M; Cerri, A; Cerrito, L; Chang, S H; Chen, Y C; Chertok, M; Chiarelli, G; Chlachidze, G; Chlebana, F; Cho, I; Cho, K; Chokheli, D; Chou, J P; Choudalakis, G; Chuang, S H; Chung, K; Chung, W H; Chung, Y S; Ciljak, M; Ciobanu, C I; Ciocci, M A; Clark, A; Clark, D; Coca, M; Compostella, G; Convery, M E; Conway, J; Cooper, B; Copic, K; Cordelli, M; Cortiana, G; Crescioli, F; Cuenca Almenar, C; Cuevas, J; Culbertson, R; Cully, J C; Cyr, D; DaRonco, S; D'Auria, S; Davies, T; D'Onofrio, M; Dagenhart, D; de Barbaro, P; De Cecco, S; Deisher, A; De Lentdecker, G; Dell'Orso, M; Delli Paoli, F; Demortier, L; Deng, J; Deninno, M; De Pedis, D; Derwent, P F; Di Giovanni, G P; Dionisi, C; Di Ruzza, B; Dittmann, J R; DiTuro, P; Dörr, C; Donati, S; Donega, M; Dong, P; Donini, J; Dorigo, T; Dube, S; Efron, J; Erbacher, R; Errede, D; Errede, S; Eusebi, R; Fang, H C; Farrington, S; Fedorko, I; Fedorko, W T; Feild, R G; Feindt, M; Fernandez, J P; Field, R; Flanagan, G; Foland, A; Forrester, S; Foster, G W; Franklin, M; Freeman, J C; Furic, I; Gallinaro, M; Galyardt, J; Garcia, J E; Garberson, F; Garfinkel, A F; Gay, C; Gerberich, H; Gerdes, D; Giagu, S; Giannetti, P; Gibson, A; Gibson, K; Gimmell, J L; Ginsburg, C; Giokaris, N; Giordani, M; Giromini, P; Giunta, M; Giurgiu, G; Glagolev, V; Glenzinski, D; Gold, M; Goldschmidt, N; Goldstein, J; Gomez, G; Gomez-Ceballos, G; Goncharov, M; González, O; Gorelov, I; Goshaw, A T; Goulianos, K; Gresele, A; Griffiths, M; Grinstein, S; Grosso-Pilcher, C; Group, R C; Grundler, U; Guimaraes da Costa, J; Gunay-Unalan, Z; Haber, C; Hahn, K; Hahn, S R; Halkiadakis, E; Hamilton, A; Han, B-Y; Han, J Y; Handler, R; Happacher, F; Hara, K; Hare, M; Harper, S; Harr, R F; Harris, R M; Hartz, M; Hatakeyama, K; Hauser, J; Heijboer, A; Heinemann, B; Heinrich, J; Henderson, C; Herndon, M; Heuser, J; Hidas, D; Hill, C S; Hirschbuehl, D; Hocker, A; Holloway, A; Hou, S; Houlden, M; Hsu, S-C; Huffman, B T; Hughes, R E; Husemann, U; Huston, J; Incandela, J; Introzzi, G; Iori, M; Ishizawa, Y; Ivanov, A; Iyutin, B; James, E; Jang, D; Jayatilaka, B; Jeans, D; Jensen, H; Jeon, E J; Jindariani, S; Jones, M; Joo, K K; Jun, S Y; Jung, J E; Junk, T R; Kamon, T; Karchin, P E; Kato, Y; Kemp, Y; Kephart, R; Kerzel, U; Khotilovich, V; Kilminster, B; Kim, D H; Kim, H S; Kim, J E; Kim, M J; Kim, S B; Kim, S H; Kim, Y K; Kimura, N; Kirsch, L; Klimenko, S; Klute, M; Knuteson, B; Ko, B R; Kondo, K; Kong, D J; Konigsberg, J; Korytov, A; Kotwal, A V; Kovalev, A; Kraan, A C; Kraus, J; Kravchenko, I; Kreps, M; Kroll, J; Krumnack, N; Kruse, M; Krutelyov, V; Kubo, T; Kuhlmann, S E; Kuhr, T; Kusakabe, Y; Kwang, S; Laasanen, A T; Labarga, L; Lai, S; Lami, S; Lammel, S; Lancaster, M; Lander, R L; Lannon, K; Lath, A; Latino, G; Lazzizzera, I; LeCompte, T; Lee, J; Lee, J; Lee, Y J; Lee, S W; Lefèvre, R; Leonardo, N; Leone, S; Levy, S; Lewis, J D; Lin, C; Lin, C S; Lindgren, M; Lipeles, E; Liss, T M; Lister, A; Litvintsev, D O; Liu, T; Lockyer, N S; Loginov, A; Loreti, M; Loverre, P; Lu, R-S; Lucchesi, D; Lujan, P; Lukens, P; Lungu, G; Lyons, L; Lys, J; Lysak, R; Lytken, E; Mack, P; MacQueen, D; Madrak, R; Maeshima, K; Makhoul, K; Maki, T; Maksimovic, P; Malde, S; Manca, G; Margaroli, F; Marginean, R; Marino, C; Marino, C P; Martin, A; Martin, M; Martin, V; Martínez, M; Maruyama, T; Mastrandrea, P; Masubuchi, T; Matsunaga, H; Mattson, M E; Mazini, R; Mazzanti, P; McFarland, K S; McIntyre, P; McNulty, R; Mehta, A; Mehtala, P; Menzemer, S; Menzione, A; Merkel, P; Mesropian, C; Messina, A; Miao, T; Miladinovic, N; Miles, J; Miller, R; Mills, C; Milnik, M; Mitra, A; Mitselmakher, G; Miyamoto, A; Moed, S; Moggi, N; Mohr, B; Moore, R; Morello, M; Movilla Fernandez, P; Mülmenstädt, J; Mukherjee, A; Muller, Th; Mumford, R; Murat, P; Nachtman, J; Nagano, A; Naganoma, J; Nahn, S; Nakano, I; Napier, A; Necula, V; Neu, C; Neubauer, M S; Nielsen, J; Nigmanov, T; Nodulman, L; Norniella, O; Nurse, E; Oh, S H; Oh, Y D; Oksuzian, I; Okusawa, T; Oldeman, R; Orava, R; Osterberg, K; Pagliarone, C; Palencia, E; Papadimitriou, V; Paramonov, A A; Parks, B; Pashapour, S; Patrick, J; Pauletta, G; Paulini, M; Paus, C; Pellett, D E; Penzo, A; Phillips, T J; Piacentino, G; Piedra, J; Pinera, L; Pitts, K; Plager, C; Pondrom, L; Portell, X; Poukhov, O; Pounder, N; Prokoshin, F; Pronko, A; Proudfoot, J; Ptochos, F; Punzi, G; Pursley, J; Rademacker, J; Rahaman, A; Ranjan, N; Rappoccio, S; Reisert, B; Rekovic, V; Renton, P; Rescigno, M; Richter, S; Rimondi, F; Ristori, L; Robson, A; Rodrigo, T; Rogers, E; Rolli, S; Roser, R; Rossi, M; Rossin, R; Ruiz, A; Russ, J; Rusu, V; Saarikko, H; Sabik, S; Safonov, A; Sakumoto, W K; Salamanna, G; Saltó, O; Saltzberg, D; Sánchez, C; Santi, L; Sarkar, S; Sartori, L; Sato, K; Savard, P; Savoy-Navarro, A; Scheidle, T; Schlabach, P; Schmidt, E E; Schmidt, M P; Schmitt, M; Schwarz, T; Scodellaro, L; Scott, A L; Scribano, A; Scuri, F; Sedov, A; Seidel, S; Seiya, Y; Semenov, A; Sexton-Kennedy, L; Sfyrla, A; Shapiro, M D; Shears, T; Shepard, P F; Sherman, D; Shimojima, M; Shochet, M; Shon, Y; Shreyber, I; Sidoti, A; Sinervo, P; Sisakyan, A; Sjolin, J; Slaughter, A J; Slaunwhite, J; Sliwa, K; Smith, J R; Snider, F D; Snihur, R; Soderberg, M; Soha, A; Somalwar, S; Sorin, V; Spalding, J; Spinella, F; Spreitzer, T; Squillacioti, P; Stanitzki, M; Staveris-Polykalas, A; St Denis, R; Stelzer, B; Stelzer-Chilton, O; Stentz, D; Strologas, J; Stuart, D; Suh, J S; Sukhanov, A; Sun, H; Suzuki, T; Taffard, A; Takashima, R; Takeuchi, Y; Takikawa, K; Tanaka, M; Tanaka, R; Tecchio, M; Teng, P K; Terashi, K; Tesarek, R J; Thom, J; Thompson, A S; Thomson, E; Tipton, P; Tiwari, V; Tkaczyk, S; Toback, D; Tokar, S; Tollefson, K; Tomura, T; Tonelli, D; Torre, S; Torretta, D; Tourneur, S; Trischuk, W; Tsuchiya, R; Tsuno, S; Turini, N; Ukegawa, F; Unverhau, T; Uozumi, S; Usynin, D; Vallecorsa, S; van Remortel, N; Varganov, A; Vataga, E; Vázquez, F; Velev, G; Veramendi, G; Veszpremi, V; Vidal, R; Vila, I; Vilar, R; Vine, T; Vollrath, I; Volobouev, I; Volpi, G; Würthwein, F; Wagner, P; Wagner, R G; Wagner, R L; Wagner, J; Wagner, W; Wallny, R; Wang, S M; Warburton, A; Waschke, S; Waters, D; Weinberger, M; Wester, W C; Whitehouse, B; Whiteson, D; Wicklund, A B; Wicklund, E; Williams, G; Williams, H H; Wilson, P; Winer, B L; Wittich, P; Wolbers, S; Wolfe, C; Wright, T; Wu, X; Wynne, S M; Yagil, A; Yamamoto, K; Yamaoka, J; Yamashita, T; Yang, C; Yang, U K; Yang, Y C; Yao, W M; Yeh, G P; Yoh, J; Yorita, K; Yoshida, T; Yu, G B; Yu, I; Yu, S S; Yun, J C; Zanello, L; Zanetti, A; Zaw, I; Zhang, X; Zhou, J; Zucchelli, S

2007-03-23

We report a measurement of the Lambda b0 lifetime in the exclusive decay Lambda b0-->J/psi Lambda 0 in pp collisions at square root s=1.96 TeV using an integrated luminosity of 1.0 fb-1 of data collected by the CDF II detector at the Fermilab Tevatron. Using fully reconstructed decays, we measure tau(Lambda b0)=1.593(-0.078)(+0.083)(stat)+/-0.033(syst) ps. This is the single most precise measurement of tau(Lambda b0) and is 3.2sigma higher than the current world average.

Cylinders vs. Spheres: Biofluid Shear Thinning in Driven Nanoparticle Transport

PubMed Central

Cribb, Jeremy A.; Meehan, Timothy D.; Shah, Sheel M.; Skinner, Kwan; Superfine, Richard

2011-01-01

Increasingly, the research community applies magnetophoresis to micro and nanoscale particles for drug delivery applications and the nanoscale rheological characterization of complex biological materials. Of particular interest is the design and transport of these magnetic particles through entangled polymeric fluids commonly found in biological systems. We report the magnetophoretic transport of spherical and rod-shaped particles through viscoelastic, entangled solutions using lambda-phage DNA (λ-DNA) as a model system. In order to understand and predict the observed phenomena, we fully characterize three fundamental components: the magnetic field and field gradient, the shape and magnetic properties of the probe particles, and the macroscopic rheology of the solution. Particle velocities obtained in Newtonian solutions correspond to macroscale rheology, with forces calculated via Stokes Law. In λ-DNA solutions, nanorod velocities are 100 times larger than predicted by measured zero-shear viscosity. These results are consistent with particles experiencing transport through a shear thinning fluid, indicating magnetically driven transport in shear thinning may be especially effective and favor narrow diameter, high aspect ratio particles. A complete framework for designing single-particle magnetic-based delivery systems results when we combine a quantified magnetic system with qualified particles embedded in a characterized viscoelastic medium. PMID:20571853

Megabase sequencing of human genome by ordered-shotgun-sequencing (OSS) strategy

NASA Astrophysics Data System (ADS)

Chen, Ellson Y.

1997-05-01

So far we have used OSS strategy to sequence over 2 megabases DNA in large-insert clones from regions of human X chromosomes with different characteristic levels of GC content. The method starts by randomly fragmenting a BAC, YAC or PAC to 8-12 kb pieces and subcloning those into lambda phage. Insert-ends of these clones are sequenced and overlapped to create a partial map. Complete sequencing is then done on a minimal tiling path of selected subclones, recursively focusing on those at the edges of contigs to facilitate mergers of clones across the entire target. To reduce manual labor, PCR processes have been adapted to prepare sequencing templates throughout the entire operation. The streamlined process can thus lend itself to further automation. The OSS approach is suitable for large- scale genomic sequencing, providing considerable flexibility in the choice of subclones or regions for more or less intensive sequencing. For example, subclones containing contaminating host cell DNA or cloning vector can be recognized and ignored with minimal sequencing effort; regions overlapping a neighboring clone already sequenced need not be redone; and segments containing tandem repeats or long repetitive sequences can be spotted early on and targeted for additional attention.

Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a. beta. -mannanase from the extremely thermophilic bacterium Caldocellum saccharolyticum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luethi, E.; Jasmat, N.B.; Grayling, R.A.

1991-03-01

A {lambda} recombinant phage expressing {beta}-mannanase activity in Escherichia coli has been isolated from a genomic library of the extremely thermophilic anaerobe Caldocellum saccharolyticum. The gene was cloned into pBR322 on a 5-kb BamHI fragment, and its location was obtained by deletion analysis. The sequence of a 2.1-kb fragment containing the mannanase gene has been determined. One open reading frame was found which could code for a protein of M{sub r} 38,904. The mannanase gene (manA) was overexpressed in E. coli by cloning the gene downstream from the lacZ promoter of pUC18. The enzyme was most active at pH 6more » and 80 C and degraded locust bean gum, guar gum, Pinus radiata glucomannan, and konjak glucomannan. The noncoding region downstream from the mannanase gene showed strong homology to celB, a gene coding for a cellulase from the same organism, suggesting that the manA gene might have been inserted into its present position on the C. saccharolyticum genome by homologous recombination.« less

Dual genetically encoded phage-displayed ligands.

PubMed

Mohan, Kritika; Weiss, Gregory A

2014-05-15

M13 bacteriophage display presents polypeptides as fusions to phage coat proteins. Such phage-displayed ligands offer useful reagents for biosensors. Here, we report a modified phage propagation protocol for the consistent and robust display of two different genetically encoded ligands on the major coat protein, P8. The results demonstrate that the phage surface reaches a saturation point for maximum peptide display. Copyright © 2014 Elsevier Inc. All rights reserved.

Improvement and Optimization of Two Engineered Phage Resistance Mechanisms in Lactococcus lactis

PubMed Central

McGrath, Stephen; Fitzgerald, Gerald F.; van Sinderen, Douwe

2001-01-01

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17. One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages. DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case. PMID:11157223

Modification of Escherichia coli–bacteriophage interactions by surfactants and antibiotics in vitro

PubMed Central

Scanlan, Pauline D.; Bischofberger, Anna M.

2017-01-01

Abstract Although experiments indicate that the abiotic environment plays an important role in bacterial interactions with their parasitic viruses (bacteriophages or phages), it is not yet clear how exposure to compounds present in nature alters the impact of phages on bacterial growth and evolution. To address this question, we exposed Escherichia coli K12 MG1655, in combination with three lytic phages, to various substances that natural and clinical microbial populations are likely to encounter: bile salts (present in mammalian gastrointestinal tracts), sodium dodecyl sulfate (SDS, a common surfactant in cleaning and hygiene products) and four antibiotics (present at variable concentrations in natural and clinical environments). Our results show that bile salts and SDS can reduce the detrimental effect of phages on bacterial growth. In some cases these compounds completely mitigated any negative effects of phages on bacterial growth and consequently bacteria did not evolve resistance to phages in these conditions. The proportional effects of phages were unaffected by antibiotics in most combinations, excepting three cases of phage-drug synergy. These results suggest that accounting for interactions between phages and environmental factors such as surfactants and antibiotics will improve understanding of both bacterial growth and resistance evolution to phages in vivo and in nature. PMID:27737900

The highly virulent 2006 Norwegian EHEC O103:H25 outbreak strain is related to the 2011 German O104:H4 outbreak strain.

PubMed

L'Abée-Lund, Trine M; Jørgensen, Hannah J; O'Sullivan, Kristin; Bohlin, Jon; Ligård, Goro; Granum, Per Einar; Lindbäck, Toril

2012-01-01

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx(2)-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages.

The Highly Virulent 2006 Norwegian EHEC O103:H25 Outbreak Strain Is Related to the 2011 German O104:H4 Outbreak Strain

PubMed Central

L'Abée-Lund, Trine M.; Jørgensen, Hannah J.; O'Sullivan, Kristin; Bohlin, Jon; Ligård, Goro; Granum, Per Einar; Lindbäck, Toril

2012-01-01

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx2-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages. PMID:22403614

Drug delivery vectors based on filamentous bacteriophages and phage-mimetic nanoparticles.

PubMed

Ju, Zhigang; Sun, Wei

2017-11-01

With the development of nanomedicine, a mass of nanocarriers have been exploited and utilized for targeted drug delivery, including liposomes, polymers, nanoparticles, viruses, and stem cells. Due to huge surface bearing capacity and flexible genetic engineering property, filamentous bacteriophage and phage-mimetic nanoparticles are attracting more and more attentions. As a rod-like bio-nanofiber without tropism to mammalian cells, filamentous phage can be easily loaded with drugs and directly delivered to the lesion location. In particular, chemical drugs can be conjugated on phage surface by chemical modification, and gene drugs can also be inserted into the genome of phage by recombinant DNA technology. Meanwhile, specific peptides/proteins displayed on the phage surface are able to conjugate with nanoparticles which will endow them specific-targeting and huge drug-loading capacity. Additionally, phage peptides/proteins can directly self-assemble into phage-mimetic nanoparticles which may be applied for self-navigating drug delivery nanovehicles. In this review, we summarize the production of phage particles, the identification of targeting peptides, and the recent applications of filamentous bacteriophages as well as their protein/peptide for targeting drug delivery in vitro and in vivo. The improvement of our understanding of filamentous bacteriophage and phage-mimetic nanoparticles will supply new tools for biotechnological approaches.

Novel approach of using a cocktail of designed bacteriophages against gut pathogenic E. coli for bacterial load biocontrol

PubMed Central

2014-01-01

Background This study was conducted to explore new approaches of animal biocontrol via biological control feed. Method White rats were subjected to 140 highly lytic designed phages specific against E. coli. Phages were fed via drinking water, oral injection, and vegetable capsules. Phage feeding was applied by 24 h feeding with 11d monitoring and 20d phage feeding and monitoring. Group of rats received external pathogenic E. coli and another group did not, namely groups A and B. Results Phage feeding for 20d via vegetable capsules yielded the highest reduction of fecal E. coli, 3.02 and 4.62 log, in rats group A and B respectively. Second best, feeding for 20d via drinking water with alkali yielded 2.78 and 4.08 log in rats groups A and B respectively. The peak reduction in E. coli output was 5–10 d after phage feeding. Phage control declined after 10th day of feeding. Conclusions The use of cocktail of designed phages succeeded in suppressing flora or external E. coli. The phage feed biocontrol is efficient in controlling E. coli at the pre-harvest period, precisely at the 6th-8th day of phage feeding when the lowest E. coli output found. PMID:25062829

Recent Trends in Salmonella Outbreaks and Emerging Technology for Biocontrol of Salmonella Using Phages in Foods: A Review.

PubMed

Oh, Jun-Hyun; Park, Mi-Kyung

2017-12-28

Salmonella is one of the principal causes of foodborne outbreaks. As traditional control methods have shown less efficacy against emerging Salmonella serotypes or antimicrobialresistant Salmonella , new approaches have been attempted. The use of lytic phages for the biocontrol of Salmonella in the food industry has become an attractive method owing to the many advantages offered by the use of phages as biocontrol agents. Phages are natural alternatives to traditional antimicrobial agents; they have proven effective in the control of bacterial pathogens in the food industry, which has led to the development of different phage products. The treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases, and ultimately promotes safe environments for animal and plant food production, processing, and handling. After an extensive investigation of the current literature, this review focuses predominantly on the efficacy of phages for the successful control of Salmonella spp. in foods. This review also addresses the current knowledge on the pathogenic characteristics of Salmonella , the prevalence of emerging Salmonella outbreaks, the isolation and characterization of Salmonella -specific phages, the effectiveness of Salmonella -specific phages as biocontrol agents, and the prospective use of Salmonella -specific phages in the food industry.

Differentiation of Salmonella enteritidis phage type 8 strains: evaluation of three additional phage typing systems, plasmid profiles, antibiotic susceptibility patterns, and biotyping.

PubMed Central

Stubbs, A D; Hickman-Brenner, F W; Cameron, D N; Farmer, J J

1994-01-01

Three additional phage typing systems for Salmonella enteritidis, plasmid analysis, biochemical tests, and antimicrobial susceptibility tests, were used in an attempt to subdivide 30 phage type 8 (phage typing system used by the WHO International Center for Enteric Phage Typing, London, England) isolates. These isolates represented 18 different egg-related outbreaks (21 strains) and 9 reference strains or strains that were not egg-associated. Only 7 of the 30 strains (28%) were subdivided by one or more of the methods used; this included 3 of the 21 strains from egg-related outbreaks. Twenty-seven strains contained a 55-kb plasmid that is associated with S. enteritidis. Of 65 additional phages tested, 2 from the phage typing system obtained from the Pasteur Institute, Paris, France, were useful in differentiating the three strains that lacked the 55-kb plasmid. Although the results obtained for the 21 strains from egg-related outbreaks showed that the strains had minor phenotypic differences, the overall results suggested that the strains may represent a single clone. Studies are planned to test additional phages and other typing methods to see whether strains of phage type 8 can be further differentiated. PMID:8126179

Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems: Variable phage-host infection interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holmfeldt, Karin; Solonenko, Natalie; Howard-Varona, Cristina

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. In this paper, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding andmore » modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.« less

Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems: Variable phage-host infection interactions

DOE PAGES

Holmfeldt, Karin; Solonenko, Natalie; Howard-Varona, Cristina; ...

2016-06-28

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. In this paper, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding andmore » modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.« less

Isolation and Characterization of Phages Infecting Bacillus subtilis

PubMed Central

Biegalska, Anna; Łoś, Marcin; Richert, Malwina

2015-01-01

Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages) or noncontractile (ARπ phage) tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0) and alkaline (9.0 and 10.0) pH. PMID:26273592

Advances in the T7 phage display system (Review).

PubMed

Deng, Xiangying; Wang, Li; You, Xiaolong; Dai, Pei; Zeng, Yanhua

2018-01-01

The present review describes the advantages and updated applications of the T7 phage display system in bioscience and medical science. Current phage display systems are based on various bacteriophage vectors, including M13, T7, T4 and f1. Of these, the M13 phage display is the most frequently used, however, the present review highlights the advantages of the T7 system. As a phage display platform, M13 contains single‑stranded DNA, while the T7 phage consists of double‑stranded DNA, which exhibits increased stability and is less prone to mutation during replication. Additional characteristics of the T7 phage include the following: The T7 phage does not depend on a protein secretion pathway in the lytic cycle; expressed peptides and proteins are usually located on the C‑terminal region of capsid protein gp10B, which avoids problems associated with steric hindrance; and T7 phage particles exhibit high stability under various extreme conditions, including high temperature and low pH, which facilitates effective high‑throughput affinity elutriation. Recent applications of the T7 phage display system have been instrumental in uncovering mechanisms of molecular interaction, particularly in the fields of antigen discovery, vaccine development, protein interaction, and cancer diagnosis and treatment.

Comparative analysis of multiple inducible phages from Mannheimia haemolytica.

PubMed

Niu, Yan D; Cook, Shaun R; Wang, Jiaying; Klima, Cassidy L; Hsu, Yu-hung; Kropinski, Andrew M; Turner, Dann; McAllister, Tim A

2015-08-30

Mannheimia haemolytica is a commensal bacterium that resides in the upper respiratory tract of cattle that can play a role in bovine respiratory disease. Prophages are common in the M. haemolytica genome and contribute significantly to host diversity. The objective of this research was to undertake comparative genomic analysis of phages induced from strains of M. haemolytica serotype A1 (535A and 2256A), A2 (587A and 1127A) and A6 (1152A and 3927A). Overall, four P2-like (535AP1, 587AP1, 1127AP1 and 2256AP1; genomes: 34.9-35.7 kb; G+C content: 41.5-42.1 %; genes: 51-53 coding sequences, CDSs), four λ-like (535AP2, 587AP2, 1152AP2 and 3927AP1; genomes: 48.6-52.1 kb; 41.1-41.4 % mol G+C; genes: 77-83 CDSs and 2 tRNAs) and one Mu-like (3927AP2; genome: 33.8 kb; 43.1 % mol G+C; encoding 50 CDSs) phages were identified. All P2-like phages are collinear with the temperate phage φMhaA1-PHL101 with 535AP1, 2256AP1 and 1152AP1 being most closely related, followed by 587AP1 and 1127AP1. Lambdoid phages are not collinear with any other known λ-type phages, with 587AP2 being distinct from 535AP2, 3927AP1 and 1152AP2. All λ-like phages contain genes encoding a toxin-antitoxin (TA) system and cell-associated haemolysin XhlA. The Mu-like phage induced from 3927A is closely related to the phage remnant φMhaMu2 from M. haemolytica PHL21, with similar Mu-like phages existing in the genomes of M. haemolytica 535A and 587A. This is among the first reports of both λ- and Mu-type phages being induced from M. haemolytica. Compared to phages induced from commensal strains of M. haemolytica serotype A2, those induced from the more virulent A1 and A6 serotypes are more closely related. Moreover, when P2-, λ- and Mu-like phages co-existed in the M. haemolytica genome, only P2- and λ-like phages were detected upon induction, suggesting that Mu-type phages may be more resistant to induction. Toxin-antitoxin gene cassettes in λ-like phages may contribute to their genomic persistence or the establishment of persister subpopulations of M. haemolytica. Further work is required to determine if the cell-associated haemolysin XhlA encoded by λ-like phages contributes to the pathogenicity and ecological fitness of M. haemolytica.

Observation of the baryonic B decay B{sup 0}{yields}{Lambda}{sub c}{sup +}{Lambda}K{sup -}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lees, J. P.; Poireau, V.; Tisserand, V.

2011-10-01

We report the observation of the baryonic B decay B{sup 0}{yields}{Lambda}{sub c}{sup +}{Lambda}K{sup -} with a significance larger than 7 standard deviations based on 471x10{sup 6} BB pairs collected with the BABAR detector at the PEP-II storage ring at SLAC. We measure the branching fraction for the decay B{sup 0}{yields}{Lambda}{sub c}{sup +}{Lambda}K{sup -} to be (3.8{+-}0.8{sub stat}{+-}0.2{sub sys}{+-}1.0{sub {Lambda}}{sub c}{sup +})x10{sup -5}. The uncertainties are statistical, systematic, and due to the uncertainty in the {Lambda}{sub c}{sup +} branching fraction. We find that the {Lambda}{sub c}{sup +}K{sup -} invariant-mass distribution shows an enhancement above 3.5 GeV/c{sup 2}.

Doublet-spacing enhancement caused by {Lambda}N-{Sigma}N coupling in {sub {Lambda}L}i hypernuclear isotopes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Umeya, Atsushi; Harada, Toru; Research Center for Physics and Mathematics, Osaka Electro-Communication University, Neyagawa, Osaka 572-8530

2011-03-15

We theoretically investigate energy spacings of doublets in {sub {Lambda}L}i hypernuclear isotopes with A=7-10 in shell-model calculations with a {Lambda}N-{Sigma}N coupling effect. The calculated results show that the energy shifts are {Delta}{epsilon}=0.09-0.28 MeV and the {Sigma}-mixing probabilities are P{sub {Sigma}}=0.10%-0.34% in {Lambda} ground states for the isotopes because of the {Lambda}N-{Sigma}N coupling in the first-order perturbation. It is found that the energy spacing of the doublet is enhanced as a neutron number N increases; the contribution of the {Lambda}N-{Sigma}N coupling interaction is comparable to that of the {Lambda}N interaction in the neutron-rich {Lambda} hypernuclei. The coherent mechanism of this doublet-spacingmore » enhancement is also discussed in terms of Fermi-type and Gamow-Teller-type {Lambda}N-{Sigma}N couplings.« less

INACTIVATION AND REACTIVATION OF B. MEGATHERIUM PHAGE

PubMed Central

Northrop, John H.

1955-01-01

Preparation of Reversibly Inactivated (R.I.) Phage.— If B. megatherium phage (of any type, or in any stage of purification) is suspended in dilute salt solutions at pH 5–6, it is completely inactivated; i.e., it does not form plaques, or give rise to more phage when mixed with a sensitive organism (Northrop, 1954). The inactivation occurs when the phage is added to the dilute salt solution. If a suspension of the inactive phage in pH 7 peptone is titrated to pH 5 and allowed to stand, the activity gradually returns. The inactivation is therefore reversible. Properties of R.I. Phage.— The R.I. phage is adsorbed by sensitive cells at about the same rate as the active phage. It kills the cells, but no active phage is produced. The R.I. phage therefore has the properties of phage "ghosts" (Herriott, 1951) or of colicines (Gratia, 1925), or phage inactivated by ultraviolet light (Luria, 1947). The R.I. phage is sedimented in the centrifuge at the same rate as active phage. It is therefore about the same size as the active phage. The R.I. phage is most stable in pH 7, 5 per cent peptone, and may be kept in this solution for weeks at 0°C. The rate of digestion of R.I. phage by trypsin, chymotrypsin, or desoxyribonuclease is about the same as that of active phage (Northrop, 1955 a). Effect of Various Substances on the Formation of R.I. Phage.— There is an equilibrium between R.I. phage and active phage. The R.I. form is the stable one in dilute salt solution, pH 5 to 6.5 and at low temperature (<20°C.). At pH >6.5, in dilute salt solution, the R.I. phage changes to the active form. The cycle, active ⇌ inactive phage, may be repeated many times at 0°C. by changing the pH of the solution back and forth between pH 7 and pH 6. Irreversible inactivation is caused by distilled water, some heavy metals, concentrated urea or quanidine solutions, and by l-arginine. Reversible inactivation is prevented by all salts tested (except those causing irreversible inactivation, above). The concentration required to prevent R.I. is lower, the higher the valency of either the anion or cation. There are great differences, however, between salts of the same valency, so that the chemical nature as well as the valency is important. Peptone, urea, and the amino acids, tryptophan, leucine, isoleucine, methionine, asparagine, dl-cystine, valine, and phenylalanine, stabilize the system at pH 7, so that no change occurs if a mixture of R.I. and active phage is added to such solutions. The active phage remains active and the R.I. phage remains inactive. The R.I. phage in pH 7 peptone becomes active if the pH is changed to 5.0. This does not occur in solutions of urea or the amino acids which stabilize at pH 7.0. Kinetics of Reversible Inactivation.— The inactivation is too rapid, even at 0° to allow the determination of an accurate time-inactivation curve. The rate is independent of the phage concentration and is complete in a few seconds, even in very dilute suspensions containing <1 x 104 particles/ml. This result rules out any type of bimolecular reaction, or any precipitation or agglutination mechanism, since the minimum theoretical time for precipitation (or agglutination) of a suspension of particles in a concentration of only 1 x 104 per ml. would be about 300 days even though every collision were effective. Mechanism of Salt Reactivation.— Addition of varying concentrations of MgSO4 (or many other salts) to a suspension of either active or R.I. phage in 0.01 M, pH 6 acetate buffer results in the establishment of an equilibrium ratio for active/R.I. phage. The higher the concentration of salt, the larger proportion of the phage is active. The results, with MgSO4, are in quantitative agreement with the following reaction: See PDF for Equation Effect of Temperature.— The rate of inactivation is too rapid to be measured with any accuracy, even at 0°C. The rate of reactivation in pH 5 peptone, at 0 and 10°, was measured and found to have a temperature coefficient Q 10 = 1.5 corresponding to a value of E (Arrhenius' constant) of 6500 cal. mole–1. This agrees very well with the temperature coefficient for the reactivation of denatured soy bean trypsin inhibitor (Kunitz, 1948). The equilibrium between R.I. and active phage is shifted toward the active side by lowering the temperature. The ratio R.I.P./AP is 4.7 at 15° and 2.8 at 2°. This corresponds to a change in free energy of –600 cal. mole–1 and a heat of reaction of 11,000. These values are much lower than the comparative one for trypsin (Anson and Mirsky, 1934 a) or soy bean trypsin inhibitor (Kunitz, 1948). Neither the inactivation nor the reactivation reactions are affected by light. The results in general indicate that there is an equilibrium between active and R.I. phage. The R.I. phage is probably an intermediate step in the formation of inactive phage. The equilibrium is shifted to the active side by lowering the temperature, adjusting the pH to 7–8 (except in the presence of high concentrations of peptone), raising the salt concentration, or increasing the valency of the ions present. The reaction may be represented by the following: See PDF for Equation The assumption that the active/R.I. phage equilibrium represents an example of native/denatured protein equilibrium predicts all the results qualitatively. Quantitatively, however, it fails to predict the relative rate of digestion of the two forms by trypsin or chymotrypsin, and also the effect of temperature on the equilibrium. PMID:13271723

Comparative Genomic and Morphological Analyses of Listeria Phages Isolated from Farm Environments

PubMed Central

Denes, Thomas; Ackermann, Hans-Wolfgang; Moreno Switt, Andrea I.; Wiedmann, Martin; den Bakker, Henk C.

2014-01-01

The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large-genome (∼135-kb) myoviruses belonging to the genus “Twort-like viruses,” three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (∼66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (∼56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes. PMID:24837381

Investigation of a Large Collection of Pseudomonas aeruginosa Bacteriophages Collected from a Single Environmental Source in Abidjan, Côte d’Ivoire

PubMed Central

Essoh, Christiane; Latino, Libera; Midoux, Cédric; Blouin, Yann; Loukou, Guillaume; Nguetta, Simon-Pierre A.; Lathro, Serge; Cablanmian, Arsher; Kouassi, Athanase K.; Vergnaud, Gilles; Pourcel, Christine

2015-01-01

Twenty two distinct bacteriophages were isolated from sewage water from five locations in the city of Abidjan, Côte d'Ivoire over a two-year period, using a collection of Pseudomonas aeruginosa strains with diverse genotypes. The phages were characterized by their virulence spectrum on a panel of selected P. aeruginosa strains from cystic fibrosis patients and by whole genome sequencing. Twelve virions representing the observed diversity were visualised by electron microscopy. The combined observations showed that 17 phages, distributed into seven genera, were virulent, and that five phages were related to temperate phages belonging to three genera. Some showed similarity with known phages only at the protein level. The vast majority of the genetic variations among virulent phages from the same genus resulted from seemingly non-random horizontal transfer events, inside a population of P. aeruginosa phages with limited diversity. This suggests the existence of a single environmental reservoir or ecotype in which continuous selection is taking place. In contrast, mostly point mutations were observed among phages potentially capable of lysogenisation. This is the first study of P. aeruginosa phage diversity in an African city and it shows that a large variety of phage species can be recovered in a limited geographical site at least when different bacterial strains are used. The relative temporal and spatial stability of the Abidjan phage population might reflect equilibrium in the microbial community from which they are released. PMID:26115051

Marine Phages As Tracers: Effects of Size, Morphology, and Physico-Chemical Surface Properties on Transport in a Porous Medium.

PubMed

Ghanem, Nawras; Kiesel, Bärbel; Kallies, René; Harms, Hauke; Chatzinotas, Antonis; Wick, Lukas Y

2016-12-06

Although several studies examined the transport of viruses in terrestrial systems only few studies exist on the use of marine phages (i.e., nonterrestrial viruses infecting marine host bacteria) as sensitively detectable microbial tracers for subsurface colloid transport and water flow. Here, we systematically quantified and compared for the first time the effects of size, morphology and physicochemical surface properties of six marine phages and two coliphages (MS2, T4) on transport in sand-filled percolated columns. Phage-sand interactions were described by colloidal filtration theory and the extended Derjaguin-Landau-Verwey-Overbeek approach (XDLVO), respectively. The phages belonged to different families and comprised four phages never used in transport studies (i.e., PSA-HM1, PSA-HP1, PSA-HS2, and H3/49). Phage transport was influenced by size, morphology and hydrophobicity in an approximate order of size > hydrophobicity ≥ morphology. Two phages PSA-HP1, PSA-HS2 (Podoviridae and Siphoviridae) exhibited similar mass recovery as commonly used coliphage MS2 and were 7-fold better transported than known marine phage vB_PSPS-H40/1. Differing properties of the marine phages may be used to trace transport of indigenous viruses, natural colloids or anthropogenic nanomaterials and, hence, contribute to better risk analysis. Our results underpin the potential role of marine phages as microbial tracer for transport of colloidal particles and water flow.

Investigation of a Large Collection of Pseudomonas aeruginosa Bacteriophages Collected from a Single Environmental Source in Abidjan, Côte d'Ivoire.

PubMed

Essoh, Christiane; Latino, Libera; Midoux, Cédric; Blouin, Yann; Loukou, Guillaume; Nguetta, Simon-Pierre A; Lathro, Serge; Cablanmian, Arsher; Kouassi, Athanase K; Vergnaud, Gilles; Pourcel, Christine

2015-01-01

Twenty two distinct bacteriophages were isolated from sewage water from five locations in the city of Abidjan, Côte d'Ivoire over a two-year period, using a collection of Pseudomonas aeruginosa strains with diverse genotypes. The phages were characterized by their virulence spectrum on a panel of selected P. aeruginosa strains from cystic fibrosis patients and by whole genome sequencing. Twelve virions representing the observed diversity were visualised by electron microscopy. The combined observations showed that 17 phages, distributed into seven genera, were virulent, and that five phages were related to temperate phages belonging to three genera. Some showed similarity with known phages only at the protein level. The vast majority of the genetic variations among virulent phages from the same genus resulted from seemingly non-random horizontal transfer events, inside a population of P. aeruginosa phages with limited diversity. This suggests the existence of a single environmental reservoir or ecotype in which continuous selection is taking place. In contrast, mostly point mutations were observed among phages potentially capable of lysogenisation. This is the first study of P. aeruginosa phage diversity in an African city and it shows that a large variety of phage species can be recovered in a limited geographical site at least when different bacterial strains are used. The relative temporal and spatial stability of the Abidjan phage population might reflect equilibrium in the microbial community from which they are released.

Mapping the Tail Fiber as the Receptor Binding Protein Responsible for Differential Host Specificity of Pseudomonas aeruginosa Bacteriophages PaP1 and JG004

PubMed Central

Le, Shuai; He, Xuesong; Tan, Yinling; Huang, Guangtao; Zhang, Lin; Lux, Renate; Shi, Wenyuan; Hu, Fuquan

2013-01-01

The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy. PMID:23874674

Lambda Red Mediated Gap Repair Utilizes a Novel Replicative Intermediate in Escherichia coli

PubMed Central

Reddy, Thimma R.; Fevat, Léna M. S.; Munson, Sarah E.; Stewart, A. Francis; Cowley, Shaun M.

2015-01-01

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination. PMID:25803509

Basics of Antibody Phage Display Technology.

PubMed

Ledsgaard, Line; Kilstrup, Mogens; Karatt-Vellatt, Aneesh; McCafferty, John; Laustsen, Andreas H

2018-06-09

Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins.

Genomic analysis of WCP30 Phage of Weissella cibaria for Dairy Fermented Foods.

PubMed

Lee, Young-Duck; Park, Jong-Hyun

2017-01-01

In this study, we report the morphogenetic analysis and genome sequence of a new WCP30 phage of Weissella cibaria , isolated from a fermented food. Based on its morphology, as observed by transmission electron microscopy, WCP30 phage belongs to the family Siphoviridae . Genomic analysis of WCP30 phage showed that it had a 33,697-bp double-stranded DNA genome with 41.2% G+C content. Bioinformatics analysis of the genome revealed 35 open reading frames. A BLASTN search showed that WCP30 phage had low sequence similarity compared to other phages infecting lactic acid bacteria. This is the first report of the morphological features and complete genome sequence of WCP30 phage, which may be useful for controlling the fermentation of dairy foods.

Molecular Characterization of Three Lactobacillus delbrueckii subsp. bulgaricus Phages

PubMed Central

Casey, Eoghan; Mahony, Jennifer; O'Connell-Motherway, Mary; Bottacini, Francesca; Cornelissen, Anneleen; Neve, Horst; Heller, Knut J.; Noben, Jean-Paul; Dal Bello, Fabio

2014-01-01

In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations. PMID:25002431

Phage display as a technology delivering on the promise of peptide drug discovery.

PubMed

Hamzeh-Mivehroud, Maryam; Alizadeh, Ali Akbar; Morris, Michael B; Church, W Bret; Dastmalchi, Siavoush

2013-12-01

Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries can be displayed on the phage surface. The phage library can be incubated with a target of interest and the phage which bind can be isolated and sequenced to reveal the displayed peptides' primary structure. In this review, we focus on the 'mechanics' of the phage display process, whilst highlighting many diverse and subtle ways it has been used to further the drug-development process, including the potential for the phage particle itself to be used as a drug carrier targeted to a particular pathogen or cell type in the body. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

PubMed Central

Vasala, A; Dupont, L; Baumann, M; Ritzenthaler, P; Alatossava, T

1993-01-01

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module. Images PMID:8497043

Genomic Characterization of a Novel Phage Found in Black Abalone (Haliotis cracherodii) Infected with Withering Syndrome

NASA Astrophysics Data System (ADS)

Closek, C. J.; Langevin, S.; Burge, C. A.; Crosson, L.; White, S.; Friedman, C. S.

2016-02-01

Withering syndrome (WS), caused by the bacterium Candidatus Xenohaliotis californiensis, a Rickettsia-like organism (RLO), infects many species of abalone. Black abalone (Haliotis cracherodii), one of two endangered species of abalone, has experienced high population losses along the California coast due to WS. Recently, we observed reduced pathogenicity and mortality events in RLO-infected abalone when a novel bacteriophage (phage) was also present. To better understand phage-bacterium dynamics and develop more informative diagnostic tools, we sequenced the genome of the novel phage associated with the RLO responsible for WS. Metagenomic sequencing libraries were prepared with extracted genomic DNA from two experimentally infected H. cracherodii and phage sequences were enriched using hydroxyapatite chromatography normalization. Normalized libraries were individually barcoded and sequenced with Illumina MiSeq. Raw sequence reads were processed using VIrominer and de novo assembly produced one single phage-like contig (35.7Kb) from the experimentally infected abalone. This highly divergent genome had closest homology with a virus associated with abalone shriveling syndrome (SS). Of the 34 predicted ORFs, overlapping homology with the SS virus ranged from 20-72%, demonstrating the phage sequenced is genetically distinct from any known phage. The phage-like sequences represented a significant portion of the total reads sequenced ( 2 million of the 12 million paired-end reads; 17%) and we obtained 94,000X coverage across the novel phage genome. Beyond characterization of this novel phage, which appears to reduce pathogenicity of the RLO, the genome enabled us to develop quantitative PCR and in situ hybridization assays as diagnostic tools. These tools allow us to detect and quantify this phage in the endangered H. cracherodii.

Efficacy of potential phage cocktails against Vibrio harveyi and closely related Vibrio species isolated from shrimp aquaculture environment in the south east coast of India.

PubMed

Stalin, Nattan; Srinivasan, Pappu

2017-08-01

A diverse set of novel phages infecting the marine pathogenic Vibrio harveyi was isolated from shrimp aquaculture environments in the south east coast of India. Based on initial screening, three phages with a broad host range revealed that the growth inhibition of phage is relatively specific to V. harveyi. They were also able to infect V. alginolyticus and V. parahemolyticus that belonged to the Harveyi clade species from shrimp pond and sea coast environment samples. However, the impact of these phages on their host bacterium are well understood; a one-step growth curve experiment and transmission electron microscope (TEM) revealed three phages grouped under the Myoviridae (VHM1 and VHM2); Siphoviridae (VHS1) family. These phages were further molecular characterized with respect to phage genomic DNA isolates. The randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) digestion with HindIII, and major structural proteins were distinguished by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that all the phage isolates were different, even when they came from the same source, giving an insight into the diversity of phages. Evaluation of microcosm studies of Penaeus monodon larvae infected with V. harveyi (105 CFU mL-1) showed that larvae survival after 96 h in the presence of phage treatment at 109 PFU mL-1 was enhanced when compared with the control. The resolution in over survival highly recommended that this study provides the phage-based therapy which could be an innovative and eco-friendly solution against Vibrio disease in shrimp aquaculture and in the natural environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Clostridium difficile phages: still difficult?

PubMed Central

Hargreaves, Katherine R.; Clokie, Martha R. J.

2014-01-01

Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however, developing suitable phages is challenging. In this review we summarize the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics. Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage–host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution. No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using “whole-phages” are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem-free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen. PMID:24808893

Production of inhalation phage powders using spray freeze drying and spray drying techniques for treatment of respiratory infections

PubMed Central

Leung, Sharon S.Y.; Parumasivam, Thaigarajan; Gao, Fiona G.; Carrigy, Nicholas B.; Vehring, Reinhard; Finlay, Warren H.; Morales, Sandra; Britton, Warwick J; Kutter, Elizabeth; Chan, Hak-Kim

2016-01-01

Purpose The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders. Method A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1 = 60:20:20 and F2 = 40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed. Results A significant titer loss (~ 2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1 = 13.1 ± 1.7 × 104 pfu and SD-F2 = 11.0 ± 1.4 × 104 pfu) than from their counterpart SFD formulations (SFD-F1 = 8.3 ± 1.8 × 104 pfu and SFD-F2 = 2.1 ± 0.3 × 104 pfu). Conclusion Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2. PMID:26928668

Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages

PubMed Central

Berg, Jordan A.; Merrill, Bryan D.; Crockett, Justin T.; Esplin, Kyle P.; Evans, Marlee R.; Heaton, Karli E.; Hilton, Jared A.; Hyde, Jonathan R.; McBride, Morgan S.; Schouten, Jordan T.; Simister, Austin R.; Thurgood, Trever L.; Ward, Andrew T.; Breakwell, Donald P.; Hope, Sandra; Grose, Julianne H.

2016-01-01

Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared. PMID:27304881

Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp.

PubMed

Colom, Joan; Cano-Sarabia, Mary; Otero, Jennifer; Cortés, Pilar; Maspoch, Daniel; Llagostera, Montserrat

2015-07-01

Bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A cocktail of in vitro efficient phages is not a guarantee for in vivo therapeutic results against avian colibacillosis.

PubMed

Tsonos, Jessica; Oosterik, Leon H; Tuntufye, Huruma N; Klumpp, Jochen; Butaye, Patrick; De Greve, Henri; Hernalsteens, Jean-Pierre; Lavigne, Rob; Goddeeris, Bruno M

2014-07-16

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, leading to important economic losses worldwide. To cure APEC-infected chickens, a cocktail of four different APEC-specific bacteriophages (phages) was composed and tested. Specific phages were selected from a collection of phages isolated in Belgium. The selection was based on their obligate lytic infection cycle, a broad host range, low cross-resistance and low frequency of development of resistant APEC mutants. Genome analysis of the phages indicated they were close relatives of T4 and N4, considered to be safe in vivo. Chickens were intratracheally infected with APEC strain CH2 (serogroup O78), causing a mortality of about 50% during the seven days following the infection. The phage cocktail was administered 2h after the infection, via three different ways: intratracheally, intra-esophageally or via the drinking water. Treated groups did not show a significant decrease in mortality, lesion scores or weight loss compared to untreated groups, although the APEC-specific phages could be re-isolated from the lung and heart of chickens that were euthanized. Moreover, the re-isolated bacteria from infected chickens had remained sensitive to the phage cocktail. Our results indicate that the efficiency of the phage cocktail used in treating CH2-infected chickens in vivo is negligible, even though in vitro, the phages in the cocktail were able to efficiently lyse the APEC strain CH2. Our results emphasize that the 'traditional' pathway of isolation, followed by phenotypical and genotypical characterization of phages composing the cocktail, does not lead to success in phage therapy in all cases. Copyright © 2013 Elsevier B.V. All rights reserved.

Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp.

PubMed Central

Colom, Joan; Cano-Sarabia, Mary; Otero, Jennifer; Cortés, Pilar

2015-01-01

Bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals. PMID:25956778

Review: elimination of bacteriophages in whey and whey products

PubMed Central

Atamer, Zeynep; Samtlebe, Meike; Neve, Horst; J. Heller, Knut; Hinrichs, Joerg

2013-01-01

As the cheese market faces strong international competition, the optimization of production processes becomes more important for the economic success of dairy companies. In dairy productions, whey from former cheese batches is frequently re-used to increase the yield, to improve the texture and to increase the nutrient value of the final product. Recycling of whey cream and particulated whey proteins is also routinely performed. Most bacteriophages, however, survive pasteurization and may re-enter the cheese manufacturing process. There is a risk that phages multiply to high numbers during the production. Contamination of whey samples with bacteriophages may cause problems in cheese factories because whey separation often leads to aerosol-borne phages and thus contamination of the factory environment. Furthermore, whey cream or whey proteins used for recycling into cheese matrices may contain thermo-resistant phages. Drained cheese whey can be contaminated with phages as high as 109 phages mL-1. When whey batches are concentrated, phage titers can increase significantly by a factor of 10 hindering a complete elimination of phages. To eliminate the risk of fermentation failure during recycling of whey, whey treatments assuring an efficient reduction of phages are indispensable. This review focuses on inactivation of phages in whey by thermal treatment, ultraviolet (UV) light irradiation, and membrane filtration. Inactivation by heat is the most common procedure. However, application of heat for inactivation of thermo-resistant phages in whey is restricted due to negative effects on the functional properties of native whey proteins. Therefore an alternative strategy applying combined treatments should be favored – rather than heating the dairy product at extreme temperature/time combinations. By using membrane filtration or UV treatment in combination with thermal treatment, phage numbers in whey can be reduced sufficiently to prevent subsequent phage accumulations. PMID:23882262

Characterization of Five Podoviridae Phages Infecting Citrobacter freundii

PubMed Central

Hamdi, Sana; Rousseau, Geneviève M.; Labrie, Simon J.; Kourda, Rim S.; Tremblay, Denise M.; Moineau, Sylvain; Slama, Karim B.

2016-01-01

Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus. PMID:27446058

Characterization and Genomic Study of Phage vB_EcoS-B2 Infecting Multidrug-Resistant Escherichia coli

PubMed Central

Xu, Yue; Yu, Xinyan; Gu, Yu; Huang, Xu; Liu, Genyan; Liu, Xiaoqiu

2018-01-01

The potential of bacteriophage as an alternative antibacterial agent has been reconsidered for control of pathogenic bacteria due to the widespread occurrence of multi-drug resistance bacteria. More and more lytic phages have been isolated recently. In the present study, we isolated a lytic phage named vB_EcoS-B2 from waste water. VB_EcoS-B2 has an icosahedral symmetry head and a long tail without a contractile sheath, indicating that it belongs to the family Siphoviridae. The complete genome of vB_EcoS-B2 is composed of a circular double stranded DNA of 44,283 bp in length, with 54.77% GC content. vB_EcoS-B2 is homologous to 14 relative phages (such as Escherichia phage SSL-2009a, Escherichia phage JL1, and Shigella phage EP23), but most of these phages exhibit different gene arrangement. Our results serve to extend our understanding toward phage evolution of family Siphoviridae of coliphages. Sixty-five putative open reading frames were predicted in the complete genome of vB_EcoS-B2. Twenty-one of proteins encoded by vB_EcoS-B2 were determined in phage particles by Mass Spectrometry. Bacteriophage genome and proteome analysis confirmed the lytic nature of vB_EcoS-B2, namely, the absence of toxin-coding genes, islands of pathogenicity, or genes through lysogeny or transduction. Furthermore, vB_EcoS-B2 significantly reduced the growth of E. coli MG1655 and also inhibited the growth of several multi-drug resistant clinical stains of E. coli. Phage vB_EcoS-B2 can kill some of the MRD E. coli entirely, strongly indicating us that it could be one of the components of phage cocktails to treat multi-drug resistant E. coli. This phage could be used to interrupt or reduce the spread of multi-drug resistant E. coli. PMID:29780362

H-NS Mutation-Mediated CRISPR-Cas Activation Inhibits Phage Release and Toxin Production of Escherichia coli Stx2 Phage Lysogen.

PubMed

Fu, Qiang; Li, Shiyu; Wang, Zhaofei; Shan, Wenya; Ma, Jingjiao; Cheng, Yuqiang; Wang, Hengan; Yan, Yaxian; Sun, Jianhe

2017-01-01

Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an Escherichia coli ( E. coli ) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune system has on Stx phage lysogen. We investigated heat-stable nucleoid-structuring (H-NS) mutation-mediated CRISPR-Cas activation and its effect on E. coli Stx2 phage lysogen. The Δ hns mutant (MG1655Δ hns ) of the E. coli K-12 strain MG1655 was obtained. The Δ hns mutant lysogen that was generated after Stx phage lysogenic infection had a repressed growth status and showed subdued group behavior, including biofilm formation and swarming motility, in comparison to the wild-type strain. The de-repression effect of the H-NS mutation on CRISPR-Cas activity was then verified. The results showed that cas gene expression was upregulated and the transformation efficiency of the wild-type CRISPR plasmids was decreased, which may indicate activation of the CRISPR-Cas system. Furthermore, the function of CRISPR-Cas on Stx2 phage lysogen was investigated by activating the CRISPR-Cas system, which contains an insertion of the protospacer regions of the Stx2 phage Min27. The phage release and toxin production of four lysogens harboring the engineered CRISPRs were investigated. Notably, in the supernatant of the Δ hns mutant lysogen harboring the Min27 spacer, both the progeny phage release and the toxin production were inhibited after mitomycin C induction. These observations demonstrate that the H-NS mutation-activated CRISPR-Cas system plays a role in modifying the effects of the Stx2 phage lysogen. Our findings indicated that H-NS mutation-mediated CRISPR-Cas activation in E. coli protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.

Liquid based formulations of bacteriophages for the management of waterborne bacterial pathogens in water microcosms.

PubMed

Ahiwale, Sangeeta; Tagunde, Sujata; Khopkar, Sushama; Karni, Mrudula; Gajbhiye, Milind; Kapadnis, Balasaheb

2013-11-01

Water resources are contaminated by life-threatening multidrug resistant pathogenic bacteria. Unfortunately, these pathogenic bacteria do not respond to the traditional water purification methods. Therefore, there is a need of environmentally friendly strategies to overcome the problems associated with the antimicrobial resistant bacterial pathogens. In the present study, highly potent lytic phages against multidrug-resistant Salmonella enterica serovar Paratyphi B, Pseudomonas aeruginosa and Klebsiella pneumoniae were isolated from the Pavana river water. They belonged to the Podoviridae and Siphoviridae families. These phages were purified and enriched in the laboratory. Monovalent formulations of phiSPB, BVPaP-3 and KPP phages were prepared in three different liquids viz., phage broth, saline and distilled water. The phages were stable for almost 8-10 months in the phage broth at 4 degrees C. The stability of the phages in saline and distilled water was 5-6 months at 4 degrees C. All of the phages were stable only for 4-6 months in the phage broth at 30 degrees C. The monovalent phage formulation of psiSPB was applied at MOI < 1, as disinfectant against an exponential and stationary phase cells of Salmonella enterica serovar Paratyphi B in various water microcosms. The results indicated that there was almost 80 % reduction in the log phase cells of Salmonella serovar Paratyphi B in 24 h. In stationary phase cells, the reduction was comparatively less within same period. At the same time, there was concomitant increase in the phage population by 80% in all the microcosms indicating that psiSPB phage is highly potent in killing pathogen in water. Results strongly support that the formulation of psiSPB in the phage broth in monovalent form could be used as an effective biological disinfectant for preventing transmission of water-borne bacterial pathogens, including antimicrobial resistant ones.

Biological responses to M13 bacteriophage modified titanium surfaces in vitro.

PubMed

Sun, Yuhua; Li, Yiting; Wu, Baohua; Wang, Jianxin; Lu, Xiong; Qu, Shuxin; Weng, Jie; Feng, Bo

2017-08-01

Phage-based materials have showed great potential in tissue engineering application. However, it is unknown what inflammation response will happen to this kind of materials. This work is to explore the biological responses to M13 bacteriophage (phage) modified titanium surfaces in vitro from the aspects of their interaction with macrophages, osteoblasts and mineralization behavior. Pretreated Ti surface, Ti surfaces with noncrosslinked phage film (APP) and crosslinked phage film (APPG) were compared. Phage films could limit the macrophage adhesion and activity due to inducing adherent-cell apoptosis. The initial inflammatory activity (24h) caused by phage films was relatively high with more production of TNF-α, but in the later stage (7-10days) inflammatory response was reduced with lower TNF-α, IL-6 and higher IL-10. In addition, phage films improved osteoblast adhesion, differentiation, and hydroapatite (HA)-forming via a combination of topographical and biochemcial cues. The noncrosslinked phage film displayed the best immunomodulatory property, osteogenic activity and HA mineralization ability. This work provides better understanding of inflammatory and osteogenetic activity of phage-based materials and contributes to their future application in tissue engineering. In vivo, the bone and immune cells share a common microenvironment, and are being affected by similar cytokines, signaling molecules, transcription factors and membrane receptors. Ideal implants should cause positive biological response, including adequate and appropriate inflammatory reaction, well-balanced bone formation and absorption. Phage-based materials have showed great potential in tissue engineering application. However, at present it is unknown what inflammation response will happen to this kind of materials. A good understanding of the immune response possibly induced by phage-based materials is needed. This work studied the osteoimmunomodulation property of phage films on titanium surface, involving inflammatory response, osteogenic activity and biomineralization ability. It provides more understanding of the phage-based materials and contributes to their future application in tissue engineering. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Exploring Viral Mediated Carbon Cycling in Thawing Permafrost Microbial Communities

NASA Astrophysics Data System (ADS)

Trubl, G. G.; Solonenko, N.; Moreno, M.; Sullivan, M. B.; Rich, V. I.

2014-12-01

Viruses are the most abundant biological entities on Earth and their impact on carbon cycling in permafrost habitats is poorly understood. Arctic C cycling is particularly important to interpret due to the rapid climate change occurring and the large amount of C stockpiled there (~1/3 of global soil C is stored in permafrost). Viruses of microbes (i.e. phages) play central roles in C cycling in the oceans, through cellular lysis (phage drive the largest ocean C flux about 150 Gt yr-1, dwarfing all others by >5-fold), production of associated DOC, as well as transport and expression during infection (1029 transduction events day-1). C cycling in thawing permafrost systems is critical in understanding the climate trajectory and phages may be as important for C cycling here as they are in the ocean. The thawed C may become a food source for microbes, producing CO2 and potentially CH4, both potent greenhouse gases. To address the potential role of phage in C cycling in these dynamic systems, we are examining phage from an arctic permafrost thaw gradient in northern Sweden. We have developed a protocol for successfully extracting phage from peat soils and are quantifying phage in 15 peat and 2 lake sediment cores, with the goal of sequencing viromes. Preliminary data suggest that phage are present at 109 g-1 across the permafrost thaw gradient (compared to the typical marine count ~105 ml-1), implying a potentially robust phage-host interaction web in these changing environments. We are examining phage from 11 depth intervals (covering the active and permafrost layer) in the cores to assess phage-host community dynamics. Phage morphology and abundance for each layer and environment are being determined using qTEM and EFM. Understanding the phage that infect bacteria and archaea in these rapidly changing habitats will provide insight into the controls on current and future CH4 and CO2 emissions in permafrost habitats.

Galaxy formation in Lambda greater than 0 Friedmann models: Consequences for the number counts versus redshift test

NASA Technical Reports Server (NTRS)

Martel, Hugo

1994-01-01

We study the effect of the cosmological constant Lambda on galaxy formation using a simple spherical top-hat overdensity model. We consider models with Omega(sub 0) = 0.2, lambda(sub 0) = 0, and Omega(sub 0) = 0.2, lambda(sub 0) = 0.8 (where Omega(sub 0) is the density parameter, and lambda(sub 0) identically equal Lambda/3 H(sub 0 exp 2) where H(sub 0) is the Hubble constant). We adjust the initial power spectrum amplitude so that both models reproduce the same large-scale structures. The galaxy formation era in the lambda(sub 0) = 0 model occurs early (z approximately 6) and is very short, whereas in the lambda(sub 0) = 0.8 model the galaxy formation era starts later (z approximately 4), and last much longer, possibly all the way to the present. Consequently, galaxies at low redshift (z less than 1) are significantly more evolved in the lambda(sub 0) = 0 model than in the lambda(sub 0) = 0.8 model. This result implies that previous attempts to determine Lambda using the number counts versus redshift test are probably unreliable.

Coevolution of CRISPR bacteria and phage in 2 dimensions

NASA Astrophysics Data System (ADS)

Han, Pu; Deem, Michael

2014-03-01

CRISPR (cluster regularly interspaced short palindromic repeats) is a newly discovered adaptive, heritable immune system of prokaryotes. It can prevent infection of prokaryotes by phage. Most bacteria and almost all archae have CRISPR. The CRISPR system incorporates short nucleotide sequences from viruses. These incorporated sequences provide a historical record of the host and predator coevolution. We simulate the coevolution of bacteria and phage in 2 dimensions. Each phage has multiple proto-spacers that the bacteria can incorporate. Each bacterium can store multiple spacers in its CRISPR. Phages can escape recognition by the CRISPR system via point mutation or recombination. We will discuss the different evolutionary consequences of point mutation or recombination on the coevolution of bacteria and phage. We will also discuss an intriguing ``dynamic phase transition'' in the number of phage as a function of time and mutation rate. We will show that due to the arm race between phages and bacteria, the frequency of spacers and proto-spacers in a population can oscillate quite rapidly.

ORF phage display to identify cellular proteins with different functions.

PubMed

Li, Wei

2012-09-01

Open reading frame (ORF) phage display is a new branch of phage display aimed at improving its efficiency to identify cellular proteins with specific binding or functional activities. Despite the success of phage display with antibody libraries and random peptide libraries, phage display with cDNA libraries of cellular proteins identifies a high percentage of non-ORF clones encoding unnatural short peptides with minimal biological implications. This is mainly because of the uncontrollable reading frames of cellular proteins in conventional cDNA libraries. ORF phage display solves this problem by eliminating non-ORF clones to generate ORF cDNA libraries. Here I summarize the procedures of ORF phage display, discuss the factors influencing its efficiency, present examples of its versatile applications, and highlight evidence of its capability of identifying biologically relevant cellular proteins. ORF phage display coupled with different selection strategies is capable of delineating diverse functions of cellular proteins with unique advantages. Copyright © 2012 Elsevier Inc. All rights reserved.

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Targeting mammalian organelles with internalizing phage (iPhage) libraries

PubMed Central

Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

2015-01-01

Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

Phages of life - the path to pharma.

PubMed

Forde, Amanda; Hill, Colin

2018-02-01

Bacteriophage (phage) therapy has encountered both enthusiasm and scepticism in the past century. New antimicrobial strategies against lethal pathogens are now a top priority for the World Health Organization, and although compassionate use of phages recently met with significant success, regulated clinical interventions seem unlikely in the near future. The hundredth anniversary of their discovery seems an appropriate time for a revival of phage therapy, particularly as the dilemma of antibiotic resistance grows. Phages are ubiquitous in the environment, on our food and in and on our bodies. Their influence on human health is currently being evaluated, and in this mini-review, we examine data from recent metagenomic studies that propose a role for phages in the structure of the microbiome and in health and disease. We assess evidence for phages as vehicles for gene transfer in the context of antibiotic resistance and discuss challenges and opportunities along the critical path from phage discovery to a patient-focused pharmaceutical intervention. © 2017 The British Pharmacological Society.

Removal of bacteriophages with different surface charges by diverse ceramic membrane materials in pilot spiking tests.

PubMed

Hambsch, B; Bösl, M; Eberhagen, I; Müller, U

2012-01-01

This study examines mechanisms for removal of bacteriophages (MS2 and phiX174) by ceramic membranes without application of flocculants. The ceramic membranes considered included ultra- and microfiltration membranes of different materials. Phages were spiked into the feed water in pilot scale tests in a waterworks. The membranes with pore sizes of 10 nm provided a 2.5-4.0 log removal of the phages. For pore sizes of 50 nm, the log removal dropped to 0.96-1.8. The membrane with a pore size of 200 nm did not remove phages. So, the removal of both MS2- and phiX174-phages depended on the pore size of the membranes. But apart from pore size also other factors influence the removal of phages. Removal was 0.5-0.9 log higher for MS2-phages compared with phiX174-phages. Size exclusion seems to be the major but not the only mechanism which influences the efficiency of phage removal by ceramic membranes.

Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector.

PubMed

Xu, Hai; Bao, Xi; Wang, Yiwei; Xu, Yue; Deng, Bihua; Lu, Yu; Hou, Jibo

2018-03-20

DNA delivery with bacteriophage by surface-displayed mammalian cell penetrating peptides has been reported. Although, various phages have been used to facilitate DNA transfer by surface displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide), no similar study has been conducted using T7 phage. In this study, we engineeredT7 phage as a DNA targeting delivery vector to facilitate cellular internalization. We constructed recombinant T7 phages that displayed Tat peptide on their surface and carried eukaryotic expression box (EEB) as a part of their genomes (T7-EEB-Tat). We demonstrated that T7 phage harboring foreign gene insertion had packaged into infective progeny phage particles. Moreover, when mammalian cells that were briefly exposed to T7-EEB-Tat, expressed a significant higher level of the marker gene with the control cells infected with the wide type phage without displaying Tat peptides. These data suggested that the potential of T7 phage as an effective delivery vector for DNA vaccine transfer.

Phage Types and Genotypes of Shiga Toxin-Producing Escherichia coli O157:H7 Isolates from Humans and Animals in Spain: Identification and Characterization of Two Predominating Phage Types (PT2 and PT8)

PubMed Central

Mora, Azucena; Blanco, Miguel; Blanco, Jesús E.; Alonso, M. Pilar; Dhabi, Ghizlane; Thomson-Carter, Fiona; Usera, Miguel A.; Bartolomé, Rosa; Prats, Guillermo; Blanco, Jorge

2004-01-01

Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused. PMID:15364983

Competitive and noncompetitive phage immunoassays for the determination of benzothiostrobin.

PubMed

Hua, Xiude; Zhou, Liangliang; Feng, Lu; Ding, Yuan; Shi, Haiyan; Wang, Limin; Gee, Shirley J; Hammock, Bruce D; Wang, Minghua

2015-08-26

Twenty-three phage-displayed peptides that specifically bind to an anti-benzothiostrobin monoclonal antibody (mAb) in the absence or presence of benzothiostrobin were isolated from a cyclic 8-residue peptide phage library. Competitive and noncompetitive phage enzyme linked immunosorbent assays (ELISAs) for benzothiostrobin were developed by using a clone C3-3 specific to the benzothiostrobin-free mAb and a clone N6-18 specific to the benzothiostrobin immunocomplex, respectively. Under the optimal conditions, the half maximal inhibition concentration (IC50) of the competitive phage ELISA and the concentration of analyte producing 50% saturation of the signal (SC50) of the noncompetitive phage ELISA for benzothiostrobin were 0.94 and 2.27 ng mL(-1), respectively. The noncompetitive phage ELISA showed higher selectivity compared to the competitive. Recoveries of the competitive and the noncompetitive phage ELISAs for benzothiostrobin in cucumber, tomato, pear and rice samples were 67.6-119.6% and 70.4-125.0%, respectively. The amounts of benzothiostrobin in the containing incurred residues samples detected by the two types of phage ELISAs were significantly correlated with that detected by high-performance liquid chromatography (HPLC). Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of phage assay for rapid phenotypic detection of rifampicin resistance in Mycobacterium tuberculosis

PubMed Central

Yzquierdo, Sergio Luis; Lemus, Dihadenys; Echemendia, Miguel; Montoro, Ernesto; McNerney, Ruth; Martin, Anandi; Palomino, Juan Carlos

2006-01-01

Background Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. Methods Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. Results Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively. Conclusion Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem. PMID:16630356

Efficient genome engineering of a virulent Klebsiella bacteriophage using CRISPR-Cas9.

PubMed

Shen, Juntao; Zhou, Jinjie; Chen, Guo-Qiang; Xiu, Zhi-Long

2018-06-13

Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens usually with multiple drug-resistance. Phage therapy, a potential new therapeutics to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well-characterized as the lack of efficient genome editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage phiKpS2. We firstly evaluated the distribution of sgRNA activity in phages and proved that it's largely inconsistent with the predicted activity from current models trained on eukaryotic cell datasets. A simple CRISPR-based phage genome editing procedure was developed based on the discovery that homologous arms as short as 30-60 bp was sufficient to introduce point mutation, gene deletion and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the above strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy. IMPORTANCE In the present study, we have addressed an efficient, time-saving and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote the applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proved enough to introduce point mutation, small frameshift deletion, gene deletion and swap into phages, and weak sgRNAs were proved useful for precise phage genome editing but failed to select random recombinants, which all make the CRISPR-based phage genome editing easier to use. Copyright © 2018 American Society for Microbiology.

Complete Genome Sequences of Four Novel Escherichia coli Bacteriophages Belonging to New Phage Groups

PubMed Central

Kot, Witold

2015-01-01

Here, we describe the sequencing and genome annotations of a set of four Escherichia coli bacteriophages (phages) belonging to newly discovered groups previously consisting of only a single phage and thus expand our knowledge of these phage groups. PMID:26184932

Complete genome sequences of three Campylobacter jejuni phage-propagating strains

USDA-ARS?s Scientific Manuscript database

Bacteriophage therapy has the potential to reduce Campylobacter jejuni numbers in livestock, but requires a detailed understanding of phage-host interactions. Some C. jejuni strains are readily infected by certain phages, and are thus designated as phage-propagating strains. Here we report the compl...

[Screening serum response special antibodies of U251 cell line from surface display phage antibody library].

PubMed

Yu, Min; Tan, De-Yong; Qian, Wei; Lai, Jian-Hua; Sun, Gui-Lin

2004-05-01

U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong response in serum recovered cells. The antibody No.2 had the distinctive response to the serum recovered cells in different incubation time (15min, 30min, 1h, 2h, 4h, 8h, 12h and 48h) after serum starvation. The results showed that No.2 antibody would be useful to research the factors of cell cycle regulation and apply to tumor diagnosis.

Characterization and complete genome sequence analysis of a novel virulent Siphoviridae phage against Staphylococcus aureus isolated from bovine mastitis in Xinjiang, China.

PubMed

Zhang, Qian; Xing, Shaozhen; Sun, Qiang; Pei, Guangqian; Cheng, Shi; Liu, Yannan; An, Xiaoping; Zhang, Xianglilan; Qu, Yonggang; Tong, Yigang

2017-06-01

Bovine mastitis is one of the most costly diseases in dairy cows worldwide. It can be caused by over 150 different microorganisms, where Staphylococcus aureus is the most frequently isolated and a major pathogen responsible for heavy economic losses in dairy industry. Although antibiotic therapy is most widely used, alternative treatments are necessary due to the increasing antibiotic resistance. Using phage for pathogen control is a promising tool in the fight against antibiotic resistance. Mainly using high-throughput sequencing, bioinformatics and our proposed phage termini identification method, we have isolated and characterized a novel virulent phage, designated as vB_SauS_IMEP5, from manure collected from dairy farms in Shihezi, Xinjiang, China, for use as a biocontrol agent against Staphylococcus aureus infections. Its latent period was about 30 min and its burst size was approximately 272PFU/cell. Phage vB_SauS_IMEP5 survives in a wide pH range between 3 and 12. A treatment at 70 °C for 20 min can inactive the phage. Morphological analysis of vB_SauS_IMEP5 revealed that phage vB_SauS_IMEP5 morphologically resembles phages in the family Siphoviridae. Among our tested multiplicity of infections (MOIs), the optimal multiplicity of infection (MOI) of this phage was determined to be 0.001, suggesting that phage vB_SauS_IMEP5 has high bacteriolytic potential and good efficiency for reducing bacterial growth. The complete genome of IME-P5 is a 44,677-bp, linear, double-stranded DNA, with a G+C content of 34.26%, containing 69 putative ORFs. The termini of genome were determined with next-generation sequencing data using our previously proposed termini identification method, which suggests that this phage has non-redundant termini with 9nt 3' protruding cohesive ends. The genomic and proteomic characteristics of IMEP5 demonstrate that this phage does not belong to any of the previously recognized Siphoviridae Staphylococcus phage groups, suggesting the creation of a new lineage, thus adding to the knowledge on the diversity of Staphylococcus phages. An N-acetylmuramoyl-L-alanine amidase gene and several conserved genes were predicted, while no virulence or antibiotic resistance genes were identified. This study isolated and characterized a novel S. aureus phage vB_SauS_IMEP5, and our findings suggest that this phage may be potentially utilized as a therapeutic or prophylactic candidate against S.aureus infections.

Development of in-vitro models to elucidate mechanisms of intrinsic cellular and tissue fluorescence

NASA Astrophysics Data System (ADS)

Savage, Howard E.; Kolli, Venkateswara; Saha, Sanjoy; Zhang, Jian C.; Glasgold, Mark; Sacks, Peter G.; Alfano, Robert R.; Schantz, Stimson P.

1995-04-01

In vitro cell model systems have been used to study the mechanisms of intrinsic cellular and tissue fluorescence as a potential biomarker for cancer. Phenotypic characteristics of cancer that are different from normal tissue include changes in histoarchitecture, proliferation rates and differentiation. a nitrosmethlybenzylamine (NMBA)/rat esophageal carcinogenesis model (NMBA), a transforming growth factor beta (TGF- (beta) )/normal epithelial cell model, and a retinoic acid (RA)/multicellular tumor spheroid model (RAMTS) were used to assess fluorescence changes associated respectively with changes in histoarchitecture, proliferation rates and differentiation. A xenon based fluorescence spectrophotometer (Mediscience Corp.) was used to collect excitation and emission spectra. Two excitation scans ((lambda) Ex 200-360 nm, (lambda) Em 380 nm; (lambda) Ex 240-430 nm, (lambda) Em 450 nm) and two emission scans ((lambda) Ex 300 nm, (lambda) Em 320-580 nm; (lambda) Ex 340 nm, (lambda) Em 360-660 nm) were used to analyze the three model systems. Using the NMBA model. Differences were seen in the excitation scan ((lambda) Ex 200-360 nm, (lambda) Em 380 nm) and the emission scan ((lambda) Ex 340 nm, (lambda) Em 360-660 nm) when normal rat esophageal tissue was compared to hyperplastic and tumor tissue. In the (TGF-(beta) ) model, differences were seen in the excitation scan ((lambda) Ex 240-430 nm, (lambda) Em 450 nm) when comparing proliferation slowed (TGF-(beta) treated) epithelial cells to their untreated controls. In the RAMTS model, differences were seen with all four scans when RA treated multicellular tumor spheroids (nondifferentiating) were compared to untreated control cells (differentiating). The data indicate that fluorescence changes seen in these model systems may relate to changes in histoarchitecture, proliferation rates and differentiation. Their relationship to in vivo fluorescence changes seen in cancer patients remains to be elucidated.

The Magistral Phage

PubMed Central

Pirnay, Jean-Paul; Verbeken, Gilbert; Ceyssens, Pieter-Jan; Huys, Isabelle; De Vos, Daniel; Ameloot, Charlotte; Fauconnier, Alan

2018-01-01

Since time immemorial, phages—the viral parasites of bacteria—have been protecting Earth’s biosphere against bacterial overgrowth. Today, phages could help address the antibiotic resistance crisis that affects all of society. The greatest hurdle to the introduction of phage therapy in Western medicine is the lack of an appropriate legal and regulatory framework. Belgium is now implementing a pragmatic phage therapy framework that centers on the magistral preparation (compounding pharmacy in the US) of tailor-made phage medicines. PMID:29415431

BACTERIOPHAGE FORMATION WITHOUT BACTERIAL GROWTH

PubMed Central

Price, Winston H.

1947-01-01

1. Iodoacetate, fluoride, and azide have been found to prevent the formation of phage and to inhibit the synthesis of ATP by Staphylococcus muscae. It is suggested that energy-rich phosphate is needed for the synthesis of phage. 2. Gramicidin prevented the formation of phage. 3. No differences were found between normal bacteria and phage-infected bacteria in the inorganic phosphate, adenosinetriphosphate, ribonucleic acid, and desoxyribonucleic acid content of the cells. 4. The mechanism of phage formation is discussed. PMID:18896936

Synergy and Order Effects of Antibiotics and Phages in Killing Pseudomonas aeruginosa Biofilms

PubMed Central

Chaudhry, Waqas Nasir; Concepción-Acevedo, Jeniffer; Park, Taehyun; Andleeb, Saadia; Bull, James J.

2017-01-01

In contrast to planktonic cells, bacteria imbedded biofilms are notoriously refractory to treatment by antibiotics or bacteriophage (phage) used alone. Given that the mechanisms of killing differ profoundly between drugs and phages, an obvious question is whether killing is improved by combining antibiotic and phage therapy. However, this question has only recently begun to be explored. Here, in vitro biofilm populations of Pseudomonas aeruginosa PA14 were treated singly and with combinations of two phages and bactericidal antibiotics of five classes. By themselves, phages and drugs commonly had only modest effects in killing the bacteria. However some phage-drug combinations reduced bacterial densities to well below that of the best single treatment; in some cases, bacterial densities were reduced even below the level expected if both agents killed independently of each other (synergy). Furthermore, there was a profound order effect in some cases: treatment with phages before drugs achieved maximum killing. Combined treatment was particularly effective in killing in Pseudomonas biofilms grown on layers of cultured epithelial cells. Phages were also capable of limiting the extent to which minority populations of bacteria resistant to the treating antibiotic ascend. The potential of combined antibiotic and phage treatment of biofilm infections is discussed as a realistic way to evaluate and establish the use of bacteriophage for the treatment of humans. PMID:28076361

Means to Facilitate the Overcoming of Gastric Juice Barrier by a Therapeutic Staphylococcal Bacteriophage A5/80

PubMed Central

Międzybrodzki, Ryszard; Kłak, Marlena; Jończyk-Matysiak, Ewa; Bubak, Barbara; Wójcik, Anna; Kaszowska, Marta; Weber-Dąbrowska, Beata; Łobocka, Małgorzata; Górski, Andrzej

2017-01-01

In this article we compare the efficacy of different pharmacological agents (ranitidine, and omeprazole) to support phage transit from stomach to distal portions of the gastrointestinal tract in rats. We show that a temporal modification of environment in the animal stomach may protect Twort-like therapeutic antistaphylococcal phage A5/80 (from bacteriophage collection of the Hirszfeld Institute of Immunology and Experimental Therapy PAS in Wroclaw, Poland) from the inactivation by gastric juice effectively enough to enable a significant fraction of orally administered A5/80 to pass to the intestine. Interestingly, we found that yogurt may be a relatively strong in enhancing phage transit. Given the immunomodulating activities of phages our data may suggest that phages and yogurt can act synergistically in mediating their probiotic activities and enhancing the effectiveness of oral phage therapy. We also demonstrate that orally applied phages of similar size, morphology, and sensitivity to acidic environment may differ in their translocation into the bloodstream. This was evident in mice in which a therapeutic staphylococcal phage A5/80 reached the blood upon oral administration combined with antacid agent whilst T4 phage was not detected even when applied in 103 times higher dose. Our findings also suggest that phage penetration from digestive tract to the blood may be species-specific. PMID:28386250

Directed synthesis of bio-inorganic vanadium oxide composites using genetically modified filamentous phage

NASA Astrophysics Data System (ADS)

Mueller, Michael; Baik, Seungyun; Jeon, Hojeong; Kim, Yuchan; Kim, Jungtae; Kim, Young Jun

2015-05-01

The growth of crystalline vanadium oxide using a filamentous bacteriophage template was investigated using sequential incubation in a V2O5 precursor. Using the genetic modification of the bacteriophage, we displayed two cysteines that constrained the RSTB-1 peptide on the major coat protein P8, resulting in vanadium oxide crystallization. The phage-driven vanadium oxide crystals with different topologies, microstructures, photodegradation and vanadium oxide composites were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), quartz microbalance and dissipation (QCM-D) and X-ray photoelectron spectroscopy (XPS). Non-specific electrostatic attraction between a wild-type phage (wt-phage) and vanadium cations in the V2O5 precursor caused phage agglomeration and fiber formation along the length of the viral scaffold. As a result, the addition of recombinant phage (re-phage) in V2O5 precursors formed heterogeneous structures, which led to efficient condensation of vanadium oxide crystal formation in lines, shown by QCM-D analysis. Furthermore, re-phage/VxOx composites showed significantly enhanced photodegradation activities compared with the synthesized wt-phage-V2O5 composite under illumination. This study demonstrates that peptide-mediated vanadium oxide mineralization is governed by a complicated interplay of peptide sequence, local structure, kinetics and the presence of a mineralizing aid, such as the two cysteine-constrained peptides on the phage surface, and has potential for use in nanotechnology applications.

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

PubMed

Dröge, Melloney J; Boersma, Ykelien L; Braun, Peter G; Buining, Robbert Jan; Julsing, Mattijs K; Selles, Karin G A; van Dijl, Jan Maarten; Quax, Wim J

2006-07-01

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

PubMed Central

Holguín, Angela V.; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C.; Barrios, Andrés Fernando González; Vives, Martha J.

2015-01-01

Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization. PMID:26274971

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

PubMed Central

Osório, Nádia; Pereira, Carla; Simões, Sara; Delgadillo, Ivonne

2018-01-01

The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells. PMID:29518018

Isolation, Characterization, and Bioinformatic Analyses of Lytic Salmonella Enteritidis Phages and Tests of Their Antibacterial Activity in Food.

PubMed

Han, Han; Wei, Xiaoting; Wei, Yi; Zhang, Xiufeng; Li, Xuemin; Jiang, Jinzhong; Wang, Ran

2017-02-01

Salmonella Enteritidis remains a major threat for food safety. To take efforts to develop phage-based biocontrol for S. Enteritidis contamination in food, in this study, the phages against S. Enteritidis were isolated from sewage samples, characterized by host range assays, DNA restriction enzyme pattern analyses, and transmission electron microscope observations, and tested for antibacterial activity in food; some potent phages were further characterized by bioinformatic analyses. Results showed that based on the plaque quality and host range, seven lytic phages targeting S. Enteritidis were selected, considered as seven distinct phages through DNA physical maps, and classified as Myoviridae or Siphoviridae family by morphologic observations; the combined use of such seven strain phages as a "food additive" could succeed in controlling the artificial S. Enteritidis contamination in the different physical forms of food at a range of temperatures; by bioinformatic analyses, both selected phage BPS 11 Q 3 and BPS 15 Q 2 seemed to be newfound obligate lytic phage strains with no indications for any potentially harmful genes in their genomes. In conclusion, our results showed a potential of isolated phages as food additives for controlling S. Enteritidis contamination in some salmonellosis outbreak-associated food vehicles, and there could be minimized potential risk associated with using BPS 11 Q 3 and BPS 15 Q 2 in food.

Bacteriophages and Phage-Derived Proteins – Application Approaches

PubMed Central

Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara

2015-01-01

Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes – peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases – that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general. PMID:25666799

First measurement of the ratio of branching fractions B({lambda}{sub b}{sup 0}{yields}{lambda}{sub c}{sup +}{mu}{sup -}{nu}{sub {mu}})/B({lambda}{sub b}{sup 0}{yields}{lambda}{sub c}{sup +}{pi}{sup -})

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aaltonen, T.; Maki, T.; Mehtala, P.

2009-02-01

This article presents the first measurement of the ratio of branching fractions B({lambda}{sub b}{sup 0}{yields}{lambda}{sub c}{sup +}{mu}{sup -}{nu}{sub {mu}})/B({lambda}{sub b}{sup 0}{yields}{lambda}{sub c}{sup +}{pi}{sup -}). Measurements in two control samples using the same technique B(B{sup 0}{yields}D{sup +}{mu}{sup -}{nu}{sub {mu}})/B(B{sup 0}{yields}D{sup +}{pi}{sup -}) and B(B{sup 0}{yields}D*(2010){sup +}{mu}{sup -}{nu}{sub {mu}})/B(B{sup 0}{yields}D*(2010){sup +}{pi}{sup -}) are also reported. The analysis uses data from an integrated luminosity of approximately 172 pb{sup -1} of pp collisions at {radical}(s)=1.96 TeV, collected with the CDF II detector at the Fermilab Tevatron. The relative branching fractions are measured to be (B({lambda}{sub b}{sup 0}{yields}{lambda}{sub c}{sup +}{mu}{sup -}{nu}{sub {mu}})/B({lambda}{sub b}{sup 0}{yields}{lambda}{sub c}{supmore » +}{pi}{sup -}))=16.6{+-}3.0(stat){+-}1.0(syst)(+2.6/-3.4)(PDG){+-}0.3 (EBR), (B(B{sup 0}{yields}D{sup +}{mu}{sup -}{nu}{sub {mu}})/B(B{sup 0}{yields}D{sup +}{pi}{sup -}))9.9{+-}1.0(stat){+-}0.6(syst){+-}0.4(PDG){+-}0.5(EBR), and (B(B{sup 0}{yields}D*(2010){sup +}{mu}{sup -}{nu}{sub {mu}})/B(B{sup 0}{yields}D*(2010){sup +}{pi}{sup -}))=16.5{+-}2.3(stat){+-} 0.6(syst){+-}0.5(PDG){+-}0.8(EBR). The uncertainties are from statistics (stat), internal systematics (syst), world averages of measurements published by the Particle Data Group or subsidiary measurements in this analysis (PDG), and unmeasured branching fractions estimated from theory (EBR), respectively. This article also presents measurements of the branching fractions of four new {lambda}{sub b}{sup 0} semileptonic decays: {lambda}{sub b}{sup 0}{yields}{lambda}{sub c}(2595){sup +}{mu}{sup -}{nu}{sub {mu}}, {lambda}{sub b}{sup 0}{yields}{lambda}{sub c}(2625){sup +}{mu}{sup -}{nu}{sub {mu}}, {lambda}{sub b}{sup 0}{yields}{sigma}{sub c}(2455){sup 0}{pi}{sup +}{mu}{sup -}{nu}{sub {mu}}, and {lambda}{sub b}{sup 0}{yields}{sigma}{sub c}(2455){sup ++}{pi}{sup -}{mu}{sup -}{nu}{sub {mu}}, relative to the branching fraction of the {lambda}{sub b}{sup 0}{yields}{lambda}{sub c}{sup +}{mu}{sup -}{nu}{sub {mu}} decay. Finally, the transverse-momentum distribution of {lambda}{sub b}{sup 0} baryons produced in pp collisions is measured and found to be significantly different from that of B{sup 0} mesons, which results in a modification in the production cross-section ratio {sigma}{sub {lambda}{sub b}{sup 0}}/{sigma}{sub B{sup 0}} with respect to the CDF I measurement.« less

M13 bacteriophage displaying DOPA on surfaces: fabrication of various nanostructured inorganic materials without time-consuming screening processes.

PubMed

Park, Joseph P; Do, Minjae; Jin, Hyo-Eon; Lee, Seung-Wuk; Lee, Haeshin

2014-01-01

M13 bacteriophage (phage) was engineered for the use as a versatile template for preparing various nanostructured materials via genetic engineering coupled to enzymatic chemical conversions. First, we engineered the M13 phage to display TyrGluGluGlu (YEEE) on the pVIII coat protein and then enzymatically converted the Tyr residue to 3,4-dihydroxyl-l-phenylalanine (DOPA). The DOPA-displayed M13 phage could perform two functions: assembly and nucleation. The engineered phage assembles various noble metals, metal oxides, and semiconducting nanoparticles into one-dimensional arrays. Furthermore, the DOPA-displayed phage triggered the nucleation and growth of gold, silver, platinum, bimetallic cobalt-platinum, and bimetallic iron-platinum nanowires. This versatile phage template enables rapid preparation of phage-based prototype devices by eliminating the screening process, thus reducing effort and time.

Molecular characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages.

PubMed

Casey, Eoghan; Mahony, Jennifer; O'Connell-Motherway, Mary; Bottacini, Francesca; Cornelissen, Anneleen; Neve, Horst; Heller, Knut J; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2014-09-01

In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Regulation of Bacteriophage T5 Development by ColI Factors

PubMed Central

Moyer, R. W.; Fu, A. S.; Szabo, C.

1972-01-01

The I-type colicinogenic factor ColIb transforms Escherichia coli from a permissive to a nonpermissive host for bacteriophage T5 reproduction by preventing complete expression of the phage genome. T5-infected ColIb+ cells synthesize only class I (early) phage protein and ribonucleic acid (RNA). Neither phage-specific class II proteins [associated with viral deoxyribonucleic acid (DNA) replication] nor class III proteins (phage structural components) are formed due to the failure of the infected ColIb+ cells to synthesize class II or class III phage-specific messenger RNA. Comparable studies with T5-infected cells colicinogenic for the related ColIa factor revealed no decrease in the yield of progeny phage although the presence of the ColIa factor leads to a significant reduction in the amount of phage-directed class III protein synthesis. Images PMID:4554465

Metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity

PubMed Central

Willner, Dana; Furlan, Mike; Schmieder, Robert; Grasis, Juris A.; Pride, David T.; Relman, David A.; Angly, Florent E.; McDole, Tracey; Mariella, Ray P.; Rohwer, Forest; Haynes, Matthew

2011-01-01

The human oropharynx is a reservoir for many potential pathogens, including streptococcal species that cause endocarditis. Although oropharyngeal microbes have been well described, viral communities are essentially uncharacterized. We conducted a metagenomic study to determine the composition of oropharyngeal DNA viral communities (both phage and eukaryotic viruses) in healthy individuals and to evaluate oropharyngeal swabs as a rapid method for viral detection. Viral DNA was extracted from 19 pooled oropharyngeal swabs and sequenced. Viral communities consisted almost exclusively of phage, and complete genomes of several phage were recovered, including Escherichia coli phage T3, Propionibacterium acnes phage PA6, and Streptococcus mitis phage SM1. Phage relative abundances changed dramatically depending on whether samples were chloroform treated or filtered to remove microbial contamination. pblA and pblB genes of phage SM1 were detected in the metagenomes. pblA and pblB mediate the attachment of S. mitis to platelets and play a significant role in S. mitis virulence in the endocardium, but have never previously been detected in the oral cavity. These genes were also identified in salivary metagenomes from three individuals at three time points and in individual saliva samples by PCR. Additionally, we demonstrate that phage SM1 can be induced by commonly ingested substances. Our results indicate that the oral cavity is a reservoir for pblA and pblB genes and for phage SM1 itself. Further studies will determine the association between pblA and pblB genes in the oral cavity and the risk of endocarditis. PMID:20547834

Soil-based systemic delivery and phyllosphere in vivo propagation of bacteriophages: Two possible strategies for improving bacteriophage persistence for plant disease control.

PubMed

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

2012-10-01

Soil-based root applications and attenuated bacterial strains were evaluated as means to enhance bacteriophage persistence on plants for bacterial disease control. In addition, the systemic nature of phage applied to tomato roots was also evaluated. Several experiments were conducted applying either single phages or phage mixtures specific for Ralstonia solanacearum , Xanthomonas perforans or X. euvesicatoria to soil surrounding tomato plants and measuring the persistence and translocation of the phages over time. In general, all phages persisted in the roots of treated plants and were detected in stems and leaves; although phage level varied and persistence in stems and leaves was at a much lower level compared with persistence in roots. Bacterial wilt control was typically best if the phage or phage mixtures were applied to the soil surrounding tomatoes at the time of inoculation, less effective if applied 3 days before inoculation, and ineffective if applied 3 days after inoculation. The use of an attenuated X. perforans strain was also evaluated to improve the persistence of phage populations on tomato leaf surfaces. In greenhouse and field experiments, foliar applications of an attenuated mutant X. perforans 91-118:∆ OPGH strain prior to phage applications significantly improved phage persistence on tomato foliage compared with untreated tomato foliage. Both the soil-based bacteriophage delivery and the use of attenuated bacterial strains improved bacteriophage persistence on respective root and foliar tissues, with evidence of translocation with soil-based bacteriophage applications. Both strategies could lead to improved control of bacterial pathogens on plants.

Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro.

PubMed

Khan Mirzaei, Mohammadali; Haileselassie, Yeneneh; Navis, Marit; Cooper, Callum; Sverremark-Ekström, Eva; Nilsson, Anders S

2016-01-01

Due to a global increase in the range and number of infections caused by multi-resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (10(9) PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

EPA worst case water microcosms for testing phage biocontrol of Salmonella.

PubMed

McLaughlin, Michael R; Brooks, John P

2008-01-01

A microplate method was developed as a tool to test phages for their ability to control Salmonella in aqueous environments. The method used EPA (U.S. Environmental Protection Agency) worst case water (WCW) in 96-well plates. The WCW provided a consistent and relatively simple defined turbid aqueous matrix, high in total organic carbon (TOC) and total dissolved salts (TDS), to simulate swine lagoon effluent, without the inconvenience of malodor and confounding effects from other biological factors. The WCW was originally defined to simulate high turbidity and organic matter in water for testing point-of-use filtration devices. Use of WCW to simulate lagoon effluent for phage testing is a new and innovative application of this matrix. Control of physical and chemical parameters (TOC, TDS, turbidity, temperature, and pH) allowed precise evaluation of microbiological parameters (Salmonella and phages). In a typical application, wells containing WCW were loaded with Salmonella enterica susp. enterica serovar Typhimurium (ATCC14028) and treated with phages alone and in cocktail combinations. Mean Salmonella inactivation rates (k, where the lower the value, the greater the inactivation) of phage treatments ranged from -0.32 to -1.60 versus -0.004 for Salmonella controls. Mean log(10) reductions (the lower the value, the greater the reduction) of Salmonella phage treatments were -1.60 for phage PR04-1, -2.14 for phage PR37-96, and -2.14 for both phages in a sequential cocktail, versus -0.08 for Salmonella controls. The WCW microcosm system was an effective tool for evaluating the biocontrol potential of Salmonella phages.

Immunocontraception: Filamentous Bacteriophage as a Platform for Vaccine Development.

PubMed

Samoylova, Tatiana I; Braden, Timothy D; Spencer, Jennifer A; Bartol, Frank F

2017-11-20

Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to address this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Filamentous bacteriophages can be used as platforms for development of such vaccines. In this review authors highlight structural and immunogenic properties of filamentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in animals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those administering the vaccines. Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be produced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas.

PubMed

Ahern, Stephen J; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry; Gonzalez, Carlos F

2014-01-01

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ~4 × 10(-12) ml cell(-1) min(-1) for X. fastidiosa strain Temecula 1 and ~5 × 10(-10) to 7 × 10(-10) ml cell(-1) min(-1) for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.

Phage display: concept, innovations, applications and future.

PubMed

Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K

2010-01-01

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field. Copyright © 2010 Elsevier Inc. All rights reserved.

Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening.

PubMed

Mazor, Yariv; Van Blarcom, Thomas; Carroll, Sean; Georgiou, George

2010-05-01

Phage display of antibody libraries is a powerful tool for antibody discovery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab')(2). We recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluorescence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol 25, 563-565]. Although, as a single-cell analysis technique, FACS is very powerful, especially for the isolation of high-affinity binders, even with state of the art instrumentation the screening of libraries with diversity > 10(8) is technically challenging. We report here a system that takes advantage of display of full-length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid-encoded IgG with the fUSE5-ZZ phage as a helper phage. These phage particles display the Fc-binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demonstrate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without undersampling, and at the same time capitalizes on the advantages of FACS for real-time monitoring and optimization of the screening process.

Immunocontraception: Filamentous Bacteriophage as a Platform for Vaccine Development

PubMed Central

Samoylova, Tatiana I.; Braden, Timothy D.; Spencer, Jennifer A.; Bartol, Frank F.

2017-01-01

Background: Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to ad-dress this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Fil-amentous bacteriophages can be used as platforms for development of such vaccines. Objective: In this review authors highlight structural and immunogenic properties of fila-mentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. Results: Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in ani-mals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those ad-ministering the vaccines. Conclusion: Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be pro-duced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents. PMID:28901276

Biological characterization of v. Cholerae-specific bacteriophages isolated from water sources in Georgia.

PubMed

Elbakidze, T; Kokashvili, T; Janelidze, N; Porchkhidze, K; Koberidze, T; Tediashvili, M

2015-03-01

Vibrio cholerae, a widely spread bacterium in various marine, fresh, and brackish water environments, can cause a devastating diarrheal disease - cholera and also mild forms of gastroenteritis. Bacterial viruses are natural controllers of bacterial population density in water systems. The goal of this study was to isolate and characterize V. cholerae-specific bacteriophages occurring in the Georgian coastal zone of the Black Sea and inland water reservoirs in the eastern part of Georgia. During 2006-2009, 71 phages lytic to V. cholerae were collected from these aquatic environments. The phage isolation rate was varying from 8% to 15%, depending on the sampling season and site, and the abundance of host bacteria. The majority of phages specific to V. cholerae were collected from freshwater sources. The phage isolation showed seasonal character covering warm period -from April to September. Based on basic characteristics of primary phage isolates (lytic spectrum, virion morphology and DNA restriction profiles) 23 V. cholerae -specific phages were selected for series of consecutive screenings. Comparatively wide spectrum of lytic activity was revealed in case of 14 phages specific to V. cholerae O1, and one phage - VchBS3, active against non-O1 V. cholerae. Three phages active against V. cholerae non-O1 and six V. cholerae O1 -specific phages have been studied in detail for a number of biological features (stability in different solutions, temperature-, pH- and UV- sensitivity, influence of high ionic strength etc.), considered to be additional important characteristics for selection of phages with therapeutic potential.

Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

PubMed Central

Ahern, Stephen J.; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry

2014-01-01

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ∼4 × 10−12 ml cell−1 min−1 for X. fastidiosa strain Temecula 1 and ∼5 × 10−10 to 7 × 10−10 ml cell−1 min−1 for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa. PMID:24214944

On the nature of the nova-like variable CD-42 deg 14462

NASA Technical Reports Server (NTRS)

Guinan, E. F.; Sion, E. M.

1981-01-01

Low dispersion long and short wavelength IUE spectra of the nova like system CD-42 deg 14462 were obtained on August 24 U.T. The short wave spectrum exhibits absorption features due to C III (lambda 1175), Lalpha 1216), NV (lambda1240), HeII (lambda 1640), SiIV (lambda1394), NIV (lambda1875) with CIV (lambda1550) as a P Cygni feature with blue shifted absorption suggesting the presence of material leaving the system. Possible interpretations of this object are discussed.

Anti p and anti Lambda production in Si + Au collisions at the AGS

NASA Technical Reports Server (NTRS)

Wu, Yue-Dong

1996-01-01

(anti (ital p)) and (anti (Lambda)) production in central Si + Au collisions has been measured by E589 at the BNL-AGS. Preliminary (ital m)(sub (perpendicular)) spectra are presented for (anti (ital p))'s and (anti (Lambda))'s. The (ital dn/dy) distribution for (anti (ital p))'s is also presented. Based on the (anti (ital p)) and (anti (Lambda)) measurements, (anti (Lambda))/(anti (ital p)) ratios are calculated in the rapidity range of 1.1-1.5.

Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

PubMed

Seo, Moon-Hyeong; Nim, Satra; Jeon, Jouhyun; Kim, Philip M

2017-01-01

Protein-protein interactions are essential to cellular functions and signaling pathways. We recently combined bioinformatics and custom oligonucleotide arrays to construct custom-made peptide-phage libraries for screening peptide-protein interactions, an approach we call proteomic peptide-phage display (ProP-PD). In this chapter, we describe protocols for phage display for the identification of natural peptide binders for a given protein. We finally describe deep sequencing for the analysis of the proteomic peptide-phage display.

ASYMMETRIC ABSORPTION PROFILES OF Ly{alpha} AND Ly{beta} IN DAMPED Ly{alpha} SYSTEMS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hee-Won, E-mail: hwlee@sejong.ac.kr

2013-08-01

Damped Ly{alpha} systems observed in the quasar spectra are characterized by a high neutral hydrogen column density, N{sub HI} > 2 x 10{sup 20} cm{sup -2}. The absorption wing profiles are often fitted using the Voigt function due to the fact that the scattering cross section near the resonant line center is approximately described by the Lorentzian function. Since a hydrogen atom has infinitely many p states that participate in the electric dipole interaction, the cross section starts to deviate from the Lorentzian in an asymmetric way in the line wing regions. We investigate this asymmetry in the absorption linemore » profiles around Ly{alpha} and Ly{beta} as a function of the neutral hydrogen column density N{sub HI}. In terms of {Delta}{lambda} {identical_to} {lambda} - {lambda}{sub {alpha}}, we expand the Kramers-Heisenberg formula around Ly{alpha} to find {sigma}({lambda}) {approx_equal} (0.5f{sub 12}){sup 2}{sigma}{sub T}({Delta}{lambda}/{lambda}{sub {alpha}}){sup -2}[1 + 3.792({Delta}{lambda}/{lambda}{sub {alpha}})], where f{sub 12} and {sigma}{sub T} are the oscillator strength of Ly{alpha} and the Thomson scattering cross section, respectively. In terms of {Delta}{lambda}{sub 2} {identical_to} {lambda} - {lambda}{sub {beta}} in the vicinity of Ly{beta}, the total scattering cross section, given as the sum of cross sections for Rayleigh and Raman scattering, is shown to be {sigma}({lambda}) {approx_equal} {sigma}{sub T}(0.5f{sub 13}){sup 2}(1 + R{sub 0})({Delta}{lambda}{sub 2}/{lambda}{sub {beta}}){sup -2}[1 - 24.68({Delta}{lambda}{sub 2}/{lambda}{sub {beta}})] with f{sub 13} and the factor R{sub 0} = 0.1342 being the oscillator strength for Ly{beta} and the ratio of the Raman cross section to Rayleigh cross section, respectively. A redward asymmetry develops around Ly{alpha}, whereas a blue asymmetry is obtained for Ly{beta}. The absorption center shifts are found to be almost proportional to the neutral hydrogen column density.« less

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.

Bacteriophages of Yersinia pestis.

PubMed

Zhao, Xiangna; Skurnik, Mikael

2016-01-01

Bacteriophage play many varied roles in microbial ecology and evolution. This chapter collates a vast body of knowledge and expertise on Yersinia pestis phages, including the history of their isolation and classical methods for their isolation and identification. The genomic diversity of Y. pestis phage and bacteriophage islands in the Y. pestis genome are also discussed because all phage research represents a branch of genetics. In addition, our knowledge of the receptors that are recognized by Y. pestis phage, advances in phage therapy for Y. pestis infections, the application of phage in the detection of Y. pestis, and clustered regularly interspaced short palindromic repeats (CRISPRs) sequences of Y. pestis from prophage DNA are all reviewed here.

Phage transposon mutagenesis.

PubMed

Siegrist, M Sloan; Rubin, Eric J

2009-01-01

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stern, Adi; Sorek, Rotem

Bacteria, the most abundant organisms on the planet, are outnumbered by a factor of 10 to 1 by phages that infect them. Faced with the rapid evolution and turnover of phage particles, bacteria have evolved various mechanisms to evade phage infection and killing, leading to an evolutionary arms race. The extensive co-evolution of both phage and host has resulted in considerable diversity on the part of both bacterial and phage defensive and offensive strategies. In this paper, we discuss the unique and common features of phage resistance mechanisms and their role in global biodiversity. Finally, the commonalities between defense mechanismsmore » suggest avenues for the discovery of novel forms of these mechanisms based on their evolutionary traits.« less

Genome sequences and characterization of the related Gordonia phages GTE5 and GRU1 and their use as potential biocontrol agents.

PubMed

Petrovski, Steve; Tillett, Daniel; Seviour, Robert J

2012-01-01

Activated sludge plants suffer frequently from the operational problem of stable foam formation on aerobic reactor surfaces, which can be difficult to prevent. Many foams are stabilized by mycolic acid-containing Actinobacteria, the mycolata. The in situ biocontrol of foaming using phages is an attractive strategy. We describe two polyvalent phages, GTE5 and GRU1, targeting Gordonia terrae and Gordonia rubrupertincta, respectively, isolated from activated sludge. Phage GRU1 also propagates on Nocardia nova. Both phages belong to the family Siphoviridae and have similar-size icosahedral heads that encapsulate double-stranded DNA genomes (∼65 kb). Their genome sequences are similar to each other but markedly different from those of other sequenced phages. Both are arranged in a modular fashion. These phages can reduce or eliminate foam formation by their host cells under laboratory conditions.

Bacteriophage-Based Pathogen Detection

NASA Astrophysics Data System (ADS)

Ripp, Steven

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

ORFeome Phage Display.

PubMed

Zantow, Jonas; Moreira, Gustavo Marçal Schmidt Garcia; Dübel, Stefan; Hust, Michael

2018-01-01

ORFeome phage display allows the efficient functional screening of entire proteomes or even metaproteomes to identify immunogenic proteins. For this purpose, randomly fragmented, whole genomes or metagenomes are cloned into a phage-display vector allowing positive selection for open reading frames (ORF) to improve the library quality. These libraries display all possible proteins encoded by a pathogen or a microbiome on the phage surface. Consequently, immunogenic proteins can be selected from these libraries using disease-related immunoglobulins from patient serum. ORFeome phage display in particular allows the identification of immunogenic proteins that are only expressed in the host-pathogen interaction but not in cultivation, as well as the detection of very low expressed and very small immunogens and immunogenic proteins of non-cultivable organisms. The identified immunogenic proteins are potential biomarkers for the development of diagnostic assays or vaccines. These articles will give an introduction to ORFeome phage-display technology and give detailed protocols to identify immunogenic proteins by phage display.

Control of Pierce's Disease by Phage

PubMed Central

Das, Mayukh; Bhowmick, Tushar Suvra; Ahern, Stephen J.; Young, Ry; Gonzalez, Carlos F.

2015-01-01

Pierce’s Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella. PMID:26107261

Advancement and applications of peptide phage display technology in biomedical science.

PubMed

Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

2016-01-19

Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

Bacteriophages and their derivatives for the treatment and control of food-producing animal infections.

PubMed

Carvalho, Carla; Costa, Ana Rita; Silva, Filipe; Oliveira, Ana

2017-09-01

Nowadays, the world is facing an increasing emergence of antibiotic resistant bacteria. Simultaneously, the banning of some existing antibiotics and the lack of development of new antimicrobials have created an urgent need to find new alternatives against animal infections. Bacteriophages (phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and harmless to animals. For these reasons, phages and their derivatives are being considered valuable antimicrobial alternatives and an opportunity to reduce the current use of antibiotics in agri-food production, increasing animal productivity and providing environmental protection. Furthermore, the possibility of combining phage genetic material with foreign genes encoding peptides of interest has enabled their use as vaccine delivery tools. In this case, besides bacterial infections, they might be used to prevent viral infections. This review explores current data regarding advances on the use of phages and phage-encoded proteins, such as endolysins, exolysins and depolymerases, either for therapeutic or prophylactic applications, in animal husbandry. The use of recombinant phage-derived particles or genetically modified phages, including phage vaccines, will also be reviewed.

Stability of bacteriophages in burn wound care products

PubMed Central

Monserez, Riet; van Belleghem, Jonas; Rose, Thomas; Jennes, Serge; De Vos, Daniel; Verbeken, Gilbert; Vaneechoutte, Mario; Pirnay, Jean-Paul

2017-01-01

Bacteriophages could be used along with burn wound care products to enhance antimicrobial pressure during treatment. However, some of the components of the topical antimicrobials that are traditionally used for the prevention and treatment of burn wound infection might affect the activity of phages. Therefore, it is imperative to determine the counteraction of therapeutic phage preparations by burn wound care products before application in patients. Five phages, representatives of two morphological families (Myoviridae and Podoviridae) and active against 3 common bacterial burn wound pathogens (Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus) were tested against 13 different products commonly used in the treatment of burn wounds. The inactivation of the phages was quite variable for different phages and different products. Majority of the anti-infective products affected phage activity negatively either immediately or in the course of time, although impact was not always significant. Products with high acidity had the most adverse effect on phages. Our findings demonstrate that during combined treatment the choice of phages and wound care products must be carefully defined in advance. PMID:28750102

Modular Approach to Select Bacteriophages Targeting Pseudomonas aeruginosa for Their Application to Children Suffering With Cystic Fibrosis

PubMed Central

Krylov, Victor; Shaburova, Olga; Pleteneva, Elena; Bourkaltseva, Maria; Krylov, Sergey; Kaplan, Alla; Chesnokova, Elena; Kulakov, Leonid; Magill, Damian; Polygach, Olga

2016-01-01

This review discusses the potential application of bacterial viruses (phage therapy) toward the eradication of antibiotic resistant Pseudomonas aeruginosa in children with cystic fibrosis (CF). In this regard, several potential relationships between bacteria and their bacteriophages are considered. The most important aspect that must be addressed with respect to phage therapy of bacterial infections in the lungs of CF patients is in ensuring the continuity of treatment in light of the continual occurrence of resistant bacteria. This depends on the ability to rapidly select phages exhibiting an enhanced spectrum of lytic activity among several well-studied phage groups of proven safety. We propose a modular based approach, utilizing both mono-species and hetero-species phage mixtures. With an approach involving the visual recognition of characteristics exhibited by phages of well-studied phage groups on lawns of the standard P. aeruginosa PAO1 strain, the simple and rapid enhancement of the lytic spectrum of cocktails is permitted, allowing the development of tailored preparations for patients capable of circumventing problems associated with phage resistant bacterial mutants. PMID:27790211

Rapid detection of bacteriophages in starter culture using water-in-oil-in-water emulsion microdroplets.

PubMed

Wang, Min S; Nitin, Nitin

2014-10-01

Bacteriophage contamination of starter culture and raw material poses a major problem in the fermentation industry. In this study, a rapid detection of lytic phage contamination in starter culture using water-in-oil-in-water (W/O/W) emulsion microdroplets was described. A model bacteria with varying concentrations of lytic phages were encapsulated in W/O/W emulsion microdroplets using a simple needle-in-tube setup. The detection of lytic phage contamination was accomplished in 1 h using the propidium iodide labeling of the phage-infected bacteria inside the W/O/W emulsion microdroplets. Using this approach, a detection limit of 10(2) PFU/mL of phages was achieved quantitatively, while 10(4) PFU/mL of phages could be detected qualitatively based on visual comparison of the fluorescence images. Given the simplicity and sensitivity of this approach, it is anticipated that this method can be adapted to any strains of bacteria and lytic phages that are commonly used for fermentation, and has potential for a rapid detection of lytic phage contamination in the fermentation industry.

Pros and cons of phage therapy

PubMed Central

Loc-Carrillo, Catherine

2011-01-01

Many publications list advantages and disadvantages associated with phage therapy, which is the use of bacterial viruses to combat populations of nuisance or pathogenic bacteria. The goal of this commentary is to discuss many of those issues in a single location. In terms of “Pros,” for example, phages can be bactericidal, can increase in number over the course of treatment, tend to only minimally disrupt normal flora, are equally effective against antibiotic-sensitive and antibiotic-resistant bacteria, often are easily discovered, seem to be capable of disrupting bacterial biofilms, and can have low inherent toxicities. In addition to these assets, we consider aspects of phage therapy that can contribute to its safety, economics, or convenience, but in ways that are perhaps less essential to the phage potential to combat bacteria. For example, autonomous phage transfer between animals during veterinary application could provide convenience or economic advantages by decreasing the need for repeated phage application, but is not necessarily crucial to therapeutic success. We also consider possible disadvantages to phage use as antibacterial agents. These “Cons,” however, tend to be relatively minor. PMID:22334867

Prospects of Phage Application in the Treatment of Acne Caused by Propionibacterium acnes

PubMed Central

Jończyk-Matysiak, Ewa; Weber-Dąbrowska, Beata; Żaczek, Maciej; Międzybrodzki, Ryszard; Letkiewicz, Sławomir; Łusiak-Szelchowska, Marzanna; Górski, Andrzej

2017-01-01

Propionibacterium acnes is associated with purulent skin infections, and it poses a global problem for both patients and doctors. Acne vulgaris (acne) remains a problem due to its chronic character and difficulty of treatment, as well as its large impact on patients' quality of life. Due to the chronic course of the disease, treatment is long lasting, and often ineffective. Currently there are data regarding isolation of P. acnes phages, and there have been numerous studies on phage killing of P. acnes, but no data are available on phage application specifically in acne treatment. In this review, we have summarized the current knowledge on the phages active against P. acnes described so far and their potential application in the treatment of acne associated with P. acnes. The treatment of acne with phages may be important in order to reduce the overuse of antibiotics, which are currently the main acne treatment. However, more detailed studies are first needed to understand phage functioning in the skin microbiome and the possibility to use phages to combat P. acnes. PMID:28228751

Genomic characteristics of vB_PpaP_PP74, a T7-like Autographivirinae bacteriophage infecting a potato pathogen of the newly proposed species Pectobacterium parmentieri.

PubMed

Kabanova, Anastasia; Shneider, Mikhail; Bugaeva, Eugenia; Ha, Vo Thi Ngoc; Miroshnikov, Kirill; Korzhenkov, Aleksei; Kulikov, Eugene; Toschakov, Stepan; Ignatov, Alexander; Miroshnikov, Konstantin

2018-06-01

Bacteriophage vB_PpaP_PP74 (PP74) is a novel virulent phage that infects members of the species Pectobacterium parmentieri, a newly established species of soft-rot-causing bacteria in the family Pectobacteriaceae, derived from potato-specific Pectobacterium wasabiae. vB_PpaP_PP74 was identified as a member of the family Podoviridae by transmission electron microscopy. The phage has a 39,790-bp dsDNA genome containing 50 open reading frames (ORFs). Because of the absence of genes encoding toxins or lysogeny factors, PP74 may be considered a candidate phage for pathogen biocontrol applications. The genome layout is similar to genomes of T7-like phages within the subfamily Autographivirinae, and therefore, functions can be attributed to most of ORFs. However, the closest nucleotide sequence homologs of phage PP74 are unclassified Escherichia phages. Based on phylogenetic analysis, vB_PpaP_PP74 is a sensu lato T7-like phage, but it forms a distant subgenus group together with homologous enterobacterial phages.

Romulus and Remus, two phage isolates representing a distinct clade within the Twortlikevirus genus, display suitable properties for phage therapy applications.

PubMed

Vandersteegen, Katrien; Kropinski, Andrew M; Nash, John H E; Noben, Jean-Paul; Hermans, Katleen; Lavigne, Rob

2013-03-01

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

Survey on the phage resistance mechanisms displayed by a dairy Lactobacillus helveticus strain.

PubMed

Zago, Miriam; Orrù, Luigi; Rossetti, Lia; Lamontanara, Antonella; Fornasari, Maria Emanuela; Bonvini, Barbara; Meucci, Aurora; Carminati, Domenico; Cattivelli, Luigi; Giraffa, Giorgio

2017-09-01

In this study the presence and functionality of phage defence mechanisms in Lactobacillus helveticus ATCC 10386, a strain of dairy origin which is sensitive to ΦLh56, were investigated. After exposure of ATCC 10386 to ΦLh56, the whole-genome sequences of ATCC 10386 and of a phage-resistant derivative (LhM3) were compared. LhM3 showed deletions in the S-layer protein and a higher expression of the genes involved in the restriction/modification (R/M) system. Genetic data were substantiated by measurements of bacteriophage adsorption rates, efficiency of plaquing, cell wall protein size and by gene expression analysis. In LhM3 two phage resistance mechanisms, the inhibition of phage adsorption and the upregulation of Type I R/M genes, take place and explain its resistance to ΦLh56. Although present in both ATCC 10386 and LhM3 genomes, the CRISPR machinery did not seem to play a role in the phage resistance of LhM3. Overall, the natural selection of phage resistant strains resulted successful in detecting variants carrying multiple phage defence mechanisms in L. helveticus. The concurrent presence of multiple phage-resistance systems should provide starter strains with increased fitness and robustness in dairy ecosystems. Copyright © 2017 Elsevier Ltd. All rights reserved.

The First Siphoviridae Family Bacteriophages Infecting Bordetella bronchiseptica Isolated from Environment.

PubMed

Petrovic, Aleksandra; Kostanjsek, Rok; Rakhely, Gabor; Knezevic, Petar

2017-02-01

Bordetella bronchiseptica is a well-known etiological agent of kennel cough in dogs and cats and one of the two causative agents of atrophic rhinitis, a serious swine disease. The aim of the study was to isolate B. bronchiseptica bacteriophages from environmental samples for the first time. A total of 29 phages from 65 water samples were isolated using the strain ATCC 10580 as a host. The lytic spectra of the phages were examined at 25 and 37 °C, using 12 strains of B. bronchiseptica. All phages were able to plaque on 25.0 % to 41.7 % of the strains. The selected phages showed similar morphology (Siphoviridae, morphotype B2), but variation of RFLP patterns and efficacy of plating on various strains. The partial genome sequence of phage vB_BbrS_CN1 showed its similarity to phages from genus Yuavirus. Using PCR, it was confirmed that the phages do not originate from the host strain, and environmental origin was additionally confirmed by the analysis of host genome sequence in silico and plating heated and unheated samples in parallel. Accordingly, this is the first isolation of B. bronchiseptica phages from environment and the first isolation and characterization of phages of B. bronchiseptica belonging to family Siphoviridae.

Impact of reducing and oxidizing agents on the infectivity of Qβ phage and the overall structure of its capsid.

PubMed

Loison, Pauline; Majou, Didier; Gelhaye, Eric; Boudaud, Nicolas; Gantzer, Christophe

2016-11-01

Qβ phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Qβ phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Spatial Vulnerability: Bacterial Arrangements, Microcolonies, and Biofilms as Responses to Low Rather than High Phage Densities

PubMed Central

Abedon, Stephen T.

2012-01-01

The ability of bacteria to survive and propagate can be dramatically reduced upon exposure to lytic bacteriophages. Study of this impact, from a bacterium’s perspective, tends to focus on phage-bacterial interactions that are governed by mass action, such as can be observed within continuous flow or similarly planktonic ecosystems. Alternatively, bacterial molecular properties can be examined, such as specific phage‑resistance adaptations. In this study I address instead how limitations on bacterial movement, resulting in the formation of cellular arrangements, microcolonies, or biofilms, could increase the vulnerability of bacteria to phages. Principally: (1) Physically associated clonal groupings of bacteria can represent larger targets for phage adsorption than individual bacteria; and (2), due to a combination of proximity and similar phage susceptibility, individual bacteria should be especially vulnerable to phages infecting within the same clonal, bacterial grouping. Consistent with particle transport theory—the physics of movement within fluids—these considerations are suggestive that formation into arrangements, microcolonies, or biofilms could be either less profitable to bacteria when phage predation pressure is high or require more effective phage-resistance mechanisms than seen among bacteria not living within clonal clusters. I consider these ideas of bacterial ‘spatial vulnerability’ in part within a phage therapy context. PMID:22754643

Treatment of in vitro enterohemorrhagic Escherichia coli infection using phage and probiotics.

PubMed

Dini, C; Bolla, P A; de Urraza, P J

2016-07-01

To assay the combination of phage and probiotics against EHEC in vitro on infected Hep-2 cells. Phage and probiotics treatments on EHEC O157:H7-infected Hep-2 cells were assayed individually or combined. The effect of freeze-drying on phage and probiotic antimicrobial activity was also studied. While treatment with phage alone increased cell detachment caused by EHEC infection, the treatments with MM alone or in combination with phage proved to effectively diminish cell damage caused by EHEC infection. Combined treatment showed a decrease in apoptotic cell count of 57·3% and a reduction in EHEC adhesion to cell monolayer of 1·2 log CFU. The simultaneous use of phage and probiotics showed no antagonistic effect, and freeze-drying did not affect their antipathogenic activity. The combination of phage and probiotics has great potential for reducing the number of pathogens adhered to epithelial cells during EHEC O157:H7 infection and attenuating the cytotoxic effect derived from it. Further in vivo assays are needed for assessing the actual effectiveness of the treatment. This study presents a freeze-dried formulation of phage and probiotics capable of controlling EHEC infections and reducing epithelial cell damage in vitro. © 2016 The Society for Applied Microbiology.

Bioinformatic analysis of phage AB3, a phiKMV-like virus infecting Acinetobacter baumannii.

PubMed

Zhang, J; Liu, X; Li, X-J

2015-01-16

The phages of Acinetobacter baumannii has drawn increasing attention because of the multi-drug resistance of A. baumanni. The aim of this study was to sequence Acinetobacter baumannii phage AB3 and conduct bioinformatic analysis to lay a foundation for genome remodeling and phage therapy. We isolated and sequenced A. baumannii phage AB3 and attempted to annotate and analyze its genome. The results showed that the genome is a double-stranded DNA with a total length of 31,185 base pairs (bp) and 97 open reading frames greater than 100 bp. The genome includes 28 predicted genes, of which 24 are homologous to phage AB1. The entire coding sequence is located on the negative strand, representing 90.8% of the total length. The G+C mol% was 39.18%, without areas of high G+C content over 200 bp in length. No GC island, tRNA gene, or repeated sequence was identified. Gene lengths were 120-3099 bp, with an average of 1011 bp. Six genes were found to be greater than 2000 bp in length. Genomic alignment and phylogenetic analysis of the RNA polymerase gene showed that similar to phage AB1, phage AB3 is a phiKMV-like virus in the T7 phage family.