The Protein Interaction Network of Bacteriophage Lambda with Its Host, Escherichia coli

PubMed Central

Blasche, Sonja; Wuchty, Stefan; Rajagopala, Seesandra V.

2013-01-01

Although most of the 73 open reading frames (ORFs) in bacteriophage λ have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens. PMID:24049175

iPHLoc-ES: Identification of bacteriophage protein locations using evolutionary and structural features.

PubMed

Shatabda, Swakkhar; Saha, Sanjay; Sharma, Alok; Dehzangi, Abdollah

2017-12-21

Bacteriophage proteins are viruses that can significantly impact on the functioning of bacteria and can be used in phage based therapy. The functioning of Bacteriophage in the host bacteria depends on its location in those host cells. It is very important to know the subcellular location of the phage proteins in a host cell in order to understand their working mechanism. In this paper, we propose iPHLoc-ES, a prediction method for subcellular localization of bacteriophage proteins. We aim to solve two problems: discriminating between host located and non-host located phage proteins and discriminating between the locations of host located protein in a host cell (membrane or cytoplasm). To do this, we extract sets of evolutionary and structural features of phage protein and employ Support Vector Machine (SVM) as our classifier. We also use recursive feature elimination (RFE) to reduce the number of features for effective prediction. On standard dataset using standard evaluation criteria, our method significantly outperforms the state-of-the-art predictor. iPHLoc-ES is readily available to use as a standalone tool from: https://github.com/swakkhar/iPHLoc-ES/ and as a web application from: http://brl.uiu.ac.bd/iPHLoc-ES/. Copyright © 2017 Elsevier Ltd. All rights reserved.

Conformational states of mutant M13 coat proteins are regulated by transmembrane residues.

PubMed

Li, Z; Glibowicka, M; Joensson, C; Deber, C M

1993-03-05

Mutational and structural analysis of the 28 viable bacteriophage M13 mutants obtained by randomized mutagenesis of the effective transmembrane (TM) segment of the 50-residue major coat (gene VIII) protein (residues 21-39) demonstrated that M13 coat protein functionality, as reflected by phage viability, is incompatible with an increase in Gly + beta-branched residue content in its TM core. SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy performed in membrane environments on purified mutant coat proteins revealed that these proteins exist in a range of state(s), identified as helical monomers and dimers and polymeric (alpha-helical and/or beta-sheet) species, of which relative populations, and thermally induced conformational transitions, were dependent uniquely upon mutation type and locus. Mutations to relatively polar residues (e.g. G23D, Y24D, Y24H, A27E, I32T, and T36S) stabilized principally monomeric species, while mutants with decreased beta-branched content in the protein TM hydrophobic core (e.g. V29A, V30A, V31A, V31L, and V33A) displayed mainly dimeric species. Mutation of Ile37-->Thr within a "Sternberg-Gullick" consensus sequence of the coat protein TM segment led to a highly oligomerized/polymerized protein. The overall results suggest that TM residues in M13 coat protein are not universal components of a hydrophobic anchor segment per se, but are further selected (i) to impart conformational flexibility to the TM segment through helix destabilization and (ii) to retain the capacity to regulate protein-protein association and packing motifs within membranes.

M13 Bacteriophage Based Protein Sensors

NASA Astrophysics Data System (ADS)

Lee, Ju Hun

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

Learning from Bacteriophages - Advantages and Limitations of Phage and Phage-Encoded Protein Applications

PubMed Central

Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grażyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

2012-01-01

The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application. PMID:23305359

BACTERIOPHAGE PRD1 AND SILICA COLLOID TRANSPORT AND RECOVERY IN AN IRON OXIDE-COATED SAND AQUIFER. (R826179)

EPA Science Inventory

Bacteriophage PRD1 and silica colloids were co-injected into
sewage-contaminated and uncontaminated zones of an iron oxide-coated sand
aquifer on Cape Cod, MA, and their transport was monitored over distances up to
6 m in three arrays. After deposition, the attache...

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

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

The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku

PubMed Central

di Fagagna, Fabrizio d'Adda; Weller, Geoffrey R.; Doherty, Aidan J.; Jackson, Stephen P.

2003-01-01

Mu bacteriophage inserts its DNA into the genome of host bacteria and is used as a model for DNA transposition events in other systems. The eukaryotic Ku protein has key roles in DNA repair and in certain transposition events. Here we show that the Gam protein of phage Mu is conserved in bacteria, has sequence homology with both subunits of Ku, and has the potential to adopt a similar architecture to the core DNA-binding region of Ku. Through biochemical studies, we demonstrate that Gam and the related protein of Haemophilus influenzae display DNA binding characteristics remarkably similar to those of human Ku. In addition, we show that Gam can interfere with Ty1 retrotransposition in Saccharomyces cerevisiae. These data reveal structural and functional parallels between bacteriophage Gam and eukaryotic Ku and suggest that their functions have been evolutionarily conserved. PMID:12524520

Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine, and Ozone.

PubMed

Choe, Jong Kwon; Richards, David H; Wilson, Corey J; Mitch, William A

2015-11-17

Proteins are important targets of chemical disinfectants. To improve the understanding of disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is driven by their disinfectant rate constants rather than their geometrical arrangement. Across the model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs with its high disinfectant rate constants and supports its hypothesized role as a sacrificial antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine. These results concur with the prevalence of geometrical motifs featuring histidines or lysines near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer pathway, histidine, tyrosine, and lysine degradation followed their relative O3 rate constants. Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did not correlate with loss of particular amino acids, suggesting the need to characterize the oxidation of specific geometric motifs to understand structural degradation.

Dehydration of bacteriophages in electrospun nanofibers: effect of excipients in polymeric solutions

NASA Astrophysics Data System (ADS)

Koo, Charmaine K. W.; Senecal, Kris; Senecal, Andre; Nugen, Sam R.

2016-12-01

Bacteriophages are viruses capable of infecting and lysing target bacterial cells; as such they have potential applications in agriculture for decontamination of foods, food contact surfaces and food rinse water. Although bacteriophages can retain infectivity long-term using lyophilized storage, the process of freeze-drying can be time consuming and expensive. In this study, electrospinning was used for dehydrating bacteriophages in polyvinylpyrrolidone polymer solutions with addition of excipients (sodium chloride, magnesium sulfate, Tris-HCl, sucrose) in deionized water. The high voltage dehydration reduced the infectivity of bacteriophages following electrospinning, with the damaging effect abated with addition of storage media (SM) buffer and sucrose. SM buffer and sucrose also provided the most protection over extended storage (8 weeks; 20 °C 1% relative humidity) by mitigating environmental effects on the dried bacteriophages. Magnesium sulfate however provided the least protection due to coagulation effects of the ion, which can disrupt the native conformation of the bacteriophage protein coat. Storage temperatures (20 °C, 4 °C and -20 °C 1% relative humidity) had a minimal effect while relative humidity had substantial effect on the infectivity of bacteriophages. Nanofibers stored in higher relative humidity (33% and 75%) underwent considerable damage due to extensive water absorption and disruption of the fibers. Overall, following storage of nanofiber mats for eight weeks at ambient temperatures, high infective phage concentrations (106-107 PFU ml-1) were retained. Therefore, this study provided valuable insights on preservation and dehydration of bacteriophages by electrospinning in comparison to freeze drying and liquid storage, and the influence of excipients on the viability of bacteriophages.

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

Detection of Intermediates And Kinetic Control During Assembly of Bacteriophage P22 Procapsid

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

Tuma, R.; Tsuruta, H.; French, K.H.

2009-05-26

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter {approx} 50 nm). In this work, we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibriummore » (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering, which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild-type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly.« less

Cytoplasmic bacteriophage display system

DOEpatents

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

1998-06-16

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

Cytoplasmic bacteriophage display system

DOEpatents

Studier, F. William; Rosenberg, Alan H.

1998-06-16

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

Non-Identity-Mediated CRISPR-Bacteriophage Interaction Mediated via the Csy and Cas3 Proteins ▿#

PubMed Central

Cady, Kyle C.; O'Toole, George A.

2011-01-01

Studies of the Escherichia, Neisseria, Thermotoga, and Mycobacteria clustered regularly interspaced short palindromic repeat (CRISPR) subtypes have resulted in a model whereby CRISPRs function as a defense system against bacteriophage infection and conjugative plasmid transfer. In contrast, we previously showed that the Yersinia-subtype CRISPR region of Pseudomonas aeruginosa strain UCBPP-PA14 plays no detectable role in viral immunity but instead is required for bacteriophage DMS3-dependent inhibition of biofilm formation by P. aeruginosa. The goal of this study is to define the components of the Yersinia-subtype CRISPR region required to mediate this bacteriophage-host interaction. We show that the Yersinia-subtype-specific CRISPR-associated (Cas) proteins Csy4 and Csy2 are essential for small CRISPR RNA (crRNA) production in vivo, while the Csy1 and Csy3 proteins are not absolutely required for production of these small RNAs. Further, we present evidence that the core Cas protein Cas3 functions downstream of small crRNA production and that this protein requires functional HD (predicted phosphohydrolase) and DEXD/H (predicted helicase) domains to suppress biofilm formation in DMS3 lysogens. We also determined that only spacer 1, which is not identical to any region of the DMS3 genome, mediates the CRISPR-dependent loss of biofilm formation. Our evidence suggests that gene 42 of phage DMS3 (DMS3-42) is targeted by CRISPR2 spacer 1 and that this targeting tolerates multiple point mutations between the spacer and DMS3-42 target sequence. This work demonstrates how the interaction between P. aeruginosa strain UCBPP-PA14 and bacteriophage DMS3 can be used to further our understanding of the diverse roles of CRISPR system function in bacteria. PMID:21398535

The allosteric switching mechanism in bacteriophage MS2

NASA Astrophysics Data System (ADS)

Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F.

2016-07-01

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.

The allosteric switching mechanism in bacteriophage MS2

PubMed Central

Perkett, Matthew R.; Mirijanian, Dina T.

2016-01-01

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates. PMID:27448905

Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

PubMed

Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

2017-11-01

The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

The structure of the bacteriophage PRD1 spike sheds light on the evolution of viral capsid architecture.

PubMed

Merckel, Michael C; Huiskonen, Juha T; Bamford, Dennis H; Goldman, Adrian; Tuma, Roman

2005-04-15

Comparisons of bacteriophage PRD1 and adenovirus protein structures and virion architectures have been instrumental in unraveling an evolutionary relationship and have led to a proposal of a phylogeny-based virus classification. The structure of the PRD1 spike protein P5 provides further insight into the evolution of viral proteins. The crystallized P5 fragment comprises two structural domains: a globular knob and a fibrous shaft. The head folds into a ten-stranded jelly roll beta barrel, which is structurally related to the tumor necrosis factor (TNF) and the PRD1 coat protein domains. The shaft domain is a structural counterpart to the adenovirus spike shaft. The structural relationships between PRD1, TNF, and adenovirus proteins suggest that the vertex proteins may have originated from an ancestral TNF-like jelly roll coat protein via a combination of gene duplication and deletion.

Bacteriophage PRD1 and silica colloid transport and recovery in an iron oxide-coated sand aquifer

USGS Publications Warehouse

Ryan, J.N.; Elimelech, M.; Ard, R.A.; Harvey, R.W.; Johnson, P.R.

1999-01-01

Bacteriophage PRD1 and silica colloids were co-injected into sewage- contaminated and uncontaminated zones of an iron oxide-coated sand aquifer on Cape Cod, MA, and their transport was monitored over distances up to 6 m in three arrays. After deposition, the attached PRD1 and silica colloids were mobilized by three different chemical perturbations (elevated pH, anionic surfactant, and reductant). PRD1 and silica colloids experienced less attenuation in the contaminated zone where adsorbed organic matter and phosphate may be hindering attachment of PRD1 and silica colloids to the iron oxide coatings. The PRD1 collision efficiencies agree well with collision efficiencies predicted by assuming favorable PRD1 deposition on iron oxide coatings for which the surface area coverage was measured by microprobe analysis of sediment thin sections. ?? potentials of the PRD1, silica colloids, and aquifer grains corroborated the transport results, indicating that electrostatic forces dominated the attachment of PRD1 and silica colloids. Elevated pH was the chemical perturbation most effective at mobilizing the attached PRD1 and silica colloids. Elevated surfactant concentration mobilized the attached PRD1 and silica colloids more effectively in the contaminated zone than in the uncontaminated zone.Bacteriophage PRD1 and silica colloids were co-injected into sewage-contaminated and uncontaminated zones of an iron oxide-coated sand aquifer on Cape Cod, MA, and their transport was monitored over distances up to 6 m in three arrays. After deposition, the attached PRD1 and silica colloids were mobilized by three different chemical perturbations (elevated pH, anionic surfactant, and reductant). PRD1 and silica colloids experienced less attenuation in the contaminated zone where adsorbed organic matter and phosphate may be hindering attachment of PRD1 and silica colloids to the iron oxide coatings. The PRD1 collision efficiencies agree well with collision efficiencies predicted by

The protein interaction map of bacteriophage lambda

PubMed Central

2011-01-01

Background Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage. Results In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome") into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%). We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda. Conclusions Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins. PMID:21943085

Nanoscale bacteriophage biosensors beyond phage display.

PubMed

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

2013-01-01

Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.

Nanoscale bacteriophage biosensors beyond phage display

PubMed Central

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

2013-01-01

Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology. PMID:24143096

Backbone dynamics of a model membrane protein: assignment of the carbonyl carbon /sup 13/C NMR resonances in detergent-solubilized M13 coat protein

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

Henry, G.D.; Weiner, J.H.; Sykes, B.D.

The major coat protein of the filamentous bacteriophage M13 is a 50-residue amphiphilic polypeptide which is inserted, as an integral membrane-spanning protein, in the inner membrane of the Escherichia coli host during infection. /sup 13/C was incorporated biosynthetically into a total of 23 of the peptide carbonyls using labeled amino acids (alanine, glycine, lysine, phenylalanine, and proline). The structure and dynamics of carbonyl-labeled M13 coat protein were monitored by /sup 13/C nuclear magnetic resonance (NMR) spectroscopy. Assignment of many resonances was achieved by using protease digestion, pH titration, or labeling of the peptide bond with both /sup 13/C and /supmore » 15/N. The carbonyl region of the natural-abundance /sup 13/C NMR spectrum of M13 coat protein in sodium dodecyl sulfate solution shows approximately eight backbone carbonyl resonances with line widths much narrower than the rest. Three of these more mobile residues correspond to assigned peaks (glycine-3, lysine-48, and alanine-49) in the individual amino acid spectra, and another almost certainly arises from glutamic acid-2. A ninth residue, alanine-1, also gives rise to a very narrow carbonyl resonance if the pH is well above or below the pK/sub a/ of the terminal amino group. These data suggest that only about four residues at either end of the protein experience large-amplitude spatial fluctuations; the rest of the molecule is essentially rigid on the time scale of the overall rotational tumbling of the protein-detergent complex. The relative exposure of different regions of detergent-bound protein was monitored by limited digestion with proteinase K. Comparable spectra and digestion patterns were obtained when the protein was solubilized in sodium deoxycholate, suggesting that the coat protein binds both amphiphiles in a similar fashion.« less

Production and Purification of Recombinant Filamentous Bacteriophages Displaying Immunogenic Heterologous Epitopes.

PubMed

Deng, Lei; Linero, Florencia; Saelens, Xavier

2016-01-01

Viruslike particles often combine high physical stability with robust immunogenicity. Furthermore, when such particles are based on bacteriophages, they can be produced in high amounts at minimal cost and typically will require only standard biologically contained facilities. We provide protocols for the characterization and purification of recombinant viruslike particles derived from filamentous bacteriophages. As an example, we focus on filamentous Escherichia coli fd phage displaying a conserved influenza A virus epitope that is fused genetically to the N-terminus of the major coat protein of this phage. A step-by-step procedure to obtain a high-titer, pure recombinant phage preparation is provided. We also describe a quality control experiment based on a biological readout of the purified fd phage preparation. These protocols together with the highlighted critical steps may facilitate generic implementation of the provided procedures for the display of other epitopes by recombinant fd phages.

Bioorthogonal Modification of the Major Sheath Protein of Bacteriophage M13: Extending the Versatility of Bionanomaterial Scaffolds.

PubMed

Urquhart, Taylor; Daub, Elisabeth; Honek, John Frank

2016-10-19

With a mass of ∼1.6 × 10 7 Daltons and composed of approximately 2700 proteins, bacteriophage M13 has been employed as a molecular scaffold in bionanomaterials fabrication. In order to extend the versatility of M13 in this area, residue-specific unnatural amino acid incorporation was employed to successfully display azide functionalities on specific solvent-exposed positions of the pVIII major sheath protein of this bacteriophage. Employing a combination of engineered mutants of the gene coding for the pVIII protein, the methionine (Met) analog, l-azidohomoalanine (Aha), and a suitable Escherichia coli Met auxotroph for phage production, conditions were developed to produce M13 bacteriophage labeled with over 350 active azides (estimated by fluorescent dye labeling utilizing a strain-promoted azide-alkyne cycloaddition) and capable of azide-selective attachment to 5 nm gold nanoparticles as visualized by transmission electron microscopy. The capability of this system to undergo dual labeling utilizing both chemical acylation and bioorthogonal cycloaddition reactions was also verified. The above stratagem should prove particularly advantageous in the preparation of assemblies of larger and more complex molecular architectures based on the M13 building block.

The allosteric switching mechanism in bacteriophage MS2

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

Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F., E-mail: hagan@brandeis.edu

2016-07-21

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we usemore » all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.« less

Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

PubMed

Rauch, Benjamin J; Silvis, Melanie R; Hultquist, Judd F; Waters, Christopher S; McGregor, Michael J; Krogan, Nevan J; Bondy-Denomy, Joseph

2017-01-12

Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

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

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2008-01-01

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

Direct interaction of the bacteriophage SPP1 packaging ATPase with the portal protein.

PubMed

Oliveira, Leonor; Cuervo, Ana; Tavares, Paulo

2010-03-05

DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.

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

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

Fokine, Andrei; Islam, Mohammad Z.; Zhang, Zhihong

2011-09-16

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

Identification of trapped and boundary lipid binding sites in M13 coat protein/lipid complexes by deuterium NMR spectroscopy

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

Van Gorkom, L.C.; Horvath, L.I.; Hemminga, M.A.

The major coat protein of M13 bacteriophage has been incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the trimethyl segments of the choline headgroup (DMPC-d9). Two-component deuterium and phosphorus-31 NMR spectra have been observed from bilayer complexes containing the coat protein, indicating slow exchange (on the deuterium quadrupole anisotropy and phosphorus-31 chemical shift averaging time scales) of lipid molecules of less than 10(3) Hz between two motionally distinct environments in the complexes. The fraction of the isotropic spectral component increases with increasing M13 protein concentration, and this component is attributed to lipid headgroups, which are disordered relative to their order inmore » protein-free bilayers. The activation energy of the fast local motions of the trimethyl groups of the choline residue in the headgroup decreases from 23 kJ mol-1 in the pure lipid bilayers to 20 kJ mol-1 for the protein-associated lipid headgroups. The chemical exchange rate of lipid molecules between the two motionally distinct environments has been estimated to be 20-50 Hz by steady-state line-shape simulations of the deuterium spectra of DMPC-d9/M13 coat protein complexes using exchange-coupled modified Bloch equations. The off-rate was, as expected from one-to-one exchange, independent of the L/P ratio; tau off -1 = 0.23 kHz. It is suggested that the protein-associated lipid may be trapped between closely packed parallel aggregates of M13 coat protein and that the high local concentration of protein in a one-dimensional arrangement in lipid bilayers may be required for the fast reassembly of phage particles before release from an infected cell.« less

STUDIES ON THE BACTERIOPHAGE OF D'HERELLE : IX. EVIDENCE OF HYDROLYSIS OF BACTERIAL PROTEIN DURING LYSIS.

PubMed

Hetler, D M; Bronfenbrenner, J

1928-07-31

1. During the process of lysis by bacteriophage, there is an appreciable increase in the amount of free amino acid present in the culture. 2. The increase of free amino acid is due to hydrolysis of bacterial protein.

Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis

PubMed Central

2012-01-01

Background Haemophilus parasuis, the causative agent of Glässer’s disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer’s disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate. Results Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome�

Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis.

PubMed

Zehr, Emilie S; Tabatabai, Louisa B; Bayles, Darrell O

2012-07-23

Haemophilus parasuis, the causative agent of Glässer's disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer's disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate. Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome's G + C content was

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs.

PubMed

Wang, C; Deber, C M

2000-05-26

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.

A Bacteriophage Capsid Protein Is an Inhibitor of a Conserved Transcription Terminator of Various Bacterial Pathogens.

PubMed

Ghosh, Gairika; Reddy, Jayavardhana; Sambhare, Susmit; Sen, Ranjan

2018-01-01

Rho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits Escherichia coli Rho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, including Mycobacterium smegmatis , Mycobacterium bovis , Mycobacterium tuberculosis , Xanthomonas campestris , Xanthomonas oryzae , Corynebacterium glutamicum , Vibrio cholerae , Salmonella enterica , and Pseudomonas syringae The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pulldown assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis , M. bovis , X. campestris , and E. coli expressing S. enterica Rho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the "universal" inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions. IMPORTANCE Bacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of the E. coli transcription

Campylobacter bacteriophages and bacteriophage therapy.

PubMed

Connerton, P L; Timms, A R; Connerton, I F

2011-08-01

Members of the genus Campylobacter are frequently responsible for human enteric disease with occasionally very serious outcomes. Much of this disease burden is thought to arise from consumption of contaminated poultry products. More than 80% of poultry in the UK harbour Campylobacter as a part of their intestinal flora. To address this unacceptably high prevalence, various interventions have been suggested and evaluated. Among these is the novel approach of using Campylobacter-specific bacteriophages, which are natural predators of the pathogen. To optimize their use as therapeutic agents, it is important to have a comprehensive understanding of the bacteriophages that infect Campylobacter, and how they can affect their host bacteria. This review will focus on many aspects of Campylobacter-specific bacteriophages including: their first isolation in the 1960s, their use in bacteriophage typing schemes, their isolation from the different biological sources and genomic characterization. As well as their use as therapeutic agents to reduce Campylobacter in poultry their future potential, including their use in bio-sanitization of food, will be explored. The evolutionary consequences of naturally occurring bacteriophage infection that have come to light through investigations of bacteriophages in the poultry ecosystem will also be discussed. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Antibacterial application of engineered bacteriophage nanomedicines: antibody-targeted, chloramphenicol prodrug loaded bacteriophages for inhibiting the growth of Staphylococcus aureus bacteria.

PubMed

Vaks, Lilach; Benhar, Itai

2011-01-01

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus there is an urgent need to develop new antimicrobial agents. To be effective, these new antimicrobials should possess novel modes of action and/or different cellular targets compared with existing antibiotics. Bacteriophages (phages) have been used for over a century as tools for the treatment of bacterial infections, for nearly half a century as tools in genetic research, for about two decades as tools for the discovery of specific target-binding proteins and peptides, and for almost a decade as tools for vaccine development. We describe a new application in the area of antibacterial nanomedicines where filamentous phages can be formulated as targeted drug-delivery vehicles of nanometric dimensions (phage nanomedicines) and used for therapeutic purposes. This protocol involves both genetic and chemical engineering of these phages. The genetic engineering of the phage coat, which results in the display of a target-specificity-conferring peptide or protein on the phage coat, can be used to design the drug-release mechanism and is not described herein. However, the methods used to chemically conjugate cytotoxic drugs at high density on the phage coat are described. Further, assays to measure the drug load on the surface of the phage and the potency of the system in the inhibition of growth of target cells as well as assessment of the therapeutic potential of the phages in a mouse disease model are discussed.

Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

PubMed Central

Deber, C M; Khan, A R; Li, Z; Joensson, C; Glibowicka, M; Wang, J

1993-01-01

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins. Images Fig. 2 Fig. 4 PMID:8265602

Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

PubMed

Deber, C M; Khan, A R; Li, Z; Joensson, C; Glibowicka, M; Wang, J

1993-12-15

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins.

Use of bacteriophage cell wall-binding proteins for rapid diagnostics of Listeria.

PubMed

Schmelcher, Mathias; Loessner, Martin J

2014-01-01

Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.

Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

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

Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid’) built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinitymore » for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging’ is a DNA-dependent symmetrization of portal protein.« less

Doxorubicin-conjugated bacteriophages carrying anti-MHC class I chain-related A for targeted cancer therapy in vitro.

PubMed

Phumyen, Achara; Jantasorn, Siriporn; Jumnainsong, Amonrat; Leelayuwat, Chanvit

2014-01-01

Cancer therapy by systemic administration of anticancer drugs, besides the effectiveness shown on cancer cells, demonstrated the side effects and cytotoxicity on normal cells. The targeted drug-carrying nanoparticles may decrease the required drug concentration at the site and the distribution of drugs to normal tissues. Overexpression of major histocompatibility complex class I chain-related A (MICA) in cancer is useful as a targeted molecule for the delivery of doxorubicin to MICA-expressing cell lines. The application of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) chemistry was employed to conjugate the major coat protein of bacteriophages carrying anti-MICA and doxorubicin in a mildly acid condition. Doxorubicin (Dox) on phages was determined by double fluorescence of phage particles stained by M13-fluorescein isothiocyanate (FITC) and drug autofluorescence by flow cytometry. The ability of anti-MICA on phages to bind MICA after doxorubicin conjugation was evaluated by indirect enzyme-linked immunosorbent assay. One cervical cancer and four cholangiocarcinoma cell lines expressing MICA were used as models to evaluate targeting activity by cell cytotoxicity test. Flow cytometry and indirect enzyme-linked immunosorbent assay demonstrated that most of the phages (82%) could be conjugated with doxorubicin, and the Dox-carrying phage-displaying anti-MICA (Dox-phage) remained the binding activity against MICA. Dox-phage was more efficient than free drugs in killing all the cell lines tested. The half maximal inhibitory concentration (IC50) values of Dox-phage were lower than those of free drugs at approximately 1.6-6 times depending on MICA expressions and the cell lines tested. Evidently, the application of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide chemistry is effective to conjugate doxorubicin and major coat protein of bacteriophages without destroying binding activity of MICA antibodies. Dox-carrying bacteriophages targeting MICA have been

Antimicrobial bacteriophage-derived proteins and therapeutic applications

USDA-ARS?s Scientific Manuscript database

Antibiotics have the remarkable power to control bacterial infections. Unfortunately, widespread use, whether regarded as prudent or not, has favored the emergence and persistence of antibiotic resistant strains of human pathogenic bacteria, resulting in a global health threat. Bacteriophages (pha...

The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd.

PubMed

Enshell-Seijffers, D; Smelyanski, L; Gershoni, J M

2001-05-15

Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides. Two phage proteins are often employed for peptide display: the infectivity protein, PIII, and the major coat protein, PVIII. The use of PVIII typically requires the expression of two pVIII genes: the wild-type and the recombinant pVIII gene, to generate mosaic phages. 'Type 88' vectors contain two pVIII genes in one phage genome. In this study a novel 'type 88' expression vector has been rationally designed and constructed. Two factors were taken into account: the insertion site and the genetic stability of the second pVIII gene. It was found that selective deletion of recombinant genes was encountered when inserts were cloned into either of the two non-coding regions of the phage genome. The deletions were independent of recA yet required a functional F-episome. Transcription was also found to be a positive factor for deletion. Taking the above into account led to the generation of a novel vector, designated fth1, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages. The fth1 vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.

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

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

Granell, Meritxell; Namura, Mikiyoshi; Alvira, Sara

2014-06-19

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

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

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®.

STUDIES ON THE PURIFICATION OF BACTERIOPHAGE

PubMed Central

Kalmanson, G.; Bronfenbrenner, J.

1939-01-01

A simple method of concentrating and purifying bacteriophage has been described. The procedure consisted essentially in collecting the active agent on a reinforced collodion membrane of a porosity that would just retain all the active agent and permit extraneous material to pass through. Advantage was taken of the fact that B. coli will proliferate and regenerate bacteriophage in a completely diffusible synthetic medium with ammonia as the only source of nitrogen, which permitted the purification of the bacteriophage by copious washing. The material thus obtained was concentrated by suction and after thorough washing possessed all the activity of the original filtrate. It was labile, losing its activity in a few days on standing, and was quickly and completely inactivated upon drying. This material contained approximately 15 per cent of nitrogen and with 2 or 3 mg. samples of inactive dry residue it was possible to obtain positive protein color tests. The concentrated and purified bacteriophage has about 10–14 mg. of nitrogen, or 6 x 10–17 gm. of protein per unit of lytic activity. Assuming that each unit of activity represents a molecule, the calculated maximum average molecular weight would be approximately 36,000,000, and on the assumption of a spherical shape of particles and a density of 1.3, the calculated radius would be about 22 millimicra. By measurement of the diffusion rate, the average radius of particle of the fraction of the purified bacteriophage which diffuses most readily through a porous plate was found to be of the order of magnitude of 9 millimicra, or of a calculated molecular weight of 2,250,000. Furthermore, when this purified bacteriophage was fractionated by forcing it through a thin collodion membrane, which permits the passage of only the smaller particles, it was possible to demonstrate in the ultrafiltrate active particles of about 2 millimicra in radius, and of a calculated molecular weight of 25,000. It was of interest to apply

A Bacteriophage-Related Chimeric Marine Virus Infecting Abalone

PubMed Central

Zhuang, Jun; Cai, Guiqin; Lin, Qiying; Wu, Zujian; Xie, Lianhui

2010-01-01

Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV) can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin). The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs), eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB) protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria. PMID:21079776

Structural Studies of MS2 Bacteriophage Virus Particle Disassembly by Nuclear Magnetic Resonance Relaxation Measurements

PubMed Central

Anobom, C. D.; Albuquerque, S. C.; Albernaz, F. P.; Oliveira, A. C.; Silva, J. L.; Peabody, D. S.; Valente, A. P.; Almeida, F. C. L.

2003-01-01

In this article we studied, by nuclear magnetic resonance relaxation measurements, the disassembly of a virus particle—the MS2 bacteriophage. MS2 is one of the single-stranded RNA bacteriophages that infect Escherichia coli. At pH 4.5, the phage turns to a metastable state, as is indicated by an increase in the observed nuclear magnetic resonance signal intensity upon decreasing the pH from 7.0 to 4.5. Steady-state fluorescence and circular dichroism spectra at pH 4.5 show that the difference in conformation and secondary structure is not pronounced if compared with the phage at pH 7.0. At pH 4.5, two-dimensional 15N-1H heteronuclear multiple quantum coherence (HMQC) spectrum shows ∼40 crosspeaks, corresponding to the most mobile residues of MS2 coat protein at pH 4.5. The 15N linewidth is ∼30 Hz, which is consistent with an intermediate with a rotational relaxation time of 100 ns. The average spin lattice relaxation time (T1) of the mobile residues was measured at different temperatures, clearly distinguishing between the dimer and the equilibrium intermediate. The results show, for the first time, the presence of intermediates in the process of dissociation of the MS2 bacteriophage. PMID:12770895

Polyglycerol coatings of glass vials for protein resistance.

PubMed

Höger, Kerstin; Becherer, Tobias; Qiang, Wei; Haag, Rainer; Friess, Wolfgang; Küchler, Sarah

2013-11-01

Proteins are surface active molecules which undergo non-specific adsorption when getting in contact with surfaces such as the primary packaging material. This process is critical as it may cause a loss of protein content or protein aggregation. To prevent unspecific adsorption, protein repellent coatings are of high interest. We describe the coating of industrial relevant borosilicate glass vials with linear methoxylated polyglycerol, hyperbranched polyglycerol, and hyperbranched methoxylated polyglycerol. All coatings provide excellent protein repellent effects. The hyperbranched, non-methoxylated coating performed best. The protein repellent properties were maintained also after applying industrial relevant sterilization methods (≥200 °C). Marginal differences in antibody stability between formulations stored in bare glass vials and coated vials were detected after 3 months storage; the protein repellent effect remained largely stable. Here, we describe a new material suitable for the coating of primary packaging material of proteins which significantly reduces the protein adsorption and thus could present an interesting new possibility for biomedical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of ferric oxyhydroxide grain coatings on the transport of bacteriophage PRD1 and Cryptosporidium parvum oocysts in saturated porous media

USGS Publications Warehouse

Abudalo, R.A.; Bogatsu, Y.G.; Ryan, J.N.; Harvey, R.W.; Metge, D.W.; Elimelech, M.

2005-01-01

To test the effect of geochemical heterogeneity on microorganism transport in saturated porous media, we measured the removal of two microorganisms, the bacteriophage PRD1 and oocysts of the protozoan parasite Cryptosporidium parvum, in flow-through columns of quartz sand coated by different amounts of a ferric oxyhydroxide. The experiments were conducted over ranges of ferric oxyhydroxide coating fraction of ?? = 0-0.12 for PRD1 and from ?? = 0-0.32 for the oocysts at pH 5.6-5.8 and 10-4 M ionic strength. To determine the effect of pH on the transport of the oocysts, experiments were also conducted over a pH range of 5.7-10.0 at a coating fraction of ?? = 0.04. Collision (attachment) efficiencies increased as the fraction of ferric oxyhydroxide coated quartz sand increased, from ?? = 0.0071 to 0.13 over ?? = 0-0.12 for PRD1 and from ?? = 0.059 to 0.75 over ?? = 0-0.32 for the oocysts. Increasing the pH from 5.7 to 10.0 resulted in a decrease in the oocyst collision efficiency as the pH exceeded the expected point of zero charge of the ferric oxyhydroxide coatings. The collision efficiencies correlated very well with the fraction of quartz sand coated by the ferric oxyhydroxide for PRD1 but not as well for the oocysts. ?? 2005 American Chemical Society.

Engineered M13 bacteriophage nanocarriers for intracellular delivery of exogenous proteins to human prostate cancer cells.

PubMed

DePorter, Sandra M; McNaughton, Brian R

2014-09-17

The size, well-defined structure, and relatively high folding energies of most proteins allow them to recognize disease-relevant receptors that present a challenge to small molecule reagents. While multiple challenges must be overcome in order to fully exploit the use of protein reagents in basic research and medicine, perhaps the greatest challenge is their intracellular delivery to a particular diseased cell. Here, we describe the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nanocarriers for the intracellular delivery of functional exogenous proteins to a human prostate cancer cell line.

Methods for Bacteriophage Preservation.

PubMed

Łobocka, Małgorzata B; Głowacka, Aleksandra; Golec, Piotr

2018-01-01

In a view of growing interest in bacteriophages as the most abundant members of microbial communities and as antibacterial agents, reliable methods for bacteriophage long-term preservation, that warrant the access to original or mutant stocks of unchanged properties, have become of crucial importance. A storage method that retains the infectivity of any kind of bacteriophage virions, either in a cell lysate or in a purified suspension, does not exist, due to the enormous diversity of bacteriophages and hence the differentiation of their sensitivity to various storage conditions. Here, we describe a method of long-term bacteriophage preservation, which is based on freezing of freshly infected susceptible bacteria at early stages of bacteriophage development. The infected bacteria release mature bacteriophages upon melting enabling the recovery of bacteriophage virions with high efficiency. The only limitation of this method is the sensitivity of bacteriophage host to deep-freezing, and thus it can be used for the long-term preservation of the vast majority of bacteriophages.

[Advance on genome research of Yersinia pestis bacteriophage].

PubMed

Tan, H L; Wang, P; Li, W

2017-04-10

Completion of the genome sequences on Yersinia pestis bacteriophage offered unprecedented opportunity for researchers to carry out related genomic studies. This review was based on the genomic sequences and provided a genomic perspective in describing the essential features of genome on Yersinia pestis bacteriophage. Based on the comparative genomics, genetic evolutionary relationship was discussed. Description of functions from the gene prediction and protein annotation provided evidence for further related studies.

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

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

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

Fokine, Andrei; Bowman, Valorie D.; Battisti, Anthony J.

2007-10-25

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

Genomic diversity of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum.

PubMed

Castillo, Daniel; Middelboe, Mathias

2016-12-01

Bacteriophages infecting the fish pathogen Flavobacterium psychrophilum can potentially be used to prevent and control outbreaks of this bacterium in salmonid aquaculture. However, the application of bacteriophages in disease control requires detailed knowledge on their genetic composition. To explore the diversity of F. pyschrophilum bacteriophages, we have analyzed the complete genome sequences of 17 phages isolated from two distant geographic areas (Denmark and Chile), including the previously characterized temperate bacteriophage 6H. Phage genome size ranged from 39 302 to 89 010 bp with a G+C content of 27%-32%. None of the bacteriophages isolated in Denmark contained genes associated with lysogeny, whereas the Chilean isolates were all putative temperate phages and similar to bacteriophage 6H. Comparative genome analysis showed that phages grouped in three different genetic clusters based on genetic composition and gene content, indicating a limited genetic diversity of F. psychrophilum-specific bacteriophages. However, amino acid sequence dissimilarity (25%) was found in putative structural proteins, which could be related to the host specificity determinants. This study represents the first analysis of genomic diversity and composition among bacteriophages infecting the fish pathogen F. psychrophilum and discusses the implications for the application of phages in disease control. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A protein coated piezoelectric crystal detector

NASA Astrophysics Data System (ADS)

Suleiman, Ahmad; Pender, Marie; Ngeh-Ngwainbi, Jerome; Lubrano, Glenn; Guilbault, George

1990-05-01

The purpose of this project was to develop a protein coated, portable piezoelectric crystal detector for organophosphorus compounds. The performance of acetylcholinesterase, GD-1 anti-soman, anti-DMMP antibody, and bovine serum albumin (BSA) coatings was evaluated. Different immobilization methods were also tested. The responses obtained with the protein coatings immobilized via cross-linking with glutaraldehyde were acceptable, provided that the reference crystal was coated with dextran. The proposed coatings showed good stability and reasonable lifetimes that ranged from approximately three weeks in the case of the antibody coatings to several months in the case of BSA. Although moisture, gasoline, and sulfur are potential interferents, their effects on the sensor were eliminated by using a sodium sulfate scrubber which did not affect the performance of the detector towards organophosphates. A small, battery operated portable instrument capable of real time measurements with alarm function was produced. The instrument can be used in a wide range of applications, depending on the coatings applied to the crystals.

Bacteriophage Mediated Killing of Staphylococcus aureus In Vitro on Orthopaedic K Wires in Presence of Linezolid Prevents Implant Colonization

PubMed Central

Kaur, Sandeep; Harjai, Kusum; Chhibber, Sanjay

2014-01-01

Background Infections of bone and joint tissues following arthroplasty surgeries remain a major challenge in orthopaedic settings. Methicillin resistant Staphylococcus aureus (MRSA) is recognised as an established pathogen in such infections. Combination therapy using linezolid and bacteriophage impregnated in biopolymer was investigated in the present study as an alternative strategy to prevent MRSA colonisation on the orthopaedic implant surface. Methodology Coating of stainless steel orthopaedic grade K-wires was achieved using hydroxypropylmethlycellulose (HPMC) mixed with phage alone, linezolid alone and phage and linezolid together. The potential of these agents to inhibit adhesion of S.aureus (MRSA) 43300 on K-wires was assessed. Coated and naked wires were analysed by scanning electron microscopy (SEM) and fluorescent staining. Result Significant reduction in bacterial adhesion was achieved on phage/linezolid wires in comparison to naked as well as HPMC coated wires. However, maximum reduction in bacterial adherence (∼4 log cycles) was observed on the wires coated with phage-linezolid combination. The frequency of emergence of resistant mutants was also negligible in presence of both the agents. Conclusion This study provides evidence to confirm that local delivery system employing linezolid (a potent protein synthesis inhibitor) along with a broad spectrum lytic bacteriophage (capable of self-multiplication) is able to attack the adhered as well as surrounding bacteria present near the implant site. Unlike other antibiotic based therapies, this combination has the potential to significantly restrict the emergence of resistant mutants, thus paving the way for effective treatment of MRSA associated infection of medical implants. PMID:24594764

A general insert label for peptide display on chimeric filamentous bacteriophages.

PubMed

Kaplan, Gilad; Gershoni, Jonathan M

2012-01-01

The foreign insert intended to be displayed via recombinant phage proteins can have a negative effect on protein expression and phage assembly. A typical example is the case of display of peptides longer than 6 amino acid residues on the major coat protein, protein VIII of the filamentous bacteriophages M13 and fd. A solution to this problem has been the use of "two-gene systems" generating chimeric phages that concomitantly express wild-type protein VIII along with recombinant protein VIII. Although the two-gene systems are much more permissive in regard to insert length and composition, some cases can still adversely affect phage assembly. Although these phages genotypically contain the desired DNA of the insert, they appear to be phenotypically wild type. To avoid false-negative results when using chimeric phages in binding studies, it is necessary to confirm that the observed lack of phage recognition is not due to faulty assembly and display of the intended insert. Here we describe a strategy for generating antibodies that specifically recognize recombinant protein VIII regardless of the nature of its foreign insert. These antibodies can be used as a general monitor of the display of recombinant protein VIII into phage particles. Copyright © 2011 Elsevier Inc. All rights reserved.

Bacteriophage P23-77 Capsid Protein Structures Reveal the Archetype of an Ancient Branch from a Major Virus Lineage

PubMed Central

Rissanen, Ilona; Grimes, Jonathan M.; Pawlowski, Alice; Mäntynen, Sari; Harlos, Karl; Bamford, Jaana K.H.; Stuart, David I.

2013-01-01

Summary It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor. PMID:23623731

Mechanisms of inactivation of bacteriophage phiX174 and its DNA in aerosols by ozone and ozonized cyclohexene.

PubMed Central

de Mik, G.; de Groot, I.

1977-01-01

The mechanisms of inactivation of aerosolized bacteriophage phiX174 in atmospheres containing ozone, cyclohexene, or ozonized cyclohexene were studied by using 32P-labelled phage. The inactivation of the aerosolized phage in clear air or in air containing cyclohexene is due to damage of the protein coat since the deoxyribonucleic acid (DNA) extracted from the inactivated phage retains its biological activity. Inactivation of the phage in air containing ozonized cyclohexene is due both to protein and DNA damage. Sucrose gradient analysis shows that aerosolized inactivated phiX174 releases unbroken DNA. In contrast, the DNA from phage phiX174 inactivated by ozonized cyclohexene is broken. The inactivation of aerosolized phage phiX174-DNA was studied in the same atmospheres using 32P-labelled DNA. phiX174-DNA aerosolized in clear air or air containing cyclohexene at 75% r.h. is inactivated by a factor of 2 in 30 min. The inactivated DNA is broken. Ozone as well as ozonized cyclohexene inactivates KNA very fast causing breaks in the molecule. This is in contrast with the intact bacteriophage in which ozone does not produce breaks in the DNA. PMID:265342

Simultaneous display of two large proteins on the head and tail of bacteriophage lambda

PubMed Central

2013-01-01

Background Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA. Results In the present work we describe an original method for dual display of large proteins on the surface of lambda particles. An anti-CEA single-chain antibody fragment and green fluorescent protein or alkaline phosphatase were simultaneously displayed by engineering both gpD and gpV lambda proteins. Conclusions Here we show that such modified phage particles can be used for the detection of target molecules in vitro and in vivo. Dual expression of functional moieties on the surface of the lambda phage might open the way to generation of a new class of diagnostic and therapeutic targeted nanoparticles. PMID:24073829

Simultaneous display of two large proteins on the head and tail of bacteriophage lambda.

PubMed

Pavoni, Emiliano; Vaccaro, Paola; D'Alessio, Valeria; De Santis, Rita; Minenkova, Olga

2013-09-30

Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA. In the present work we describe an original method for dual display of large proteins on the surface of lambda particles. An anti-CEA single-chain antibody fragment and green fluorescent protein or alkaline phosphatase were simultaneously displayed by engineering both gpD and gpV lambda proteins. Here we show that such modified phage particles can be used for the detection of target molecules in vitro and in vivo. Dual expression of functional moieties on the surface of the lambda phage might open the way to generation of a new class of diagnostic and therapeutic targeted nanoparticles.

Hyperexpansion of RNA Bacteriophage Diversity

PubMed Central

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

2016-01-01

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

Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

PubMed

Munoz, L; Sadaie, Y; Doi, R H

1978-10-10

The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.

Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions.

PubMed

Kang, Yu; Gohlke, Ulrich; Engström, Olof; Hamark, Christoffer; Scheidt, Tom; Kunstmann, Sonja; Heinemann, Udo; Widmalm, Göran; Santer, Mark; Barbirz, Stefanie

2016-07-27

Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system.

Bacteriophage T4 capsid packaging and unpackaging of DNA and proteins.

PubMed

Mullaney, Julienne M; Black, Lindsay W

2014-01-01

Bacteriophage T4 has proven itself readily amenable to phage-based DNA and protein packaging, expression, and display systems due to its physical resiliency and genomic flexibility. As a large dsDNA phage with dispensable internal proteins and dispensable outer capsid proteins it can be adapted to package both DNA and proteins of interest within the capsid and to display peptides and proteins externally on the capsid. A single 170 kb linear DNA, or single or multiple copies of shorter linear DNAs, of any sequence can be packaged by the large terminase subunit in vitro into protein-containing proheads and give full or partially full capsids. The prohead receptacles for DNA packaging can also display peptides or full-length proteins from capsid display proteins HOC and SOC. Our laboratory has also developed a protein expression, packaging, and processing (PEPP) system which we have found to have advantages over mammalian and bacterial cell systems, including high yield, increased stability, and simplified downstream processing. Proteins that we have produced by the phage PEPP platform include human HIV-1 protease, micrococcal endonuclease from Staphylococcus aureus, restriction endonuclease EcoRI, luciferase, human granulocyte colony stimulating factor (GCSF), green fluorescent protein (GFP), and the 99 amino acid C-terminus of amyloid precursor protein (APP). Difficult to produce proteins that are toxic in mammalian protein expression systems are easily produced, packaged, and processed with the PEPP platform. APP is one example of such a highly refractory protein that has been produced successfully. The methods below describe the procedures for in vitro packaging of proheads with DNA and for producing recombinant T4 phage that carry a gene of interest in the phage genome and produce and internally package the corresponding protein of interest.

Computational Determination of the Effects of Bacteriophage Bacteriophage Interactions in Human body.

PubMed

Mostafa, Marwa Mostafa; Nassef, Mohammad; Badr, Amr

2017-10-19

Chronic diseases are becoming more serious and widely spreading and this carries a heavy burden on doctors to deal with such patients. Although many of these diseases can be treated by bacteriophages, the situation is significantly dangerous in patients having concomitant more than one chronic disease, where conflicts between phages used in treating these diseases are very closer to happen. This research paper presents a method to detecting the Bacteriophage-Bacteriophage Interaction. This method is implemented based on Domain-Domain Interactions model and it was used to infer Domain-Domain Interactions between the bacteriophages injected in the human body at the same time. By testing the method over bacteriophages that are used to treat tuberculosis, salmonella and virulent E.coli, many interactions have been inferred and detected between these bacteriophages. Several effects were detected for the resulted interactions such as: playing a role in DNA repair such as non-homologous end joining, playing a role in DNA replication, playing a role in the interaction between the immune system and the tumor cells and playing a role in the stiff man syndrome. We revised all patents relating to bacteriophage bacteriophage interactions and phage therapy. The proposed method is developed to help doctors to realize the effect of simultaneously injecting different bacteriophages into the human body to treat different diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates.

PubMed

Benjamin, Christopher J; Wright, Kyle J; Hyun, Seok-Hee; Krynski, Kyle; Yu, Guimei; Bajaj, Ruchika; Guo, Fei; Stauffacher, Cynthia V; Jiang, Wen; Thompson, David H

2016-01-19

We report the preparation and performance of TEM grids bearing stabilized nonfouling lipid monolayer coatings. These films contain NTA capture ligands of controllable areal density at the distal end of a flexible poly(ethylene glycol) 2000 (PEG2000) spacer to avoid preferred orientation of surface-bound histidine-tagged (His-tag) protein targets. Langmuir-Schaefer deposition at 30 mN/m of mixed monolayers containing two novel synthetic lipids-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(5-amido-1-carboxypentyl)iminodiacetic acid]polyethylene glycolamide 2000) (NTA-PEG2000-DSPE) and 1,2-(tricosa-10',12'-diynoyl)-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycolamide 350) (mPEG350-DTPE)-in 1:99 and 5:95 molar ratios prior to treatment with a 5 min, 254 nm light exposure was used for grid fabrication. These conditions were designed to limit nonspecific protein adsorption onto the stabilized lipid coating by favoring the formation of a mPEG350 brush layer below a flexible, mushroom conformation of NTA-PEG2000 at low surface density to enable specific immobilization and random orientation of the protein target on the EM grid. These grids were then used to capture His6-T7 bacteriophage and RplL from cell lysates, as well as purified His8-green fluorescent protein (GFP) and nanodisc solubilized maltose transporter, His6-MalFGK2. Our findings indicate that TEM grid supported, polymerized NTA lipid monolayers are capable of capturing His-tag protein targets in a manner that controls their areal densities, while efficiently blocking nonspecific adsorption and limiting film degradation, even upon prolonged detergent exposure.

Photodynamic inactivation of antigenic determinants of single-stranded DNA bacteriophage phiX174

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

Khan, N.C.; Poddar, R.K.

1974-05-01

Bacteriophage phi X174 when photodynamically inactivated (i.e., when rendered unable to produce plaques as a result of exposure to visible light in air in the presence of proflavine) progressively lost their capacity to bind efficiently with homologous antiserum. Such loss of serum-blocking power was evident with heat-inactivated but not with uv-irradiated phage. The ability of the phages to adsorb to host cells, however, remained practically unaltered even after photodynamic inactivation. It thus appears that photodynamic damages in the so-called ''jacket'' component of the phi X174 coat proteins are partly responsible for the loss of plaque-forming ability, whereas the ''spikes'' aremore » either poor antigens or insensitive to photodynamic treatment. (auth)« less

Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

PubMed

Yu, Xiaohua; Biedrzycki, Adam H; Khalil, Andrew S; Hess, Dalton; Umhoefer, Jennifer M; Markel, Mark D; Murphy, William L

2017-09-01

Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacteriophages and Biofilms

PubMed Central

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

2014-01-01

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

Two New Lytic Bacteriophages of the Myoviridae Family Against Carbapenem-Resistant Acinetobacter baumannii

PubMed Central

Zhou, Weilong; Feng, Yu; Zong, Zhiyong

2018-01-01

Two lytic bacteriophages, WCHABP1 and WCHABP12, were recovered from hospital sewage and were able to infect 9 and 12 out of 18 carbapenem-resistant Acinetobacter baumannii clinical strains, which belonged to different clones. Electron microscopy scan showed that both bacteriophages had the similar morphology as those of the Myoviridae family. Whole genomic sequencing revealed 45.4- or 45.8-kb genome with a 37.6% GC content for WCHABP1 and WCHABP12, both of which showed significant DNA sequence similarity with bacteriophages of the Ap22virus genus within the Myoviridae family. Taxonomic analysis was therefore performed following the proposal approved by the International Committee on Taxonomy of Viruses, which confirmed that WCHABP1 and WCHABP12 represented two new species of the Ap22virus genus. No tRNAs but 88 and 89 open reading frames (ORFs) were predicted for the two bacteriophages, among which 22 and 21 had known function and encoded proteins for morphogenesis, packaging, lysis, and nucleiotide metabolism. The C-terminal amino acids of the large unit of fiber tail proteins varied between the bacteriophages, which may explain their different host ranges. For most lytic bacteriophages, a set of holin and endolysin are required for lysis. However, no known holin-encoding genes were identified in WCHABP1 and WCHABP12, suggesting that they may use alternative, yet-to-be-identified, novel holins for host cell membrane lysis. To test the efficacy of the bacteriophages in protecting against A. baumannii infection, a Galleria mellonella larva model was used. Only <20% G. mellonella larvae survived at 96 h after being infected by carbapenem-resistant A. baumannii strains, from which the two bacteriophages were recovered. With the administration of WCHABP1 and WCHABP12, the survival of larvae increased to 75%, while the treatment of polymyxin B only slightly increased the survival rate to 25%. The isolation of two new lytic bacteriophages in this study could expand our

Stabilization of T4 bacteriophage at acidic and basic pH by adsorption on paper.

PubMed

Meyer, Abigail; Greene, Melissa; Kimmelshue, Chad; Cademartiri, Rebecca

2017-12-01

Bacteriophages find applications in agriculture, medicine, and food safety. Many of these applications can expose bacteriophages to stresses that inactivate them including acidic and basic pH. Bacteriophages can be stabilized against these stresses by materials including paper, a common material in packaging and consumer products. Combining paper and bacteriophages creates antibacterial materials, which can reduce the use of antibiotics. Here we show that adsorption on paper protects T4, T5, and T7 bacteriophage from acidic and basic pH. We added bacteriophages to filter paper functionalized with carboxylic acid (carboxyl methyl cellulose) or amine (chitosan) groups, and exposed them to pH from 5.6 to 14. We determined the number of infective bacteriophages after exposure directly on the paper. All papers extended the lifetime of infective bacteriophage by at least a factor of four with some papers stabilizing bacteriophages for up to one week. The degree of stabilization depended on five main factors (i) the family of the bacteriophage, (ii) the charge of the paper and bacteriophages, (iii) the location of the bacteriophages within the paper, (iv) the ability of the paper to prevent bacteriophage-bacteriophage aggregation, and (v) the sensitivity of the bacteriophage proteins to the tested pH. Even when adsorbed on paper the bacteriophages were able to remove E. coli in milk. Choosing the right paper modification or material will protect bacteriophages adsorbed on that material against detrimental pH and other environmental challenges increasing the range of applications of bacteriophages on materials. Copyright © 2017 Elsevier B.V. All rights reserved.

Comprehensive mutational analysis of the M13 major coat protein: improved scaffolds for C-terminal phage display.

PubMed

Held, Heike A; Sidhu, Sachdev S

2004-07-09

A peptide was fused to the C terminus of the M13 bacteriophage major coat protein (P8), and libraries of P8 mutants were screened to select for variants that displayed the peptide with high efficiency. Over 600 variants were sequenced to compile a comprehensive database of P8 sequence diversity compatible with assembly into the wild-type phage coat. The database reveals that, while the alpha-helical P8 molecule was highly tolerant to mutations, certain functional epitopes were required for efficient incorporation. Three hydrophobic epitopes were located approximately equidistantly along the length of the alpha-helix. In addition, a positively charged epitope was required directly opposite the most C-terminal hydrophobic epitope and on the same side as the other two epitopes. Both ends of the protein were highly tolerant to mutations, consistent with the use of P8 as a scaffold for both N and C-terminal phage display. Further rounds of selection were used to enrich for P8 variants that supported higher levels of C-terminal peptide display. The largest improvements in display resulted from mutations around the junction between P8 and the C-terminal linker, and additional mutations in the N-terminal region were selected for further improvements in display. The best P8 variants improved C-terminal display more than 100-fold relative to the wild-type, and these variants could support the simultaneous display of N and C-terminal fusions. These finding provide information on the requirements for filamentous phage coat assembly, and provide improved scaffolds for phage display technology. Copyright 2004 Elsevier Ltd.

Protein-Coated Microcrystals, Combi-Protein-Coated Microcrystals, and Cross-Linked Protein-Coated Microcrystals of Enzymes for Use in Low-Water Media.

PubMed

Mukherjee, Joyeeta; Gupta, Munishwar N

2017-01-01

Protein-coated microcrystals (PCMC) are a high-activity preparation of enzymes for use in low-water media. The protocols for the preparation of PCMCs of Subtilisin Carlsberg and Candida antarctica lipase B (CAL B) are described. The combi-PCMC concept is useful both for cascade and non-cascade reactions. It can also be beneficial to combine two different specificities of a lipase when the substrate requires it. Combi-PCMC of CALB and Palatase used for the conversion of coffee oil present in spent coffee grounds to biodiesel is described. Cross-linked protein-coated microcrystals (CL-PCMC) in some cases can give better results than PCMC. Protocols for the CLPCMC of Subtilisin Carlsberg and Candida antarctica lipase B (CAL B) are described. A discussion of their applications is also provided.

[POSSIBILITIES OF APPLICATION OF MALDI-TOF MASS-SPECTROMETRY FOR STUDY OF CARBOHYDRATE-SPECIFIC RECEPTORS FOR DIAGNOSTIC BACTERIOPHAGE EL TOR].

PubMed

Telesmanich, N R; Goncharenko, E V; Chaika, S O; Chaika, I A; Telicheva, V O

2016-01-01

Study mechanisms of interaction of diagnostic bacteriophage El Tor with sensitive strain Vibrio cholerae El Tor 18507 using direct protein profiling, identification of constant and variable proteins, taking part in interaction of the phage and cell, as well as carbohydrate-specific phage receptors. . A commercial preparation of cholera diagnostic bacteriophage El Tor, strain V. cholerae El Tor 18507 were used. Effect of carbohydrates on bacteriophage activity was determined in experiments with phage by a classic and modified by us method. Protein profiles of the studied objects were studied using MSP-analysis method. Sucrose was shown to inhibit lytic activity of bacteriophage. Proteome profiles of El Tor bacteriophage and sensitive indicator strains were studied, identification of constant and variable proteins of the studied objects by MSP Peak-list program was carried out. Analysis of changes of profiles of phage and microbial cell during interaction with sucrose gave a basis for assuming, that sucrose in the mixture of culture-phage enters interaction namely with phage protein receptors, blocking receptors specific for cholera vibrio, that subsequently manifests in a sharp decrease of phage activity against the sensitive strain.

Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold

PubMed Central

Henry, Kevin A.; Arbabi-Ghahroudi, Mehdi; Scott, Jamie K.

2015-01-01

For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. PMID:26300850

Interaction of Bacteriophages with the Immune System: Induction of Bacteriophage-Specific Antibodies.

PubMed

Dąbrowska, Krystyna

2018-01-01

In all cases when a bacteriophage makes direct contact with a mammalian organism, it may challenge the mammalian immunological system. Its major consequence is production of antibodies specific to the bacteriophage. Here we present protocols applicable in studies of bacteriophage ability to induce specific antibodies. The protocols have been divided into three parts: purification, immunization, and detection (ELISA).

Evaluation of the factors controlling the time-dependent inactivation rate coefficients of bacteriophage MS2 and PRD1

USGS Publications Warehouse

Anders, R.; Chrysikopoulos, C.V.

2006-01-01

Static and dynamic batch experiments were conducted to study the effects of temperature and the presence of sand on the inactivation of bacteriophage MS2 and PRD1. The experimental data suggested that the inactivation process can be satisfactorily represented by a pseudo-first-order expression with time-dependent rate coefficients. The time-dependent rate coefficients were used to determine pertinent thermodynamic properties required for the analysis of the molecular processes involved in the inactivation of each bacteriophage. A combination of high temperature and the presence of sand appears to produce the greatest disruption to the surrounding protein coat of MS2. However, the lower activation energies for PRD1 indicate a weaker dependence of the inactivation rate on temperature. Instead, the presence of air-liquid and air-solid interfaces appears to produce the greatest damage to specific viral components that are related to infection. These results indicate the importance of using thermodynamic parameters based on the time-dependent inactivation model to better predict the inactivation of viruses in groundwater. ?? 2006 American Chemical Society.

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

PubMed Central

Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F.

2017-01-01

ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. PMID:29233895

Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6.

PubMed

Makeyev, E V; Bamford, D H

2000-01-04

In nature, synthesis of both minus- and plus-sense RNA strands of all the known double-stranded RNA viruses occurs in the interior of a large protein assembly referred to as the polymerase complex. In addition to other proteins, the complex contains a putative polymerase possessing characteristic sequence motifs. However, none of the previous studies has shown template-dependent RNA synthesis directly with an isolated putative polymerase protein. In this report, recombinant protein P2 of double-stranded RNA bacteriophage phi6 was purified and demonstrated in an in vitro enzymatic assay to act as the replicase. The enzyme efficiently utilizes phage-specific, positive-sense RNA substrates to produce double-stranded RNA molecules, which are formed by newly synthesized, full-length minus-strands base paired with the plus-strand templates. P2-catalyzed replication is also shown to be very effective with a broad range of heterologous single-stranded RNA templates. The importance and implications of these results are discussed.

M13 bacteriophage-activated superparamagnetic beads for affinity separation.

PubMed

Muzard, Julien; Platt, Mark; Lee, Gil U

2012-08-06

The growth of the biopharmaceutical industry has created a demand for new technologies for the purification of genetically engineered proteins.The efficiency of large-scale, high-gradient magnetic fishing could be improved if magnetic particles offering higher binding capacity and magnetization were available. This article describes several strategies for synthesizing microbeads that are composed of a M13 bacteriophage layer assembled on a superparamagnetic core. Chemical cross-linking of the pVIII proteins to a carboxyl-functionalized bead produces highly responsive superparamagnetic particles (SPM) with a side-on oriented, adherent virus monolayer. Also, the genetic manipulation of the pIII proteins with a His(6) peptide sequence allows reversible assembly of the bacteriophage on a nitrilotriacetic-acid-functionalized core in an end-on configuration. These phage-magnetic particles are successfully used to separate antibodies from high-protein concentration solutions in a single step with a >90% purity. The dense magnetic core of these particles makes them five times more responsive to magnetic fields than commercial materials composed of polymer-(iron oxide) composites and a monolayer of phage could produce a 1000 fold higher antibody binding capacity. These new bionanomaterials appear to be well-suited to large-scale high-gradient magnetic fishing separation and promise to be cost effective as a result of the self-assembling and self-replicating properties of genetically engineered M13 bacteriophage. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lytic bacteriophages

PubMed Central

Sharma, Manan

2013-01-01

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

[RATIONAL ASPECTS OF BACTERIOPHAGES USE].

PubMed

Vakarina, A A; Kataeva, L V; Karpukhina, N F

2015-01-01

Analysis of existing aspects of bacteriophage use and study features of their lytic activity by using various techniques. Effect of monophages and associated bacteriophages (staphylococci, piopolyvalent and piocombined, intestiphage, pneumonia klebsiella and polyvalent klebsiella produced by "Microgen") was studied with 380 strains of Staphylococcus aureus and 279 cultures of Klebsiella pneumoniae in liquid and solid nutrient media. From patients with intestinal disorder, sensitivity was analyzed to 184 strains of Salmonella genus bacteria 18 serological variants to salmonella bacteriophages, 137 strains of Escherichia coli (lactose-negative, hemolytic), as well as some members of OKA groups (21 serovars) to coli-proteic and piopolyvalent bacteriophages. Lytic ability of the piobacteriophage against Klebsiella and Proteus genus bacteria was determined. Staphylococcus aureus was sensitive to staphylococcus bacteriophage in 71.6% of cases and to piobacteriophage--in 86.15% of cases. A 100% lytic ability of salmonella bacteriophage against Salmonella spp. was established. Sensitivity of E. coli of various serogroups to coli-proteic and piobacteriophage was 66 - 100%. Klebsiella, Proteus genus bacteria were sensitive to piobacteriophage in only 35% and 43.15% of cases, respectively. A more rational use of bacteriophages is necessary: development of a technique, evaluation of sensitivity of bacteria to bacteriophage, introduction of corrections into their production (expansion of bacteriophage spectra, determination and indication of their concentration in accompanying documents).

Convection-enhanced delivery of M13 bacteriophage to the brain

PubMed Central

Ksendzovsky, Alexander; Walbridge, Stuart; Saunders, Richard C.; Asthagiri, Ashok R.; Heiss, John D.; Lonser, Russell R.

2013-01-01

Object Recent studies indicate that M13 bacteriophage, a very large nanoparticle, binds to β-amyloid and α-synuclein proteins, leading to plaque disaggregation in models of Alzheimer and Parkinson disease. To determine the feasibility, safety, and characteristics of convection-enhanced delivery (CED) of M13 bacteriophage to the brain, the authors perfused primate brains with bacteriophage. Methods Four nonhuman primates underwent CED of M13 bacteriophage (900 nm) to thalamic gray matter (4 infusions) and frontal white matter (3 infusions). Bacteriophage was coinfused with Gd-DTPA (1 mM), and serial MRI studies were performed during infusion. Animals were monitored for neurological deficits and were killed 3 days after infusion. Tissues were analyzed for bacteriophage distribution. Results Real-time T1-weighted MRI studies of coinfused Gd-DTPA during infusion demonstrated a discrete region of perfusion in both thalamic gray and frontal white matter. An MRI-volumetric analysis revealed that the mean volume of distribution (Vd) to volume of infusion (Vi) ratio of M13 bacteriophage was 2.3 ± 0.2 in gray matter and 1.9 ± 0.3 in white matter. The mean values are expressed ± SD. Immunohistochemical analysis demonstrated mean Vd:Vi ratios of 2.9 ± 0.2 in gray matter and 2.1 ± 0.3 in white matter. The Gd-DTPA accurately tracked M13 bacteriophage distribution (the mean difference between imaging and actual bacteriophage Vd was insignificant [p > 0.05], and was −2.2% ± 9.9% in thalamic gray matter and 9.1% ± 9.5% in frontal white matter). Immunohistochemical analysis revealed evidence of additional spread from the initial delivery site in white matter (mean Vd:Vi, 16.1 ± 9.1). All animals remained neurologically intact after infusion during the observation period, and histological studies revealed no evidence of toxicity. Conclusions The CED method can be used successfully and safely to distribute M13 bacteriophage in the brain. Furthermore, additional white

Convection-enhanced delivery of M13 bacteriophage to the brain.

PubMed

Ksendzovsky, Alexander; Walbridge, Stuart; Saunders, Richard C; Asthagiri, Ashok R; Heiss, John D; Lonser, Russell R

2012-08-01

Recent studies indicate that M13 bacteriophage, a very large nanoparticle, binds to β-amyloid and α-synuclein proteins, leading to plaque disaggregation in models of Alzheimer and Parkinson disease. To determine the feasibility, safety, and characteristics of convection-enhanced delivery (CED) of M13 bacteriophage to the brain, the authors perfused primate brains with bacteriophage. Four nonhuman primates underwent CED of M13 bacteriophage (900 nm) to thalamic gray matter (4 infusions) and frontal white matter (3 infusions). Bacteriophage was coinfused with Gd-DTPA (1 mM), and serial MRI studies were performed during infusion. Animals were monitored for neurological deficits and were killed 3 days after infusion. Tissues were analyzed for bacteriophage distribution. Real-time T1-weighted MRI studies of coinfused Gd-DTPA during infusion demonstrated a discrete region of perfusion in both thalamic gray and frontal white matter. An MRI-volumetric analysis revealed that the mean volume of distribution (Vd) to volume of infusion (Vi) ratio of M13 bacteriophage was 2.3 ± 0.2 in gray matter and 1.9 ± 0.3 in white matter. The mean values are expressed ± SD. Immunohistochemical analysis demonstrated mean Vd:Vi ratios of 2.9 ± 0.2 in gray matter and 2.1 ± 0.3 in white matter. The Gd-DTPA accurately tracked M13 bacteriophage distribution (the mean difference between imaging and actual bacteriophage Vd was insignificant [p > 0.05], and was -2.2% ± 9.9% in thalamic gray matter and 9.1% ± 9.5% in frontal white matter). Immunohistochemical analysis revealed evidence of additional spread from the initial delivery site in white matter (mean Vd:Vi, 16.1 ± 9.1). All animals remained neurologically intact after infusion during the observation period, and histological studies revealed no evidence of toxicity. The CED method can be used successfully and safely to distribute M13 bacteriophage in the brain. Furthermore, additional white matter spread after infusion cessation

Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage ϕ29

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

Badasso, Mohammed O., E-mail: badas001@umn.edu; Anderson, Dwight L.; Department of Oral Science, University of Minnesota, Minneapolis, MN 55455

2005-04-01

ϕ29 bacteriophage scaffolding protein (gp7) has been overproduced in E. coli, purified, crystallized and characterized by X-ray diffraction. Two distinct crystal forms were obtained and a diffraction data set was collected to 1.8 Å resolution. The Bacillus subtilis bacteriophage ϕ29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 77.13, c = 37.12 Å.more » Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 Å. Complete data sets have been collected to 1.78 and 1.80 Å for forms I and II, respectively, at 100 K using Cu Kα X-rays from a rotating-anode generator. Calculation of a V{sub M} value of 2.46 Å{sup 3} Da{sup −1} for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a V{sub M} of 4.80 Å{sup 3} Da{sup −1} with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.« less

Bacteriophages Infecting Propionibacterium acnes

PubMed Central

2013-01-01

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

Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein.

PubMed

Urban, Johannes H; Moosmeier, Markus A; Aumüller, Tobias; Thein, Marcus; Bosma, Tjibbe; Rink, Rick; Groth, Katharina; Zulley, Moritz; Siegers, Katja; Tissot, Kathrin; Moll, Gert N; Prassler, Josef

2017-11-15

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.

Enhanced protein adsorption and patterning on nanostructured latex-coated paper.

PubMed

Juvonen, Helka; Määttänen, Anni; Ihalainen, Petri; Viitala, Tapani; Sarfraz, Jawad; Peltonen, Jouko

2014-06-01

Specific interactions of extracellular matrix proteins with cells and their adhesion to the substrate are important for cell growth. A nanopatterned latex-coated paper substrate previously shown to be an excellent substrate for cell adhesion and 2D growth was studied for directed immobilization of proteins. The nanostructured latex surface was formed by short-wavelength IR irradiation of a two-component latex coating consisting of a hydrophilic film-forming styrene butadiene acrylonitrile copolymer and hydrophobic polystyrene particles. The hydrophobic regions of the IR-treated latex coating showed strong adhesion of bovine serum albumin (cell repelling protein), fibronectin (cell adhesive protein) and streptavidin. Opposite to the IR-treated surface, fibronectin and streptavidin had a poor affinity toward the untreated pristine latex coating. Detailed characterization of the physicochemical surface properties of the latex-coated substrates revealed that the observed differences in protein affinity were mainly due to the presence or absence of the protein repelling polar and charged surface groups. The protein adsorption was assisted by hydrophobic (dehydration) interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

STUDIES ON THE BACTERIOPHAGE OF D'HÉRELLE

PubMed Central

Hetler, D. M.; Bronfenbrenner, J.

1928-01-01

1. During the process of lysis by bacteriophage, there is an appreciable increase in the amount of free amino acid present in the culture. 2. The increase of free amino acid is due to hydrolysis of bacterial protein. PMID:19869482

Toward modern inhalational bacteriophage therapy: nebulization of bacteriophages of Burkholderia cepacia complex.

PubMed

Golshahi, Laleh; Seed, Kimberley D; Dennis, Jonathan J; Finlay, Warren H

2008-12-01

Antibiotic-resistant bacterial infections have renewed interest in finding substitute methods of treatment. The purpose of the present in vitro study was to investigate the possibility of respiratory delivery of a Burkholderia cepacia complex (BCC) bacteriophage by nebulized aerosol administration. Bacteriophages in isotonic saline were aerosolized with Pari LC star and eFlow nebulizers, at titers with mean value (standard deviation) of 2.15 x 10(8) (1.63 x 10(8)) plaque-forming unit (PFU)/mL in 2.5-mL nebulizer fills. The breathing pattern of an adult was simulated using a pulmonary waveform generator. During breath simulation, the size distributions of the nebulized aerosol were measured using phase doppler anemometry (PDA). Efficiency of nebulizer delivery was subsequently determined by collection of aerosol on low resistance filters and measurement of bacteriophage titers. These filter titers were used as input data to a mathematical lung deposition model to predict regional deposition of bacteriophages in the lung and initial bacteriophage titers in the liquid surface layer of each conducting airway generation. The results suggest that BCC bacteriophages can be nebulized successfully within a reasonable delivery time and predicted titers in the lung indicate that this method may hold potential for treatment of bacterial lung infections common among cystic fibrosis patients.

Engineered enzymatically active bacteriophages and methods of uses thereof

DOEpatents

Collins, James J [Newton, MA; Kobayashi, Hideki [Yokohama, JP; Kearn, Mads [Ottawa, CA; Araki, Michihiro [Minatoku, JP; Friedland, Ari [Boston, MA; Lu, Timothy Kuan-Ta [Palo Alto, CA

2012-05-22

The present invention provides engineered bacteriophages that express at least one biofilm degrading enzyme on their surface and uses thereof for degrading bacterial biofilms. The invention also provides genetically engineered bacteriophages expressing the biofilm degrading enzymes and proteins necessary for the phage to replicate in different naturally occurring biofilm producing bacteria. The phages of the invention allow a method of biofilm degradation by the use of one or only a few administration of the phage because the system using these phages is self perpetuating, and capable of degrading biofilm even when the concentration of bacteria within the biofilm is low.

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

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

Cardarelli, Lia; Lam, Robert; Tuite, Ashleigh

2010-08-17

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 {angstrom} crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophagemore » SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.« less

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

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

Cardarelli, Lia; Lam, Robert; Tuite, Ashleigh

2011-11-23

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophagemore » SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.« less

Bacteriophage in polar inland waters

USGS Publications Warehouse

Säwström, Christin; Lisle, John; Anesio, A.M.; Priscu, John C.; Laybourn-Parry, J.

2008-01-01

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

Exploring the interaction network of the Bacillus subtilis outer coat and crust proteins.

PubMed

Krajčíková, Daniela; Forgáč, Vladimír; Szabo, Adam; Barák, Imrich

2017-11-01

Bacillus subtilis spores, representatives of an exceptionally resistant dormant cell type, are encircled by a thick proteinaceous layer called the spore coat. More than 80 proteins assemble into four distinct coat layers: a basement layer, an inner coat, an outer coat and a crust. As the spore develops inside the mother cell, spore coat proteins synthesized in the cytoplasm are gradually deposited onto the prespore surface. A small set of morphogenetic proteins necessary for spore coat morphogenesis are thought to form a scaffold to which the rest of the coat proteins are attached. Extensive localization and proteomic studies using wild type and mutant spores have revealed the arrangement of individual proteins within the spore coat layers. In this study we examined the interactions between the proteins localized to the outer coat and crust using a bacterial two hybrid system. These two layers are composed of at least 25 components. Self-interactions were observed for most proteins and numerous novel interactions were identified. The most interesting contacts are those made with the morphogenetic proteins CotE, CotY and CotZ; these could serve as a basis for understanding the specific roles of particular proteins in spore coat morphogenesis. Copyright © 2017 Elsevier GmbH. All rights reserved.

Differential bacteriophage mortality on exposure to copper.

PubMed

Li, Jinyu; Dennehy, John J

2011-10-01

Many studies report that copper can be used to control microbial growth, including that of viruses. We determined the rates of copper-mediated inactivation for a wide range of bacteriophages. We used two methods to test the effect of copper on bacteriophage survival. One method involved placing small volumes of bacteriophage lysate on copper and stainless steel coupons. Following exposure, metal coupons were rinsed with lysogeny broth, and the resulting fluid was serially diluted and plated on agar with the corresponding bacterial host. The second method involved adding copper sulfate (CuSO(4)) to bacteriophage lysates to a final concentration of 5 mM. Aliquots were removed from the mixture, serially diluted, and plated with the appropriate bacterial host. Significant mortality was observed among the double-stranded RNA (dsRNA) bacteriophages Φ6 and Φ8, the single-stranded RNA (ssRNA) bacteriophage PP7, the ssDNA bacteriophage ΦX174, and the dsDNA bacteriophage PM2. However, the dsDNA bacteriophages PRD1, T4, and λ were relatively unaffected by copper. Interestingly, lipid-containing bacteriophages were most susceptible to copper toxicity. In addition, in the first experimental method, the pattern of bacteriophage Φ6 survival over time showed a plateau in mortality after lysates dried out. This finding suggests that copper's effect on bacteriophage is mediated by the presence of water.

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.

Peering down the barrel of a bacteriophage portal: the genome packaging and release valve in p22.

PubMed

Tang, Jinghua; Lander, Gabriel C; Olia, Adam S; Olia, Adam; Li, Rui; Casjens, Sherwood; Prevelige, Peter; Cingolani, Gino; Baker, Timothy S; Johnson, John E

2011-04-13

The encapsidated genome in all double-strand DNA bacteriophages is packaged to liquid crystalline density through a unique vertex in the procapsid assembly intermediate, which has a portal protein dodecamer in place of five coat protein subunits. The portal orchestrates DNA packaging and exit, through a series of varying interactions with the scaffolding, terminase, and closure proteins. Here, we report an asymmetric cryoEM reconstruction of the entire P22 virion at 7.8 Å resolution. X-ray crystal structure models of the full-length portal and of the portal lacking 123 residues at the C terminus in complex with gene product 4 (Δ123portal-gp4) obtained by Olia et al. (2011) were fitted into this reconstruction. The interpreted density map revealed that the 150 Å, coiled-coil, barrel portion of the portal entraps the last DNA to be packaged and suggests a mechanism for head-full DNA signaling and transient stabilization of the genome during addition of closure proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding.

PubMed

Newcomer, Rebecca L; Fraser, LaTasha C R; Teschke, Carolyn M; Alexandrescu, Andrei T

2015-12-15

The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining (3)JNC' couplings transmitted through H-bonds, the temperature and urea-concentration dependence of (1)HN and (15)N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and (3)JNC' H-bond couplings, are identified with an accuracy of 90% by (1)HN temperature coefficients. The accuracy is improved to 95% when (15)N temperature coefficients are also included. In contrast, the urea dependence of (1)HN and (15)N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding

PubMed Central

Newcomer, Rebecca L.; Fraser, LaTasha C.R.; Teschke, Carolyn M.; Alexandrescu, Andrei T.

2015-01-01

The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining 3JNC’ couplings transmitted through H-bonds, the temperature and urea-concentration dependence of 1HN and 15N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and 3JNC’ H-bond couplings, are identified with an accuracy of 90% by 1HN temperature coefficients. The accuracy is improved to 95% when 15N temperature coefficients are also included. In contrast, the urea dependence of 1HN and 15N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. PMID:26682823

[Strategies to prevent bacteriophage infection in industrial fermentation].

PubMed

Shen, Juntao; Xiu, Zhilong

2017-12-25

During the development of bacteria-based biotechnology, bacteriophage infection is one of the constant threats and troublesome problems in industrial fermentation. The core of puzzled bacteriophage infection is a complex arm race of coevolution between bacteriophages and their hosts where bacteriophage has evolved lots of escaped ways against bacterial resistance mechanisms. The strategies of rationally designing factories and rotation of starter strains could reduce the risk of bacteriophage infection, but often fail to avoid. Genetic engineering to increase bacterial resistance is one of the strategies to prevent bacteriophage infection and more knowledge about bacteriophage and its host is needed. Recently, there are some new findings on bacterial resistance mechanisms which provide new solutions for bacteriophage infection. For example, it is possible for a rational design of resistant strains to use CRISPR-Cas based technologies just based on the sequences of bacteriophages. Moreover, it is also possible to avoid the escape of bacteriophage by iteratively building up resistance levels to generate robust industrial starter cultures. Quorum-sensing signal molecules have recently been proved to be involved in the interactions between bacteria and bacteriophages, which provides a possible way to solve bacteriophage infection from a population level. Finally, the rapid development of bacteriophage genome editing and synthetic biology will bring some new cues for preventing bacteriophage infection in industrial fermentation.

Antigenic properties of HCMV peptides displayed by filamentous bacteriophages vs. synthetic peptides.

PubMed

Ulivieri, Cristina; Citro, Alessandra; Ivaldi, Federico; Mascolo, Dina; Ghittoni, Raffaella; Fanigliulo, Daniela; Manca, Fabrizio; Baldari, Cosima Tatiana; Li Pira, Giuseppina; Del Pozzo, Giovanna

2008-08-15

Several efforts have been invested in the identification of CTL and Th epitopes, as well as in the characterization of their immunodominance and MHC restriction, for the generation of a peptide-based HCMV vaccine. Small synthetic peptides are, however, poor antigens and carrier proteins are important for improving the efficacy of synthetic peptide vaccines. Recombinant bacteriophages appear as promising tools in the design of subunit vaccines. To investigate the antigenicity of peptides carried by recombinant bacteriophages we displayed different HCMV MHCII restricted peptides on the capsid of filamentous bacteriophage (fd) and found that hybrid bacteriophages are processed by human APC and activate HCMV-specific CD4 T-cells. Furthermore we constructed a reporter T-cell hybridoma expressing a chimeric TCR comprising murine alphabeta constant regions and human variable regions specific for the HLA-A2 restricted immunodominant NLV peptide of HCMV. Using the filamentous bacteriophage as an epitope carrier, we detected a more robust and long lasting response of the reporter T-cell hybridoma compared to peptide stimulation. Our results show a general enhancement of T-cell responses when antigenic peptides are carried by phages.

Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages

PubMed Central

Yang, Hongyan; Makeyev, Eugene V.; Bamford, Dennis H.

2001-01-01

The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage φ6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3′ termini from the φ6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from φ6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the φ6-related bacteriophages φ8 and φ13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from φ6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3′-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit. PMID:11602748

TF1, the bacteriophage SPO1-encoded type II DNA-binding protein, is essential for viral multiplication.

PubMed

Sayre, M H; Geiduschek, E P

1988-09-01

The lytic Bacillus subtilis bacteriophage SPO1 encodes an abundant, 99-amino-acid type II DNA-binding protein, transcription factor 1 (TF1). TF1 is special in this family of procaryotic chromatin-forming proteins in its preference for hydroxymethyluracil-containing DNA, such as SPO1 DNA, and in binding with high affinity to specific sites in the SPO1 chromosome. We constructed recessive null alleles of the TF1 gene and introduced them into SPO1 chromosomes. Segregation analysis with partially diploid phage heterozygous for TF1 showed that phage bearing only these null alleles was inviable. Deletion of the nine C-proximal amino acids of TF1 prohibited phage multiplication in vivo and abolished its site-specific DNA-binding activity in vitro.

Identification and Characterization of Arabidopsis Seed Coat Mucilage Proteins.

PubMed

Tsai, Allen Yi-Lun; Kunieda, Tadashi; Rogalski, Jason; Foster, Leonard J; Ellis, Brian E; Haughn, George W

2017-02-01

Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features. © 2017 American Society of Plant Biologists. All Rights Reserved.

Screening of Pro-Asp Sequences Exposed on Bacteriophage M13 as an Ideal Anchor for Gold Nanocubes.

PubMed

Lee, Hwa Kyoung; Lee, Yujean; Kim, Hyori; Lee, Hye-Eun; Chang, Hyejin; Nam, Ki Tae; Jeong, Dae Hong; Chung, Junho

2017-09-15

Bacteriophages are thought to be ideal vehicles for linking antibodies to nanoparticles. Here, we define the sequence of peptides exposed as a fusion protein on M13 bacteriophages to yield optimal binding of gold nanocubes and efficient bacteriophage amplification. We generated five helper bacteriophage libraries using AE(X) 2 DP, AE(X) 3 DP, AE(X) 4 DP, AE(X) 5 DP, and AE(X) 6 DP as the exposed portion of pVIII, in which X was a randomized amino acid residue encoded by the nucleotide sequence NNK. Efficient phage amplification was achievable only in the AE(X) 2 DP, AE(X) 3 DP, and AE(X) 4 DP libraries. Through biopanning with gold nanocubes, we enriched the phage clones and selected the clone with the highest fold change after enrichment. This clone displayed Pro-Asp on the surface of the bacteriophage and had amplification yields similar to those of the wild-type helper bacteriophage (VCSM13). The clone displayed even binding of gold nanocubes along its length and minimal aggregation after binding. We conclude that, for efficient amplification, the exposed pVIII amino acid length should be limited to six residues and Ala-Glu-Pro-Asp-Asp-Pro (AEPDDP) is the ideal fusion protein sequence for guaranteeing the optimal formation of a complex with gold nanocubes.

Re-directing bacterial microcompartment systems to enhance recombinant expression of lysis protein E from bacteriophage ΦX174 in Escherichia coli

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

Yung, Mimi C.; Bourguet, Feliza A.; Carpenter, Timothy S.

Recombinant expression of toxic proteins remains a challenging problem. Furthermore, one potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ΦX174 inside recombinant BMCs to enhance its expression and achieve higher yields duringmore » downstream purification.« less

Re-directing bacterial microcompartment systems to enhance recombinant expression of lysis protein E from bacteriophage ΦX174 in Escherichia coli

DOE PAGES

Yung, Mimi C.; Bourguet, Feliza A.; Carpenter, Timothy S.; ...

2017-04-26

Recombinant expression of toxic proteins remains a challenging problem. Furthermore, one potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ΦX174 inside recombinant BMCs to enhance its expression and achieve higher yields duringmore » downstream purification.« less

The Small Viral Membrane-Associated Protein P32 Is Involved in Bacteriophage PRD1 DNA Entry

PubMed Central

Grahn, A. Marika; Daugelavičius, Rimantas; Bamford, Dennis H.

2002-01-01

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32− particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior. PMID:11967303

The Bacteriophage P1 HumD Protein Is a Functional Homolog of the Prokaryotic UmuD′-Like Proteins and Facilitates SOS Mutagenesis in Escherichia coli

PubMed Central

McLenigan, Mary P.; Kulaeva, Olga I.; Ennis, Don G.; Levine, Arthur S.; Woodgate, Roger

1999-01-01

The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD′. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD′ protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD′-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD′ functions in vivo as well as examined HumD’s physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive ΔumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD′, HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD′-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo. PMID:10559166

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro.

PubMed

Cherwa, James E; Tyson, Joshua; Bedwell, Gregory J; Brooke, Dewey; Edwards, Ashton G; Dokland, Terje; Prevelige, Peter E; Fane, Bentley A

2017-01-01

During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical

Incorporation of T4 bacteriophage in electrospun fibres.

PubMed

Korehei, R; Kadla, J

2013-05-01

Antibacterial food packaging materials, such as bacteriophage-activated electrospun fibrous mats, may address concerns triggered by waves of bacterial food contamination. To address this, we investigated several efficient methods for incorporating T4 bacteriophage into electrospun fibrous mats. The incorporation of T4 bacteriophage using simple suspension electrospinning led to more than five orders of magnitude decrease in bacteriophage activity. To better maintain bacteriophage viability, emulsion electrospinning was developed where the T4 bacteriophage was pre-encapsulated in an alginate reservoir via an emulsification process and subsequently electrospun into fibres. This resulted in an increase in bacteriophage viability, but there was still two orders of magnitude drop in activity. Using a coaxial electrospinning process, full bacteriophage activity could be maintained. In this process, a core/shell fibre structure was formed with the T4 bacteriophage being directly incorporated into the fibre core. The core/shell fibre encapsulated bacteriophage exhibited full bacteriophage viability after storing for several weeks at +4°C. Coaxial electrospinning was shown to be capable of encapsulating bacteriophages with high loading capacity, high viability and long storage time. These results are significant in the context of controlling and preventing bacterial infections in perishable foods during storage. © 2013 The Society for Applied Microbiology.

Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles

PubMed Central

Antrobus, Robin; Hirst, Jennifer; Bhumbra, Gary S.; Kozik, Patrycja; Jackson, Lauren P.; Sahlender, Daniela A.

2012-01-01

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions. PMID:22472443

Bacteriophages as Potential Treatment for Urinary Tract Infections

PubMed Central

Sybesma, Wilbert; Zbinden, Reinhard; Chanishvili, Nino; Kutateladze, Mzia; Chkhotua, Archil; Ujmajuridze, Aleksandre; Mehnert, Ulrich; Kessler, Thomas M.

2016-01-01

Background: Urinary tract infections (UTIs) are among the most prevalent microbial diseases and their financial burden on society is substantial. The continuing increase of antibiotic resistance worldwide is alarming so that well-tolerated, highly effective therapeutic alternatives are urgently needed. Objective: To investigate the effect of bacteriophages on Escherichia coli and Klebsiella pneumoniae strains isolated from the urine of patients suffering from UTIs. Material and methods: Forty-one E. coli and 9 K. pneumoniae strains, isolated from the urine of patients suffering from UTIs, were tested in vitro for their susceptibility toward bacteriophages. The bacteriophages originated from either commercially available bacteriophage cocktails registered in Georgia or from the bacteriophage collection of the George Eliava Institute of Bacteriophage, Microbiology and Virology. In vitro screening of bacterial strains was performed by use of the spot-test method. The experiments were implemented three times by different groups of scientists. Results: The lytic activity of the commercial bacteriophage cocktails on the 41 E. coli strains varied between 66% (Pyo bacteriophage) and 93% (Enko bacteriophage). After bacteriophage adaptation of the Pyo bacteriophage cocktail, its lytic activity was increased from 66 to 93% and only one E. coli strain remained resistant. One bacteriophage of the Eliava collection could lyse all 9 K. pneumoniae strains. Conclusions: Based on the high lytic activity and the potential of resistance optimization by direct adaption of bacteriophages as reported in this study, and in view of the continuing increase of antibiotic resistance worldwide, bacteriophage therapy is a promising treatment option for UTIs highly warranting randomized controlled trials. PMID:27148173

Bacteriophages as Potential Treatment for Urinary Tract Infections.

PubMed

Sybesma, Wilbert; Zbinden, Reinhard; Chanishvili, Nino; Kutateladze, Mzia; Chkhotua, Archil; Ujmajuridze, Aleksandre; Mehnert, Ulrich; Kessler, Thomas M

2016-01-01

Urinary tract infections (UTIs) are among the most prevalent microbial diseases and their financial burden on society is substantial. The continuing increase of antibiotic resistance worldwide is alarming so that well-tolerated, highly effective therapeutic alternatives are urgently needed. To investigate the effect of bacteriophages on Escherichia coli and Klebsiella pneumoniae strains isolated from the urine of patients suffering from UTIs. Forty-one E. coli and 9 K. pneumoniae strains, isolated from the urine of patients suffering from UTIs, were tested in vitro for their susceptibility toward bacteriophages. The bacteriophages originated from either commercially available bacteriophage cocktails registered in Georgia or from the bacteriophage collection of the George Eliava Institute of Bacteriophage, Microbiology and Virology. In vitro screening of bacterial strains was performed by use of the spot-test method. The experiments were implemented three times by different groups of scientists. The lytic activity of the commercial bacteriophage cocktails on the 41 E. coli strains varied between 66% (Pyo bacteriophage) and 93% (Enko bacteriophage). After bacteriophage adaptation of the Pyo bacteriophage cocktail, its lytic activity was increased from 66 to 93% and only one E. coli strain remained resistant. One bacteriophage of the Eliava collection could lyse all 9 K. pneumoniae strains. Based on the high lytic activity and the potential of resistance optimization by direct adaption of bacteriophages as reported in this study, and in view of the continuing increase of antibiotic resistance worldwide, bacteriophage therapy is a promising treatment option for UTIs highly warranting randomized controlled trials.

Spatially controlled coating of continuous liquid Interface production microneedles for transdermal protein delivery.

PubMed

Caudill, Cassie L; Perry, Jillian L; Tian, Shaomin; Luft, J Christopher; DeSimone, Joseph M

2018-06-09

Microneedle patches, arrays of micron-scale projections that penetrate skin in a minimally invasive manner, are a promising tool for transdermally delivering therapeutic proteins. However, current microneedle fabrication techniques are limited in their ability to fabricate microneedles rapidly and with a high degree of control over microneedle design parameters. We have previously demonstrated the ability to fabricate microneedle patches with a range of compositions and geometries using the novel additive manufacturing technique Continuous Liquid Interface Production (CLIP). Here, we establish a method for dip coating CLIP microneedles with protein cargo in a spatially controlled manner. Microneedle coating mask devices were fabricated with CLIP and utilized to coat polyethylene glycol-based CLIP microneedles with model proteins bovine serum albumin, ovalbumin, and lysozyme. The design of the coating mask device was used to control spatial deposition and loading of coated protein cargo on the microneedles. CLIP microneedles rapidly released coated protein cargo both in solution and upon insertion into porcine skin. The model enzyme lysozyme was shown to retain its activity throughout the CLIP microneedle coating process, and permeation of bovine serum albumin across full thickness porcine skin was observed after application with coated CLIP microneedles. Protein-coated CLIP microneedles were applied to live mice and showed sustained retention of protein cargo in the skin over 72 h. These results demonstrate the utility of a versatile coating platform for preparation of precisely coated microneedles for transdermal therapeutic delivery. Copyright © 2018. Published by Elsevier B.V.

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

PubMed Central

Culver, J N; Lehto, K; Close, S M; Hilf, M E; Dawson, W O

1993-01-01

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes. Images Fig. 2 Fig. 3 Fig. 4 PMID:8446627

Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive spore forming bacteria.

PubMed

Nakonieczna, A; Cooper, C J; Gryko, R

2015-09-01

Since their discovery in 1915, bacteriophages have been routinely used within Eastern Europe to treat a variety of bacterial infections. Although initially ignored by the West due to the success of antibiotics, increasing levels and diversity of antibiotic resistance is driving a renaissance for bacteriophage-derived therapy, which is in part due to the highly specific nature of bacteriophages as well as their relative abundance. This review focuses on the bacteriophages and derived lysins of relevant Gram-positive spore formers within the Bacillus cereus group and Clostridium genus that could have applications within the medical, food and environmental sectors. © 2015 The Society for Applied Microbiology.

Detection of a Bacteriophage Gene Encoding a Mu-like Portal Protein in Haemophilus parasuis Reference Strains and Field Isolates by Nested Polymerase Chain Reaction

USDA-ARS?s Scientific Manuscript database

A nested PCR assay was developed to determine the presence of a gene encoding a bacteriophage Mu-like portal protein, gp29, in 15 reference strains and 31 field isolates of Haemophilus parasuis. Specific primers, based on the gene’s sequence, were utilized. A majority of the virulent reference strai...

Gene Expression During the Development of Bacteriophage φ29 III. Analysis of Viral-Specific Protein Synthesis with Suppressible Mutants

PubMed Central

McGuire, Jeffrey C.; Pène, Jacques J.; Barrow-Carraway, Joyce

1974-01-01

Fifty-four suppressible mutants of bacteriophage φ29 have been isolated with a variety of mutagens and assigned to eight complementation groups. Viral-specific protein synthesis in UV light-irradiated, nonsuppressing Bacillus subtilis 60084 was analyzed with exponential acrylamide gels. Four additional φ29 proteins which were undetected on ordinary acrylamide gels are reported in this paper. Five phage φ29 proteins have been unambiguously assigned to specific cistrons. Two cistrons had pleiotropic effects on viral protein synthesis. Mutants in cistrons I or II were unable to synthesize DNA in nonsuppressing bacteria. Mutants in cistron I were unable to attach viral chromosomes to the host cell membrane, and the protein responsible for this function has been identified. The other viral protein playing a role in phage φ29 DNA synthesis is also identified and assigned to cistron II. Mutants in cistron II can attach viral chromosomes to membrane, but cannot synthesize DNA in nonsuppressing bacteria. Images PMID:4362871

Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1.

PubMed

Schneider, G J; Geiduschek, E P

1990-06-25

The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.

Computational determination of the effects of virulent Escherichia coli and salmonella bacteriophages on human gut.

PubMed

Mostafa, Marwa Mostafa; Nassef, Mohammad; Badr, Amr

2016-10-01

Salmonella and Escherichia coli are different types of bacteria that cause food poisoning in humans. In the elderly, infants and people with chronic conditions, it is very dangerous if Salmonella or E. coli gets into the bloodstream and then they must be treated by phage therapy. Treating Salmonella and E. coli by phage therapy affects the gut flora. This research paper presents a system for detecting the effects of virulent E. coli and Salmonella bacteriophages on human gut. A method based on Domain-Domain Interactions (DDIs) model is implemented in the proposed system to determine the interactions between the proteins of human gut bacteria and the proteins of bacteriophages that infect virulent E. coli and Salmonella. The system helps gastroenterologists to realize the effect of injecting bacteriophages that infect virulent E. coli and Salmonella on the human gut. By testing the system over Enterobacteria phage 933W, Enterobacteria phage VT2-Sa and Enterobacteria phage P22, it resulted in four interactions between the proteins of the bacteriophages that infect E. coli O157:H7, E. coli O104:H4 and Salmonella typhimurium and the proteins of human gut bacterium strains. Several effects were detected such as: antibacterial activity against a number of bacterial species in human gut, regulation of cellular differentiation and organogenesis during gut, lung, and heart development, ammonia assimilation in bacteria, yeasts, and plants, energizing defense system and its function in the detoxification of lipopolysaccharide, and in the prevention of bacterial translocation in human gut. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

High efficiency protein separation with organosilane assembled silica coated magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Chang, Jeong Ho; Kang, Ki Ho; Choi, Jinsub; Jeong, Young Keun

2008-10-01

This work describes the development of high efficiency protein separation with functionalized organosilanes on the surface of silica coated magnetic nanoparticles. The magnetic nanoparticles were synthesized with average particle size of 9 nm and silica coated magnetic nanoparticles were obtained by controlling the coating thicknesses on magnetic nanoparticles. The silica coating thickness could be uniformly sized with a diameter of 10-40 nm by a sol-gel approach. The surface modification was performed with four kinds of functionalized organosilanes such as carboxyl, aldehyde, amine, and thiol groups. The protein separation work with organosilane assembled silica coated magnetic nanoparticles was achieved for model proteins such as bovine serum albumin (BSA) and lysozyme (LSZ) at different pH conditions. Among the various functionalities, the thiol group showed good separation efficiency due to the change of electrostatic interactions and protein conformational structure. The adsorption efficiency of BSA and LSZ was up to 74% and 90% corresponding pH 4.65 and pH 11.

Insights into Bacteriophage T5 Structure from Analysis of Its Morphogenesis Genes and Protein Components

PubMed Central

Zivanovic, Yvan; Confalonieri, Fabrice; Ponchon, Luc; Lurz, Rudi; Chami, Mohamed; Flayhan, Ali; Renouard, Madalena; Huet, Alexis; Decottignies, Paulette; Davidson, Alan R.; Breyton, Cécile

2014-01-01

Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm. PMID:24198424

Lipid-coated mannitol core microparticles for sustained release of protein.

PubMed

Wang, Bifeng; Friess, Wolfgang

2018-07-01

Parenteral sustained release systems for proteins which provide therapeutic levels over a longer period avoiding frequent administration, which preserve protein stability during manufacturing, storage and application and which are biodegradable and highly biocompatible in the body are intensively sought after. The aim of this study was to generate and study mannitol core microparticles loaded with a monoclonal antibody IgG1 and coated with lipid either hard fat or glyceryl stearate at different coating levels. The protein was stabilized with 22.5 mg/mL sucrose, 0.1% PS 80, 10 mM methionine in 10 mM His buffer pH 7.2 during the spray loading process. 30 g protein-loaded mannitol carrier microparticles were coated with 5 g, 10 g, 20 g and 30 g of lipid, respectively. Placing more lipid onto the protein-loaded microparticles reduced both burst and release rate, and the particles maintained their geometric form during the release test. The IgG1 release from microparticles covered with a hard fat layer extended up to 6 weeks. The IgG1 was released in its monomeric form and maintained its secondary structure as shown by FTIR. Incomplete release of IgG1 from glyceryl stearate-coated microparticles was observed, which may be due to the small pore sizes of the glyceryl stearate layer or a detrimental surfactant character of glyceryl stearate to protein. Hence, these hard fat-coated mannitol core microparticles have high potential for protein delivery. Copyright © 2018 Elsevier B.V. All rights reserved.

[TL, the new bacteriophage of Pseudomonas aeruginosa and its application for the search of halo-producing bacteriophages].

PubMed

Pleteneva, E A; Burkal'tseva, M V; Shaburova, O V; Krylov, S V; Pechnikova, E V; Sokolova, O S; Krylov, V N

2011-01-01

The properties of new virulent bacteriophage TL of Pseudomonas aeruginosa belonging to the family Podoviridae (genome size of 46 kb) were investigated. This bacteriophage is capable of lysogenizing the bacterial lawn in halo zones around negative colonies (NC) of other bacteriophages. TL forms large NC, that are hardly distinguishable on the lawn of P. aeruginisa PAO1. At the same time, on the lawns of some phage-resistant PAO1 mutants, as well as on those produced by a number of clinical isolates, TL forms more transparent NC. It is suggested that more effective growth of the bacteriophage TL NC is associated with the differences in outer lipopolysaccharide (LPS) layer of the cell walls of different bacterial strains, as well as of the bacteria inside and outside of the halos. This TL property was used to optimize selection of bacteriophages producing halos around NC on the lawn of P. aeruginosa PAO1. As a result, a group of bacteriophages differing in the patterns of interaction between their halos and TL bacteriophage, as well as in some characters was identified. Taking into consideration the importance of cell-surfaced structures of P. aeruginosa in manifestation of virulence and pathogenicity, possible utilization of specific phage enzymes, polysacchadide depolymerases, for more effective treatment of P. aeruginosa infections is discussed.

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis.

PubMed

Wang, Katherine H; Isidro, Anabela L; Domingues, Lia; Eskandarian, Haig A; McKenney, Peter T; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O; Eichenberger, Patrick

2009-11-01

Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.

Bacteriophages as scaffolds for bipartite display: designing swiss army knives on a nanoscale.

PubMed

Molek, Peter; Bratkovič, Tomaž

2015-03-18

Bacteriophages have been exploited as cloning vectors and display vehicles for decades owing to their genetic and structural simplicity. In bipartite display setting, phage takes on the role of a handle to which two modules are attached, each endowing it with specific functionality, much like the Swiss army knife. This concept offers unprecedented potential for phage applications in nanobiotechnology. Here, we compare common phage display platforms and discuss approaches to simultaneously append two or more different (poly)peptides or synthetic compounds to phage coat using genetic fusions, chemical or enzymatic conjugations, and in vitro noncovalent decoration techniques. We also review current reports on design of phage frameworks to link multiple effectors, and their use in diverse scientific disciplines. Bipartite phage display had left its mark in development of biosensors, vaccines, and targeted delivery vehicles. Furthermore, multifunctionalized phages have been utilized to template assembly of inorganic materials and protein complexes, showing promise as scaffolds in material sciences and structural biology, respectively.

Novel bacteriophages containing a genome of another bacteriophage within their genomes.

PubMed

Swanson, Maud M; Reavy, Brian; Makarova, Kira S; Cock, Peter J; Hopkins, David W; Torrance, Lesley; Koonin, Eugene V; Taliansky, Michael

2012-01-01

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

M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins.

PubMed

Hess, Gaelen T; Cragnolini, Juan J; Popp, Maximilian W; Allen, Mark A; Dougan, Stephanie K; Spooner, Eric; Ploegh, Hidde L; Belcher, Angela M; Guimaraes, Carla P

2012-07-18

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis

PubMed Central

Wang, Katherine H.; Isidro, Anabela L.; Domingues, Lia; Eskandarian, Haig A.; McKenney, Peter T.; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N.; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O.; Eichenberger, Patrick

2009-01-01

SUMMARY Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to GFP and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homolog of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement. PMID:19775244

Use of hydrophilic polymer coatings for control of electroosmosis and protein adsorption

NASA Technical Reports Server (NTRS)

Harris, J. Milton

1987-01-01

The purpose of this project was to examine the utility of polyethylene glycol (PEG) and dextran coatings for control of electroosmosis and protein adsorption; electroosmosis is an important, deleterious process affecting electrophoretic separations, and protein adsorption is a factor which needs to be controlled during protein crystal growth to avoid multiple nucleation sites. Performance of the project required use of X-ray photoelectron spectroscopy to refine previously developed synthetic methods. The results of this spectroscopic examination are reported. Measurements of electroosmotic mobility of charged particles in appropriately coated capillaries reveals that a new, one-step route to coating capillaries gives a surface in which electroosmosis is dramatically reduced. Similarly, both PEG and dextran coatings were shown by protein adsorption measurements to be highly effective at reducing protein adsorption on solid surfaces. These results should have impact on future low-g electrophoretic and protein crystal growth experiments.

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

Magic-angle spinning NMR of a class I filamentous bacteriophage virus.

PubMed

Abramov, Gili; Morag, Omry; Goldbourt, Amir

2011-08-11

The fd bacteriophage is a filamentous virus that is widely used for bio- and nanotechnology applications ranging from phage display to battery materials. The possibility of obtaining a detailed description of its structural properties regardless of its state is therefore essential not only for understanding its physical arrangement and its bacterial infection process but also for many other applications. Here we present a study of the fd phage by magic-angle spinning solid-state NMR. While current structures rely on a Y21M mutant, experiments performed on a strain bearing a wild-type capsid report on high symmetry of the phage and lack of explicit subunit polymorphism. Chemical shift analysis confirmed that the coat protein mostly consists of a rigid right-handed curved α-helix (residues 6-47 of 50), preceded by a flexible loop-structured N-terminus. We were able to qualitatively assign the resonances belonging to the DNA, including the deoxyribose sugars and the thymine bases. These chemical shifts are consistent with base stacking and a C2'-endo/C3'-exo sugar pucker. © 2011 American Chemical Society

Genetic evolution of bacteriophage. I. Hybrids between unrelated bacteriophages P22 and Fels 2.

PubMed

Yamamoto, N

1969-01-01

A new bacteriophage species, designated F22, was isolated from phage P22 stocks grown on Salmonella typhimurium Q1 lysogenic for Fels 2 at a frequency of less than 10(-11). P22 has a very short tail with a hexagonal base plate and six spikes. Phage Fels 2 is morphologically similar to E. coli T-even phages, having a long tail with a contractile sheath and carrying no genetic region related to P22. Phage F22 is morphologically and serologically indistinguishable from Fels 2, but carries the c(c(1), c(2), and c(3)) markers of P22. The color markers h(21), g, and m(3) of P22 do not appear in F22. Thus, F22 is evidently a recombinant between the unrelated bacteriophages P22 and Fels 2. The recombination between unrelated bacteriophages could play an important role in the evolution of bacteriophages.

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

PubMed

Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H

1978-09-01

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

Structure and dynamics of a detergent-solubilized membrane protein: measurement of amide hydrogen exchange rates in M13 coat protein by /sub 1/H NMR spectroscopy

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

O'Neil, J.D.J.; Sykes, B.D.

The coat protein of bacteriophage M13 is inserted into the inner membrane of Escherichia coli where it exists as an integral membrane protein during the reproductive cycle of the phage. The protein sequence consists of a highly hydrophobic 19-residue central segment flanked by an acidic 20-residue N-terminus and a basic 11-residue C-terminus. The authors have measured backbone amide hydrogen exchange of the protein solubilized in perdeuteriated sodium dodecyl sulfate using /sup 1/H nuclear magnetic resonance (NMR) spectroscopy. Direct proton exchange-out measurements in D/sub 2/O at 24 /sup 0/C were used to follow the exchange of the slowest amides in themore » protein. Multiple exponential fitting of the exchange data showed that these amides exchanged in two kinetic sets with exchange rates that differed by more than 100-fold. Steady-state saturation-transfer techniques were also used to measure exchange. These methods showed that 15-20 amides in the protein are very stable at 55/sup 0/C and that bout 30 amides have exchange rates retarded by at least 10/sup 5/-fold at 24/sup 0/C. Saturation-transfer studies also showed that the pH dependence of exchange in the hydrophilic termini was unusual. Relaxation and solid-state NMR experiments have previously shown that the majority of the protein backbone is rigid on the picosecond to microsecond time scale, except for the extreme ends of the molecule which are mobile. The hydrogen exchange results, which are sensitive to a much longer time scale, suggest a stable core with a progressive increase in amplitude or frequency of motions as the ends of the protein are approached.« less

Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling

PubMed Central

Baker, Matthew L.; Hryc, Corey F.; Zhang, Qinfen; Wu, Weimin; Jakana, Joanita; Haase-Pettingell, Cameron; Afonine, Pavel V.; Adams, Paul D.; King, Jonathan A.; Jiang, Wen; Chiu, Wah

2013-01-01

High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all bacteria and archaea, making them excellent potential alternatives to antibiotics and therapies for multidrug-resistant bacteria. Here, we improved upon our previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution sufficient to determine the tertiary structures of both gp7 and gp10 protein subunits that form the T = 7 icosahedral lattice. This study utilizes recently established best practice for near-atomic to high-resolution (3–5 Å) electron cryomicroscopy data evaluation. The resolution and reliability of the density map were cross-validated by multiple reconstructions from truly independent data sets, whereas the models of the individual protein subunits were validated adopting the best practices from X-ray crystallography. Some sidechain densities are clearly resolved and show the subunit–subunit interactions within and across the capsomeres that are required to stabilize the virus. The presence of the canonical phage and jellyroll viral protein folds, gp7 and gp10, respectively, in the same virus suggests that epsilon15 may have emerged more recently relative to other bacteriophages. PMID:23840063

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.

Elucidating the pH-Dependent Structural Transition of T7 Bacteriophage Endolysin.

PubMed

Sharma, Meenakshi; Kumar, Dinesh; Poluri, Krishna Mohan

2016-08-23

Bacteriophages are the most abundant and diverse biological entities on earth. Bacteriophage endolysins are unique peptidoglycan hydrolases and have huge potential as effective enzybiotics in various infectious models. T7 bacteriophage endolysin (T7L), also known as N-acetylmuramoyl-l-alanine amidase or T7 lysozyme, is a 17 kDa protein that lyses a range of Gram-negative bacteria by hydrolyzing the amide bond between N-acetylmuramoyl residues and the l-alanine of the peptidoglycan layer. Although the activity profiles of several of the T7 family members have been known for many years, the molecular basis for their pH-dependent differential activity is not clear. In this study, we explored the pH-induced structural, stability, and activity characteristics of T7L by applying a variety of biophysical techniques and protein nuclear magnetic resonance (NMR) spectroscopy. Our studies established a reversible structural transition of T7L below pH 6 and the formation of a partially denatured conformation at pH 3. This low-pH conformation is thermally stable and exposed its hydrophobic pockets. Further, NMR relaxation measurements and structural analysis unraveled that T7L is highly dynamic in its native state and a network of His residues are responsible for the observed pH-dependent conformational dynamics and transitions. As bacteriophage chimeric and engineered endolysins are being developed as novel therapeutics against multiple drug resistance pathogens, we believe that our results are of great help in designing these entities as broadband antimicrobial and/or antibacterial agents.

GENETIC EVOLUTION OF BACTERIOPHAGE, I. HYBRIDS BETWEEN UNRELATED BACTERIOPHAGES P22 AND FELS 2*

PubMed Central

Yamamoto, Nobuto

1969-01-01

A new bacteriophage species, designated F22, was isolated from phage P22 stocks grown on Salmonella typhimurium Q1 lysogenic for Fels 2 at a frequency of less than 10-11. P22 has a very short tail with a hexagonal base plate and six spikes. Phage Fels 2 is morphologically similar to E. coli T-even phages, having a long tail with a contractile sheath and carrying no genetic region related to P22. Phage F22 is morphologically and serologically indistinguishable from Fels 2, but carries the c(c1, c2, and c3) markers of P22. The color markers h21, g, and m3 of P22 do not appear in F22. Thus, F22 is evidently a recombinant between the unrelated bacteriophages P22 and Fels 2. The recombination between unrelated bacteriophages could play an important role in the evolution of bacteriophages. Images PMID:4890254

Fine Structure Genetic and Physical Map of the Gene 3 to 10 Region of the Bacteriophage P22 Chromosome

PubMed Central

Casjens, S.; Eppler, K.; Sampson, L.; Parr, R.; Wyckoff, E.

1991-01-01

The mechanism by which dsDNA is packaged by viruses is not yet understood in any system. Bacteriophage P22 has been a productive system in which to study the molecular genetics of virus particle assembly and DNA packaging. Only five phage encoded proteins, the products of genes 3, 2, 1, 8 and 5, are required for packaging the virus chromosome inside the coat protein shell. We report here the construction of a detailed genetic and physical map of these genes, the neighboring gene 4 and a portion of gene 10, in which 289 conditional lethal amber, opal, temperature sensitive and cold sensitive mutations are mapped into 44 small (several hundred base pair) intervals of known sequence. Knowledge of missense mutant phenotypes and information on the location of these mutations allows us to begin the assignment of partial protein functions to portions of these genes. The map and mapping strains will be of use in the further genetic dissection of the P22 DNA packaging and prohead assembly processes. PMID:2029965

Two-Stage Dynamics of In Vivo Bacteriophage Genome Ejection

NASA Astrophysics Data System (ADS)

Chen, Yi-Ju; Wu, David; Gelbart, William; Knobler, Charles M.; Phillips, Rob; Kegel, Willem K.

2018-04-01

Biopolymer translocation is a key step in viral infection processes. The transfer of information-encoding genomes allows viruses to reprogram the cell fate of their hosts. Constituting 96% of all known bacterial viruses [A. Fokine and M. G. Rossmann, Molecular architecture of tailed double-stranded DNA phages, Bacteriophage 4, e28281 (2014)], the tailed bacteriophages deliver their DNA into host cells via an "ejection" process, leaving their protein shells outside of the bacteria; a similar scenario occurs for mammalian viruses like herpes, where the DNA genome is ejected into the nucleus of host cells, while the viral capsid remains bound outside to a nuclear-pore complex. In light of previous experimental measurements of in vivo bacteriophage λ ejection, we analyze here the physical processes that give rise to the observed dynamics. We propose that, after an initial phase driven by self-repulsion of DNA in the capsid, the ejection is driven by anomalous diffusion of phage DNA in the crowded bacterial cytoplasm. We expect that this two-step mechanism is general for phages that operate by pressure-driven ejection, and we discuss predictions of our theory to be tested in future experiments.

Valyl-tRNA synthetase modification-dependent restriction of bacteriophage T4.

PubMed Central

Olson, N J; Marchin, G L

1984-01-01

A strain of Escherichia coli, CP 790302, severely restricts the growth of wild-type bacteriophage T4. In broth culture, most infections of single cells are abortive, although a few infected cells exhibit reduced burst sizes. In contrast, bacteriophage T4 mutants impaired in the ability to modify valyl-tRNA synthetase develop normally on this strain. Biochemical evidence indicates that the phage-modified valyl-tRNA synthetase in CP 790302 is different from that previously described. Although the enzyme is able to support normal protein synthesis, a disproportionate amount of phage structural protein (serum blocking power) fails to mature into particles of the appropriate density. The results with host strain CP 790302 are consistent with either a gratuitous inhibition of phage assembly by faulty modification or abrogation of an unknown role that valyl-tRNA synthetase might normally play in viral assembly. PMID:6374167

Plating Bacteriophage M13.

PubMed

Green, Michael R; Sambrook, Joseph

2017-10-03

A plaque of bacteriophage M13 derives from infection of a single bacterium by a single virus particle. The progeny particles infect neighboring bacteria, which, in turn, release another generation of daughter virus particles. If the bacteria are growing in semisolid medium (e.g., containing agar or agarose), then the diffusion of the progeny particles is limited. Cells infected with bacteriophage M13 are not killed, but have a longer generation time than uninfected Escherichia coli In consequence, plaques appear as areas of slower-growing cells on a faster-growing lawn of bacterial cells. This protocol describes plating of bacteriophage M13 stocks. Plaques are readily detectable on top agar after 4-8 h of incubation at 37°C. © 2017 Cold Spring Harbor Laboratory Press.

A first step toward liposome-mediated intracellular bacteriophage therapy.

PubMed

Nieth, Anita; Verseux, Cyprien; Barnert, Sabine; Süss, Regine; Römer, Winfried

2015-01-01

The emergence of antibiotic-resistant bacteria presents a severe challenge to medicine and public health. While bacteriophage therapy is a promising alternative to traditional antibiotics, the general inability of bacteriophages to penetrate eukaryotic cells limits their use against resistant bacteria, causing intracellular diseases like tuberculosis. Bacterial vectors show some promise in carrying therapeutic bacteriophages into cells, but also bring a number of risks like an overload of bacterial antigens or the acquisition of virulence genes from the pathogen. As a first step in the development of a non-bacterial vector for bacteriophage delivery into pathogen-infected cells, we attempted to encapsulate bacteriophages into liposomes. Here we report effective encapsulation of the model bacteriophage λeyfp and the mycobacteriophage TM4 into giant liposomes. Furthermore, we show that liposome-associated bacteriophages are taken up into eukaryotic cells more efficiently than free bacteriophages. These are important milestones in the development of an intracellular bacteriophage therapy that might be useful in the fight against multi-drug-resistant intracellular pathogens like Mycobacterium tuberculosis.

No effect of femtosecond laser pulses on M13, E. coli, DNA, or protein.

PubMed

Wigle, Jeffrey C; Holwitt, Eric A; Estlack, Larry E; Noojin, Gary D; Saunders, Katharine E; Yakovlev, Valdislav V; Rockwell, Benjamin A

2014-01-01

Data showing what appears to be nonthermal inactivation of M13 bacteriophage (M13), Tobacco mosaic virus, Escherichia coli (E. coli), and Jurkatt T-cells following exposure to 80-fs pulses of laser radiation have been published. Interest in the mechanism led to attempts to reproduce the results for M13 and E. coli. Bacteriophage plaque-forming and bacteria colony-forming assays showed no inactivation of the microorganisms; therefore, model systems were used to see what, if any, damage might be occurring to biologically important molecules. Purified plasmid DNA (pUC19) and bovine serum albumin were exposed to and analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively, and no effect was found. DNA and coat proteins extracted from laser-exposed M13 and analyzed by AGE or PAGE found no effect. Raman scattering by M13 in phosphate buffered saline was measured to determine if there was any physical interaction between M13 and femtosecond laser pulses, and none was found. Positive controls for the endpoints measured produced the expected results with the relevant assays. Using the published methods, we were unable to reproduce the inactivation results or to show any interaction between ultrashort laser pulses and buffer/water, DNA, protein, M13 bacteriophage, or E. coli.

No effect of femtosecond laser pulses on M13, E. coli, DNA, or protein

NASA Astrophysics Data System (ADS)

Wigle, Jeffrey C.; Holwitt, Eric A.; Estlack, Larry E.; Noojin, Gary D.; Saunders, Katharine E.; Yakovlev, Valdislav V.; Rockwell, Benjamin A.

2014-01-01

Data showing what appears to be nonthermal inactivation of M13 bacteriophage (M13), Tobacco mosaic virus, Escherichia coli (E. coli), and Jurkatt T-cells following exposure to 80-fs pulses of laser radiation have been published. Interest in the mechanism led to attempts to reproduce the results for M13 and E. coli. Bacteriophage plaque-forming and bacteria colony-forming assays showed no inactivation of the microorganisms; therefore, model systems were used to see what, if any, damage might be occurring to biologically important molecules. Purified plasmid DNA (pUC19) and bovine serum albumin were exposed to and analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively, and no effect was found. DNA and coat proteins extracted from laser-exposed M13 and analyzed by AGE or PAGE found no effect. Raman scattering by M13 in phosphate buffered saline was measured to determine if there was any physical interaction between M13 and femtosecond laser pulses, and none was found. Positive controls for the endpoints measured produced the expected results with the relevant assays. Using the published methods, we were unable to reproduce the inactivation results or to show any interaction between ultrashort laser pulses and buffer/water, DNA, protein, M13 bacteriophage, or E. coli.

Collagen mimetic peptide engineered M13 bacteriophage for collagen targeting and imaging in cancer.

PubMed

Jin, Hyo-Eon; Farr, Rebecca; Lee, Seung-Wuk

2014-11-01

Collagens are over-expressed in various human cancers and subsequently degraded and denatured by proteolytic enzymes, thus making them a target for diagnostics and therapeutics. Genetically engineered bacteriophage (phage) is a promising candidate for the development of imaging or therapeutic materials for cancer collagen targeting due to its promising structural features. We genetically engineered M13 phages with two functional peptides, collagen mimetic peptide and streptavidin binding peptide, on their minor and major coat proteins, respectively. The resulting engineered phage functions as a therapeutic or imaging material to target degraded and denatured collagens in cancerous tissues. We demonstrated that the engineered phages are able to target and label abnormal collagens expressed on A549 human lung adenocarcinoma cells after the conjugation with streptavidin-linked fluorescent agents. Our engineered collagen binding phage could be a useful platform for abnormal collagen imaging and drug delivery in various collagen-related diseases. Published by Elsevier Ltd.

Identification and Characterization of Arabidopsis Seed Coat Mucilage Proteins1[OPEN

PubMed Central

Tsai, Allen Yi-Lun; Kunieda, Tadashi; Rogalski, Jason; Foster, Leonard J.; Ellis, Brian E.

2017-01-01

Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features. PMID:28003327

Phosphorylation of spore coat proteins by a family of atypical protein kinases

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

Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

Phosphorylation of spore coat proteins by a family of atypical protein kinases

DOE PAGES

Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; ...

2016-05-16

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

Novel Podoviridae Family Bacteriophage Infecting Weissella cibaria Isolated from Kimchi

PubMed Central

Holo, Helge; Jeon, Sang-Rok; Nes, Ingolf F.; Yoon, Sung-Sik

2012-01-01

The first complete genome sequence of a phage infecting Weissella cibaria (Weissella kimchii) is presented. The bacteriophage ϕYS61 was isolated from kimchi, a Korean fermented vegetable dish. Bacteriophages are recognized as a serious problem in industrial fermentations; however, ϕYS61 differed from many virulent phages associated with food fermentations since it was difficult to propagate and was very susceptible to resistance development. Sequence analysis revealed that ϕYS61 resembles Podoviridae of the subfamily Picovirinae. Within the subfamily Picovirinae, the ϕ29-like phages have been extensively studied, and their terminal protein-primed DNA replication is well characterized. Our data strongly suggest that ϕYS61 also replicates by a protein-primed mechanism. Weissella phage ϕYS61 is, however, markedly different from members of the Picovirinae with respect to genome size and morphology. Picovirinae are characterized by small (approximately 20-kb) genomes which contrasts with the 33,594-bp genome of ϕYS61. Based on electron microscopy analysis, ϕYS61 was classified as a member of the Podoviridae of morphotype C2, similar to the ϕ29-like phages, but its capsid dimensions are significantly larger than those reported for these phages. The novelty of ϕYS61 was also emphasized by the low number of open reading frames (ORFs) showing significant similarity to database sequences. We propose that the bacteriophage ϕYS61 should represent a new subfamily within the family Podoviridae. PMID:22885743

Taking Bacteriophage Therapy Seriously: A Moral Argument

PubMed Central

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

2014-01-01

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

Disposable amperometric biosensor based on nanostructured bacteriophages for glucose detection

NASA Astrophysics Data System (ADS)

Kang, Yu Ri; Hwang, Kyung Hoon; Kim, Ju Hwan; Nam, Chang Hoon; Kim, Soo Won

2010-10-01

The selection of electrode material profoundly influences biosensor science and engineering, as it heavily influences biosensor sensitivity. Here we propose a novel electrochemical detection method using a working electrode consisting of bio-nanowires from genetically modified filamentous phages and nanoparticles. fd-tet p8MMM filamentous phages displaying a three-methionine (MMM) peptide on the major coat protein pVIII (designated p8MMM phages) were immobilized on the active area of an electrochemical sensor through physical adsorption and chemical bonding. Bio-nanowires composed of p8MMM phages and silver nanoparticles facilitated sensitive, rapid and selective detection of particular molecules. We explored whether the composite electrode with bio-nanowires was an effective platform to detect the glucose oxidase. The current response of the bio-nanowire sensor was high at various glucose concentrations (0.1 µm-0.1 mM). This method provides a considerable advantage to demonstrate analyte detection over low concentration ranges. Especially, phage-enabled bio-nanowires can serve as receptors with high affinity and specificity for the detection of particular biomolecules and provide a convenient platform for designing site-directed multifunctional scaffolds based on bacteriophages and may serve as a simple method for label-free detection.

Cationic antimicrobial peptides inactivate Shiga toxin-encoding bacteriophages

NASA Astrophysics Data System (ADS)

Del Cogliano, Manuel E.; Hollmann, Axel; Martinez, Melina; Semorile, Liliana; Ghiringhelli, Pablo D.; Maffía, Paulo C.; Bentancor, Leticia V.

2017-12-01

Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: 1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, 2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and 3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

An M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins

PubMed Central

Hess, Gaelen T.; Cragnolini, Juan J.; Popp, Maximilian W.; Allen, Mark A.; Dougan, Stephanie K.; Spooner, Eric; Ploegh, Hidde L.; Belcher, Angela M.; Guimaraes, Carla P.

2013-01-01

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool. PMID:22759232

Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu

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

Kondou, Youhei; Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama; Kitazawa, Daisuke

2005-01-01

Bacteriophage Mu baseplate protein gene product 44 was crystallized. The crystal belongs to space group R3, with unit-cell parameters a = b = 126.6, c = 64.2 Å. Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Å. The crystals diffract X-rays to at least 2.1 Å resolution andmore » are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Å resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.« less

Bacteriophages in dairy products: pros and cons.

PubMed

Mc Grath, Stephen; Fitzgerald, Gerald F; van Sinderen, Douwe

2007-04-01

Since the time bacteriophages were first identified as a major cause of fermentation failure in the dairy industry, researchers have been struggling to develop strategies to exclude them from the dairy environment. Over 70 years of research has led to huge improvements in the consistency and quality of fermented dairy products, while also facilitating an appreciation of the beneficial properties of bacteriophages with respect to dairy product development. With specific reference to Lactococcus lactis and cheese production, this review outlines some recently reported novel methods aimed at limiting the bacteriophage infection as well as highlighting some beneficial aspects of bacteriophage activity.

Exploiting Radiation Damage to Map Proteins in Nucleoprotein Complexes: The Internal Structure of Bacteriophage T7

PubMed Central

Cheng, Naiqian; Wu, Weimin; Watts, Norman R.; Steven, Alasdair C.

2014-01-01

In the final stage of radiation damage in cryo-electron microscopy of proteins, bubbles of hydrogen gas are generated. Proteins embedded in DNA bubble sooner than free-standing proteins and DNA does not bubble under the same conditions. These properties make it possible to distinguish protein from DNA. Here we explored the scope of this technique (“bubblegram imaging”) by applying it to bacteriophage T7, viewed as a partially defined model system. T7 has a thin-walled icosahedral capsid, 60 nm in diameter, with a barrel-shaped protein core under one of its twelve vertices (the portal vertex). The core is densely wrapped with DNA but details of their interaction and how their injection into a host bacterium is coordinated are lacking. With short (10 sec) intervals between exposures of 17 electrons/Å2 each, bubbling starts in the third exposure, with 1 – 4 bubbles nucleating in the core: in subsequent exposures, these bubbles grow and merge. A 3D reconstruction from fifth-exposure images depicts a bipartite cylindrical gas cloud in the core. In its portal-proximal half, the axial region is gaseous whereas in the portal-distal half, it is occupied by a 3 nm-wide dense rod. We propose that they respectively represent core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles at other sites may represent residual scaffolding protein. Thus, bubbling depends on dose rate, protein amount, and tightness of the DNA seal. PMID:24345345

Bacteriophage 5' untranslated regions for control of plastid transgene expression.

PubMed

Yang, Huijun; Gray, Benjamin N; Ahner, Beth A; Hanson, Maureen R

2013-02-01

Expression of foreign proteins from transgenes incorporated into plastid genomes requires regulatory sequences that can be recognized by the plastid transcription and translation machinery. Translation signals harbored by the 5' untranslated region (UTR) of plastid transcripts can profoundly affect the level of accumulation of proteins expressed from chimeric transgenes. Both endogenous 5' UTRs and the bacteriophage T7 gene 10 (T7g10) 5' UTR have been found to be effective in combination with particular coding regions to mediate high-level expression of foreign proteins. We investigated whether two other bacteriophage 5' UTRs could be utilized in plastid transgenes by fusing them to the aadA (aminoglycoside-3'-adenyltransferase) coding region that is commonly used as a selectable marker in plastid transformation. Transplastomic plants containing either the T7g1.3 or T4g23 5' UTRs fused to Myc-epitope-tagged aadA were successfully obtained, demonstrating the ability of these 5' UTRs to regulate gene expression in plastids. Placing the Thermobifida fusca cel6A gene under the control of the T7g1.3 or T4g23 5' UTRs, along with a tetC downstream box, resulted in poor expression of the cellulase in contrast with high-level accumulation while using the T7g10 5' UTR. However, transplastomic plants with the bacteriophage 5' UTRs controlling the aadA coding region exhibited fewer undesired recombinant species than plants containing the same marker gene regulated by the Nicotiana tabacum psbA 5' UTR. Furthermore, expression of the T7g1.3 and T4g23 5' UTR::aadA fusions downstream of the cel6A gene provided sufficient spectinomycin resistance to allow selection of homoplasmic transgenic plants and had no effect on Cel6A accumulation.

Gold-Coated M13 Bacteriophage as a Template for Glucose Oxidase Biofuel Cells with Direct Electron Transfer.

PubMed

Blaik, Rita A; Lan, Esther; Huang, Yu; Dunn, Bruce

2016-01-26

Glucose oxidase-based biofuel cells are a promising source of alternative energy for small device applications, but still face the challenge of achieving robust electrical contact between the redox enzymes and the current collector. This paper reports on the design of an electrode consisting of glucose oxidase covalently attached to gold nanoparticles that are assembled onto a genetically engineered M13 bacteriophage using EDC-NHS chemistry. The engineered phage is modified at the pIII protein to attach onto a gold substrate and serves as a high-surface-area template. The resulting "nanomesh" architecture exhibits direct electron transfer (DET) and achieves a higher peak current per unit area of 1.2 mA/cm(2) compared to most other DET attachment schemes. The final enzyme surface coverage on the electrode was calculated to be approximately 4.74 × 10(-8) mol/cm(2), which is a significant improvement over most current glucose oxidase (GOx) DET attachment methods.

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

PubMed

Pawluk, April; Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F; Davidson, Alan R; Maxwell, Karen L

2017-12-12

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. Copyright © 2017 Pawluk et al.

The Search for Therapeutic Bacteriophages Uncovers One New Subfamily and Two New Genera of Pseudomonas-Infecting Myoviridae

PubMed Central

Henry, Marine; Bobay, Louis-Marie; Chevallereau, Anne; Saussereau, Emilie; Ceyssens, Pieter-Jan; Debarbieux, Laurent

2015-01-01

In a previous study, six virulent bacteriophages PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5 and CHA_P1 were evaluated for their in vivo efficacy in treating Pseudomonas aeruginosa infections using a mouse model of lung infection. Here, we show that their genomes are closely related to five other Pseudomonas phages and allow a subdivision into two clades, PAK_P1-like and KPP10-like viruses, based on differences in genome size, %GC and genomic contents, as well as number of tRNAs. These two clades are well delineated, with a mean of 86% and 92% of proteins considered homologous within individual clades, and 25% proteins considered homologous between the two clades. By ESI-MS/MS analysis we determined that their virions are composed of at least 25 different proteins and electron microscopy revealed a morphology identical to the hallmark Salmonella phage Felix O1. A search for additional bacteriophage homologs, using profiles of protein families defined from the analysis of the 11 genomes, identified 10 additional candidates infecting hosts from different species. By carrying out a phylogenetic analysis using these 21 genomes we were able to define a new subfamily of viruses, the Felixounavirinae within the Myoviridae family. The new Felixounavirinae subfamily includes three genera: Felixounalikevirus, PAK_P1likevirus and KPP10likevirus. Sequencing genomes of bacteriophages with therapeutic potential increases the quantity of genomic data on closely related bacteriophages, leading to establishment of new taxonomic clades and the development of strategies for analyzing viral genomes as presented in this article. PMID:25629728

Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128

PubMed Central

Saravanan, Sanjeev Rajagopalan; Paul, Vivek Daniel; George, Shilpa; Sundarrajan, Sudarson; Kumar, Nirmal; Hebbur, Madhavi; Kumar, Naveen; Veena, Ananda; Maheshwari, Uma; Appaiah, Chemira Biddappa; Chidambaran, Muralidharan; Bhat, Anuradha Gopal; Hariharan, Sukumar; Padmanabhan, Sriram

2013-01-01

P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a Staphylococcus bacteriophage K tail associated structural protein and a cell wall targeting domain from the Staphylococcus bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain S. simulans biovar staphylolyticus, P128 was thermostable and was lytic towards S. simulans biovar staphylolyticus demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the Vmax (∆OD600 [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser Vmax value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent Km (Km APP) and apparent Kcat (KcatAPP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin. PMID:24251076

Protein-resistant polymer coatings obtained by matrix assisted pulsed laser evaporation

NASA Astrophysics Data System (ADS)

Rusen, L.; Mustaciosu, C.; Mitu, B.; Filipescu, M.; Dinescu, M.; Dinca, V.

2013-08-01

Adsorption of proteins and polysaccharides is known to facilitate microbial attachment and subsequent formation of biofilm on surfaces that ultimately results in its biofouling. Therefore, protein repellent modified surfaces are necessary to block the irreversible attachment of microorganisms. Within this context, the feasibility of using the Poly(ethylene glycol)-block-poly(ɛ-caprolactone) methyl ether (PEG-block-PCL Me) copolymer as potential protein-resistant coating was explored in this work. The films were deposited using Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique that allows good control of composition, thickness and homogeneity. The chemical and morphological characteristics of the films were examined using Fourier Transform Infrared Spectroscopy (FTIR), contact angle measurements and Atomic Force Microscopy (AFM). The FTIR data demonstrates that the functional groups in the MAPLE-deposited films remain intact, especially for fluences below 0.5 J cm-2. Optical Microscopy and AFM images show that the homogeneity and the roughness of the coatings are related to both laser parameters (fluence, number of pulses) and target composition. Protein adsorption tests were performed on the PEG-block-PCL Me copolymer coated glass and on bare glass surface as a control. The results show that the presence of copolymer as coating significantly reduces the adsorption of proteins.

Bacteriophages and Their Immunological Applications against Infectious Threats.

PubMed

Criscuolo, Elena; Spadini, Sara; Lamanna, Jacopo; Ferro, Mattia; Burioni, Roberto

2017-01-01

Bacteriophage therapy dates back almost a century, but the discovery of antibiotics led to a rapid decline in the interests and investments within this field of research. Recently, the novel threat of multidrug-resistant bacteria highlighted the alarming drop in research and development of new antibiotics: 16 molecules were discovered during 1983-87, 10 new therapeutics during the nineties, and only 5 between 2003 and 2007. Phages are therefore being reconsidered as alternative therapeutics. Phage display technique has proved to be extremely promising for the identification of effective antibodies directed against pathogens, as well as for vaccine development. At the same time, conventional phage therapy uses lytic bacteriophages for treatment of infections and recent clinical trials have shown great potential. Moreover, several other approaches have been developed in vitro and in vivo using phage-derived proteins as antibacterial agents. Finally, their use has also been widely considered for public health surveillance, as biosensor phages can be used to detect food and water contaminations and prevent bacterial epidemics. These novel approaches strongly promote the idea that phages and their proteins can be exploited as an effective weapon in the near future, especially in a world which is on the brink of a "postantibiotic era."

Bacteriophages and Their Immunological Applications against Infectious Threats

PubMed Central

Lamanna, Jacopo; Ferro, Mattia; Burioni, Roberto

2017-01-01

Bacteriophage therapy dates back almost a century, but the discovery of antibiotics led to a rapid decline in the interests and investments within this field of research. Recently, the novel threat of multidrug-resistant bacteria highlighted the alarming drop in research and development of new antibiotics: 16 molecules were discovered during 1983–87, 10 new therapeutics during the nineties, and only 5 between 2003 and 2007. Phages are therefore being reconsidered as alternative therapeutics. Phage display technique has proved to be extremely promising for the identification of effective antibodies directed against pathogens, as well as for vaccine development. At the same time, conventional phage therapy uses lytic bacteriophages for treatment of infections and recent clinical trials have shown great potential. Moreover, several other approaches have been developed in vitro and in vivo using phage-derived proteins as antibacterial agents. Finally, their use has also been widely considered for public health surveillance, as biosensor phages can be used to detect food and water contaminations and prevent bacterial epidemics. These novel approaches strongly promote the idea that phages and their proteins can be exploited as an effective weapon in the near future, especially in a world which is on the brink of a “postantibiotic era.” PMID:28484722

Protein Corona Composition of Superparamagnetic Iron Oxide Nanoparticles with Various Physico-Chemical Properties and Coatings

NASA Astrophysics Data System (ADS)

Sakulkhu, Usawadee; Mahmoudi, Morteza; Maurizi, Lionel; Salaklang, Jatuporn; Hofmann, Heinrich

2014-05-01

Because of their biocompatibility and unique magnetic properties, superparamagnetic iron oxide nanoparticles NPs (SPIONs) are recognized as some of the most prominent agents for theranostic applications. Thus, understanding the interaction of SPIONs with biological systems is important for their safe design and efficient applications. In this study, SPIONs were coated with 2 different polymers: polyvinyl alcohol polymer (PVA) and dextran. The obtained NPs with different surface charges (positive, neutral, and negative) were used as a model study of the effect of surface charges and surface polymer materials on protein adsorption using a magnetic separator. We found that the PVA-coated SPIONs with negative and neutral surface charge adsorbed more serum proteins than the dextran-coated SPIONs, which resulted in higher blood circulation time for PVA-coated NPs than the dextran-coated ones. Highly abundant proteins such as serum albumin, serotransferrin, prothrombin, alpha-fetoprotein, and kininogen-1 were commonly found on both PVA- and dextran-coated SPIONs. By increasing the ionic strength, soft- and hard-corona proteins were observed on 3 types of PVA-SPIONs. However, the tightly bound proteins were observed only on negatively charged PVA-coated SPIONs after the strong protein elution.

Growing Bacteriophage M13 in Liquid Culture.

PubMed

Green, Michael R; Sambrook, Joseph

2017-11-01

Stocks of bacteriophage M13 are usually grown in liquid culture. The infected bacteria do not lyse but, instead, grow at a slower than normal rate to form a dilute suspension. The inoculum of bacteriophage is almost always a freshly picked plaque or a suspension of bacteriophage particles obtained from a single plaque, as described here. Infected cells contain up to 200 copies of double-stranded, replicative-form DNA and extrude several hundred bacteriophage particles per generation. Thus, a 1-mL culture of infected cells can produce enough double-stranded viral DNA (1-2 mg) for restriction mapping and recovery of cloned DNA inserts and sufficient single-stranded DNA (∼5-10 mg) for site-directed mutagenesis, DNA sequencing, or synthesis of radiolabeled probes. The titer of bacteriophages in the supernatant from infected cells is so high (∼10 12 pfu/mL) that a small aliquot serves as a permanent stock of the starting plaque. © 2017 Cold Spring Harbor Laboratory Press.

Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry.

PubMed

Gutiérrez, Diana; Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

2016-01-01

Microbiological contamination in the food industry is often attributed to the presence of biofilms in processing plants. Bacterial biofilms are complex communities of bacteria attached to a surface and surrounded by an extracellular polymeric material. Their extreme resistance to cleaning and disinfecting processes is related to a unique organization, which implies a differential bacterial growth and gene expression inside the biofilm. The impact of biofilms on health, and the economic consequences, has promoted the development of different approaches to control or remove biofilm formation. Recently, successful results in phage therapy have boosted new research in bacteriophages and phage lytic proteins for biofilm eradication. In this regard, this review examines the environmental factors that determine biofilm development in food-processing equipment. In addition, future perspectives for the use of bacteriophage-derived tools as disinfectants are discussed.

Physical Regulation of the Self-Assembly of Tobacco Mosaic Virus Coat Protein

PubMed Central

Kegel, Willem K.; van der Schoot, Paul

2006-01-01

We present a statistical mechanical model based on the principle of mass action that explains the main features of the in vitro aggregation behavior of the coat protein of tobacco mosaic virus (TMV). By comparing our model to experimentally obtained stability diagrams, titration experiments, and calorimetric data, we pin down three competing factors that regulate the transitions between the different kinds of aggregated state of the coat protein. These are hydrophobic interactions, electrostatic interactions, and the formation of so-called “Caspar” carboxylate pairs. We suggest that these factors could be universal and relevant to a large class of virus coat proteins. PMID:16731551

Evidence for a lineage of virulent bacteriophages that target Campylobacter.

PubMed

Timms, Andrew R; Cambray-Young, Joanna; Scott, Andrew E; Petty, Nicola K; Connerton, Phillippa L; Clarke, Louise; Seeger, Kathy; Quail, Mike; Cummings, Nicola; Maskell, Duncan J; Thomson, Nicholas R; Connerton, Ian F

2010-03-30

Our understanding of the dynamics of genome stability versus gene flux within bacteriophage lineages is limited. Recently, there has been a renewed interest in the use of bacteriophages as 'therapeutic' agents; a prerequisite for their use in such therapies is a thorough understanding of their genetic complement, genome stability and their ecology to avoid the dissemination or mobilisation of phage or bacterial virulence and toxin genes. Campylobacter, a food-borne pathogen, is one of the organisms for which the use of bacteriophage is being considered to reduce human exposure to this organism. Sequencing and genome analysis was performed for two Campylobacter bacteriophages. The genomes were extremely similar at the nucleotide level (> or = 96%) with most differences accounted for by novel insertion sequences, DNA methylases and an approximately 10 kb contiguous region of metabolic genes that were dissimilar at the sequence level but similar in gene function between the two phages. Both bacteriophages contained a large number of radical S-adenosylmethionine (SAM) genes, presumably involved in boosting host metabolism during infection, as well as evidence that many genes had been acquired from a wide range of bacterial species. Further bacteriophages, from the UK Campylobacter typing set, were screened for the presence of bacteriophage structural genes, DNA methylases, mobile genetic elements and regulatory genes identified from the genome sequences. The results indicate that many of these bacteriophages are related, with 10 out of 15 showing some relationship to the sequenced genomes. Two large virulent Campylobacter bacteriophages were found to show very high levels of sequence conservation despite separation in time and place of isolation. The bacteriophages show adaptations to their host and possess genes that may enhance Campylobacter metabolism, potentially advantaging both the bacteriophage and its host. Genetic conservation has been shown to extend to other

Complete genome sequence of 285P, a novel T7-like polyvalent E. coli bacteriophage.

PubMed

Xu, Bin; Ma, Xiangyu; Xiong, Hongyan; Li, Yafei

2014-06-01

Bacteriophages are considered potential biological agents for the control of infectious diseases and environmental disinfection. Here, we describe a novel T7-like polyvalent Escherichia coli bacteriophage, designated "285P," which can lyse several strains of E. coli. The genome, which consists of 39,270 base pairs with a G+C content of 48.73 %, was sequenced and annotated. Forty-three potential open reading frames were identified using bioinformatics tools. Based on whole-genome sequence comparison, phage 285P was identified as a novel strain of subgroup T7. It showed strongest sequence similarity to Kluyvera phage Kvp1. The phylogenetic analyses of both non-structural proteins (endonuclease gp3, amidase gp3.5, DNA primase/helicase gp4, DNA polymerase gp5, and exonuclease gp6) and structural protein (tail fiber protein gp17) led to the identification of 285P as T7-like phage. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses verified the annotation of the structural proteins (major capsid protein gp10a, tail protein gp12, and tail fiber protein gp17).

From Bits and Pieces to Whole Phage to Nanomachines: Pathogen Detection Using Bacteriophages.

PubMed

Anany, H; Chou, Y; Cucic, S; Derda, R; Evoy, S; Griffiths, M W

2017-02-28

The innate specificity of bacteriophages toward their hosts makes them excellent candidates for the development of detection assays. They can be used in many ways to detect pathogens, and each has its own advantages and disadvantages. Whole bacteriophages can carry reporter genes to alter the phenotype of the target. Bacteriophages can act as staining agents or the progeny of the infection process can be detected, which further increases the sensitivity of the detection assay. Compared with whole-phage particles, use of phage components as probes offers other advantages: for example, smaller probe size to enhance binding activity, phage structures that can be engineered for better affinity, as well as specificity, binding properties, and robustness. When no natural binding with the target exists, phages can be used as vehicles to identify new protein-ligand interactions necessary for diagnostics. This review comprehensively summarizes many uses of phages as detection tools and points the way toward how phage-based technologies may be improved.

Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens

PubMed Central

Attai, Hedieh; Rimbey, Jeanette; Smith, George P.

2017-01-01

ABSTRACT To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogen Agrobacterium tumefaciens. Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of A. tumefaciens. The coinoculation of A. tumefaciens with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages. In silico attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative phage peptidoglycan hydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of pph in the absence of additional phage genes causes a block in cell division and subsequent lysis of A. tumefaciens cells. When the presumed active site of the N-acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic. IMPORTANCE The characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as Agrobacterium tumefaciens, may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The

Bacteriophages as indicators of faecal pollution and enteric ...

EPA Pesticide Factsheets

Bacteriophages are an attractive alternative to fecal indicator bacteria (FIB), particularly as surrogates of enteric virus fate and transport due to their closer morphological and biological properties compared to FIB. Based on a meta-analysis of published data, we summarize concentrations of coliphages (F+ and somatic), Bacteroides spp. and enterococci bacteriophages (phages) in human waste, non-human waste, fresh and marine waters as well as removal through wastewater treatment processes. We also provide comparisons with FIB and enteric viruses whenever possible. Lastly, we examine fate and transport characteristics in the environment and provide an overview of the methods available for detection and enumeration of bacteriophages. In summary, concentrations of FIB bacteriophages in various sources were consistently lower than FIB, but more reflective of infectious enteric virus levels. Our investigation supports use of bacteriophages as viral surrogates especially for wastewater treatment processes, while additional research is needed to clarify their utility as indicators of viral fate and transport in the ambient water. Describes concentrations and removal through environmental and engineered systems of bacteriophages, fecal indicator bacteria and viral pathogens.

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

PubMed

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

2013-09-15

Norovirus (NoV), the leading cause of adult non-bacterial gastroenteritis can be commonly detected in wastewater but the extent of NoV removal provided by wastewater treatment plants (WWTPs) is unclear. We monitored a newly commissioned WWTP with UV disinfection on a weekly basis over a six month period for NoV using RT-qPCR and for FRNA bacteriophage GA using both RT-qPCR (total concentration) and a plaque assay (infectious concentration). Mean concentrations of NoV GI and GII in influent wastewater were reduced by 0.25 and 0.41 log10 genome copies 100 ml(-1), respectively by the WWTP. The mean concentration of total FRNA bacteriophage GA was reduced by 0.35 log genome copies 100 ml(-1) compared to a reduction of infectious FRNA bacteriophage GA of 2.13 log PFU 100 ml(-1). A significant difference between concentrations of infectious and total FRNA bacteriophage GA was observed in treated, but not in untreated wastewaters. We conclude that RT-qPCR in isolation underestimates the reduction of infectious virus during wastewater treatment. We further compared the concentrations of infectious virus in combined sewer overflow (CSO) and UV treated effluents using FRNA bacteriophage GA. A greater percentage (98%) of infectious virus is released in CSO discharges than UV treated effluent (44%). Following a CSO discharge, concentrations of NoV GII and infectious FRNA bacteriophage GA in oysters from less than the limit of detection to 3150 genome copies 100 g(-1) and 1050 PFU 100 g(-1) respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

Call for a dedicated European legal framework for bacteriophage therapy.

PubMed

Verbeken, Gilbert; Pirnay, Jean-Paul; Lavigne, Rob; Jennes, Serge; De Vos, Daniel; Casteels, Minne; Huys, Isabelle

2014-04-01

The worldwide emergence of antibiotic resistances and the drying up of the antibiotic pipeline have spurred a search for alternative or complementary antibacterial therapies. Bacteriophages are bacterial viruses that have been used for almost a century to combat bacterial infections, particularly in Poland and the former Soviet Union. The antibiotic crisis has triggered a renewed clinical and agricultural interest in bacteriophages. This, combined with new scientific insights, has pushed bacteriophages to the forefront of the search for new approaches to fighting bacterial infections. But before bacteriophage therapy can be introduced into clinical practice in the European Union, several challenges must be overcome. One of these is the conceptualization and classification of bacteriophage therapy itself and the extent to which it constitutes a human medicinal product regulated under the European Human Code for Medicines (Directive 2001/83/EC). Can therapeutic products containing natural bacteriophages be categorized under the current European regulatory framework, or should this framework be adapted? Various actors in the field have discussed the need for an adapted (or entirely new) regulatory framework for the reintroduction of bacteriophage therapy in Europe. This led to the identification of several characteristics specific to natural bacteriophages that should be taken into consideration by regulators when evaluating bacteriophage therapy. One important consideration is whether bacteriophage therapy development occurs on an industrial scale or a hospital-based, patient-specific scale. More suitable regulatory standards may create opportunities to improve insights into this promising therapeutic approach. In light of this, we argue for the creation of a new, dedicated European regulatory framework for bacteriophage therapy.

Development of bioactive coatings based on γ-irradiated proteins to preserve strawberries

NASA Astrophysics Data System (ADS)

Vu, K. D.; Hollingsworth, R. G.; Salmieri, S.; Takala, P. N.; Lacroix, M.

2012-08-01

Gamma irradiation was applied for creating cross-linked proteins to enhance the physicochemical properties of edible films made of calcium caseinate, whey protein isolate and glycerol. The characteristics of γ irradiated cross-linked proteins were analyzed by Fourier Transform Infrared spectroscopy. A second derivative spectra exhibited changes in band intensities that were correlated to an increase of β-sheet structure and a decrease of α-helix and unordered fractions of γ irradiated-cross-linked proteins as compared to the control without irradiation. Furthermore, on addition of methylcellulose to the irradiated protein matrix it was found that it has potential in enhancing the puncture strength and has no detrimental effect on water vapor permeability of protein based films. Finally, these film formulations were used as bioactive edible coatings containing natural antimicrobial agents (limonene and peppermint) to preserve the shelf life of fresh strawberries during storage. The bioactive coatings containing peppermint was found to be more efficient as preserving coatings than the formulations containing limonene. Irradiated proteins/methylcellulose/peppermint formulation had only 40% of decay at day 8 while it was 65% for the control.

[Isolation and characterization of a lytic bacteriophage from Mingyong glacier melt water].

PubMed

Li, Mingyuan; Ji, Xiuling; Wang, Baoqiang; Zhang, Qi; Lin, Lianbing; Zhang, Bing; Wei, Yunlin

2012-02-04

Glacier is a unique ecological system. This study focused on the isolation and characterization of a cold-active bateriophage from Mingyong glacier area in northwest Yunnan. Bacterial strains isolated from glacial melt water were used as host cells to isolate and purify bacteriophages by double-layer plate method. The morphology of the isolated phages and their host strains were observed by electron microscope. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA, constituent proteins and physiological analysis of the bacteriophages were further carried out to characterize the phages. A lytic cold-active bacteriophage, designated as MYSP03, was isolated from Mingyong glacier. Its host strain MYB03 was identified as a member of genus Flavobacterium, based on the 16S rRNA sequence analysis. The bacteriophage MYSP03 has a isometric head (about 72 nm in diameter) and a long tail (about 240 nm in length and 10 nm in width), but no envelope was detected. Physiological analysis results showed that MYSP03 had infection activity at 4 degrees C, and clear and transparent plaques were formed on double-layer plates between 4 and 20 degrees C. Its optimum infection temperature was 10 degrees C and optimal pH 9.4, respectively. It is insensitive to chloroform. Furthermore, the genome of MYSP03 consists of double-stranded DNA and is approximately 66 kb.

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.

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

M13 Bacteriophage-Polymer Nanoassemblies as Drug Delivery Vehicles

DTIC Science & Technology

2011-01-01

the M13 bacteriophage is well-defined and can be genetically engineered to produce conductive fibers [35] or display peptides or proteins in...36], and fluorescent dyes [42, 43], can be chemically anchored on the surface of M13 bacterio- phage . Our group has systematically investigated the...bioconjugation chemistry of M13 , and used such modified M13 phages to produce conductive nanofibers [35], direct cell growth [36], and target

Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry

PubMed Central

Gutiérrez, Diana; Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

2016-01-01

Microbiological contamination in the food industry is often attributed to the presence of biofilms in processing plants. Bacterial biofilms are complex communities of bacteria attached to a surface and surrounded by an extracellular polymeric material. Their extreme resistance to cleaning and disinfecting processes is related to a unique organization, which implies a differential bacterial growth and gene expression inside the biofilm. The impact of biofilms on health, and the economic consequences, has promoted the development of different approaches to control or remove biofilm formation. Recently, successful results in phage therapy have boosted new research in bacteriophages and phage lytic proteins for biofilm eradication. In this regard, this review examines the environmental factors that determine biofilm development in food-processing equipment. In addition, future perspectives for the use of bacteriophage-derived tools as disinfectants are discussed. PMID:27375566

Bacteriophage-based nanoprobes for rapid bacteria separation

NASA Astrophysics Data System (ADS)

Chen, Juhong; Duncan, Bradley; Wang, Ziyuan; Wang, Li-Sheng; Rotello, Vincent M.; Nugen, Sam R.

2015-10-01

The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying concentrations were determined. The results indicated a similar bacteria capture efficiency between the two nanoprobes.The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying

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

Isolation and characterization of bacteriophages of Salmonella enterica serovar Pullorum.

PubMed

Bao, H; Zhang, H; Wang, R

2011-10-01

In this study, 2 bacteriophages of Salmonella Pullorum were isolated using an enrichment protocol and the double agar layer method. They were named PSPu-95 and PSPu-4-116, respectively, against clinical isolates of Salmonella Pullorum SPu-95 and SPu-116. The host ranges of the 2 bacteriophages were determined by performing spot tests with 20 bacteria strains. Both bacteriophages had wide host ranges. Bacteriophage PSPu-95 had a lytic effect on 17 of the 20 isolates (85%), and PSPu-4-116 produced a lytic effect on 14 isolates (70%) and was the only bacteriophage that produced a clear plaque on enterotoxigenic Escherichia coli K88. Transmission electron microscopy revealed the bacteriophages belonged to the order Caudovirales. Bacteriophage PSPu-95 was a member of the family Siphoviridae, but bacteriophage PSPu-4-116 belonged to the family Myoviridae. Both had a double-stranded DNA, which was digested with HindIII or EcoRI, that was estimated to be 58.3 kbp (PSPu-95) and 45.2 kbp (PSPu-4-116) by 1% agar electrophoresis. One-step growth kinetics showed that the latent periods were all less than 20 min, and the burst size was 77.5 pfu/cell for PSPu-95 and 86 pfu/cell for PSPu-4-116. The bacteriophages were able to survive in a pH range between 4 and 10, and they were able to survive in a treatment of 70°C for 60 min. The characterizations of these 2 bacteriophages were helpful in establishing a basis for adopting the most effective bacteriophage to control bacteria in the poultry industry.

Bacterial adhesion to protein-coated surfaces: An AFM and QCM-D study

NASA Astrophysics Data System (ADS)

Strauss, Joshua; Liu, Yatao; Camesano, Terri A.

2009-09-01

Bacterial adhesion to biomaterials, mineral surfaces, or other industrial surfaces is strongly controlled by the way bacteria interact with protein layers or organic matter and other biomolecules that coat the materials. Despite this knowledge, many studies of bacterial adhesion are performed under clean conditions, instead of in the presence of proteins or organic molecules. We chose fetal bovine serum (FBS) as a model protein, and prepared FBS films on quartz crystals. The thickness of the FBS layer was characterized using atomic force microscopy (AFM) imaging under liquid and quartz crystal microbalance with dissipation (QCM-D). Next, we characterized how the model biomaterial surface would interact with the nocosomial pathogen Staphylococcus epidermidis. An AFM probe was coated with S. epidermidis cells and used to probe a gold slide that had been coated with FBS or another protein, fibronectin (FN). These experiments show that AFM and QCM-D can be used in complementary ways to study the complex interactions between bacteria, proteins, and surfaces.

Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

PubMed

Masuda, Hiro-taka; Ishihara, Seiichiro; Harada, Ichiro; Mizutani, Takeomi; Ishikawa, Masayori; Kawabata, Kazushige; Haga, Hisashi

2014-01-01

We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

Detection specificity studies of bacteriophage adhesin-coated long-period grating-based biosensor

NASA Astrophysics Data System (ADS)

Koba, Marcin; Śmietana, Mateusz; Brzozowska, Ewa; Górska, Sabina; Mikulic, Predrag; Cusano, Andrea; Bock, Wojtek J.

2015-09-01

In this work, we present a label-free detection specificity study of an optical fiber long-period grating (LPG) biosensor working near the dispersion turning point of higher order cladding modes. The LPG sensor functionalized with bacteriophage adhesin is tested with specific and non-specific bacteria dry weight. We show that such biosensor is able to selectively bind, thus recognize different bacteria. We use bacteria dry weights of E. coli B as positive test and E. coli K12 and Salmonella enterica as negative tests. The resonance wavelength shift induced by E. coli B reaches over 90 nm, while for E. coli K12 and Salmonella enterica approximately 40 and 20 nm, respectively.

Challenging the state of the art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10.

PubMed

Kryshtafovych, Andriy; Moult, John; Bales, Patrick; Bazan, J Fernando; Biasini, Marco; Burgin, Alex; Chen, Chen; Cochran, Frank V; Craig, Timothy K; Das, Rhiju; Fass, Deborah; Garcia-Doval, Carmela; Herzberg, Osnat; Lorimer, Donald; Luecke, Hartmut; Ma, Xiaolei; Nelson, Daniel C; van Raaij, Mark J; Rohwer, Forest; Segall, Anca; Seguritan, Victor; Zeth, Kornelius; Schwede, Torsten

2014-02-01

For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins. Copyright © 2013 The Authors. Wiley Periodicals, Inc.

Challenging the state-of-the-art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10

PubMed Central

Kryshtafovych, Andriy; Moult, John; Bales, Patrick; Bazan, J. Fernando; Biasini, Marco; Burgin, Alex; Chen, Chen; Cochran, Frank V.; Craig, Timothy K.; Das, Rhiju; Fass, Deborah; Garcia-Doval, Carmela; Herzberg, Osnat; Lorimer, Donald; Luecke, Hartmut; Ma, Xiaolei; Nelson, Daniel C.; van Raaij, Mark J.; Rohwer, Forest; Segall, Anca; Seguritan, Victor; Zeth, Kornelius; Schwede, Torsten

2014-01-01

For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, over 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this paper, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict trans-membrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin IL-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fibre protein gp17 from bacteriophage T7; the Bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins. PMID:24318984

Real-Time Protein and Cell Binding Measurements on Hydroxyapatite Coatings

PubMed Central

Vilardell, A. M.; Cinca, N.; Jokinen, A.; Garcia-Giralt, N.; Dosta, S.; Cano, I. G.; Guilemany, J. M.

2016-01-01

Although a lot of in vitro and in vivo assays have been performed during the last few decades years for hydroxyapatite bioactive coatings, there is a lack of exploitation of real-time in vitro interaction measurements. In the present work, real-time interactions for a plasma sprayed hydroxyapatite coating were measured by a Multi-Parametric Surface Plasmon Resonance (MP-SPR), and the results were compared with standard traditional cell viability in vitro assays. MP-SPR is proven to be suitable not only for measurement of molecule–molecule interactions but also molecule–material interaction measurements and cell interaction. Although SPR is extensively utilized in interaction studies, recent research of protein or cell adsorption on hydroxyapatite coatings for prostheses applications was not found. The as-sprayed hydroxyapatite coating resulted in 62.4% of crystalline phase and an average thickness of 24 ± 6 μm. The MP-SPR was used to measure lysozyme protein and human mesenchymal stem cells interaction to the hydroxyapatite coating. A comparison between the standard gold sensor and Hydroxyapatite (HA)-plasma coated sensor denoted a clearly favourable cell attachment on HA coated sensor as a significantly higher signal of cell binding was detected. Moreover, traditional cell viability and proliferation tests showed increased activity with culture time indicating that cells were proliferating on HA coating. Cells show homogeneous distribution and proliferation along the HA surface between one and seven days with no significant mortality. Cells were flattened and spread on rough surfaces from the first day, with increasing cytoplasmatic extensions during the culture time. PMID:27618911

Construction and properties of a temperature-sensitive mutation in the gene for the bacteriophage SPO1 DNA-binding protein TF1.

PubMed

Sayre, M H; Geiduschek, E P

1990-08-01

The Bacillus subtilis bacteriophage SPO1 encodes the DNA-binding protein TF1, a homolog of the ubiquitous type II DNA-binding proteins that are components of bacterial chromatin. The known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the TF1 gene. At the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, altered the kinetics of accumulation of at least one viral transcript, and prohibited the production of infective progeny phage. We suggest that TF1 function is required to shut off the expression of several early-middle and middle viral genes and that TF1 plays a role in phage head morphogenesis. Spontaneous second-site mutations of the temperature-sensitive mutant TF1 allele that suppressed its associated phenotypes were analyzed. These suppressor mutations conferred greater amino acid sequence homology with the type II DNA-binding protein from the thermophile Bacillus stearothermophilus.

Polymer-based delivery systems for support and delivery of bacteriophages

NASA Astrophysics Data System (ADS)

Brown, Alyssa Marie

One of the most urgent problems in the fields of medicine and agriculture is the decreasing effectiveness of antibiotics. Once a miracle drug, antibiotics have recently become associated with the creation of antibiotic-resistant bacteria. The main limitations of these treatments include lack of both adaptability and specificity. To overcome these shortcomings of current antibiotic treatments, there has been a renewed interest in bacteriophage research. Bacteriophages are naturally-occurring viruses that lyse bacteria. They are highly specific, with each bacteriophage type lysing a narrow range of bacteria strains. Bacteriophages are also ubiquitous biological entities, populating environments where bacterial growth is supported. Just as humans are exposed to bacteria in their daily lives, we are exposed to bacteriophages as well. To use bacteriophages in practical applications, they must be delivered to the site of an infection in a controlled-release system. Two systems were studied to observe their support of bacteriophage lytic activity, as well as investigate the possibility of controlling bacteriophage release rates. First, hydrogels were studied, using crosslinking and blending techniques to achieve a range of release profiles. Second, polyanhydride microparticles were studied, evaluating release rates as a function of monomer chemistries.

RNA-dependent RNA polymerases of dsRNA bacteriophages.

PubMed

Makeyev, Eugene V; Grimes, Jonathan M

2004-04-01

Genome replication and transcription of riboviruses are catalyzed by an RNA-dependent RNA polymerase (RdRP). RdRPs are normally associated with other virus- or/and host-encoded proteins that modulate RNA polymerization activity and template specificity. The polymerase complex of double-stranded dsRNA viruses is a large icosahedral particle (inner core) containing RdRP as a minor constituent. In phi6 and other dsRNA bacteriophages from the Cystoviridae family, the inner core is composed of four virus-specific proteins. Of these, protein P2, or Pol subunit, has been tentatively identified as RdRP by sequence comparisons, but the role of this protein in viral RNA synthesis has not been studied until recently. Here, we overview the work on the Pol subunits of phi6 and related viruses from the standpoints of function, structure and evolution.

Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

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

Bielmann, Regula; Habann, Matthias; Eugster, Marcel R.

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wallmore » teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.« less

Bacteriophage Applications for Food Production and Processing

PubMed Central

Moye, Zachary D.; Woolston, Joelle; Sulakvelidze, Alexander

2018-01-01

Foodborne illnesses remain a major cause of hospitalization and death worldwide despite many advances in food sanitation techniques and pathogen surveillance. Traditional antimicrobial methods, such as pasteurization, high pressure processing, irradiation, and chemical disinfectants are capable of reducing microbial populations in foods to varying degrees, but they also have considerable drawbacks, such as a large initial investment, potential damage to processing equipment due to their corrosive nature, and a deleterious impact on organoleptic qualities (and possibly the nutritional value) of foods. Perhaps most importantly, these decontamination strategies kill indiscriminately, including many—often beneficial—bacteria that are naturally present in foods. One promising technique that addresses several of these shortcomings is bacteriophage biocontrol, a green and natural method that uses lytic bacteriophages isolated from the environment to specifically target pathogenic bacteria and eliminate them from (or significantly reduce their levels in) foods. Since the initial conception of using bacteriophages on foods, a substantial number of research reports have described the use of bacteriophage biocontrol to target a variety of bacterial pathogens in various foods, ranging from ready-to-eat deli meats to fresh fruits and vegetables, and the number of commercially available products containing bacteriophages approved for use in food safety applications has also been steadily increasing. Though some challenges remain, bacteriophage biocontrol is increasingly recognized as an attractive modality in our arsenal of tools for safely and naturally eliminating pathogenic bacteria from foods. PMID:29671810

Comparative Genomics of Bacteriophage of the Genus Seuratvirus

PubMed Central

Sazinas, Pavelas; Redgwell, Tamsin; Rihtman, Branko; Grigonyte, Aurelija; Michniewski, Slawomir; Scanlan, David J; Hobman, Jon

2018-01-01

Abstract Despite being more abundant and having smaller genomes than their bacterial host, relatively few bacteriophages have had their genomes sequenced. Here, we isolated 14 bacteriophages from cattle slurry and performed de novo genome sequencing, assembly, and annotation. The commonly used marker genes polB and terL showed these bacteriophages to be closely related to members of the genus Seuratvirus. We performed a core-gene analysis using the 14 new and four closely related genomes. A total of 58 core genes were identified, the majority of which has no known function. These genes were used to construct a core-gene phylogeny, the results of which confirmed the new isolates to be part of the genus Seuratvirus and expanded the number of species within this genus to four. All bacteriophages within the genus contained the genes queCDE encoding enzymes involved in queuosine biosynthesis. We suggest these genes are carried as a mechanism to modify DNA in order to protect these bacteriophages against host endonucleases. PMID:29272407

Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface

NASA Astrophysics Data System (ADS)

Abramov, Gili; Morag, Omry; Goldbourt, Amir

2015-04-01

Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses.

Dissecting binding of a β-barrel membrane protein by phage display.

PubMed

Meneghini, Luz M; Tripathi, Sarvind; Woodworth, Marcus A; Majumdar, Sudipta; Poulos, Thomas L; Weiss, Gregory A

2017-07-25

Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein's initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.

Bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection.

PubMed

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

2011-11-01

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

Direction of Translation and Size of Bacteriophage φX174 Cistrons

PubMed Central

Benbow, Robert M.; Mayol, Robert F.; Picchi, Joanna C.; Sinsheimer, Robert L.

1972-01-01

Translation of the bacteriophage φX174 genome follows cistron order D-E-F-G-H-A-B-C. To establish this, the position of a nonsense mutation on the genetic map was compared with the physical size (molecular weight) of the appropriate protein fragment generated in nonpermissive cells. Distances on the φX174 genetic map and distances on a physical map constructed from the molecular weights of φX174 proteins and protein fragments are proportional over most of the genome with the exception of the high recombination region within cistron A. Images PMID:16789133

Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens.

PubMed

Attai, Hedieh; Rimbey, Jeanette; Smith, George P; Brown, Pamela J B

2017-12-01

To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogen Agrobacterium tumefaciens Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of A. tumefaciens The coinoculation of A. tumefaciens with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages. In silico attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative p hage p eptidoglycan h ydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of pph in the absence of additional phage genes causes a block in cell division and subsequent lysis of A. tumefaciens cells. When the presumed active site of the N -acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic. IMPORTANCE The characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as Agrobacterium tumefaciens , may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The

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

RNA packaging device of double-stranded RNA bacteriophages, possibly as simple as hexamer of P4 protein.

PubMed

Kainov, Denis E; Pirttimaa, Markus; Tuma, Roman; Butcher, Sarah J; Thomas, George J; Bamford, Dennis H; Makeyev, Eugene V

2003-11-28

Genomes of complex viruses have been demonstrated, in many cases, to be packaged into preformed empty capsids (procapsids). This reaction is performed by molecular motors translocating nucleic acid against the concentration gradient at the expense of NTP hydrolysis. At present, the molecular mechanisms of packaging remain elusive due to the complex nature of packaging motors. In the case of the double-stranded RNA bacteriophage phi 6 from the Cystoviridae family, packaging of single-stranded genomic precursors requires a hexameric NTPase, P4. In the present study, the purified P4 proteins from two other cystoviruses, phi 8 and phi 13, were characterized and compared with phi 6 P4. All three proteins are hexameric, single-stranded RNA-stimulated NTPases with alpha/beta folds. Using a direct motor assay, we found that phi 8 and phi 13 P4 hexamers translocate 5' to 3' along ssRNA, whereas the analogous activity of phi 6 P4 requires association with the procapsid. This difference is explained by the intrinsically high affinity of phi 8 and phi 13 P4s for nucleic acids. The unidirectional translocation results in RNA helicase activity. Thus, P4 proteins of Cystoviridae exhibit extensive similarity to hexameric helicases and are simple models for studying viral packaging motor mechanisms.

Sensing of heavy metal ions by intrinsic TMV coat protein fluorescence.

PubMed

Bayram, Serene S; Green, Philippe; Blum, Amy Szuchmacher

2018-04-15

We propose the use of a cysteine mutant of TMV coat protein as a signal transducer for the selective sensing and quantification of the heavy metal ions, Cd 2+ , Pb 2+ , Zn 2+ and Ni 2+ based on intrinsic tryptophan quenching. TMV coat protein is inexpensive, can be mass-produced since it is expressed and extracted from E-coli. It also displays several different functional groups, enabling a wide repertoire of bioconjugation chemistries; thus it can be easily integrated into functional devices. In addition, TMV-ion interactions have been widely reported and utilized for metallization to generate organic-inorganic hybrid composite novel materials. Building on these previous observations, we herein determine, for the first time, the TMV-ion binding constants assuming the static fluorescence quenching model. We also show that by comparing TMV-ion interactions between native and denatured coat protein, we can distinguish between chemically similar heavy metal ions such as cadmium and zinc ions. Copyright © 2018 Elsevier B.V. All rights reserved.

Sensing of heavy metal ions by intrinsic TMV coat protein fluorescence

NASA Astrophysics Data System (ADS)

Bayram, Serene S.; Green, Philippe; Blum, Amy Szuchmacher

2018-04-01

We propose the use of a cysteine mutant of TMV coat protein as a signal transducer for the selective sensing and quantification of the heavy metal ions, Cd2+, Pb2+, Zn2+ and Ni2+ based on intrinsic tryptophan quenching. TMV coat protein is inexpensive, can be mass-produced since it is expressed and extracted from E-coli. It also displays several different functional groups, enabling a wide repertoire of bioconjugation chemistries; thus it can be easily integrated into functional devices. In addition, TMV-ion interactions have been widely reported and utilized for metallization to generate organic-inorganic hybrid composite novel materials. Building on these previous observations, we herein determine, for the first time, the TMV-ion binding constants assuming the static fluorescence quenching model. We also show that by comparing TMV-ion interactions between native and denatured coat protein, we can distinguish between chemically similar heavy metal ions such as cadmium and zinc ions.

40 CFR 174.515 - Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

40 CFR 174.516 - Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

40 CFR 174.512 - Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Potato Virus Y...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.512 Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance. Residues of Coat Protein of Potato Virus Y are exempt from...

40 CFR 174.512 - Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Coat Protein of Potato Virus Y...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.512 Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance. Residues of Coat Protein of Potato Virus Y are exempt from...

40 CFR 174.515 - Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

40 CFR 174.516 - Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

40 CFR 174.515 - Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

40 CFR 174.516 - Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

40 CFR 174.516 - Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

40 CFR 174.512 - Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Coat Protein of Potato Virus Y...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.512 Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance. Residues of Coat Protein of Potato Virus Y are exempt from...

40 CFR 174.512 - Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Coat Protein of Potato Virus Y...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.512 Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance. Residues of Coat Protein of Potato Virus Y are exempt from...

40 CFR 174.515 - Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

40 CFR 174.515 - Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

40 CFR 174.512 - Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Coat Protein of Potato Virus Y...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.512 Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance. Residues of Coat Protein of Potato Virus Y are exempt from...

40 CFR 174.516 - Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

Occurrence of Propionibacterium freudenreichii bacteriophages in swiss cheese.

PubMed Central

Gautier, M; Rouault, A; Sommer, P; Briandet, R

1995-01-01

We isolated bacteriophages active against Propionibacterium freudenreichii from 16 of 32 swiss cheese samples. Bacteriophage concentrations ranged from 14 to 7 x 10(5) PFU/g, depending on the sample and the sensitive strain used for detection. Only a few strains, 8 of the 44 strains of P. freudenreichii in our collection, were sensitive. We observed that multiplication of bacteriophages occurred in the cheese loaf during multiplication of propionibacteria in a warm curing room, but it seems that these bacteriophages have no adverse effect on the development of the propionic flora. We also found that sensitive cells, originating from either the starter or the cheese-making milk, were present at a high level (10(9) CFU/g) in the cheese. PMID:7618869

40 CFR 174.531 - Coat protein of plum pox virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of plum pox virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.531 Coat protein of plum pox virus; exemption from the requirement of a tolerance. Residues of the coat protein of plum pox virus in or on the...

40 CFR 174.531 - Coat protein of plum pox virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Coat protein of plum pox virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.531 Coat protein of plum pox virus; exemption from the requirement of a tolerance. Residues of the coat protein of plum pox virus in or on the...

40 CFR 174.531 - Coat protein of plum pox virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Coat protein of plum pox virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.531 Coat protein of plum pox virus; exemption from the requirement of a tolerance. Residues of the coat protein of plum pox virus in or on the...

40 CFR 174.531 - Coat protein of plum pox virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Coat protein of plum pox virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.531 Coat protein of plum pox virus; exemption from the requirement of a tolerance. Residues of the coat protein of plum pox virus in or on the...

40 CFR 174.531 - Coat protein of plum pox virus; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Coat protein of plum pox virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.531 Coat protein of plum pox virus; exemption from the requirement of a tolerance. Residues of the coat protein of plum pox virus in or on the...

Reduction of Salmonella in ground chicken using a bacteriophage.

PubMed

Grant, Ar'Quette; Parveen, Salina; Schwarz, Jurgen; Hashem, Fawzy; Vimini, Bob

2017-08-01

This study's goal was to ascertain the effectiveness of a commercially available Salmonella bacteriophage during ground chicken production focusing on: water source, different Salmonella serovars, and time. Salmonella-free boneless, skinless chicken meat was inoculated with 4.0 Log CFU/cm2 of either a cocktail of 3 Salmonella isolates derived from ground chicken (GC) or a cocktail of 3 Salmonella strains not isolated from ground chicken (non-GC). Bacteriophages were spread onto the chicken using sterile tap or filtered water for 30 min or 8 h. Salmonella was recovered using standard plating method. Greater Salmonella reduction was observed when the bacteriophage was diluted in sterile tap water than in sterile filtered water: 0.39 Log CFU/cm2 and 0.23 Log CFU/cm2 reduction after 30 min, respectively (P < 0.05). The non-GC isolates showed reductions of 0.71 Log CFU/cm2 and 0.90 Log CFU/cm2 after 30 min and 8 h, respectively (P < 0.05). The GC isolates were less sensitive to the bacteriophage: 0.39 Log CFU/cm2 and 0.67 Log CFU/cm2 reductions after 30 min and 8 h, respectively (P < 0.05). In conclusion, bacteriophage reduction was dependent on water used to dilute the bacteriophage, Salmonella's susceptibility to the bacteriophage, and treatment time. © 2017 Poultry Science Association Inc.

Susceptibility of Escherichia coli isolated from uteri of postpartum dairy cows to antibiotic and environmental bacteriophages. Part I: Isolation and lytic activity estimation of bacteriophages.

PubMed

Bicalho, R C; Santos, T M A; Gilbert, R O; Caixeta, L S; Teixeira, L M; Bicalho, M L S; Machado, V S

2010-01-01

The objective of this study was to isolate bacteriophages from environmental samples of 2 large commercial dairy farms using Escherichia coli isolated from the uteri of postpartum Holstein dairy cows as hosts. A total of 11 bacteriophage preparations were isolated from manure systems of commercial dairy farms and characterized for in vitro antimicrobial activity. In addition, a total of 57 E. coli uterine isolates from 5 dairy cows were phylogenetically grouped by triplex PCR. Each E. coli bacterial host from the uterus was inoculated with their respective bacteriophage preparation at several different multiplicities of infections (MOI) to determine minimum inhibitory MOI. The effect of a single dose (MOI=10(2)) of bacteriophage on the growth curve of all 57 E. coli isolates was assessed using a microplate technique. Furthermore, genetic diversity within and between the different bacteriophage preparations was assessed by bacteriophage purification followed by DNA extraction, restriction, and agarose gel electrophoresis. Phylogenetic grouping based on triplex PCR showed that all isolates of E. coli belonged to phylogroup B1. Bacterial growth was completely inhibited at considerably low MOI, and the effect of a single dose (MOI=10(2)) of bacteriophage preparations on the growth curve of all 57 E. coli isolates showed that all bacteriophage preparations significantly decreased the growth rate of the isolates. Bacteriophage preparation 1230-10 had the greatest antimicrobial activity and completely inhibited the growth of 71.7% (n=57) of the isolates. The combined action of bacteriophage preparations 1230-10, 6375-10, 2540-4, and 6547-2, each at MOI=10(2), had the broadest spectrum of action and completely inhibited the growth (final optical density at 600 nm

Magic-angle spinning NMR of intact bacteriophages: insights into the capsid, DNA and their interface.

PubMed

Abramov, Gili; Morag, Omry; Goldbourt, Amir

2015-04-01

Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses. Copyright © 2015 Elsevier Inc. All rights reserved.

Genomic Diversity of Type B3 Bacteriophages of Caulobacter crescentus.

PubMed

Ash, Kurt T; Drake, Kristina M; Gibbs, Whitney S; Ely, Bert

2017-07-01

The genomes of the type B3 bacteriophages that infect Caulobacter crescentus are among the largest phage genomes thus far deposited into GenBank with sizes over 200 kb. In this study, we introduce six new bacteriophage genomes which were obtained from phage collected from various water systems in the southeastern United States and from tropical locations across the globe. A comparative analysis of the 12 available genomes revealed a "core genome" which accounts for roughly 1/3 of these bacteriophage genomes and is predominately localized to the head, tail, and lysis gene regions. Despite being isolated from geographically distinct locations, the genomes of these bacteriophages are highly conserved in both genome sequence and gene order. We also identified the insertions, deletions, translocations, and horizontal gene transfer events which are responsible for the genomic diversity of this group of bacteriophages and demonstrated that these changes are not consistent with the idea that modular reassortment of genomes occurs in this group of bacteriophages.

Protein-adsorption and Ca-phosphate formation on chitosan-bioactive glass composite coatings

NASA Astrophysics Data System (ADS)

Wagener, V.; Boccaccini, A. R.; Virtanen, S.

2017-09-01

In the last years, chitosan-bioactive glass (BG) composites have been developed and investigated as bioactive coatings for orthopedic applications. The increase of bioactivity occurs due to the stimulation of calcium-phosphate/hydroxyapatite formation on the surface while the coating is degrading. In the present work, protein adsorption and its influence on calcium-phosphate precipitation was studied for the first time on such composite coatings. The experiments involved coating of 316L stainless steel substrates with chitosan (Ch) and chitosan-bioactive glass (Ch-BG) and immersion of the coated samples in two different bovine serum albumin (BSA) containing solutions, namely DI H2O (with pH adjusted to about 7.2 with diluted NaOH) and simulated body fluid (SBF). In order to investigate the influence of protein adsorption on calcium-phosphate precipitation, samples were also immersed in DI H2O and in SBF without BSA. Samples were analyzed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Surface analysis revealed that adsorption of BSA takes place on all studied samples and that protein adsorption is influenced by the presence of Ca2+ and PO43- ions. Bioactivity in the form of hydroxyapatite pre-stage formation is significantly increased on Ch-BG composite coating as compared with bare stainless steel surface. However, calcium-phosphate precipitation in SBF is reduced by the presence of BSA.

Bacteriophage Taxonomy: An Evolving Discipline.

PubMed

Tolstoy, Igor; Kropinski, Andrew M; Brister, J Rodney

2018-01-01

While taxonomy is an often-unappreciated branch of science it serves very important roles. Bacteriophage taxonomy has evolved from a mainly morphology-based discipline, characterized by the work of David Bradley and Hans-Wolfgang Ackermann, to the holistic approach that is taken today. The Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) takes a comprehensive approach to classifying prokaryote viruses measuring overall DNA and protein identity and phylogeny before making decisions about the taxonomic position of a new virus. The huge number of complete genomes being deposited with NCBI and other public databases has resulted in a reassessment of the taxonomy of many viruses, and the future will see the introduction of new viral families and higher orders.

M13 Bacteriophage/Silver Nanowire Surface-Enhanced Raman Scattering Sensor for Sensitive and Selective Pesticide Detection.

PubMed

Koh, Eun Hye; Mun, ChaeWon; Kim, ChunTae; Park, Sung-Gyu; Choi, Eun Jung; Kim, Sun Ho; Dang, Jaejeung; Choo, Jaebum; Oh, Jin-Woo; Kim, Dong-Ho; Jung, Ho Sang

2018-03-28

A surface-enhanced Raman scattering (SERS) sensor comprising silver nanowires (AgNWs) and genetically engineered M13 bacteriophages expressing a tryptophan-histidine-tryptophan (WHW) peptide sequence (BPWHW) was fabricated by simple mixing of BPWHW and AgNW solutions, followed by vacuum filtration onto a glass-fiber filter paper (GFFP) membrane. The AgNWs stacked on the GFFP formed a high density of SERS-active hot spots at the points of nanowire intersections, and the surface-coated BPWHW functioned as a bioreceptor for selective pesticide detection. The BPWHW-functionalized AgNW (BPWHW/AgNW) sensor was characterized by scanning electron microscopy, confocal scanning fluorescence microscopy, atomic force microscopy, and Fourier transform infrared spectroscopy. The Raman signal enhancement and the selective pesticide SERS detection properties of the BPWHW/AgNW sensor were investigated in the presence of control substrates such as wild-type M13 bacteriophage-decorated AgNWs (BPWT/AgNW) and undecorated AgNWs (AgNW). The BPWHW/AgNW sensor exhibited a significantly higher capture capability for pesticides, especially paraquat (PQ), than the control SERS substrates, and it also showed a relatively higher selectivity for PQ than for other bipyridylium pesticides such as diquat and difenzoquat. Furthermore, as a field application test, PQ was detected on the surface of PQ-pretreated apple peels, and the results demonstrated the feasibility of using a paper-based SERS substrate for on-site residual pesticide detection. The developed M13 bacteriophage-functionalized AgNW SERS sensor might be applicable for the detection of various pesticides and chemicals through modification of the M13 bacteriophage surface peptide sequence.

First steps of bacteriophage SPP1 entry into Bacillus subtilis.

PubMed

Jakutytė, Lina; Lurz, Rudi; Baptista, Catarina; Carballido-Lopez, Rut; São-José, Carlos; Tavares, Paulo; Daugelavičius, Rimantas

2012-01-20

The mechanism of genome transfer from the virion to the host cytoplasm is critical to understand and control the beginning of viral infection. The initial steps of bacteriophage SPP1 infection of the Gram-positive bacterium Bacillus subtilis were monitored by following changes in permeability of the cytoplasmic membrane (CM). SPP1 leads to a distinctively faster CM depolarization than the one caused by podovirus ϕ29 or myovirus SP01 during B. subtilis infection. Depolarization requires interaction of SPP1 infective virion to its receptor protein YueB. The amplitude of depolarization depends on phage input and concentration of YueB at the cell surface. Sub-millimolar concentrations of Ca(2+) are necessary and sufficient for SPP1 reversible binding to the host envelope and thus to trigger depolarization while DNA delivery to the cytoplasm depends on millimolar concentrations of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of goethite coating and humic acid on the transport of bacteriophage PRD1 in columns of saturated sand

NASA Astrophysics Data System (ADS)

Foppen, J. W. A.; Okletey, S.; Schijven, J. F.

2006-05-01

The transport of bacteriophage PRD1, a model virus, was studied in columns containing sediment mixtures of quartz sand with goethite-coated sand and using various solutions consisting of monovalent and divalent salts and humic acid (HA). Without HA and in the absence of sand, the inactivation rate of PRD1 was found to be as low as 0.014 day - 1 (at 5 ± 3 °C), but in the presence of HA it was much lower (0.0009 day - 1 ), indicating that HA helps PRD1 to survive. When the fraction of goethite in the sediment was increased, the removal of PRD1 also increased. However, in the presence of HA, C/ C0 values of PRD1 increased by as much as 5 log units, thereby almost completely eliminating the effect of addition of goethite. The sticking efficiency was not linearly dependent on the amount of goethite added to the quartz sand; this is apparently due to surface charge heterogeneity of PRD1. Our results imply that, in the presence of dissolved organic matter (DOM), viruses can be transported for long distances thanks to two effects: attachment is poor because DOM has occupied favourable sites for attachment and inactivation of virus may have decreased. This conclusion justifies making conservative assumptions about the attachment of viruses when calculating protection zones for groundwater wells.

Molecular interactions of mussel protective coating protein, mcfp-1, from Mytilus californianus.

PubMed

Lu, Qingye; Hwang, Dong Soo; Liu, Yang; Zeng, Hongbo

2012-02-01

Protective coating of the byssus of mussels (Mytilus sp.) has been suggested as a new paradigm of medical coating due to its high extensibility and hardness co-existence without their mutual detriment. The only known biomacromolecule in the extensible and tough coating on the byssus is mussel foot protein-1 (mfp-1), which is made up with positively charged residues (~20 mol%) and lack of negatively charged residues. Here, adhesion and molecular interaction mechanisms of Mytilus californianus foot protein-1 (mcfp-1) from California blue mussel were investigated using a surface forces apparatus (SFA) in buffer solutions of different ionic concentrations (0.2-0.7 M) and pHs (3.0-5.5). Strong and reversible cohesion between opposed positively charged mcfp-1 films was measured in 0.1 M sodium acetate buffer with 0.1 M KNO(3). Cohesion of mcfp-1 was gradually reduced with increasing the ionic strength, but was not changed with pH variations. Oxidation of 3,4-dihydroxyphenylalanine (DOPA) residues of mcfp-1, a key residue for adhesive and coating proteins of mussel, didn't change the cohesion strength of mcfp-1 films, but the addition of chemicals with aromatic groups (i.e., aspirin and 4-methylcatechol) increased the cohesion. These results suggest that the cohesion of mcfp-1 films is mainly mediated by cation-π interactions between the positively charged residues and benzene rings of DOPA and other aromatic amino acids (~20 mol% of total amino acids of mcfp-1), and π-π interactions between the phenyl groups in mcfp-1. The adhesion mechanism obtained for the mcfp-1 proteins provides important insight into the design and development of functional biomaterials and coatings mimicking the extensible and robust mussel cuticle coating. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bacteriophages as indicators of faecal pollution and enteric virus removal.

PubMed

McMinn, B R; Ashbolt, N J; Korajkic, A

2017-07-01

Bacteriophages are an attractive alternative to faecal indicator bacteria (FIB), particularly as surrogates of enteric virus fate and transport, due to their closer morphological and biological properties. Based on a review of published data, we summarize densities of coliphages (F+ and somatic), Bacteroides spp. and enterococci bacteriophages (phages) in individual human waste, raw wastewater, ambient fresh and marine waters and removal through wastewater treatment processes utilizing traditional treatments. We also provide comparisons with FIB and enteric viruses whenever possible. Lastly, we examine fate and transport characteristics in the aquatic environment and provide an overview of the environmental factors affecting their survival. In summary, concentrations of bacteriophages in various sources were consistently lower than FIB, but more reflective of infectious enteric virus levels. Overall, our investigation indicates that bacteriophages may be adequate viral surrogates, especially in built systems, such as wastewater treatment plants. Bacteriophage are alternative fecal indicators that may be better surrogates for viral pathogens than fecal indicator bacteria (FIB). This report offers a summary of the existing literature concerning the utility of bacteriophage as indicators of viral presence (fecal sources and surface waters) and persistence (in built infrastructure and aquatic environments). Our findings indicate that bacteriophage levels in all matrices examined are consistently lower than FIB, but similar to viral pathogens. Furthermore, in built infrastructure (e.g. wastewater treatment systems) bacteriophage closely mimic viral pathogen persistence suggesting they may be adequate sentinels of enteric virus removal. © 2017 The Society for Applied Microbiology.

The Nun protein of bacteriophage HK022 inhibits translocation of Escherichia coli RNA polymerase without abolishing its catalytic activities

PubMed Central

Hung, Siu Chun; Gottesman, Max E.

1997-01-01

Bacteriophage HK022 Nun protein blocks transcription elongation by Escherichia coli RNA polymerase in vitro without dissociating the transcription complex. Nun is active on complexes located at any template site tested. Ultimately, only the 3′-OH terminal nucleotide of the nascent transcript in an arrested complex can turn over; it is removed by pyrophosphate and restored with NTPs. This suggests that Nun inhibits the translocation of RNA polymerase without abolishing its catalytic activities. Unlike spontaneously arrested complexes, Nun-arrested complexes cannot be reactivated by transcription factor GreB. The various complexes show distinct patterns of nucleotide incorporation and pyrophosphorolysis before or after treatment with Nun, suggesting that the configuration of RNAP, transcript, and template DNA is different in each complex. PMID:9334329

Anisotropic biodegradable lipid coated particles for spatially dynamic protein presentation.

PubMed

Meyer, Randall A; Mathew, Mohit P; Ben-Akiva, Elana; Sunshine, Joel C; Shmueli, Ron B; Ren, Qiuyin; Yarema, Kevin J; Green, Jordan J

2018-05-01

There has been growing interest in the use of particles coated with lipids for applications ranging from drug delivery, gene delivery, and diagnostic imaging to immunoengineering. To date, almost all particles with lipid coatings have been spherical despite emerging evidence that non-spherical shapes can provide important advantages including reduced non-specific elimination and increased target-specific binding. We combine control of core particle geometry with control of particle surface functionality by developing anisotropic, biodegradable ellipsoidal particles with lipid coatings. We demonstrate that these lipid coated ellipsoidal particles maintain advantageous properties of lipid polymer hybrid particles, such as the ability for modular protein conjugation to the particle surface using versatile bioorthogonal ligation reactions. In addition, they exhibit biomimetic membrane fluidity and demonstrate lateral diffusive properties characteristic of natural membrane proteins. These ellipsoidal particles simultaneously provide benefits of non-spherical particles in terms of stability and resistance to non-specific phagocytosis by macrophages as well as enhanced targeted binding. These biomaterials provide a novel and flexible platform for numerous biomedical applications. The research reported here documents the ability of non-spherical polymeric particles to be coated with lipids to form anisotropic biomimetic particles. In addition, we demonstrate that these lipid-coated biodegradable polymeric particles can be conjugated to a wide variety of biological molecules in a "click-like" fashion. This is of interest due to the multiple types of cellular mimicry enabled by this biomaterial based technology. These features include mimicry of the highly anisotropic shape exhibited by cells, surface presentation of membrane bound protein mimetics, and lateral diffusivity of membrane bound substrates comparable to that of a plasma membrane. This platform is demonstrated to

Screening of Potential Inhibitor against Coat Protein of Apple Chlorotic Leaf Spot Virus.

PubMed

Purohit, Rituraj; Kumar, Sachin; Hallan, Vipin

2018-06-01

In this study, we analyzed Coat protein (CP) of Apple chlorotic leaf spot virus (ACLSV), an important latent virus on Apple. Incidence of the virus is upto 60% in various apple cultivars, affecting yield losses of the order of 10-40% (depending upon the cultivar). CP plays an important role as the sole building block of the viral capsid. Homology approach was used to model 193 amino acid sequence of the coat protein. We used various servers such as ConSurf, TargetS, OSML, COACH, COFACTOR for the prediction of active site residues in coat protein. Virtual screening strategy was employed to search potential inhibitors for CP. Top twenty screened molecules considered for drugability, and toxicity analysis and one potential molecule was further analyzed by docking analysis. Here, we reported a potent molecule which could inhibit the formation of viron assembly by targeting the CP protein of virus.

Protein trans-splicing on an M13 bacteriophage: towards directed evolution of a semisynthetic split intein by phage display.

PubMed

Garbe, Daniel; Thiel, Ilka V; Mootz, Henning D

2010-10-01

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties. © 2010 European Peptide Society and John Wiley & Sons, Ltd.

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

The effects of bacteriophage and nanoparticles on microbial processes

NASA Astrophysics Data System (ADS)

Moody, Austin L.

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

Bacteriophage Infectivity Against Pseudomonas aeruginosa in Saline Conditions

PubMed Central

Scarascia, Giantommaso; Yap, Scott A.; Kaksonen, Anna H.; Hong, Pei-Ying

2018-01-01

Pseudomonas aeruginosa is a ubiquitous member of marine biofilm, and reduces thiosulfate to produce toxic hydrogen sulfide gas. In this study, lytic bacteriophages were isolated and applied to inhibit the growth of P. aeruginosa in planktonic mode at different temperature, pH, and salinity. Bacteriophages showed optimal infectivity at a multiplicity of infection of 10 in saline conditions, and demonstrated lytic abilities over all tested temperature (25, 30, 37, and 45°C) and pH 6–9. Planktonic P. aeruginosa exhibited significantly longer lag phase and lower specific growth rates upon exposure to bacteriophages. Bacteriophages were subsequently applied to P. aeruginosa-enriched biofilm and were determined to lower the relative abundance of Pseudomonas-related taxa from 0.17 to 5.58% in controls to 0.01–0.61% in treated microbial communities. The relative abundance of Alphaproteobacteria, Pseudoalteromonas, and Planococcaceae decreased, possibly due to the phage-induced disruption of the biofilm matrix. Lastly, when applied to mitigate biofouling of ultrafiltration membranes, bacteriophages were determined to reduce the transmembrane pressure increase by 18% when utilized alone, and by 49% when used in combination with citric acid. The combined treatment was more effective compared with the citric acid treatment alone, which reported ca. 30% transmembrane pressure reduction. Collectively, the findings demonstrated that bacteriophages can be used as a biocidal agent to mitigate undesirable P. aeruginosa-associated problems in seawater applications. PMID:29770130

Microneedle-mediated transdermal bacteriophage delivery

PubMed Central

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

2012-01-01

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

Temperature-Triggered Protein Adsorption on Polymer-Coated Nanoparticles in Serum.

PubMed

Koshkina, Olga; Lang, Thomas; Thiermann, Raphael; Docter, Dominic; Stauber, Roland H; Secker, Christian; Schlaad, Helmut; Weidner, Steffen; Mohr, Benjamin; Maskos, Michael; Bertin, Annabelle

2015-08-18

The protein corona, which forms on the nanoparticle's surface in most biological media, determines the nanoparticle's physicochemical characteristics. The formation of the protein corona has a significant impact on the biodistribution and clearance of nanoparticles in vivo. Therefore, the ability to influence the formation of the protein corona is essential to most biomedical applications, including drug delivery and imaging. In this study, we investigate the protein adsorption on nanoparticles with a hydrodynamic radius of 30 nm and a coating of thermoresponsive poly(2-isopropyl-2-oxazoline) in serum. Using multiangle dynamic light scattering (DLS) we demonstrate that heating of the nanoparticles above their phase separation temperature induces the formation of agglomerates, with a hydrodynamic radius of 1 μm. In serum, noticeably stronger agglomeration occurs at lower temperatures compared to serum-free conditions. Cryogenic transmission electron microscopy (cryo-TEM) revealed a high packing density of agglomerates when serum was not present. In contrast, in the presence of serum, agglomerated nanoparticles were loosely packed, indicating that proteins are intercalated between them. Moreover, an increase in protein content is observed upon heating, confirming that protein adsorption is induced by the alteration of the surface during phase separation. After cooling and switching the surface back, most of the agglomerates were dissolved and the main fraction returned to the original size of approximately 30 nm as shown by asymmetrical flow-field flow fractionation (AF-FFF) and DLS. Furthermore, the amounts of adsorbed proteins are similar before and after heating the nanoparticles to above their phase-separation temperature. Overall, our results demonstrate that the thermoresponsivity of the polymer coating enables turning the corona formation on nanoparticles on and off in situ. As the local heating of body areas can be easily done in vivo, the thermoresponsive

Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries.

PubMed

Liu, Shaorong; Gao, Lin; Pu, Qiaosheng; Lu, Joann J; Wang, Xingjia

2006-02-01

We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.

Coating nanoparticles with tunable surfactants facilitates control over the protein corona.

PubMed

Müller, J; Bauer, K N; Prozeller, D; Simon, J; Mailänder, V; Wurm, F R; Winzen, S; Landfester, K

2017-01-01

Nanoparticles with long blood circulation time are a prerequisite for targeted drug delivery. To make the nanoparticles invisible for phagocytizing cells, functional moieties on the particle surface are believed to be necessary to attract specific so-called 'stealth' proteins forming a protein 'corona'. Currently, covalent attachment of those moieties represents the only way to achieve that attraction. However, that approach requires a high synthetic effort and is difficult to control. Therefore, we present the coating of model nanoparticles with biodegradable polymeric surfactants as an alternative method. The thermodynamic parameters of the coating process can be tuned by adjusting the surfactants' block lengths and hydrophilicity. Consequently, the unspecific protein adsorption and aggregation tendency of the particles can be controlled, and stealth proteins inhibiting cell uptake are enriched on their surface. This non-covalent approach could be applied to any particle type and thus facilitates tuning the protein corona and its biological impact. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda.

PubMed Central

Zabeau, M; Stanley, K K

1982-01-01

Hybrid plasmids carrying cro-lacZ gene fusions have been constructed by joining DNA segments carrying the PR promoter and the start of the cro gene of bacteriophage lambda to the lacZ gene fragment carried by plasmid pLG400 . Plasmids in which the translational reading frames of the cro and lacZ genes are joined in-register (type I) direct the synthesis of elevated levels of cro-beta-galactosidase fusion protein amounting to 30% of the total cellular protein, while plasmids in which the genes are fused out-of-register (type II) produce a low level of beta-galactosidase protein. Sequence rearrangements downstream of the cro initiator AUG were found to influence the efficiency of translation, and have been correlated with alterations in the RNA secondary structure of the ribosome-binding site. Plasmids which direct the synthesis of high levels of beta-galactosidase are conditionally lethal and can only be propagated when the PR promoter is repressed. Deletion of sequences downstream of the lacZ gene restored viability, indicating that this region of the plasmid encodes a function which inhibits the growth of the cells. The different applications of these plasmids for expression of cloned genes are discussed. Images Fig. 6. PMID:6327257

Processing of alkaline phosphatase precursor to the mature enzyme by an Escherichia coli inner membrane preparation.

PubMed Central

Chang, C N; Inouye, H; Model, P; Beckwith, J

1980-01-01

An inner membrane preparation co-translationally cleaved both the alkaline phosphatase and bacteriophage f1 coat protein precursors to the mature proteins. Post-translational outer membrane proteolysis of pre-alkaline phosphatase generated a protein smaller than the authentic monomer. Images PMID:6991486

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

PubMed

Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

2016-02-01

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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.

Bacteriophage-based synthetic biology for the study of infectious diseases

PubMed Central

Lu, Timothy K.

2014-01-01

Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic engineering and synthetic biology. Here, we discuss bacteriophage-based technologies and their application to the study of infectious diseases. New strategies for engineering genomes have the potential to accelerate the design of novel phages as therapies, diagnostics, and tools. Though almost a century has elapsed since their discovery, bacteriophages continue to have a major impact on modern biological sciences, especially with the growth of multidrug-resistant bacteria and interest in the microbiome. PMID:24997401

Uptake and processing of modified bacteriophage M13 in mice: implications for phage display.

PubMed

Molenaar, Tom J M; Michon, Ingrid; de Haas, Sonja A M; van Berkel, Theo J C; Kuiper, Johan; Biessen, Erik A L

2002-02-01

Internalization and degradation of filamentous bacteriophage M13 by a specific target cell may have major consequences for the recovery of phage in in vivo biopanning of phage libraries. Therefore, we investigated the pharmacokinetics and processing of native and receptor-targeted phage in mice. (35)S-radiolabeled M13 was chemically modified by conjugation of either galactose (lacM13) or succinic acid groups (sucM13) to the coat protein of the phage to stimulate uptake by galactose recognizing hepatic receptors and scavenger receptors, respectively. Receptor-mediated endocytosis of modified phage reduced the plasma half-life of native M13 (t(1/2) = 4.5 h) to 18 min for lactosylated and 1.5 min for succinylated bacterophage. Internalization of sucM13 was complete within 30 min after injection and resulted in up to 5000-fold reduction of bioactive phage within 90 min. In conclusion, these data provide information on the in vivo behavior of wild-type and receptor-targeted M13, which has important implications for future in vivo phage display experiments and for the potential use of M13 as a viral gene delivery vehicle.

Visualizing cell extracellular matrix (ECM) deposited by cells cultured on aligned bacteriophage M13 thin films.

PubMed

Wu, Laying; Lee, L Andrew; Niu, Zhongwei; Ghoshroy, Soumitra; Wang, Qian

2011-08-02

Topographical features ranging from micro- to nanometers can affect cell orientation and migratory pathways, which are important factors in tissue engineering and tumor migration. In our previous study, a convective assembly of bacteriophage M13 resulted in thin films which could be used to control the alignment of cells. However, several questions regarding its underlying reasons to dictate cell alignment remained unanswered. Here, we further study the nanometer topographical features generated by the bacteriophage M13 crystalline film, which results in the alignment of the cells and extracellular matrix (ECM) proteins. Sequential imaging analyses at micro- and nanoscale levels of aligned cells and fibrillar matrix proteins were documented using scanning electron microscopy and immunofluorescence microscopy. As a result, we observed baby hamster kidney cells with higher degree of alignment on the ordered M13 substrates than NIH-3T3 fibroblasts, a difference which could be attributed to the intrinsic nature of the cells' production of ECM proteins. The results from this study provide a crucial insight into the topographical features of a biological thin film, which can be utilized to control the orientation of cells and surrounding ECM proteins.

Crystallization and preliminary crystallographic characterization of the origin-binding domain of the bacteriophage λ O replication initiator

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

Struble, E. B., E-mail: evi.struble@nist.gov; Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205; Center for Advanced Research in Biotechnology/NIST, 9600 Gudelsky Drive, Rockville, MD 20850

2007-06-01

Crystallization and preliminary diffraction data of the N-terminal 19–139 fragment of the origin-binding domain of bacteriophage λ O replication initiator are reported. The bacteriophage λ O protein binds to the λ replication origin (oriλ) and serves as the primary replication initiator for the viral genome. The binding energy derived from the binding of O to oriλ is thought to help drive DNA opening to facilitate initiation of DNA replication. Detailed understanding of this process is severely limited by the lack of high-resolution structures of O protein or of any lambdoid phage-encoded paralogs either with or without DNA. The production ofmore » crystals of the origin-binding domain of λ O that diffract to 2.5 Å is reported. Anomalous dispersion methods will be used to solve this structure.« less

Carbohydrate-protein interactions investigated on plastic chips statically coated with hydrophobically modified hydroxyethylcellulose.

PubMed

Dang, Fuquan; Maeda, Eiki; Osafune, Tomo; Nakajima, Kazuki; Kakehi, Kazuaki; Ishikawa, Mitsuru; Baba, Yoshinobu

2009-12-15

We developed a novel method for rapid screening of carbohydrate-protein interactions using poly(methyl methacrylate) (PMMA) channels statically coated with hydrophobically modified hydroxyethylcellulose (HM-HEC). We found that a self-assembled monolayer (SAM) of HM-HEC on a PMMA surface intact by water allows rapid and reproducible separations of glycan samples using a 20 mM phosphate without HM-HEC. The underlying mechanism for dynamic and static coatings on the PMMA surface is discussed. Simultaneous analysis of the molecular interaction between a complex mixture of carbohydrates from alpha1-acid glycoprotein and proteins has been successfully achieved in PMMA channels statically coated with a SAM of HM-HEC.

Evolution and the complexity of bacteriophages.

PubMed

Serwer, Philip

2007-03-13

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

A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor σ32 of Escherichia coli

PubMed Central

Perrody, Elsa; Cirinesi, Anne-Marie; Desplats, Carine; Keppel, France; Schwager, Françoise; Tranier, Samuel; Georgopoulos, Costa; Genevaux, Pierre

2012-01-01

The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ32 facilitates RB43 bacteriophage proliferation is discussed. PMID:23133404

Bacteriophage sensitivity patterns among bacteria isolated from marine waters

NASA Astrophysics Data System (ADS)

Moebus, K.; Nattkemper, H.

1981-09-01

Phage-host cross-reaction tests were performed with 774 bacterial strains and 298 bacteriophages. The bacteria (bacteriophages) were isolated at different times from water samples collected in the Atlantic Ocean between the European continental shelf and the Sargasso Sea: 733 (258) strains; in the North Sea near Helgoland: 31 (31) strains; and in the Bay of Biscay: 10 (9) strains. Of the Atlantic Ocean bacteria 326 were found to be susceptible to one or more Atlantic Ocean bacteriophage(s). The bacteriophage sensitivity patterns of these bacteria vary considerably, placing 225 of them in two large clusters of bacteriophage-host systems. Taking all into account, 250 of the 326 Atlantic Ocean bacteria are different from each other. This high degree of variation among the bacteria distinguishes microbial populations derived from widely separated eastern and western regions of the Atlantic Ocean. It also sets apart from each other the populations derived from samples collected at successive stations some 200 miles apart, although to a lesser degree. With bacterial populations found from samples collected on the way back and forth between Europe and the Sargasso Sea a gradual change was observed from "western" phage sensitivity patterns to "eastern" ones. Sixty-nine Atlantic Ocean bacteria are sensitive to bacteriophages isolated from the North Sea and the Bay of Biscay; of these only 26 strains are also susceptible to Atlantic Ocean phages. The interpretation of the results is based on the hydrographical conditions prevailing in the northern Atlantic Ocean including the North Sea, and on the assumption that the microbial populations investigated have undergone genetic changes while being transported within water masses from west to east.

Simulated hatchery system to assess bacteriophage efficacy against Vibrio harveyi.

PubMed

Raghu Patil, J; Desai, Srividya Narayanamurthy; Roy, Panchali; Durgaiah, Murali; Saravanan, R Sanjeev; Vipra, Aradhana

2014-12-02

Vibriosis caused by luminous Vibrio harveyi commonly contributes to poor survival in shrimp hatcheries and aquaculture ponds. Lytic bacteriophages pathogenic for V. harveyi are currently being investigated as an alternative to antibiotics to prevent vibriosis. Here, 8 bacteriophages were isolated from oysters and clams using V. harveyi strains as baiting hosts. Among these bacteriophages, 1 strain (VHP6b) identified as broadly pathogenic for 27 V. harveyi strains examined was further characterized by electron microscopy and genome sequence analysis. Phage VHP6b possessed a tail and morphology consistent with it being a member of the family Siphoviridae, and its genome and proteome were most closely related to the Vibrio phages SSP02 and MAR10. An integrase gene essential for lysogeny was not evident. The ability of bacteriophage VHP6b to protect shrimp postlarvae against vibriosis caused by V. harveyi strain VH6 was demonstrated in a model system designed to simulate typical hatchery conditions. Bacteriophage treatment improved survival of postlarvae by 40 to 60% under these conditions, so therapies based on this or other bacteriophages may be useful in shrimp hatcheries.

40 CFR 174.514 - Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Watermelon Mosaic... Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. Residues of Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic...

Lysogenic bacteriophage isolated from acidophilium

DOEpatents

Ward, Thomas W.; Bruhn, Debby F.; Bulmer, Deborah K.

1992-01-01

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

Genomic and proteomic characterization of a thermophilic Geobacillus bacteriophage GBSV1.

PubMed

Liu, Bin; Zhou, Fengfeng; Wu, Suijie; Xu, Ying; Zhang, Xiaobo

2009-03-01

Phages are present wherever life is found, and play roles in many biogeochemical and ecological processes. The thermophilic bacteriophages, however, have not been well studied. In this study, phage GBSV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k51 isolated from a hot spring. GBSV1 contains a double-stranded linear DNA of 34,683bp, which encodes 54 putative open reading frames (ORFs). Thirty three of these 54 ORFs exhibit sequence similarities to genes from 7 species of Geobacillus or Bacillus bacteria, as well as of bacteriophages infecting these bacteria. Twenty-two ORFs have been functionally annotated based on both their sequence similarities to known genes and predicted Pfam protein domains. Five structural proteins of the purified GBSV1 virion have been identified by proteomic analyses. Surprisingly, 7 of the GBSV1 ORFs share sequence similarities with genes from bacteria relevant to human diseases. This is the first report that genes of human disease-inducing bacteria are found in a thermophilic phage. It is suggested that thermophilic phages may be the potential evolutionary link between thermophiles and human pathogens. The characterization of GBSV1 may possibly lead to new insights into virus-host interactions and to a better understanding of gene transfers and evolution of life on earth in general.

Plant viruses and bacteriophages for drug delivery in medicine and biotechnology.

PubMed

Czapar, Anna E; Steinmetz, Nicole F

2017-06-01

There are a wide variety of synthetic and naturally occurring nanomaterials under development for nanoscale cargo-delivery applications. Viruses play a special role in these developments, because they can be regarded as naturally occurring nanomaterials evolved to package and deliver cargos. While any nanomaterial has its advantage and disadvantages, viral nanoparticles (VNPs), in particular the ones derived from plant viruses and bacteriophages, are attractive options for cargo-delivery as they are biocompatible, biodegradable, and non-infectious to mammals. Their protein-based structures are often understood at atomic resolution and are amenable to modification with atomic-level precision through chemical and genetic engineering. Here we present a focused review of the emerging technology development of plant viruses and bacteriophages targeting human health and agricultural applications. Key target areas of development are their use in chemotherapy, photodynamic therapy, pesticide-delivery, gene therapy, vaccine carriers, and immunotherapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bufo arenarum egg jelly coat: purification and characterization of two highly glycosylated proteins.

PubMed Central

Arranz, S E; Albertali, I E; Cabada, M O

1997-01-01

Egg jelly coats from Bufo arenarum are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they transit along the different oviductal portions. In this study, we have isolated two highly glycosylated proteins of the jelly coat, which are secreted almost all the way along the oviduct. Both glycoproteins [designated as highly glycosylated protein (HGP) and low-molecular-mass highly glycosylated protein (L-HGP)] were purified to homogeneity, from the secretion of the caudal oviduct portion, by CsCl density gradient ultracentrifugation. HGP is a high-molecular-mass protein with mucin-like characteristics: high viscosity, a high content of serine and threonine, about 70% carbohydrate by weight, and a protease-resistant domain. Cleavage of disulphide bridges with reducing agents resulted in the release of a single subunit (300000 Da). L-HGP is also a disulphide-cross-linked protein with lower apparent monomeric molecular mass, in the range 100-120 kDa and containing 50% carbohydrate by weight. HGP contains galactose, fucose, N-acetylgalactosamine and sialic acid, but no mannose, suggesting the presence of O-linked oligosaccharides exclusively. The secretion ratio of HGP increases from cephalic (16% of total protein in pars preconvoluta) to caudal (40% of total protein in pars convoluta) oviductal portions. It appears to be the major structural component of the jelly coat. Our purification data suggest that HGP is non-covalently linked to the other egg jelly proteins. Polyclonal antiserum to each purified glycoprotein from secretion was raised in rabbits and used to localize both glycoproteins in the different oviductal portions, total egg jelly and the aqueous medium where oocyte strings were incubated. HGP forms a stable fibre matrix around the oocyte. L-HGP is present in the jelly coat and is released into the incubation medium. PMID:9173897

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

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

Xiang, Ye; Morais, Marc C.; Cohen, Daniel N.

2009-08-28

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

T4 bacteriophage conjugated magnetic particles for E. coli capturing: Influence of bacteriophage loading, temperature and tryptone.

PubMed

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

2017-03-01

This work demonstrates the use of bacteriophage conjugated magnetic particles (Fe 3 O 4 ) for the rapid capturing and isolation of Escherichia coli. The investigation of T4 bacteriophage adsorption to silane functionalised Fe 3 O 4 with amine (NH 2 ), carboxylic (COOH) and methyl (CH 3 ) surface functional groups reveals the domination of net electrostatic and hydrophobic interactions in governing bacteriophage adsorption. The bare Fe 3 O 4 and Fe 3 O 4 -NH 2 with high T4 loading captured 3-fold more E. coli (∼70% capturing efficiency) compared to the low loading T4 on Fe 3 O 4 -COOH, suggesting the significance of T4 loading in E. coli capturing efficiency. Importantly, it is further revealed that E. coli capture is highly dependent on the incubation temperature and the presence of tryptone in the media. Effective E. coli capturing only occurs at 37°C in tryptone-containing media with the absence of either conditions resulted in poor bacteria capture. The incubation temperature dictates the capturing ability of Fe 3 O 4 /T4, whereby T4 and E. coli need to establish an irreversible binding that occurred at 37°C. The presence of tryptophan-rich tryptone in the suspending media was also critical, as shown by a 3-fold increase in E. coli capture efficiency of Fe 3 O 4 /T4 in tryptone-containing media compared to that in tryptone-free media. This highlights for the first time that successful bacteria capturing requires not only an optimum tailoring of the particle's surface physicochemical properties for favourable bacteriophage loading, but also an in-depth understanding of how factors, such as temperature and solution chemistry influence the subsequent bacteriophage-bacteria interactions. Copyright © 2016 Elsevier B.V. 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

A fully decompressed synthetic bacteriophage øX174 genome assembled and archived in yeast.

PubMed

Jaschke, Paul R; Lieberman, Erica K; Rodriguez, Jon; Sierra, Adrian; Endy, Drew

2012-12-20

The 5386 nucleotide bacteriophage øX174 genome has a complicated architecture that encodes 11 gene products via overlapping protein coding sequences spanning multiple reading frames. We designed a 6302 nucleotide synthetic surrogate, øX174.1, that fully separates all primary phage protein coding sequences along with cognate translation control elements. To specify øX174.1f, a decompressed genome the same length as wild type, we truncated the gene F coding sequence. We synthesized DNA encoding fragments of øX174.1f and used a combination of in vitro- and yeast-based assembly to produce yeast vectors encoding natural or designer bacteriophage genomes. We isolated clonal preparations of yeast plasmid DNA and transfected E. coli C strains. We recovered viable øX174 particles containing the øX174.1f genome from E. coli C strains that independently express full-length gene F. We expect that yeast can serve as a genomic 'drydock' within which to maintain and manipulate clonal lineages of other obligate lytic phage. Copyright © 2012 Elsevier Inc. All rights reserved.

Phage lytic proteins: biotechnological applications beyond clinical antimicrobials

USDA-ARS?s Scientific Manuscript database

Most bacteriophages encode two types of cell wall lytic proteins: Endolysins (lysins) and virion-associated peptidoglycan hydrolases. Both enzymes have the ability to degrade the peptidoglycan of Gram positive bacteria resulting in cell lysis when they are applied externally. Bacteriophage lytic p...

[Biological properties of bacteriophages, active to Yersinia enterocolitica].

PubMed

Darsavelidze, M A; Kapanadze, Zh S; Chanishvili, T G

2004-01-01

The biological properties of 16 clones of Y. enterolitica bacteriophages were tested to select the most active for subsequent use. For the first time Y. enterocolitica virulent phages belonging to the family of Podoviridae were described and 7 serological groups of phages with no cross reactions were registered. The technology for the production of new therapeutic and prophylactic Y. enterocolitica polyvalent bacteriophage under laboratory conditions was developed. The effective multiplicity of contamination ensuring the maximum release of phages from bacterial cells, the optimum incubation temperature and the time of exposure were established. The experimental batches of therapeutic and prophylactic Y. enterocolitica polyvalent bacteriophage thus obtained met the requirements for antibacterial preparations.

Photoreactive elastin-like proteins for use as versatile bioactive materials and surface coatings

PubMed Central

Raphel, Jordan; Parisi-Amon, Andreina; Heilshorn, Sarah

2012-01-01

Photocrosslinkable, protein-engineered biomaterials combine a rapid, controllable, cytocompatible crosslinking method with a modular design strategy to create a new family of bioactive materials. These materials have a wide range of biomedical applications, including the development of bioactive implant coatings, drug delivery vehicles, and tissue engineering scaffolds. We present the successful functionalization of a bioactive elastin-like protein with photoreactive diazirine moieties. Scalable synthesis is achieved using a standard recombinant protein expression host followed by site-specific modification of lysine residues with a heterobifunctional N-hydroxysuccinimide ester-diazirine crosslinker. The resulting biomaterial is demonstrated to be processable by spin coating, drop casting, soft lithographic patterning, and mold casting to fabricate a variety of two- and three-dimensional photocrosslinked biomaterials with length scales spanning the nanometer to millimeter range. Protein thin films proved to be highly stable over a three-week period. Cell-adhesive functional domains incorporated into the engineered protein materials were shown to remain active post-photo-processing. Human adipose-derived stem cells achieved faster rates of cell adhesion and larger spread areas on thin films of the engineered protein compared to control substrates. The ease and scalability of material production, processing versatility, and modular bioactive functionality make this recombinantly engineered protein an ideal candidate for the development of novel biomaterial coatings, films, and scaffolds. PMID:23015764

Photoreactive elastin-like proteins for use as versatile bioactive materials and surface coatings.

PubMed

Raphel, Jordan; Parisi-Amon, Andreina; Heilshorn, Sarah

2012-10-07

Photocrosslinkable, protein-engineered biomaterials combine a rapid, controllable, cytocompatible crosslinking method with a modular design strategy to create a new family of bioactive materials. These materials have a wide range of biomedical applications, including the development of bioactive implant coatings, drug delivery vehicles, and tissue engineering scaffolds. We present the successful functionalization of a bioactive elastin-like protein with photoreactive diazirine moieties. Scalable synthesis is achieved using a standard recombinant protein expression host followed by site-specific modification of lysine residues with a heterobifunctional N-hydroxysuccinimide ester-diazirine crosslinker. The resulting biomaterial is demonstrated to be processable by spin coating, drop casting, soft lithographic patterning, and mold casting to fabricate a variety of two- and three-dimensional photocrosslinked biomaterials with length scales spanning the nanometer to millimeter range. Protein thin films proved to be highly stable over a three-week period. Cell-adhesive functional domains incorporated into the engineered protein materials were shown to remain active post-photo-processing. Human adipose-derived stem cells achieved faster rates of cell adhesion and larger spread areas on thin films of the engineered protein compared to control substrates. The ease and scalability of material production, processing versatility, and modular bioactive functionality make this recombinantly engineered protein an ideal candidate for the development of novel biomaterial coatings, films, and scaffolds.

Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation.

PubMed Central

McGregor, A; Rao, M V; Duckworth, G; Stockley, P G; Connolly, B A

1996-01-01

The preparation of a 4-thiouridine phosphoramidite suitable for RNA synthesis and its subsequent incorporation into oligoribonucleotides is described. The thiol group is protected with a 2-cyanoethyl group and the 2'-OH with a 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl function. Thiouridine-containing oligoribonucleotides were used as 350 nm UV crosslinking probes for the photoaffinity labelling of RNA binding proteins. Specific crosslinking was demonstrated between the Rev protein of HIV-1 (as a glutathione S-transferase fusion protein) and its RNA target, the Rev-responsive element. It was not possible to generate crosslinks between the RNA bacteriophage MS2 coat protein and the initiator stem-loop of the replicase gene, to which it binds. These results are consistent with the structural data available on both systems. PMID:8774897

Determinants of affinity and mode of DNA binding at the carboxy terminus of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1.

PubMed

Andera, L; Geiduschek, E P

1994-03-01

The role of the carboxy-terminal amino acids of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1, in DNA binding was analyzed. Chain-terminating mutations truncating the normally 99-amino-acid TF1 at amino acids 96, 97, and 98 were constructed, as were missense mutations substituting cysteine, arginine, and serine for phenylalanine at amino acid 97 and tryptophan for lysine at amino acid 99. The binding of the resulting proteins to a synthetic 44-bp binding site in 5-(hydroxymethyl)uracil DNA, to binding sites in larger SPO1 [5-(hydroxymethyl)uracil-containing] DNA fragments, and to thymine-containing homologous DNA was analyzed by gel retardation and also by DNase I and hydroxy radical footprinting. We conclude that the C tail up to and including phenylalanine at amino acid 97 is essential for DNA binding and that the two C-terminal amino acids, 98 and 99, are involved in protein-protein interactions between TF1 dimers bound to DNA.

Albizia lebbeck Seed Coat Proteins Bind to Chitin and Act as a Defense against Cowpea Weevil Callosobruchus maculatus.

PubMed

Silva, Nadia C M; De Sá, Leonardo F R; Oliveira, Eduardo A G; Costa, Monique N; Ferreira, Andre T S; Perales, Jonas; Fernandes, Kátia V S; Xavier-Filho, Jose; Oliveira, Antonia E A

2016-05-11

The seed coat is an external tissue that participates in defense against insects. In some nonhost seeds, including Albizia lebbeck, the insect Callosobruchus maculatus dies during seed coat penetration. We investigated the toxicity of A. lebbeck seed coat proteins to C. maculatus. A chitin-binding protein fraction was isolated from seed coat, and mass spectrometry showed similarity to a C1 cysteine protease. By ELM program an N-glycosylation interaction motif was identified in this protein, and by molecular docking the potential to interact with N-acetylglucosamine (NAG) was shown. The chitin-binding protein fraction was toxic to C. maculatus and was present in larval midgut and feces but not able to hydrolyze larval gut proteins. It did not interfere, though, with the intestinal cell permeability. These results indicate that the toxicity mechanism of this seed coat fraction may be related to its binding to chitin, present in the larvae gut, disturbing nutrient absorption.

Genome Sequences of 19 Novel Erwinia amylovora Bacteriophages

PubMed Central

Esplin, Ian N. D.; Berg, Jordan A.; Sharma, Ruchira; Allen, Robert C.; Arens, Daniel K.; Ashcroft, Cody R.; Bairett, Shannon R.; Beatty, Nolan J.; Bickmore, Madeline; Bloomfield, Travis J.; Brady, T. Scott; Bybee, Rachel N.; Carter, John L.; Choi, Minsey C.; Duncan, Steven; Fajardo, Christopher P.; Foy, Brayden B.; Fuhriman, David A.; Gibby, Paul D.; Grossarth, Savannah E.; Harbaugh, Kala; Harris, Natalie; Hilton, Jared A.; Hurst, Emily; Hyde, Jonathan R.; Ingersoll, Kayleigh; Jacobson, Caitlin M.; James, Brady D.; Jarvis, Todd M.; Jaen-Anieves, Daniella; Jensen, Garrett L.; Knabe, Bradley K.; Kruger, Jared L.; Merrill, Bryan D.; Pape, Jenny A.; Payne Anderson, Ashley M.; Payne, David E.; Peck, Malia D.; Pollock, Samuel V.; Putnam, Micah J.; Ransom, Ethan K.; Ririe, Devin B.; Robinson, David M.; Rogers, Spencer L.; Russell, Kerri A.; Schoenhals, Jonathan E.; Shurtleff, Christopher A.; Simister, Austin R.; Smith, Hunter G.; Stephenson, Michael B.; Staley, Lyndsay A.; Stettler, Jason M.; Stratton, Mallorie L.; Tateoka, Olivia B.; Tatlow, P. J.; Taylor, Alexander S.; Thompson, Suzanne E.; Townsend, Michelle H.; Thurgood, Trever L.; Usher, Brittian K.; Whitley, Kiara V.; Ward, Andrew T.; Ward, Megan E. H.; Webb, Charles J.; Wienclaw, Trevor M.; Williamson, Taryn L.; Wells, Michael J.; Wright, Cole K.; Breakwell, Donald P.; Hope, Sandra

2017-01-01

ABSTRACT Erwinia amylovora is the causal agent of fire blight, a devastating disease affecting some plants of the Rosaceae family. We isolated bacteriophages from samples collected from infected apple and pear trees along the Wasatch Front in Utah. We announce 19 high-quality complete genome sequences of E. amylovora bacteriophages. PMID:29146842

Seven Bacteriophages Isolated from the Female Urinary Microbiota

PubMed Central

Malki, Kema; Sible, Emily; Cooper, Alexandria; Garretto, Andrea; Bruder, Katherine; Watkins, Siobhan C.

2016-01-01

Recent research has debunked the myth that urine is sterile, having uncovered bacteria within the bladders of healthy individuals. However, the identity, diversity, and putative roles of bacteriophages in the bladder are unknown. We report the draft genome sequences of seven bacteriophages isolated from microbial communities from adult female bladders. PMID:27881533

Dual Function of Novel Pollen Coat (Surface) Proteins: IgE-binding Capacity and Proteolytic Activity Disrupting the Airway Epithelial Barrier

PubMed Central

Bashir, Mohamed Elfatih H.; Ward, Jason M.; Cummings, Matthew; Karrar, Eltayeb E.; Root, Michael; Mohamed, Abu Bekr A.; Naclerio, Robert M.; Preuss, Daphne

2013-01-01

Background The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., “de-fatted”), and, as a result, their involvement in allergy has not been explored. Methodology/Principal Findings Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. Conclusions/Significance Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is

Methods of expanding bacteriophage host-range and bacteriophage produced by the methods

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

Crown, Kevin K.; Santarpia, Joshua

A method of producing novel bacteriophages with expanded host-range and bacteriophages with expanded host ranges are disclosed. The method produces mutant phage strains which are infectious to a second host and can be more infectious to their natural host than in their natural state. The method includes repeatedly passaging a selected phage strain into bacterial cultures that contain varied ratios of its natural host bacterial strain with a bacterial strain that the phage of interest is unable to infect; the target-host. After each passage the resulting phage are purified and screened for activity against the target-host via double-overlay assays. Whenmore » mutant phages that are shown to infect the target-host are discovered, they are further propagated in culture that contains only the target-host to produce a stock of the resulting mutant phage.« less

Coat Protein Regulation by CK2, CPIP, HSP70, and CHIP Is Required for Potato Virus A Replication and Coat Protein Accumulation

PubMed Central

Lõhmus, Andres; Hafrén, Anders

2016-01-01

ABSTRACT We demonstrate here that both coat protein (CP) phosphorylation by protein kinase CK2 and a chaperone system formed by two heat shock proteins, CP-interacting protein (CPIP) and heat shock protein 70 (HSP70), are essential for potato virus A (PVA; genus Potyvirus) replication and that all these host proteins have the capacity to contribute to the level of PVA CP accumulation. An E3 ubiquitin ligase called carboxyl terminus Hsc70-interacting protein (CHIP), which may participate in the CPIP-HSP70-mediated CP degradation, is also needed for robust PVA gene expression. Residue Thr243 within the CK2 consensus sequence of PVA CP was found to be essential for viral replication and to regulate CP protein stability. Substitution of Thr243 either with a phosphorylation-mimicking Asp (CPADA) or with a phosphorylation-deficient Ala (CPAAA) residue in CP expressed from viral RNA limited PVA gene expression to the level of nonreplicating PVA. We found that both the CPAAA mutant and CK2 silencing inhibited, whereas CPADA mutant and overexpression of CK2 increased, PVA translation. From our previous studies, we know that phosphorylation reduces the RNA binding capacity of PVA CP and an excess of CP fully blocks viral RNA translation. Together, these findings suggest that binding by nonphosphorylated PVA CP represses viral RNA translation, involving further CP phosphorylation and CPIP-HSP70 chaperone activities as prerequisites for PVA replication. We propose that this mechanism contributes to shifting potyvirus RNA from translation to replication. IMPORTANCE Host protein kinase CK2, two host chaperones, CPIP and HSP70, and viral coat protein (CP) phosphorylation at Thr243 are needed for potato virus A (PVA) replication. Our results show that nonphosphorylated CP blocks viral translation, likely via binding to viral RNA. We propose that this translational block is needed to allow time and space for the formation of potyviral replication complex around the 3′ end of

Whey protein solution coating for fat-uptake reduction in deep-fried chicken breast strips.

PubMed

Dragich, Ann M; Krochta, John M

2010-01-01

This study investigated the use of whey protein, as an additional coating, in combination with basic, well-described predust, batter, and breading ingredients, for fat-uptake reduction in fried chicken. Chicken breasts were cut into strips (1 x 5 x 10 cm) and coated with wheat flour (WF) as a predust, dipped in batter, coated with WF as a breading, then dipped in 10% denatured whey protein isolate (DWPI) aqueous solution (wet basis). A WF-batter-WF treatment with no DWPI solution dip was included as a control. Coated chicken strips were deep-fried at 160 degrees C for 5 min. A Soxhlet-type extraction was performed to determine the fat content of the meat fraction of fried samples, the coating fraction of fried samples, raw chicken, and raw coating ingredients. The WF-batter-WF-10% DWPI solution had significantly lower fat uptake than the WF-batter-WF control, by 30.67% (dry basis). This article describes applied research involving fat reduction in coated deep-fried chicken. The methods used in this article were intended to achieve maximized fat reduction while maintaining a simple procedure applicable to actual food processing lines.

Bacteriophage cocktail for biocontrol of Salmonella in dried pet food.

PubMed

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

2015-01-01

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

Langevin Dynamics Simulations of Genome Packing in Bacteriophage

PubMed Central

Forrey, Christopher; Muthukumar, M.

2006-01-01

We use Langevin dynamics simulations to study the process by which a coarse-grained DNA chain is packaged within an icosahedral container. We focus our inquiry on three areas of interest in viral packing: the evolving structure of the packaged DNA condensate; the packing velocity; and the internal buildup of energy and resultant forces. Each of these areas has been studied experimentally, and we find that we can qualitatively reproduce experimental results. However, our findings also suggest that the phage genome packing process is fundamentally different than that suggested by the inverse spool model. We suggest that packing in general does not proceed in the deterministic fashion of the inverse-spool model, but rather is stochastic in character. As the chain configuration becomes compressed within the capsid, the structure, energy, and packing velocity all become dependent upon polymer dynamics. That many observed features of the packing process are rooted in condensed-phase polymer dynamics suggests that statistical mechanics, rather than mechanics, should serve as the proper theoretical basis for genome packing. Finally we suggest that, as a result of an internal protein unique to bacteriophage T7, the T7 genome may be significantly more ordered than is true for bacteriophage in general. PMID:16617089

Langevin dynamics simulations of genome packing in bacteriophage.

PubMed

Forrey, Christopher; Muthukumar, M

2006-07-01

We use Langevin dynamics simulations to study the process by which a coarse-grained DNA chain is packaged within an icosahedral container. We focus our inquiry on three areas of interest in viral packing: the evolving structure of the packaged DNA condensate; the packing velocity; and the internal buildup of energy and resultant forces. Each of these areas has been studied experimentally, and we find that we can qualitatively reproduce experimental results. However, our findings also suggest that the phage genome packing process is fundamentally different than that suggested by the inverse spool model. We suggest that packing in general does not proceed in the deterministic fashion of the inverse-spool model, but rather is stochastic in character. As the chain configuration becomes compressed within the capsid, the structure, energy, and packing velocity all become dependent upon polymer dynamics. That many observed features of the packing process are rooted in condensed-phase polymer dynamics suggests that statistical mechanics, rather than mechanics, should serve as the proper theoretical basis for genome packing. Finally we suggest that, as a result of an internal protein unique to bacteriophage T7, the T7 genome may be significantly more ordered than is true for bacteriophage in general.

Optimizing the European regulatory framework for sustainable bacteriophage therapy in human medicine.

PubMed

Verbeken, Gilbert; Pirnay, Jean-Paul; De Vos, Daniel; Jennes, Serge; Zizi, Martin; Lavigne, Rob; Casteels, Minne; Huys, Isabelle

2012-06-01

For practitioners at hospitals seeking to use natural (not genetically modified, as appearing in nature) bacteriophages for treatment of antibiotic-resistant bacterial infections (bacteriophage therapy), Europe's current regulatory framework for medicinal products hinders more than it facilitates. Although many experts consider bacteriophage therapy to be a promising complementary (or alternative) treatment to antibiotic therapy, no bacteriophage-specific framework for documentation exists to date. Decades worth of historical clinical data on bacteriophage therapy (from Eastern Europe, particularly Poland, and the former Soviet republics, particularly Georgia and Russia, as well as from today's 27 EU member states and the US) have not been taken into account by European regulators because these data have not been validated under current Western regulatory standards. Consequently, applicants carrying out standard clinical trials on bacteriophages in Europe are obliged to initiate clinical work from scratch. This paper argues for a reduced documentation threshold for Phase 1 clinical trials of bacteriophages and maintains that bacteriophages should not be categorized as classical medicinal products for at least two reasons: (1) such a categorization is scientifically inappropriate for this specific therapy and (2) such a categorization limits the marketing authorization process to industry, the only stakeholder with sufficient financial resources to prepare a complete dossier for the competent authorities. This paper reflects on the current regulatory framework for medicines in Europe and assesses possible regulatory pathways for the (re-)introduction of bacteriophage therapy in a way that maintains its effectiveness and safety as well as its inherent characteristics of sustainability and in situ self-amplification and limitation.

Monitoring of phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage phiRSS1.

PubMed

Kawasaki, Takeru; Satsuma, Hideki; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

2007-12-01

A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.

A bacteriophage for Myxococcus xanthus: isolation, characterization and relation of infectivity to host morphogenesis.

PubMed

Burchard, R P; Dworkin, M

1966-03-01

Burchard, Robert P. (University of Minnesota, Minneapolis), and M. Dworkin. A bacteriophage for Myxococcus xanthus: isolation, characterization and relation of infectivity to host morphogenesis. J. Bacteriol. 91:1305-1313. 1966.-A bacteriophage (MX-1) infecting Myxococcus xanthus FB(t) has been isolated from cow dung. The bacteriophage particle is approximately 175 mmu long. A tail about 100 mmu in length is encased in a contractile sheath and terminates in a tail plate. The head is polyhedral with a width of about 75 mmu. The nucleic acid of the bacteriophage is deoxyribonucleic acid and has a guanine plus cytosine content of 55.5%. The bacteriophage requires 10(-3)m Ca(++) and 10(-2)m monovalent cation for optimal adsorption. Grown on vegetative cells of M. xanthus FB(t) at 30 C in 2% Casitone medium, the bacteriophage has a latent period of 120 min and a burst size of approximately 100. Host range studies indicate that three strains of M. xanthus including a morphogenetic mutant are sensitive to the bacteriophage, whereas M. fulvus, Cytophaga, Sporocytophaga myxococcoides, and a fourth strain of M. xanthus are not. Of the two cellular forms characteristic of the Myxococcus life cycle, the bacteriophage infect only the vegetative cells; they do not adsorb to microcysts. Ability to adsorb bacteriophage is lost between 65 and 75 min after initiation of the relatively synchronous conversion of vegetative cells to microcysts. The bacteriophage does not adsorb to spheroplasts. After the appearance of visible morphogenesis and before the loss of bacteriophage receptor sites, addition of bacteriophage results in the formation of microcysts which give rise to infective centers only upon germination. The possibility that the infected microcysts are harboring intact bacteriophages has been eliminated.

Potential of a lytic bacteriophage to disrupt Acinetobacter baumannii biofilms in vitro.

PubMed

Liu, Yannan; Mi, Zhiqiang; Niu, Wenkai; An, Xiaoping; Yuan, Xin; Liu, Huiying; Wang, Yong; Feng, Yuzhong; Huang, Yong; Zhang, Xianglilan; Zhang, Zhiyi; Fan, Hang; Peng, Fan; Li, Puyuan; Tong, Yigang; Bai, Changqing

2016-10-01

The ability of Acinetobacter baumannii to form biofilms and develop antibiotic resistance makes it difficult to control infections caused by this bacterium. In this study, we explored the potential of a lytic bacteriophage to disrupt A. baumannii biofilms. The potential of the lytic bacteriophage to disrupt A. baumannii biofilms was assessed by performing electron microscopy, live/dead bacterial staining, crystal violet staining and by determining adenosine triphosphate release. The bacteriophage inhibited the formation of and disrupted preformed A. baumannii biofilms. Results of disinfection assay showed that the lytic bacteriophage lysed A. baumannii cells suspended in blood or grown on metal surfaces. These results suggest the potential of the lytic bacteriophage to disrupt A. baumannii biofilms.

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation.

PubMed

Ribis, John W; Ravichandran, Priyanka; Putnam, Emily E; Pishdadian, Keyan; Shen, Aimee

2017-01-01

The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis , only two of these morphogenetic proteins have homologs in the Clostridia : SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis . Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis , C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia , but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and

Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice.

PubMed

Maura, Damien; Morello, Eric; du Merle, Laurence; Bomme, Perrine; Le Bouguénec, Chantal; Debarbieux, Laurent

2012-08-01

Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Evaluation of aerosol spray and intramuscular injection of bacteriophage to treat an Escherichia coli respiratory infection.

PubMed

Huff, W E; Huff, G R; Rath, N C; Balog, J M; Donoghue, A M

2003-07-01

Two studies were conducted to determine the efficacy of either aerosol or i.m. injection of bacteriophage to treat an Escherichia coli respiratory infection in broiler chickens. An additional two studies were conducted to enumerate the bacteriophage in the blood of birds at 1, 2, 3, 4, 5, 6, 24, and 48 h after being sprayed or injected i.m. with bacteriophage. Five birds were bled at each period. In study 1, there were 10 treatments with three replicate pens of 10 birds. The treatments consisted of an untreated control, heat-killed bacteriophage spray, active bacteriophage spray, E. coli challenge at 7 d of age, and E. coli challenge followed by spraying the birds with heat-killed bacteriophage or active bacteriophage at 2, 24, or 48 h after challenge. In study 2 there were 11 treatments with three replicate pens of 10 birds per pen. The treatments were untreated controls, birds injected i.m. in the thigh with heat-killed or active bacteriophage, E. coli challenge at 7 d of age, PBS challenge, E. coli challenge followed by injection of heat-killed or active bacteriophage immediately after challenge or at 24 or 48 h after challenge. In both studies the E. coli challenge consisted of injecting 10(4) cfu into the thoracic air sac. Treatment of this severe E. coli infection with the bacteriophage aerosol spray significantly reduced mortality from 50 to 20% when given immediately after the challenge but had little treatment efficacy when administered 24 or 48 h after challenge. The i.m. injection of bacteriophage significantly reduced mortality from 53 to 17%, 46 to 10%, and 44 to 20% when given immediately, 24, or 48 h after challenge, respectively. Only a few birds sprayed with bacteriophage had detectable bacteriophage in their blood with an average of 96 pfu/mL 1 h after bacteriophage administration, and no bacteriophage was detected 24 and 48 h after bacteriophage administration. All birds injected i.m. with bacteriophage had detectable levels of bacteriophage in

Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

PubMed Central

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

2012-01-01

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

Genome Sequences of 19 Novel Erwinia amylovora Bacteriophages.

PubMed

Esplin, Ian N D; Berg, Jordan A; Sharma, Ruchira; Allen, Robert C; Arens, Daniel K; Ashcroft, Cody R; Bairett, Shannon R; Beatty, Nolan J; Bickmore, Madeline; Bloomfield, Travis J; Brady, T Scott; Bybee, Rachel N; Carter, John L; Choi, Minsey C; Duncan, Steven; Fajardo, Christopher P; Foy, Brayden B; Fuhriman, David A; Gibby, Paul D; Grossarth, Savannah E; Harbaugh, Kala; Harris, Natalie; Hilton, Jared A; Hurst, Emily; Hyde, Jonathan R; Ingersoll, Kayleigh; Jacobson, Caitlin M; James, Brady D; Jarvis, Todd M; Jaen-Anieves, Daniella; Jensen, Garrett L; Knabe, Bradley K; Kruger, Jared L; Merrill, Bryan D; Pape, Jenny A; Payne Anderson, Ashley M; Payne, David E; Peck, Malia D; Pollock, Samuel V; Putnam, Micah J; Ransom, Ethan K; Ririe, Devin B; Robinson, David M; Rogers, Spencer L; Russell, Kerri A; Schoenhals, Jonathan E; Shurtleff, Christopher A; Simister, Austin R; Smith, Hunter G; Stephenson, Michael B; Staley, Lyndsay A; Stettler, Jason M; Stratton, Mallorie L; Tateoka, Olivia B; Tatlow, P J; Taylor, Alexander S; Thompson, Suzanne E; Townsend, Michelle H; Thurgood, Trever L; Usher, Brittian K; Whitley, Kiara V; Ward, Andrew T; Ward, Megan E H; Webb, Charles J; Wienclaw, Trevor M; Williamson, Taryn L; Wells, Michael J; Wright, Cole K; Breakwell, Donald P; Hope, Sandra; Grose, Julianne H

2017-11-16

Erwinia amylovora is the causal agent of fire blight, a devastating disease affecting some plants of the Rosaceae family. We isolated bacteriophages from samples collected from infected apple and pear trees along the Wasatch Front in Utah. We announce 19 high-quality complete genome sequences of E. amylovora bacteriophages. Copyright © 2017 Esplin et al.

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

Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni

PubMed Central

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

2014-01-01

Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage. PMID:24671947

Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni.

PubMed

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

2014-03-26

Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage.

Temperature-responsive peptide-mimetic coating based on poly(N-methacryloyl-l-leucine): properties, protein adsorption and cell growth.

PubMed

Raczkowska, Joanna; Ohar, Mariya; Stetsyshyn, Yurij; Zemła, Joanna; Awsiuk, Kamil; Rysz, Jakub; Fornal, Katarzyna; Bernasik, Andrzej; Ohar, Halyna; Fedorova, Svitlana; Shtapenko, Oksana; Polovkovych, Svyatoslav; Novikov, Volodymyr; Budkowski, Andrzej

2014-06-01

Poly(N-methacryloyl-l-leucine) (PNML) coatings were successfully fabricated via polymerization from peroxide initiator grafted to premodified glass substrate. Chemical composition and thickness of PNML coatings were determined using time of flight-secondary ion mass spectrometry (TOF- SIMS) and ellipsometry, respectively. PNML coatings exhibit thermal response of the wettability, between 4 and 28°C, which indicates a transition between hydrated loose coils and hydrophobic collapsed chains. Morphology of the PNML coating was observed with the AFM, transforming with increasing temperature from initially relatively smooth surface to rough and more structured surface. Protein adsorption observed by fluorescence microscopy for model proteins (bovine serum albumin and lentil lectin labeled with fluorescein isothiocyanate) at transition from 5 to 25°C, showed high affinity of PNML coating to proteins at all investigated temperatures and pH. Thus, PNML coating have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). The high proliferation growth of the human embryonic kidney cell (HEK 293) onto PNML coating was demonstrated, indicating its excellent cytocompatibility. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular Characterization of Podoviridae Bacteriophages Virulent for Clostridium perfringens and Comparison of Their Predicted Lytic Proteins

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control ba...

Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles.

PubMed

Dedrick, Rebekah M; Marinelli, Laura J; Newton, Gerald L; Pogliano, Kit; Pogliano, Joseph; Hatfull, Graham F

2013-05-01

Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions - such as virion proteins and repressors - cannot always be recognized. Here we describe a functional genomic dissection of mycobacteriophage Giles, in which the virion proteins are identified, genes required for lytic growth are determined, the repressor is identified, and the transcription patterns determined. We find that although all of the predicted phage genes are expressed either in lysogeny or in lytic growth, 45% of the predicted genes are non-essential for lytic growth. We also describe genes required for DNA replication, show that recombination is required for lytic growth, and that Giles encodes a novel repressor. RNAseq analysis reveals abundant expression of a small non-coding RNA in a lysogen and in late lytic growth, although it is non-essential for lytic growth and does not alter lysogeny. © 2013 Blackwell Publishing Ltd.

Development of prototypes of bioactive packaging materials based on immobilized bacteriophages for control of growth of bacterial pathogens in foods.

PubMed

Lone, Ayesha; Anany, Hany; Hakeem, Mohammed; Aguis, Louise; Avdjian, Anne-Claire; Bouget, Marina; Atashi, Arash; Brovko, Luba; Rochefort, Dominic; Griffiths, Mansel W

2016-01-18

Due to lack of adequate control methods to prevent contamination in fresh produce and growing consumer demand for natural products, the use of bacteriophages has emerged as a promising approach to enhance safety of these foods. This study sought to control Listeria monocytogenes in cantaloupes and RTE meat and Escherichia coli O104:H4 in alfalfa seeds and sprouts under different storage conditions by using specific lytic bacteriophage cocktails applied either free or immobilized. Bacteriophage cocktails were introduced into prototypes of packaging materials using different techniques: i) immobilizing on positively charged modified cellulose membranes, ii) impregnating paper with bacteriophage suspension, and iii) encapsulating in alginate beads followed by application of beads onto the paper. Phage-treated and non-treated samples were stored for various times and at temperatures of 4°C, 12°C or 25°C. In cantaloupe, when free phage cocktail was added, L. monocytogenes counts dropped below the detection limit of the plating technique (<1 log CFU/g) after 5 days of storage at both 4°C and 12°C. However, at 25°C, counts below the detection limit were observed after 3 and 6h and a 2-log CFU/g reduction in cell numbers was seen after 24h. For the immobilized Listeria phage cocktail, around 1-log CFU/g reduction in the Listeria count was observed by the end of the storage period for all tested storage temperatures. For the alfalfa seeds and sprouts, regardless of the type of phage application technique (spraying of free phage suspension, bringing in contact with bacteriophage-based materials (paper coated with encapsulated bacteriophage or impregnated with bacteriophage suspension)), the count of E. coli O104:H4 was below the detection limit (<1 log CFU/g) after 1h in seeds and about a 1-log cycle reduction in E. coli count was observed on the germinated sprouts by day 5. In ready-to-eat (RTE) meat, LISTEX™ P100, a commercial phage product, was able to

Isolation, characterization and comparative genomics of bacteriophage SfIV: a novel serotype converting phage from Shigella flexneri

PubMed Central

2013-01-01

Background Shigella flexneri is the major cause of shigellosis in the developing countries. The O-antigen component of the lipopolysaccharide is one of the key virulence determinants required for the pathogenesis of S. flexneri. The glucosyltransferase and/or acetyltransferase genes responsible for the modification of the O-antigen are encoded by temperate serotype converting bacteriophage present in the S. flexneri genome. Several serotype converting phages have previously been isolated and characterized, however, attempts to isolate a serotype converting phage which encodes the modification genes of serotypes 4a strain have not been successful. Results In this study, a novel temperate serotype converting bacteriophage SfIV was isolated. Lysogenisation of phage SfIV converted serotype Y strain to serotype 4a. Electron microscopy indicated that SfIV belongs to Myoviridae family. The 39,758 bp genome of phage SfIV encompasses 54 open reading frames (orfs). Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. The comparative analysis also revealed that SfIV phage contained five proteins which were not found in any other phages of S. flexneri. These proteins were: a tail fiber assembly protein, two hypothetical proteins with no clear function, and two other unknown proteins which were encoded by orfs present on a moron, that presumably got introduced in SfIV genome from another species via a transposon. These unique proteins of SfIV may play a role in the pathogenesis of the host. Conclusions This study reports the isolation and complete genome sequence analysis of bacteriophage SfIV. The SfIV phage has a host range significantly different from the other phages of Shigella. Comparative genome analysis identified several proteins unique to SfIV, which may potentially be involved in the survival and pathogenesis of its host. These findings will further our understanding on the

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

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

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

2012-04-05

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

Revisiting the NMR structure of the ultrafast downhill folding protein gpW from bacteriophage λ.

PubMed

Sborgi, Lorenzo; Verma, Abhinav; Muñoz, Victor; de Alba, Eva

2011-01-01

GpW is a 68-residue protein from bacteriophage λ that participates in virus head morphogenesis. Previous NMR studies revealed a novel α+β fold for this protein. Recent experiments have shown that gpW folds in microseconds by crossing a marginal free energy barrier (i.e., downhill folding). These features make gpW a highly desirable target for further experimental and computational folding studies. As a step in that direction, we have re-determined the high-resolution structure of gpW by multidimensional NMR on a construct that eliminates the purification tags and unstructured C-terminal tail present in the prior study. In contrast to the previous work, we have obtained a full manual assignment and calculated the structure using only unambiguous distance restraints. This new structure confirms the α+β topology, but reveals important differences in tertiary packing. Namely, the two α-helices are rotated along their main axis to form a leucine zipper. The β-hairpin is orthogonal to the helical interface rather than parallel, displaying most tertiary contacts through strand 1. There also are differences in secondary structure: longer and less curved helices and a hairpin that now shows the typical right-hand twist. Molecular dynamics simulations starting from both gpW structures, and calculations with CS-Rosetta, all converge to our gpW structure. This confirms that the original structure has strange tertiary packing and strained secondary structure. A comparison of NMR datasets suggests that the problems were mainly caused by incomplete chemical shift assignments, mistakes in NOE assignment and the inclusion of ambiguous distance restraints during the automated procedure used in the original study. The new gpW corrects these problems, providing the appropriate structural reference for future work. Furthermore, our results are a cautionary tale against the inclusion of ambiguous experimental information in the determination of protein structures.

Cellular responses in Bacillus thuringiensis CS33 during bacteriophage BtCS33 infection.

PubMed

Wu, Dandan; Yuan, Yihui; Liu, Pengming; Wu, Yan; Gao, Meiying

2014-04-14

Bacillus thuringiensis (Bt) has been widely used for 50years as a biopesticide for controlling insect pests. However, bacteriophage infection can cause failures in 50%-80% of the batches during Bt fermentation, resulting in severe losses. In the present work, the physiological and biochemical impacts of Bt strain CS33 have been studied during bacteriophage infection. This study adopted a gel-based proteomics approach to probe the sequential changed proteins in phage-infected Bt cells. To phage, it depressed the host energy metabolism by suppressing the respiration chain, the TCA cycle, and the utilization of PHB on one hand; on the other hand, it hijacked the host translational machine for its own macromolecular synthesis. To host, superinfection exclusion might be triggered by the changes of S-layer protein and flagella related proteins, which were located on the cell surface and might play as the candidates for the phage recognition. More importantly, the growth rate, cell mass, and ICPs yield were significantly decreased. The low yield of ICPs was mainly due to the suppressed utilization of PHB granules. Further functional study on these altered proteins may lead to a better understanding of the pathogenic mechanisms and the identification of new targets for phage control. B. thuringiensis (Bt) has been widely used for 50years as a safe biopesticide for controlling agricultural and sanitary insect pests. However, bacteriophage infection can cause severe losses during B. thuringiensis fermentation. The processes and consequences of interactions between bacteriophage and Bt were still poorly understood, and the molecular mechanisms involved were more unknown. This study adopted a gel-based proteomics approach to probe the physiological and biochemical impacts of Bt strain CS33 after phage-infection. The interactions between phage BtCS33 and its host Bt strain CS33 occurred mainly on four aspects. First, phage synthesized its nucleic acids through metabolic

Evolution of the Quorum network and the mobilome (plasmids and bacteriophages) in clinical strains of Acinetobacter baumannii during a decade.

PubMed

López, M; Rueda, A; Florido, J P; Blasco, L; Fernández-García, L; Trastoy, R; Fernández-Cuenca, F; Martínez-Martínez, L; Vila, J; Pascual, A; Bou, G; Tomas, M

2018-02-06

In this study, we compared eighteen clinical strains of A. baumannii belonging to the ST-2 clone and isolated from patients in the same intensive care unit (ICU) in 2000 (9 strains referred to collectively as Ab_GEIH-2000) and 2010 (9 strains referred to collectively as Ab_GEIH-2010), during the GEIH-REIPI project (Umbrella BioProject PRJNA422585). We observed two main molecular differences between the Ab_GEIH-2010 and the Ab_GEIH-2000 collections, acquired over the course of the decade long sampling interval and involving the mobilome: i) a plasmid harbouring genes for bla OXA 24/40 ß-lactamase and abKA/abkB proteins of a toxin-antitoxin system; and ii) two temperate bacteriophages, Ab105-1ϕ (63 proteins) and Ab105-2ϕ (93 proteins), containing important viral defence proteins. Moreover, all Ab_GEIH-2010 strains contained a Quorum functional network of Quorum Sensing (QS) and Quorum Quenching (QQ) mechanisms, including a new QQ enzyme, AidA, which acts as a bacterial defence mechanism against the exogenous 3-oxo-C12-HSL. Interestingly, the infective capacity of the bacteriophages isolated in this study (Ab105-1ϕ and Ab105-2ϕ) was higher in the Ab_GEIH-2010 strains (carrying a functional Quorum network) than in the Ab_GEIH-2000 strains (carrying a deficient Quorum network), in which the bacteriophages showed little or no infectivity. This is the first study about the evolution of the Quorum network and the mobilome in clinical strains of Acinetobacter baumannii during a decade.

Development of a novel and highly efficient method of isolating bacteriophages from water.

PubMed

Liu, Weili; Li, Chao; Qiu, Zhi-Gang; Jin, Min; Wang, Jing-Feng; Yang, Dong; Xiao, Zhong-Hai; Yuan, Zhao-Kang; Li, Jun-Wen; Xu, Qun-Ying; Shen, Zhi-Qiang

2017-08-01

Bacteriophages are widely used to the treatment of drug-resistant bacteria and the improvement of food safety through bacterial lysis. However, the limited investigations on bacteriophage restrict their further application. In this study, a novel and highly efficient method was developed for isolating bacteriophage from water based on the electropositive silica gel particles (ESPs) method. To optimize the ESPs method, we evaluated the eluent type, flow rate, pH, temperature, and inoculation concentration of bacteriophage using bacteriophage f2. The quantitative detection reported that the recovery of the ESPs method reached over 90%. The qualitative detection demonstrated that the ESPs method effectively isolated 70% of extremely low-concentration bacteriophage (10 0 PFU/100L). Based on the host bacteria composed of 33 standard strains and 10 isolated strains, the bacteriophages in 18 water samples collected from the three sites in the Tianjin Haihe River Basin were isolated by the ESPs and traditional methods. Results showed that the ESPs method was significantly superior to the traditional method. The ESPs method isolated 32 strains of bacteriophage, whereas the traditional method isolated 15 strains. The sample isolation efficiency and bacteriophage isolation efficiency of the ESPs method were 3.28 and 2.13 times higher than those of the traditional method. The developed ESPs method was characterized by high isolation efficiency, efficient handling of large water sample size and low requirement on water quality. Copyright © 2017. Published by Elsevier B.V.

Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

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

Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon

2012-03-16

The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosenmore » DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.« less

Predicting In Vivo Efficacy of Therapeutic Bacteriophages Used To Treat Pulmonary Infections

PubMed Central

Henry, Marine; Lavigne, Rob

2013-01-01

The potential of bacteriophage therapy to treat infections caused by antibiotic-resistant bacteria has now been well established using various animal models. While numerous newly isolated bacteriophages have been claimed to be potential therapeutic candidates on the basis of in vitro observations, the parameters used to guide their choice among billions of available bacteriophages are still not clearly defined. We made use of a mouse lung infection model and a bioluminescent strain of Pseudomonas aeruginosa to compare the activities in vitro and in vivo of a set of nine different bacteriophages (PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5, CHA_P1, LBL3, LUZ19, and PhiKZ). For seven bacteriophages, a good correlation was found between in vitro and in vivo activity. While the remaining two bacteriophages were active in vitro, they were not sufficiently active in vivo under similar conditions to rescue infected animals. Based on the bioluminescence recorded at 2 and 8 h postinfection, we also define for the first time a reliable index to predict treatment efficacy. Our results showed that the bacteriophages isolated directly on the targeted host were the most efficient in vivo, supporting a personalized approach favoring an optimal treatment. PMID:24041900

Arthrobacter globiformis and its bacteriophage in soil

NASA Technical Reports Server (NTRS)

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

1974-01-01

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

Huntingtin interacting protein 1 Is a clathrin coat binding protein required for differentiation of late spermatogenic progenitors.

PubMed

Rao, D S; Chang, J C; Kumar, P D; Mizukami, I; Smithson, G M; Bradley, S V; Parlow, A F; Ross, T S

2001-11-01

Huntingtin-interacting protein 1 (HIP1) interacts with huntingtin, the protein whose gene is mutated in Huntington's disease. In addition, a fusion between HIP1 and platelet-derived growth factor beta receptor causes chronic myelomonocytic leukemia. The HIP1 proteins, including HIP1 and HIP1-related (HIP1r), have an N-terminal polyphosphoinositide-interacting epsin N-terminal homology, domain, which is found in proteins involved in clathrin-mediated endocytosis. HIP1 and HIP1r also share a central leucine zipper and an actin binding TALIN homology domain. Here we show that HIP1, like HIP1r, colocalizes with clathrin coat components. We also show that HIP1 physically associates with clathrin and AP-2, the major components of the clathrin coat. To further understand the putative biological role(s) of HIP1, we have generated a targeted deletion of murine HIP1. HIP1(-/-) mice developed into adulthood, did not develop overt neurologic symptoms in the first year of life, and had normal peripheral blood counts. However, HIP1-deficient mice exhibited testicular degeneration with increased apoptosis of postmeiotic spermatids. Postmeiotic spermatids are the only cells of the seminiferous tubules that express HIP1. These findings indicate that HIP1 is required for differentiation, proliferation, and/or survival of spermatogenic progenitors. The association of HIP1 with clathrin coats and the requirement of HIP1 for progenitor survival suggest a role for HIP1 in the regulation of endocytosis.

Huntingtin Interacting Protein 1 Is a Clathrin Coat Binding Protein Required for Differentiation of late Spermatogenic Progenitors

PubMed Central

Rao, Dinesh S.; Chang, Jenny C.; Kumar, Priti D.; Mizukami, Ikuko; Smithson, Glennda M.; Bradley, Sarah V.; Parlow, A. F.; Ross, Theodora S.

2001-01-01

Huntingtin-interacting protein 1 (HIP1) interacts with huntingtin, the protein whose gene is mutated in Huntington's disease. In addition, a fusion between HIP1 and platelet-derived growth factor β receptor causes chronic myelomonocytic leukemia. The HIP1 proteins, including HIP1 and HIP1-related (HIP1r), have an N-terminal polyphosphoinositide-interacting epsin N-terminal homology, domain, which is found in proteins involved in clathrin-mediated endocytosis. HIP1 and HIP1r also share a central leucine zipper and an actin binding TALIN homology domain. Here we show that HIP1, like HIP1r, colocalizes with clathrin coat components. We also show that HIP1 physically associates with clathrin and AP-2, the major components of the clathrin coat. To further understand the putative biological role(s) of HIP1, we have generated a targeted deletion of murine HIP1. HIP1−/− mice developed into adulthood, did not develop overt neurologic symptoms in the first year of life, and had normal peripheral blood counts. However, HIP1-deficient mice exhibited testicular degeneration with increased apoptosis of postmeiotic spermatids. Postmeiotic spermatids are the only cells of the seminiferous tubules that express HIP1. These findings indicate that HIP1 is required for differentiation, proliferation, and/or survival of spermatogenic progenitors. The association of HIP1 with clathrin coats and the requirement of HIP1 for progenitor survival suggest a role for HIP1 in the regulation of endocytosis. PMID:11604514

Evidence for lysine acetylation in the coat protein of a polerovirus.

PubMed

Cilia, Michelle; Johnson, Richard; Sweeney, Michelle; DeBlasio, Stacy L; Bruce, James E; MacCoss, Michael J; Gray, Stewart M

2014-10-01

Virions of the RPV strain of Cereal yellow dwarf virus-RPV were purified from infected oat tissue and analysed by MS. Two conserved residues, K147 and K181, in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional data are available for K147, K181 lies within an interfacial region critical for virion assembly and stability. The signature immonium ion at m/z 126.0919 demonstrated the presence of N-acetyllysine, and the sequence fragment ions enabled an unambiguous assignment of the epsilon-N-acetyl modification on K181. We hypothesize that selection favours acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.

Aligning the unalignable: bacteriophage whole genome alignments.

PubMed

Bérard, Sèverine; Chateau, Annie; Pompidor, Nicolas; Guertin, Paul; Bergeron, Anne; Swenson, Krister M

2016-01-13

In recent years, many studies focused on the description and comparison of large sets of related bacteriophage genomes. Due to the peculiar mosaic structure of these genomes, few informative approaches for comparing whole genomes exist: dot plots diagrams give a mostly qualitative assessment of the similarity/dissimilarity between two or more genomes, and clustering techniques are used to classify genomes. Multiple alignments are conspicuously absent from this scene. Indeed, whole genome aligners interpret lack of similarity between sequences as an indication of rearrangements, insertions, or losses. This behavior makes them ill-prepared to align bacteriophage genomes, where even closely related strains can accomplish the same biological function with highly dissimilar sequences. In this paper, we propose a multiple alignment strategy that exploits functional collinearity shared by related strains of bacteriophages, and uses partial orders to capture mosaicism of sets of genomes. As classical alignments do, the computed alignments can be used to predict that genes have the same biological function, even in the absence of detectable similarity. The Alpha aligner implements these ideas in visual interactive displays, and is used to compute several examples of alignments of Staphylococcus aureus and Mycobacterium bacteriophages, involving up to 29 genomes. Using these datasets, we prove that Alpha alignments are at least as good as those computed by standard aligners. Comparison with the progressive Mauve aligner - which implements a partial order strategy, but whose alignments are linearized - shows a greatly improved interactive graphic display, while avoiding misalignments. Multiple alignments of whole bacteriophage genomes work, and will become an important conceptual and visual tool in comparative genomics of sets of related strains. A python implementation of Alpha, along with installation instructions for Ubuntu and OSX, is available on bitbucket (https://bitbucket.org/thekswenson/alpha).

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation

PubMed Central

Ribis, John W.; Ravichandran, Priyanka; Putnam, Emily E.; Pishdadian, Keyan

2017-01-01

ABSTRACT The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis, only two of these morphogenetic proteins have homologs in the Clostridia: SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis. Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis, C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia, but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and

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.

Natural supramolecular building blocks: from virus coat proteins to viral nanoparticles.

PubMed

Liu, Zhi; Qiao, Jing; Niu, Zhongwei; Wang, Qian

2012-09-21

Viruses belong to a fascinating class of natural supramolecular structures, composed of multiple copies of coat proteins (CPs) that assemble into different shapes with a variety of sizes from tens to hundreds of nanometres. Because of their advantages including simple/economic production, well-defined structural features, unique shapes and sizes, genetic programmability and robust chemistries, recently viruses and virus-like nanoparticles (VLPs) have been used widely in biomedical applications and materials synthesis. In this critical review, we highlight recent advances in the use of virus coat proteins (VCPs) and viral nanoparticles (VNPs) as building blocks in self-assembly studies and materials development. We first discuss the self-assembly of VCPs into VLPs, which can efficiently incorporate a variety of different materials as cores inside the viral protein shells. Then, the self-assembly of VNPs at surfaces or interfaces is summarized. Finally, we discuss the co-assembly of VNPs with different functional materials (178 references).

Versatile de novo enzyme activity in capsid proteins from an engineered M13 bacteriophage library.

PubMed

Casey, John P; Barbero, Roberto J; Heldman, Nimrod; Belcher, Angela M

2014-11-26

Biocatalysis has grown rapidly in recent decades as a solution to the evolving demands of industrial chemical processes. Mounting environmental pressures and shifting supply chains underscore the need for novel chemical activities, while rapid biotechnological progress has greatly increased the utility of enzymatic methods. Enzymes, though capable of high catalytic efficiency and remarkable reaction selectivity, still suffer from relative instability, high costs of scaling, and functional inflexibility. Herein, we developed a biochemical platform for engineering de novo semisynthetic enzymes, functionally modular and widely stable, based on the M13 bacteriophage. The hydrolytic bacteriophage described in this paper catalyzes a range of carboxylic esters, is active from 25 to 80 °C, and demonstrates greater efficiency in DMSO than in water. The platform complements biocatalysts with characteristics of heterogeneous catalysis, yielding high-surface area, thermostable biochemical structures readily adaptable to reactions in myriad solvents. As the viral structure ensures semisynthetic enzymes remain linked to the genetic sequences responsible for catalysis, future work will tailor the biocatalysts to high-demand synthetic processes by evolving new activities, utilizing high-throughput screening technology and harnessing M13's multifunctionality.

Modified Filamentous Bacteriophage as a Scaffold for Carbon Nanofiber.

PubMed

Szot-Karpińska, Katarzyna; Golec, Piotr; Leśniewski, Adam; Pałys, Barbara; Marken, Frank; Niedziółka-Jönsson, Joanna; Węgrzyn, Grzegorz; Łoś, Marcin

2016-12-21

With the advent of nanotechnology, carbon nanomaterials such as carbon nanofibers (CNF) have aroused substantial interest in various research fields, including energy storage and sensing. Further improvement of their properties might be achieved via the application of viral particles such as bacteriophages. In this report, we present a filamentous M13 bacteriophage with a point mutation in gene VII (pVII-mutant-M13) that selectively binds to the carbon nanofibers to form 3D structures. The phage-display technique was utilized for the selection of the pVII-mutant-M13 phage from the phage display peptide library. The properties of this phage make it a prospective candidate for a scaffold material for CNFs. The results for binding of CNF by mutant phage were compared with those for maternal bacteriophage (pVII-M13). The efficiency of binding between pVII-mutant-M13 and CNF is about 2 orders of magnitude higher compared to that of the pVII-M13. Binding affinity between pVII-mutant-M13 and CNF was also characterized using atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, which confirmed the specificity of the interaction of the phage pVII-mutant-M13 and the CNF; the binding occurs via the phage's ending, where the mutated pVII protein is located. No similar behavior has been observed for other carbon nanomaterials such as graphite, reduced graphene oxide, single-walled carbon nanotubes, and multiwalled carbon nanotubes. Infrared spectra confirmed differences in the interaction with CNF between the pVII-mutant-M13 and the pVII-M13. Basing on conducted research, we hypothesize that the interactions are noncovalent in nature, with π-π interactions playing the dominant role. Herein, the new bioconjugate material is introduced.

The role of bacteriophages in periodontal health and disease.

PubMed

Pinto, Graça; Silva, Maria Daniela; Peddey, Mark; Sillankorva, Sanna; Azeredo, Joana

2016-10-01

The human periodontium health is commonly compromised by chronic inflammatory conditions and has become a major public health concern. Dental plaque, the precursor of periodontal disease, is a complex biofilm consisting mainly of bacteria, but also archaea, protozoa, fungi and viruses. Viruses that specifically infect bacteria - bacteriophages - are most common in the oral cavity. Despite this, their role in the progression of periodontal disease remains poorly explored. This review aims to summarize how bacteriophages interact with the oral microbiota, their ability to increase bacterial virulence and mediate the transfer of resistance genes and suggests how bacteriophages can be used as an alternative to the current periodontal disease therapies.

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase of coat protein subgroup II gene from Cucumber mosaic virus

USDA-ARS?s Scientific Manuscript database

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These...

Intravascular local gene transfer mediated by protein-coated metallic stent.

PubMed

Yuan, J; Gao, R; Shi, R; Song, L; Tang, J; Li, Y; Tang, C; Meng, L; Yuan, W; Chen, Z

2001-10-01

To assess the feasibility, efficiency and selectivity of adenovirus-mediated gene transfer to local arterial wall by protein-coated metallic stent. A replication-defective recombinant adenovirus carrying the Lac Z reporter gene for nuclear-specific beta-galactosidase (Ad-beta gal) was used in this study. The coating for metallic stent was made by immersing it in a gelatin solution containing crosslinker. The coated stents were mounted on a 4.0 or 3.0 mm percutaneous transluminal coronary angioplasty (PTCA) balloon and submersed into a high-titer Ad-beta gal viral stock (2 x 10(10) pfu/ml) for 3 min, and then implanted into the carotid arteries in 4 mini-swines and into the left anterior descending branch of the coronary artery in 2 mini-swines via 8F large lumen guiding catheters. The animals were sacrificed 7 (n = 4), 14 (n = 1) and 21 (n = 1) days after implantation, respectively. The beta-galactosidase expression was assessed by X-gal staining. The results showed that the expression of transgene was detected in all animal. In 1 of carotid artery with an intact intima, the beta-gal expression was limited to endothelial cells. In vessels with denuded endothelium, gene expression was found in the sub-intima, media and adventitia. The transfection efficiency of medial smooth muscle cells was 38.6%. In 2 animals sacrificed 7 days after transfection, a microscopic examination of X-gal-stained samples did not show evidence of transfection in remote organs and arterial segments adjacent to the treated arterial site. Adenovirus-mediated arterial gene transfer to endothelial, smooth muscle cells and adventitia by protein-coated metallic stent is feasible. The transfection efficiency is higher. The coated stent may act as a good carrier of adenovirus-mediated gene transfer and have a potential to prevent restenosis following PTCA.

Potential impact of environmental bacteriophages in spreading antibiotic resistance genes.

PubMed

Muniesa, Maite; Colomer-Lluch, Marta; Jofre, Juan

2013-06-01

The idea that bacteriophage transduction plays a role in the horizontal transfer of antibiotic resistance genes is gaining momentum. Such transduction might be vital in horizontal transfer from environmental to human body-associated biomes and here we review many lines of evidence supporting this notion. It is well accepted that bacteriophages are the most abundant entities in most environments, where they have been shown to be quite persistent. This fact, together with the ability of many phages to infect bacteria belonging to different taxa, makes them suitable vehicles for gene transfer. Metagenomic studies confirm that substantial percentages of the bacteriophage particles present in most environments contain bacterial genes, including mobile genetic elements and antibiotic resistance genes. When specific genes of resistance to antibiotics are detected by real-time PCR in the bacteriophage populations of different environments, only tenfold lower numbers of these genes are observed, compared with those found in the corresponding bacterial populations. In addition, the antibiotic resistance genes from these bacteriophages are functional and generate resistance to the bacteria when these genes are transfected. Finally, reports about the transduction of antibiotic resistance genes are on the increase.

Bacteriophage-based Probiotic Preparation for Managing Shigella Infections

DTIC Science & Technology

2015-04-16

for a probiotic preparation – based on naturally occurring bacteriophages – as a way to condition the GI tract’s microflora gently and favorably...10-Apr-2013 Approved for Public Release; Distribution Unlimited Final Report: Bacteriophage-based Probiotic Preparation for Managing Shigella...Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 Phage, Shigella, probiotics REPORT DOCUMENTATION PAGE 11. SPONSOR/MONITOR’S

Alterations in nanoparticle protein corona by biological surfactants: impact of bile salts on β-lactoglobulin-coated gold nanoparticles.

PubMed

Winuprasith, Thunnalin; Chantarak, Sirinya; Suphantharika, Manop; He, Lili; McClements, David Julian

2014-07-15

The impact of biological surfactants (bile salts) on the protein (β-lactoglobulin) corona surrounding gold nanoparticles (200 nm) was studied using a variety of analytical techniques at pH 7: dynamic light scattering (DLS); particle electrophoresis (ζ-potential); UV-visible (UV) spectroscopy; transmission electron microscopy (TEM); and surface-enhanced Raman scattering (SERS). The bile salts adsorbed to the protein-coated nanoparticle surfaces and altered their interfacial composition, charge, and structure. SERS spectra of protein-coated nanoparticles after bile salt addition contained bands from both protein and bile salts, indicating that the protein was not fully displaced by the bile salts. UV, DLS and TEM techniques also indicated that the protein coating was not fully displaced from the nanoparticle surfaces. The impact of bile salts could be described by an orogenic mechanism: mixed interfaces were formed that consisted of islands of aggregated proteins surrounded by a sea of bile salts. This knowledge is useful for understanding the interactions of bile salts with protein-coated colloidal particles, which may be important for controlling the fate of colloidal delivery systems in the human gastrointestinal tract, or the gastrointestinal fate of ingested inorganic nanoparticles. Copyright © 2014 Elsevier Inc. All rights reserved.

A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2.

PubMed

Tumban, Ebenezer; Peabody, Julianne; Peabody, David S; Chackerian, Bryce

2011-01-01

Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin. L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV. VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.

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

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacteriophage of Clavibacter... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1307 Bacteriophage of... exemption from the requirement of a tolerance is established for residues of lytic bacteriophage of...

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

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacteriophage of Clavibacter... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1307 Bacteriophage of... exemption from the requirement of a tolerance is established for residues of lytic bacteriophage of...

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

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacteriophage of Clavibacter... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1307 Bacteriophage of... exemption from the requirement of a tolerance is established for residues of lytic bacteriophage of...

Template reporter bacteriophage platform and multiple bacterial detection assays based thereon

NASA Technical Reports Server (NTRS)

Goodridge, Lawrence (Inventor)

2007-01-01

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

Newly Isolated Bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae Families Have Variable Effects on Putative Novel Dickeya spp.

PubMed

Alič, Špela; Naglič, Tina; Tušek-Žnidarič, Magda; Ravnikar, Maja; Rački, Nejc; Peterka, Matjaž; Dreo, Tanja

2017-01-01

Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya -infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales , belonging to three different families, Podoviridae , Myoviridae , and Siphoviridae . The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20°C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral

Newly Isolated Bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae Families Have Variable Effects on Putative Novel Dickeya spp.

PubMed Central

Alič, Špela; Naglič, Tina; Tušek-Žnidarič, Magda; Ravnikar, Maja; Rački, Nejc; Peterka, Matjaž; Dreo, Tanja

2017-01-01

Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya-infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales, belonging to three different families, Podoviridae, Myoviridae, and Siphoviridae. The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20°C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral and

Expression of a bioactive bacteriophage endolysin in Nicotiana benthamiana plants

USDA-ARS?s Scientific Manuscript database

The emergence and spread of antibiotic-resistant pathogens has led to an increased interest in alternative antimicrobial treatments, such as bacteriophage, bacteriophage-encoded peptidoglycan hydrolases (endolysins) and antimicrobial peptides. In our study, the antimicrobial activity of the CP933 en...

Metavirome Sequencing of the Termite Gut Reveals the Presence of an Unexplored Bacteriophage Community

PubMed Central

Tikhe, Chinmay V.; Husseneder, Claudia

2018-01-01

The Formosan subterranean termite; Coptotermes formosanus is nutritionally dependent on the complex and diverse community of bacteria and protozoa in their gut. Although, there have been many studies to decipher the taxonomic and functional diversity of bacterial communities in the guts of termites, their bacteriophages remain unstudied. We sequenced the metavirome of the guts of Formosan subterranean termite workers to study the diversity of bacteriophages and other associated viruses. Results showed that the termites harbor a virome in their gut comprised of varied and previously unknown bacteriophages. Between 87–90% of the predicted dsDNA virus genes by Metavir showed similarity to the tailed bacteriophages (Caudovirales). Many predicted genes from the virome matched to bacterial prophage regions. These data are suggestive of a virome dominated by temperate bacteriophages. We predicted the genomes of seven novel Caudovirales bacteriophages from the termite gut. Three of these predicted bacteriophage genomes were found in high proportions in all the three termite colonies tested. Two bacteriophages are predicted to infect endosymbiotic bacteria of the gut protozoa. The presence of these putative bacteriophages infecting endosymbionts of the gut protozoa, suggests a quadripartite relationship between the termites their symbiotic protozoa, endosymbiotic bacteria of the protozoa and their bacteriophages. Other than Caudovirales, ss-DNA virus related genes were also present in the termite gut. We predicted the genomes of 12 novel Microviridae phages from the termite gut and seven of those possibly represent a new proposed subfamily. Circovirus like genomes were also assembled from the termite gut at lower relative abundance. We predicted 10 novel circovirus genomes in this study. Whether these circoviruses infect the termites remains elusive at the moment. The functional and taxonomical annotations suggest that the termites may harbor a core virome comprised of

Bacteriophage T5 encodes a homolog of the eukaryotic transcription coactivator PC4 implicated in recombination-dependent DNA replication.

PubMed

Steigemann, Birthe; Schulz, Annina; Werten, Sebastiaan

2013-11-15

The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors. © 2013.

Physical, Chemical and Biochemical Modifications of Protein-Based Films and Coatings: An Extensive Review

PubMed Central

Zink, Joël; Wyrobnik, Tom; Prinz, Tobias; Schmid, Markus

2016-01-01

Protein-based films and coatings are an interesting alternative to traditional petroleum-based materials. However, their mechanical and barrier properties need to be enhanced in order to match those of the latter. Physical, chemical, and biochemical methods can be used for this purpose. The aim of this article is to provide an overview of the effects of various treatments on whey, soy, and wheat gluten protein-based films and coatings. These three protein sources have been chosen since they are among the most abundantly used and are well described in the literature. Similar behavior might be expected for other protein sources. Most of the modifications are still not fully understood at a fundamental level, but all the methods discussed change the properties of the proteins and resulting products. Mastering these modifications is an important step towards the industrial implementation of protein-based films. PMID:27563881

Bacteriophage lambda: The path from biology to theranostic agent.

PubMed

Catalano, Carlos E

2018-03-13

Viral particles provide an attractive platform for the engineering of semisynthetic therapeutic nanoparticles. They can be modified both genetically and chemically in a defined manner to alter their surface characteristics, for targeting specific cell types, to improve their pharmacokinetic features and to attenuate (or enhance) their antigenicity. These advantages derive from a detailed understanding of virus biology, gleaned from decades of fundamental genetic, biochemical, and structural studies that have provided mechanistic insight into virus assembly pathways. In particular, bacteriophages offer significant advantages as nanoparticle platforms and several have been adapted toward the design and engineering of "designer" nanoparticles for therapeutic and diagnostic (theranostic) applications. The present review focuses on one such virus, bacteriophage lambda; I discuss the biology of lambda, the tools developed to faithfully recapitulate the lambda assembly reactions in vitro and the observations that have led to cooptation of the lambda system for nanoparticle design. This discussion illustrates how a fundamental understanding of virus assembly has allowed the rational design and construction of semisynthetic nanoparticles as potential theranostic agents and illustrates the concept of benchtop to bedside translational research. This article is categorized under: Biology-Inspired Nanomaterials> Protein and Virus-Based Structures Biology-Inspired Nanomaterials> Nucleic Acid-Based Structures. © 2018 Wiley Periodicals, Inc.

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction.

PubMed Central

Dodson, M; Echols, H; Wickner, S; Alfano, C; Mensa-Wilmot, K; Gomes, B; LeBowitz, J; Roberts, J D; McMacken, R

1986-01-01

The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda. By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages of this prepriming reaction, the first two of which we have characterized previously. First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites. Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T-rich region to the right of the binding sites. We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control. Images PMID:3020552

Genomics of Three New Bacteriophages Useful in the Biocontrol of Salmonella

PubMed Central

Bardina, Carlota; Colom, Joan; Spricigo, Denis A.; Otero, Jennifer; Sánchez-Osuna, Miquel; Cortés, Pilar; Llagostera, Montserrat

2016-01-01

Non-typhoid Salmonella is the principal pathogen related to food-borne diseases throughout the world. Widespread antibiotic resistance has adversely affected human health and has encouraged the search for alternative antimicrobial agents. The advances in bacteriophage therapy highlight their use in controlling a broad spectrum of food-borne pathogens. One requirement for the use of bacteriophages as antibacterials is the characterization of their genomes. In this work, complete genome sequencing and molecular analyses were carried out for three new virulent Salmonella-specific bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) able to infect a broad range of Salmonella strains. Sequence analysis of the genomes of UAB_Phi20, UAB_Phi78, and UAB_Phi87 bacteriophages did not evidence the presence of known virulence-associated and antibiotic resistance genes, and potential immunoreactive food allergens. The UAB_Phi20 genome comprised 41,809 base pairs with 80 open reading frames (ORFs); 24 of them with assigned function. Genome sequence showed a high homology of UAB_Phi20 with Salmonella bacteriophage P22 and other P22likeviruses genus of the Podoviridae family, including ST64T and ST104. The DNA of UAB_Phi78 contained 44,110 bp including direct terminal repeats (DTR) of 179 bp and 58 putative ORFs were predicted and 20 were assigned function. This bacteriophage was assigned to the SP6likeviruses genus of the Podoviridae family based on its high similarity not only with SP6 but also with the K1-5, K1E, and K1F bacteriophages, all of which infect Escherichia coli. The UAB_Phi87 genome sequence consisted of 87,669 bp with terminal direct repeats of 608 bp; although 148 ORFs were identified, putative functions could be assigned to only 29 of them. Sequence comparisons revealed the mosaic structure of UAB_Phi87 and its high similarity with bacteriophages Felix O1 and wV8 of E. coli with respect to genetic content and functional organization. Phylogenetic analysis of large

Dual Active Site in the Endolytic Transglycosylase gp144 of Bacteriophage phiKZ.

PubMed

Chertkov, O V; Armeev, G A; Uporov, I V; Legotsky, S A; Sykilinda, N N; Shaytan, A K; Klyachko, N L; Miroshnikov, K A

2017-01-01

Lytic transglycosylases are abundant peptidoglycan lysing enzymes that degrade the heteropolymers of bacterial cell walls in metabolic processes or in the course of a bacteriophage infection. The conventional catalytic mechanism of transglycosylases involves only the Glu or Asp residue. Endolysin gp144 of Pseudomonas aeruginosa bacteriophage phiKZ belongs to the family of Gram-negative transglycosylases with a modular composition and C -terminal location of the catalytic domain. Glu115 of gp144 performs the predicted role of a catalytic residue. However, replacement of this residue does not completely eliminate the activity of the mutant protein. Site-directed mutagenesis has revealed the participation of Tyr197 in the catalytic mechanism, as well as the presence of a second active site involving Glu178 and Tyr147. The existence of the dual active site was supported by computer modeling and monitoring of the molecular dynamics of the changes in the conformation and surface charge distribution as a consequence of point mutations.

Bacteriophage immobilized graphene electrodes for impedimetric sensing of bacteria (Staphylococcus arlettae).

PubMed

Bhardwaj, Neha; Bhardwaj, Sanjeev K; Mehta, Jyotsana; Mohanta, Girish C; Deep, Akash

2016-07-15

Bacteriophages are a class of viruses that specifically infect and replicate within a bacterium. They possess inherent affinity and specificity to the particular bacterial cells. This property of bacteriophages makes them an attractive biorecognition element in the field of biosensor development. In this work, we report the use of an immobilized bacteriophage for the development of a highly sensitive electrochemical sensor for Staphylococcus arlettae, bacteria from the pathogenic family of coagulase-negative staphylococci (CNS). The specific bacteriophages were covalently immobilized on the screen-printed graphene electrodes. Thus, the fabricated bacteriophage biosensor displayed quantitative response for the target bacteria (S. arlettae) for a broad detection range (2.0-2.0 × 10(6) cfu). A fast response time (2 min), low limit of detection (2 cfu), specificity, and stability over a prolonged period (3 months) are some of the important highlights of the proposed sensor. The practical utility of the developed sensor has been demonstrated by the analysis of S. arlettae in spiked water and apple juice samples. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Bacteriophage-nanocomposites: an easy and reproducible method for the construction, handling, storage and transport of conjugates for deployment of bacteriophages active against Pseudomonas aeruginosa.

PubMed

Cooper, Ian R; Illsley, Matthew; Korobeinyk, Alina V; Whitby, Raymond L D

2015-04-01

The purpose of this work was proof of concept to develop a novel, cost effective protocol for the binding of bacteriophages to a surface without loss of function, after storage in various media. The technology platform involved covalently bonding bacteriophage 13 (a Pseudomonas aeruginosa bacteriophage) to two magnetised multiwalled carbon nanotube scaffolds using a series of buffers; bacteriophage-nanotube (B-N) conjugates were efficacious after storage at 20 °C for six weeks. B-N conjugates were added to human cell culture in vitro for 9 days without causing necrosis and apoptosis. B-N conjugates were frozen (-20 °C) in cell culture media for several weeks, after which recovery from the human cell culture medium was possible using a simple magnetic separation technique. The retention of viral infective potential was demonstrated by subsequent spread plating onto lawns of susceptible P. aeruginosa. Analysis of the human cell culture medium revealed the production of interleukins by the human fibroblasts upon exposure to the bacteriophage. One day after exposure, IL-8 levels transitorily increased between 60 and 100 pg/mL, but this level was not found on any subsequent days, suggesting an initial but not long lasting response. This paper outlines the development of a method to deliver antimicrobial activity to a surface that is small enough to be combined with other materials. To our knowledge at time of publication, this is the first report of magnetically coupled bacteriophages specific to human pathogens which can be recovered from test systems, and could represent a novel means to conditionally deploy antibacterial agents into living eukaryotic systems without the risks of some antibiotic therapies. Copyright © 2015. Published by Elsevier B.V.

Bacteriophage SP6 encodes a second tailspike protein that recognizes Salmonella enterica serogroups C{sub 2} and C{sub 3}

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

Gebhart, Dana; Williams, Steven R.; Scholl, Dean,

SP6 is a salmonella phage closely related to coliphage K1-5. K1-5 is notable in that it encodes two polysaccharide-degrading tailspike proteins, an endosialidase that allows it to infect E. coli K1, and a lyase that enables it to infect K5 strains. SP6 is similar to K1-5 except that it encodes a P22-like endorhamnosidase tailspike, gp46, allowing it to infect group B Salmonella. We show here that SP6 can also infect Salmonella serogroups C{sub 2} and C{sub 3} and that a mutation in a putative second tailspike, gp47, eliminates this specificity. Gene 47 was fused to the coding region of themore » N-terminal portion of the Pseudomonas aeruginosa R2 pyocin tail fiber and expressed in trans such that the fusion protein becomes incorporated into pyocin particles. These pyocins, termed AvR2-SP47, killed serogroups C{sub 2} and C{sub 3}Salmonella. We conclude that SP6 encodes two tail proteins providing it a broad host range among Salmonella enterica. - Highlights: • SP6 is a “dual specificity” bacteriophage that encodes two different receptor binding proteins giving it a broad host range. • These receptor binding proteins can be used to re-target the spectrum of R-type bacteriocins to Salmonella enterica. • Both SP6 and the engineered R-type bacteriocins can kill the Salmonella serovars most associated with human disease making them attractive for development as antimicrobial agents.« less

Genomic characterization of bacteriophage vB_PcaP_PP2 infecting Pectobacterium carotovorum subsp. carotovorum, a new member of a proposed genus in the subfamily Autographivirinae.

PubMed

Lim, Jeong-A; Heu, Sunggi; Park, Jinwoo; Roh, Eunjung

2017-08-01

Bacteriophage vB_PcaP_PP2 (PP2) is a novel virulent phage that infects the plant-pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. PP2 phage has a 41,841-bp double-stranded DNA encoding 47 proteins, and it was identified as a member of the family Podoviridae by transmission electron microscopy. Nineteen of its open reading frames (ORFs) show homology to functional proteins, and 28 ORFs have been characterized as hypothetical proteins. PP2 phage is homologous to Cronobacter phage vB_CskP_GAP227 and Dev-CD-23823. Based on phylogenetic analysis, PP2 and its homologous bacteriophages form a new group within the subfamily Autographivirinae in the family Podoviridae, suggesting the need to establish a new genus. No lysogenic-cycle-related genes or bacterial toxins were identified.

Bacteriophage endolysins as novel antimicrobials

PubMed Central

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

2013-01-01

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

[Virus resistance in transgenic watermelon plants containing a WMV-2 coat protein gene].

PubMed

Wang, Hui-Zhong; Zhao, Pei-Jie; Xu, Ji-Chen; Zhao, Huai; Zhang, Hong-Sheng

2003-01-01

Virus disease is a major cause that affects the quality and output of watermelon which is an important fruit in summer. So it is really urgent to develop disease resistance plants. But it takes a long time to breed such plants in conventional ways, and it is very difficult to get ideal result. With the development of plant genetic engineering, new ways have been found to breed plants with disease resistance. By using plant transgenic technique, much progress was been made in plant improvement. There are many successful cases of transgenic plants against corresponding virus disease through transferring coat protein gene. This paper reports the results of inheritance, segregation, expression of WMV-2 coat protein gene in inbred transgenic watermelon and its resistance to virus. Through PCR analysis of inbred plants, we found WMV-2 coat protein gene in the genome of progeny R1 separated with 3:1. After successive selection and identification of 4 generations, 8 transgenic pure lines with almost the same agronomic traits were obtained from 3 independent transformants of T7, T11 and T32. The result of Western blotting shows all 3 different transgenic lines of R4T7-1, R4T11-3 and R4T32-7 can produce coat protein. Disease resistance experiment on transgenic plants with WMV-2 shows that, compared with the control groups, transgenic plants can delay the disease infection and reduce the incidence and the symptoms of virus disease. And the transgenic line R4T32-7 expressed high resistance to infection by WMV-2, which lays a foundation for breeding of disease resistant varieties through plant transgenic technique.

Formation of the outer layer of the Dictyostelium spore coat depends on the inner-layer protein SP85/PsB.

PubMed

Metcalf, Talibah; Kelley, Karen; Erdos, Gregory W; Kaplan, Lee; West, Christopher M

2003-02-01

The Dictyostelium spore is surrounded by a 220 microm thick trilaminar coat that consists of inner and outer electron-dense layers surrounding a central region of cellulose microfibrils. In previous studies, a mutant strain (TL56) lacking three proteins associated with the outer layer exhibited increased permeability to macromolecular tracers, suggesting that this layer contributes to the coat permeability barrier. Electron microscopy now shows that the outer layer is incomplete in the coats of this mutant and consists of a residual regular array of punctate electron densities. The outer layer is also incomplete in a mutant lacking a cellulose-binding protein associated with the inner layer, and these coats are deficient in an outer-layer protein and another coat protein. To examine the mechanism by which this inner-layer protein, SP85, contributes to outer-layer formation, various domain fragments were overexpressed in forming spores. Most of these exert dominant negative effects similar to the deletion of outer-layer proteins, but one construct, consisting of a fusion of the N-terminal and Cys-rich C1 domain, induces a dense mat of novel filaments at the surface of the outer layer. Biochemical studies show that the C1 domain binds cellulose, and a combination of site-directed mutations that inhibits its cellulose-binding activity suppresses outer-layer filament induction. The results suggest that, in addition to a previously described early role in regulating cellulose synthesis, SP85 subsequently contributes a cross-bridging function between cellulose and other coat proteins to organize previously unrecognized structural elements in the outer layer of the coat.

Improved bacteriophage genome data is necessary for integrating viral and bacterial ecology.

PubMed

Bibby, Kyle

2014-02-01

The recent rise in "omics"-enabled approaches has lead to improved understanding in many areas of microbial ecology. However, despite the importance that viruses play in a broad microbial ecology context, viral ecology remains largely not integrated into high-throughput microbial ecology studies. A fundamental hindrance to the integration of viral ecology into omics-enabled microbial ecology studies is the lack of suitable reference bacteriophage genomes in reference databases-currently, only 0.001% of bacteriophage diversity is represented in genome sequence databases. This commentary serves to highlight this issue and to promote bacteriophage genome sequencing as a valuable scientific undertaking to both better understand bacteriophage diversity and move towards a more holistic view of microbial ecology.

Proteomic Analysis of Interactions between a Deep-Sea Thermophilic Bacteriophage and Its Host at High Temperature ▿ †

PubMed Central

Wei, Dahai; Zhang, Xiaobo

2010-01-01

The virus-host interaction is essential to understanding the role that viruses play in ecological and geochemical processes in deep-sea vent ecosystems. Virus-induced changes in cellular gene expression and host physiology have been studied extensively. However, the molecular mechanism of interaction between a bacteriophage and its host at high temperature remains poorly understood. In the present study, the virus-induced gene expression profile of Geobacillus sp. E263, a thermophile isolated from a deep-sea hydrothermal ecosystem, was characterized. Based on proteomic analysis and random arbitrarily primed PCR (RAP-PCR) of Geobacillus sp. E263 cultured under non-bacteriophage GVE2 infection and GVE2 infection conditions, there were two types of protein/gene profiles in response to GVE2 infection. Twenty differentially expressed genes and proteins were revealed that could be grouped into 3 different categories based on cellular function, suggesting a coordinated response to infection. These differentially expressed genes and proteins were further confirmed by Northern blot analysis. To characterize the host proteins in response to virus infection, aspartate aminotransferase (AST) was inactivated to construct the AST mutant of Geobacillus sp. E263. The results showed that the AST protein was essential in virus infection. Thus, transcriptional and proteomic analyses and functional analysis revealed previously unknown host responses to deep-sea thermophilic virus infection. PMID:20015994

Utilization of specific and non-specific peptide interactions with inorganic nanomaterials on the surface of bacteriophage M13: Methodologies towards phage supported bi-functional materials

NASA Astrophysics Data System (ADS)

Avery, Kendra Nicole

Many types of organisms create a variety of nano and micro scale materials from precursors available in their surrounding environments by a process called biomineralization. As scientists begin to understand how these organisms utilize specific and non-specific interactions with a variety of biopolymers such as chitin, peptides, proteins and nucleic acids with these precursors to create inorganic/organic composite materials, they have begun to wonder about the synthesis of other types of non-biologically templated synthetic techniques that might be possible. Bioengineered organisms and biopolymers have begun to be used for these types of studies. A variety of selection techniques exist for discovering biopolymers with an affinity for a target material, however, one of the most notable is a technique called peptide phage display. This is a technique that utilizes a commercially available randomized peptide library attached at the tip of the filamentous bacteriophage M13. In this dissertation capabilities of bacteriophage M13 are explored in regard to the creation of bi-functional nano materials by exploiting both specific peptide interactions as well as non-specific peptide interactions on the surface of the organism. Chapter 2 focuses on utilizing the specific peptide interactions of the randomized library at pIII in order to discover peptides with high binding affinity for a variety of nanomaterials. Selection studies called biopanning are performed on a variety of nanomaterials such as CaMoO4, allotropes of Ni, Fe2O3 and Fe3O4, and Rh and Pt with the fcc type crystal structure. Similarities and differences between peptides discovered for these materials are discussed. Chapter 3 focuses on utilizing the non-specific peptide interactions on the long axis of M13 called pVIII. The pVIII region consists of 2700 copies of the same 50 amino acid protein which as a negatively charged domain which is exposed to solution. The pVIII region therefore provides the surface of

Application of bacteriophages in post-harvest control of human pathogenic and food spoiling bacteria.

PubMed

Pérez Pulido, Rubén; Grande Burgos, Maria José; Gálvez, Antonio; Lucas López, Rosario

2016-10-01

Bacteriophages have attracted great attention for application in food biopreservation. Lytic bacteriophages specific for human pathogenic bacteria can be isolated from natural sources such as animal feces or industrial wastes where the target bacteria inhabit. Lytic bacteriophages have been tested in different food systems for inactivation of main food-borne pathogens including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Salmonella enterica, Shigella spp., Campylobacter jejuni and Cronobacter sakazkii, and also for control of spoilage bacteria. Application of lytic bacteriophages could selectively control host populations of concern without interfering with the remaining food microbiota. Bacteriophages could also be applied for inactivation of bacteria attached to food contact surfaces or grown as biofilms. Bacteriophages may receive a generally recognized as safe status based on their lack of toxicity and other detrimental effects to human health. Phage preparations specific for L. monocytogenes, E. coli O157:H7 and S. enterica serotypes have been commercialized and approved for application in foods or as part of surface decontamination protocols. Phage endolysins have a broader host specificity compared to lytic bacteriophages. Cloned endolysins could be used as natural preservatives, singly or in combination with other antimicrobials such as bacteriocins.

Biomimetic surface coatings from modular amphiphilic proteins

NASA Astrophysics Data System (ADS)

Harden, James; Wan, Fan; Fischer, Stephen; Dick, Scott

2010-03-01

Recombinant DNA methods have been used to develop a library of diblock protein polymers for creating designer biofunctional interfaces. These proteins are composed of a surface-active, amphiphilic block joined to a disordered, water soluble block with an end terminal bioactive domain. The amphiphilic block has a strong affinity for many synthetic polymer surfaces, providing a facile means of imparting biological functionality to otherwise bio-neutral materials through physical self-assembly. We have incorporated a series of bioactive end domains into this diblock motif, including sequences that encode specific cell binding and signaling functions of extracellular matrix constituents (e.g. RGD and YIGSR). In this talk, we show that these diblock constructs self-assemble into biofunctional surface coatings on several model synthetic polymer materials. We demonstrate that surface adsorption of the proteins has minimal impacts on the presentation of the bioactive domains in the soluble block, and through the use of microscopic and cell proliferation assays, we show that the resulting biofunctional interfaces are capable of inducing appropriate cellular responses in a variety of human cell types.

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

PubMed

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

2016-04-01

In this study, which was carried out within the ChEsSO consortium project (Chemosynthetically driven ecosystems south of the Polar Front), we sampled two hydrothermal vent sites on the East Scotia Ridge, Scotia Sea, one in the Kemp Caldera, South Sandwich Arc and one in the Bransfield Strait, north-west of the Antarctic Peninsula, which exhibit strong differences in their chemical characteristics. We compared a subset of their bacteriophage population by Sanger- and 454-sequencing of g23, which codes for the major capsid protein of T4likeviruses. We found that the sites differ vastly in their bacteriophage diversity, which reflects the differences in the chemical conditions and therefore putatively the differences in microbial hosts living at these sites. Comparing phage diversity in the vent samples to other aquatic samples, the vent samples formed a distinct separate cluster, which also included the non-vent control samples that were taken several hundred meters above the vent chimneys. This indicates that the influence of the vents on the microbial population and therefore also the bacteriophage population extends much further than anticipated. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

Isolation, characterization, and application of bacteriophages for Salmonella spp. biocontrol in pigs.

PubMed

Albino, Luiz A A; Rostagno, Marcos H; Húngaro, Humberto M; Mendonça, Regina C S

2014-08-01

Foodborne illness due to Salmonella-contaminated pork products is an important public health problem, causing significant economic losses worldwide. The use of bacteriophages is a potential intervention tool that has attracted interest for the control of foodborne pathogens. The objective of this study was to detect the presence of Salmonella in commercial pig farms and to isolate specific autochthonous bacteriophages against Salmonella Typhimurium, to characterize them and to evaluate their lytic capacity against Salmonella Typhimurium in vivo and in vitro. Salmonella was isolated on 50% (4/8) of the farms, with serotype Typhimurium being the most prevalent, detected in 48.2% of samples (13/27). The isolated Salmonella Typhimurium bacteriophages belong to the Podoviridae family, were active against serotypes Abony, Enteritidis, Typhi, and Typhimurium, but not against serotypes Arizonae, Cholerasuis, Gallinarum, and Pullorum. In in vitro tests, bacteriophage at 10(7) PFU/mL and 10(9) PFU/mL significantly reduced (p<0.05) Salmonella Typhimurium counts in 1.6 and 2.5 log10 colony-forming units (CFU)/mL, respectively, after 24 h. Before the in vivo treatment with bacteriophages, Salmonella was identified in 93.3% (28/30) of the fecal samples from the pigs inoculated with 10(6) CFU/mL, and only in 56.6% (17/30) after the treatment consisting of oral administration of the pool of the bacteriophages after the fasting period, simulating a common preslaughter practice. These results indicate that the pool of bacteriophages administered was capable of reducing the colonization of Salmonella in pigs.

Significance of the Bacteriophage Treatment Schedule in Reducing Salmonella Colonization of Poultry

PubMed Central

Bardina, Carlota; Spricigo, Denis A.; Cortés, Pilar

2012-01-01

Salmonella remains the major cause of food-borne diseases worldwide, with chickens known to be the main reservoir for this zoonotic pathogen. Among the many approaches to reducing Salmonella colonization of broilers, bacteriophage offers several advantages. In this study, three bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) obtained from our collection that exhibited a broad host range against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium were characterized with respect to morphology, genome size, and restriction patterns. A cocktail composed of the three bacteriophages was more effective in promoting the lysis of S. Enteritidis and S. Typhimurium cultures than any of the three bacteriophages alone. In addition, the cocktail was able to lyse the Salmonella enterica serovars Virchow, Hadar, and Infantis. The effectiveness of the bacteriophage cocktail in reducing the concentration of S. Typhimurium was tested in two animal models using different treatment schedules. In the mouse model, 50% survival was obtained when the cocktail was administered simultaneously with bacterial infection and again at 6, 24, and 30 h postinfection. Likewise, in the White Leghorn chicken specific-pathogen-free (SPF) model, the best results, defined as a reduction of Salmonella concentration in the chicken cecum, were obtained when the bacteriophage cocktail was administered 1 day before or just after bacterial infection and then again on different days postinfection. Our results show that frequent treatment of the chickens with bacteriophage, and especially prior to colonization of the intestinal tract by Salmonella, is required to achieve effective bacterial reduction over time. PMID:22773654

Characterization of the endolysin from the Enterococcus faecalis bacteriophage VD13

USDA-ARS?s Scientific Manuscript database

Bacteriophage infecting bacteria produce endolysins (peptidoglycan hydrolases) to lyse the host cell from within and release nascent bacteriophage particles. Recombinant endolysins can also lyse Gram-positive bacteria when added exogenously. As a potential alternative to antibiotics, we cloned and...

Evolution of double-stranded DNA viruses of eukaryotes: from bacteriophages to transposons to giant viruses

PubMed Central

Koonin, Eugene V; Krupovic, Mart; Yutin, Natalya

2015-01-01

Diverse eukaryotes including animals and protists are hosts to a broad variety of viruses with double-stranded (ds) DNA genomes, from the largest known viruses, such as pandoraviruses and mimiviruses, to tiny polyomaviruses. Recent comparative genomic analyses have revealed many evolutionary connections between dsDNA viruses of eukaryotes, bacteriophages, transposable elements, and linear DNA plasmids. These findings provide an evolutionary scenario that derives several major groups of eukaryotic dsDNA viruses, including the proposed order “Megavirales,” adenoviruses, and virophages from a group of large virus-like transposons known as Polintons (Mavericks). The Polintons have been recently shown to encode two capsid proteins, suggesting that these elements lead a dual lifestyle with both a transposon and a viral phase and should perhaps more appropriately be named polintoviruses. Here, we describe the recently identified evolutionary relationships between bacteriophages of the family Tectiviridae, polintoviruses, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order “Megavirales,” and linear mitochondrial and cytoplasmic plasmids. We outline an evolutionary scenario under which the polintoviruses were the first group of eukaryotic dsDNA viruses that evolved from bacteriophages and became the ancestors of most large DNA viruses of eukaryotes and a variety of other selfish elements. Distinct lines of origin are detectable only for herpesviruses (from a different bacteriophage root) and polyoma/papillomaviruses (from single-stranded DNA viruses and ultimately from plasmids). Phylogenomic analysis of giant viruses provides compelling evidence of their independent origins from smaller members of the putative order “Megavirales,” refuting the speculations on the evolution of these viruses from an extinct fourth domain of cellular life. PMID:25727355

[Genetic study of bacteriophage phi81. I. Isolation, study of complementation and preliminary mapping of amber-mutants of bacteriophage phi81].

PubMed

Sineokiĭ, S P; Pogosov, V Z; Iankovskiĭ, N K; Krylov, V N

1976-01-01

123 Amber mutants of lambdoid bacteriophage phi81 are isolated and distributed into 19 complementation groups. Deletion mapping made possible to locate 5 gene groups on the genetic map of bacteriophage phi81 and to determine a region of possible location of mm' sticky ends on the prophage genetic map. A gene of phage phi81 is localized, which controls the adsorption specificity, and which functional similarity to a respective gene of phage phi80 is demonstrated.

Adsorption of bacteriophages on clay minerals

USGS Publications Warehouse

Chattopadhyay, Sandip; Puls, Robert W.

1999-01-01

The ability to predict the fate of microorganisms in soil is dependent on an understanding of the process of their sorption on soil and subsurface materials. Presently, we have focused on studying the thermodynamics of sorption of bacteriophages (T-2, MS-2, and φX-174) on clays (hectorite, saponite, kaolinite, and clay fraction of samples collected from a landfill site). The thermodynamic study not only determines the feasibility of the process but also provides information on the relative magnitudes of the different forces under a particular set of conditions. The total free energy of interaction during sorption of bacteriophages on clays (ΔG) has been assumed to be the summation of ΔGH (ΔG due to hydrophobic interactions) and ΔGEL (ΔG due to electrostatic interactions). The magnitude of ΔGH was determined from the different interfacial tensions (γ) present in the system, while ΔGEL was calculated from ζ-potentials of the colloidal particles. Calculated results show that surface hydrophobicities of the selected sorbents and sorbates dictate sorption. Among the selected bacteriophages, maximum sorption was observed with T-2, while hectorite has the maximum sorption capacity. Experimental results obtained from the batch adsorption studies also corroborated those obtained from the theoretical study.

Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

PubMed

Urban-Chmiel, Renata; Wernicki, Andrzej; Stęgierska, Diana; Dec, Marta; Dudzic, Anna; Puchalski, Andrzej

2015-01-01

The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves. The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR). Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101. The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages

[Determination of Azospirillum Brasilense Cells With Bacteriophages via Electrooptical Analysis of Microbial Suspensions].

PubMed

Gulii, O I; Karavayeva, O A; Pavlii, S A; Sokolov, O I; Bunin, V D; Ignatov, O V

2015-01-01

The dependence-of changes in the electrooptical properties of Azospirillum brasilense cell suspension Sp7 during interaction with bacteriophage ΦAb-Sp7 on the number and time of interactions was studied. Incubation of cells with bacteriophage significantly changed the electrooptical signal within one minute. The selective effect of bacteriophage ΦAb on 18 strains of bacteria of the genus Azospirillum was studied: A. amazonense Ami4, A. brasilense Sp7, Cd, Sp107, Sp245, Jm6B2, Brl4, KR77, S17, S27, SR55, SR75, A. halopraeferans Au4, A. irakense KBC1, K A3, A. lipoferum Sp59b, SR65 and RG20a. We determined the limit of reliable determination of microbial cells infected with bacteriophage: - 10(4) cells/mL. The presence of foreign cell cultures of E. coli B-878 and E. coli XL-1 did not complicate the detection of A brasilense Sp7 cells with the use of bacteriophage ΦAb-Sp7. The results demonstrated that bacteriophage (ΦAb-Sp7 can be used for the detection of Azospirillum microbial cells via t electrooptical analysis of cell suspensions.

Formulating food protein-stabilized indomethacin nanosuspensions into pellets by fluid-bed coating technology: physical characterization, redispersibility, and dissolution.

PubMed

He, Wei; Lu, Yi; Qi, Jianping; Chen, Lingyun; Yin, Lifang; Wu, Wei

2013-01-01

Drug nanosuspensions are very promising for enhancing the dissolution and bioavailability of drugs that are poorly soluble in water. However, the poor stability of nanosuspensions, reflected in particle growth, aggregation/agglomeration, and change in crystallinity state greatly limits their applications. Solidification of nanosuspensions is an ideal strategy for addressing this problem. Hence, the present work aimed to convert drug nanosuspensions into pellets using fluid-bed coating technology. Indomethacin nanosuspensions were prepared by the precipitation-ultrasonication method using food proteins (soybean protein isolate, whey protein isolate, β-lactoglobulin) as stabilizers. Dried nanosuspensions were prepared by coating the nanosuspensions onto pellets. The redispersibility, drug dissolution, solid-state forms, and morphology of the dried nanosuspensions were evaluated. The mean particle size for the nanosuspensions stabilized using soybean protein isolate, whey protein isolate, and β-lactoglobulin was 588 nm, 320 nm, and 243 nm, respectively. The nanosuspensions could be successfully layered onto pellets with high coating efficiency. Both the dried nanosuspensions and nanosuspensions in their original amorphous state and not influenced by the fluid-bed coating drying process could be redispersed in water, maintaining their original particle size and size distribution. Both the dried nanosuspensions and the original drug nanosuspensions showed similar dissolution profiles, which were both much faster than that of the raw crystals. Fluid-bed coating technology has potential for use in the solidification of drug nanosuspensions.

Formulating food protein-stabilized indomethacin nanosuspensions into pellets by fluid-bed coating technology: physical characterization, redispersibility, and dissolution

PubMed Central

He, Wei; Lu, Yi; Qi, Jianping; Chen, Lingyun; Yin, Lifang; Wu, Wei

2013-01-01

Background Drug nanosuspensions are very promising for enhancing the dissolution and bioavailability of drugs that are poorly soluble in water. However, the poor stability of nanosuspensions, reflected in particle growth, aggregation/agglomeration, and change in crystallinity state greatly limits their applications. Solidification of nanosuspensions is an ideal strategy for addressing this problem. Hence, the present work aimed to convert drug nanosuspensions into pellets using fluid-bed coating technology. Methods Indomethacin nanosuspensions were prepared by the precipitation-ultrasonication method using food proteins (soybean protein isolate, whey protein isolate, β-lactoglobulin) as stabilizers. Dried nanosuspensions were prepared by coating the nanosuspensions onto pellets. The redispersibility, drug dissolution, solid-state forms, and morphology of the dried nanosuspensions were evaluated. Results The mean particle size for the nanosuspensions stabilized using soybean protein isolate, whey protein isolate, and β-lactoglobulin was 588 nm, 320 nm, and 243 nm, respectively. The nanosuspensions could be successfully layered onto pellets with high coating efficiency. Both the dried nanosuspensions and nanosuspensions in their original amorphous state and not influenced by the fluid-bed coating drying process could be redispersed in water, maintaining their original particle size and size distribution. Both the dried nanosuspensions and the original drug nanosuspensions showed similar dissolution profiles, which were both much faster than that of the raw crystals. Conclusion Fluid-bed coating technology has potential for use in the solidification of drug nanosuspensions. PMID:23983465

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

PubMed Central

Dziewit, Lukasz; Oscik, Karolina; Bartosik, Dariusz

2014-01-01

ABSTRACT ΦLM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that ΦLM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of ΦLM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage ΦLM21 shows very limited similarity to other known phage genome sequences and is thus considered unique. IMPORTANCE Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, ΦLM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of

The Genome Sequence of Bacteriophage CPV1 Virulent for Clostridium perfringens

USDA-ARS?s Scientific Manuscript database

Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents. P...

M13 Bacteriophage-Based Self-Assembly Structures and Their Functional Capabilities.

PubMed

Moon, Jong-Sik; Kim, Won-Geun; Kim, Chuntae; Park, Geun-Tae; Heo, Jeong; Yoo, So Y; Oh, Jin-Woo

2015-06-01

Controlling the assembly of basic structural building blocks in a systematic and orderly fashion is an emerging issue in various areas of science and engineering such as physics, chemistry, material science, biological engineering, and electrical engineering. The self-assembly technique, among many other kinds of ordering techniques, has several unique advantages and the M13 bacteriophage can be utilized as part of this technique. The M13 bacteriophage (Phage) can easily be modified genetically and chemically to demonstrate specific functions. This allows for its use as a template to determine the homogeneous distribution and percolated network structures of inorganic nanostructures under ambient conditions. Inexpensive and environmentally friendly synthesis can be achieved by using the M13 bacteriophage as a novel functional building block. Here, we discuss recent advances in the application of M13 bacteriophage self-assembly structures and the future of this technology.

M13 Bacteriophage-Based Self-Assembly Structures and Their Functional Capabilities

PubMed Central

Moon, Jong-Sik; Kim, Won-Geun; Kim, Chuntae; Park, Geun-Tae; Heo, Jeong; Yoo, So Y; Oh, Jin-Woo

2015-01-01

Controlling the assembly of basic structural building blocks in a systematic and orderly fashion is an emerging issue in various areas of science and engineering such as physics, chemistry, material science, biological engineering, and electrical engineering. The self-assembly technique, among many other kinds of ordering techniques, has several unique advantages and the M13 bacteriophage can be utilized as part of this technique. The M13 bacteriophage (Phage) can easily be modified genetically and chemically to demonstrate specific functions. This allows for its use as a template to determine the homogeneous distribution and percolated network structures of inorganic nanostructures under ambient conditions. Inexpensive and environmentally friendly synthesis can be achieved by using the M13 bacteriophage as a novel functional building block. Here, we discuss recent advances in the application of M13 bacteriophage self-assembly structures and the future of this technology. PMID:26146494

Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels

PubMed Central

Scott, Evan A.; Nichols, Michael D.; Cordova, Lee H.; George, Brandon J.; Jun, Young-Shin; Elbert, Donald L.

2008-01-01

Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically-active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with <≈100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating. PMID:18771802

Comparative genomic analysis of bacteriophages specific to the channel catfish pathogen Edwardsiella ictaluri

PubMed Central

2011-01-01

Background The bacterial pathogen Edwardsiella ictaluri is a primary cause of mortality in channel catfish raised commercially in aquaculture farms. Additional treatment and diagnostic regimes are needed for this enteric pathogen, motivating the discovery and characterization of bacteriophages specific to E. ictaluri. Results The genomes of three Edwardsiella ictaluri-specific bacteriophages isolated from geographically distant aquaculture ponds, at different times, were sequenced and analyzed. The genomes for phages eiAU, eiDWF, and eiMSLS are 42.80 kbp, 42.12 kbp, and 42.69 kbp, respectively, and are greater than 95% identical to each other at the nucleotide level. Nucleotide differences were mostly observed in non-coding regions and in structural proteins, with significant variability in the sequences of putative tail fiber proteins. The genome organization of these phages exhibit a pattern shared by other Siphoviridae. Conclusions These E. ictaluri-specific phage genomes reveal considerable conservation of genomic architecture and sequence identity, even with considerable temporal and spatial divergence in their isolation. Their genomic homogeneity is similarly observed among E. ictaluri bacterial isolates. The genomic analysis of these phages supports the conclusion that these are virulent phages, lacking the capacity for lysogeny or expression of virulence genes. This study contributes to our knowledge of phage genomic diversity and facilitates studies on the diagnostic and therapeutic applications of these phages. PMID:21214923

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

Application of bacteriophages to reduce Salmonella contamination on workers' boots in rendering-processing environment.

PubMed

Gong, C; Jiang, X; Wang, J

2017-10-01

Workers' boots are considered one of the re-contamination routes of Salmonella for rendered meals in the rendering-processing environment. This study was conducted to evaluate the efficacy of a bacteriophage cocktail for reducing Salmonella on workers' boots and ultimately for preventing Salmonella re-contamination of rendered meals. Under laboratory conditions, biofilms of Salmonella Typhimurium avirulent strain 8243 formed on rubber templates or boots were treated with a bacteriophage cocktail of 6 strains (ca. 9 log PFU/mL) for 6 h at room temperature. Bacteriophage treatments combined with sodium hypochlorite (400 ppm) or 30-second brush scrubbing also were investigated for a synergistic effect on reducing Salmonella biofilms. Sodium magnesium (SM) buffer and sodium hypochlorite (400 ppm) were used as controls. To reduce indigenous Salmonella on workers' boots, a field study was conducted to apply a bacteriophage cocktail and other combined treatments 3 times within one wk in a rendering-processing environment. Prior to and after bacteriophage treatments, Salmonella populations on the soles of rubber boots were swabbed and enumerated on XLT-4, Miller-Mallinson or CHROMagar™ plates. Under laboratory conditions, Salmonella biofilms formed on rubber templates and boots were reduced by 95.1 to 99.999% and 91.5 to 99.2%, respectively. In a rendering-processing environment (ave. temperature: 19.3°C; ave. relative humidity: 48%), indigenous Salmonella populations on workers' boots were reduced by 84.2, 92.9, and 93.2% after being treated with bacteriophages alone, bacteriophages + sodium hypochlorite, and bacteriophages + scrubbing for one wk, respectively. Our results demonstrated the effectiveness of bacteriophage treatments in reducing Salmonella contamination on the boots in both laboratory and the rendering-processing environment. © 2017 Poultry Science Association Inc.

Bacteriophage ecology in environmental biotechnology processes.

PubMed

Shapiro, Orr H; Kushmaro, Ariel

2011-06-01