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Sample records for integrating phage vhml

  1. Replication and Maintenance of Linear Phage-Plasmid N15.

    PubMed

    Ravin, Nikolai V

    2015-02-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into the chromosome but is a linear plasmid molecule with covalently closed ends (telomeres). Upon infection, the phage DNA circularizes via cohesive ends, and then a special phage enzyme of the tyrosine recombinase family, protelomerase, cuts at another site and joins the ends, forming hairpin telomeres of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally, resulting in the formation of duplicated telomeres. The N15 protelomerase cuts them, generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by a partitioning operon similar to the F factor sop operon. Unlike the F centromere, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in the N15 genome regions involved in phage replication and control of lytic development, and binding of partition proteins at these sites regulates these processes. The family of N15-like linear phage-plasmids includes lambdoid phages ɸKO2 and pY54, as well as Myoviridae phages ΦHAP-1, VHML, VP882, Vp58.5, and vB_VpaM_MAR of marine gamma-proteobacteria. The genomes of these phages contain similar protelomerase genes, lysogeny control modules, and replication genes, suggesting that these phages may belong to a group diverged from a common ancestor.

  2. Genome, integration, and transduction of a novel temperate phage of Helicobacter pylori.

    PubMed

    Luo, Cheng-Hung; Chiou, Pei-Yu; Yang, Chiou-Ying; Lin, Nien-Tsung

    2012-08-01

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

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

    PubMed Central

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

    2012-01-01

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

  4. Analysis of the site-specific integration system of the Streptomyces aureofaciens phage μ1/6.

    PubMed

    Farkašovská, Jarmila; Godány, Andrej

    2012-03-01

    The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

  5. A proposed integrated approach for the preclinical evaluation of phage therapy in Pseudomonas infections.

    PubMed

    Danis-Wlodarczyk, Katarzyna; Vandenheuvel, Dieter; Jang, Ho Bin; Briers, Yves; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Drabik, Marcin; Higgins, Gerard; Tyrrell, Jean; Harvey, Brian J; Noben, Jean-Paul; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2016-01-01

    Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications. PMID:27301427

  6. A proposed integrated approach for the preclinical evaluation of phage therapy in Pseudomonas infections

    NASA Astrophysics Data System (ADS)

    Danis-Wlodarczyk, Katarzyna; Vandenheuvel, Dieter; Jang, Ho Bin; Briers, Yves; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Drabik, Marcin; Higgins, Gerard; Tyrrell, Jean; Harvey, Brian J.; Noben, Jean-Paul; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2016-06-01

    Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications.

  7. A proposed integrated approach for the preclinical evaluation of phage therapy in Pseudomonas infections

    PubMed Central

    Danis-Wlodarczyk, Katarzyna; Vandenheuvel, Dieter; Jang, Ho Bin; Briers, Yves; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Drabik, Marcin; Higgins, Gerard; Tyrrell, Jean; Harvey, Brian J.; Noben, Jean-Paul; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2016-01-01

    Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications. PMID:27301427

  8. Phage Transduction.

    PubMed

    Goh, Shan

    2016-01-01

    Bacteriophages mediate horizontal gene transfer through a mechanism known as transduction. Phage transduction carried out in the laboratory involves a bacterial donor and a recipient, both of which are susceptible to infection by the phage of interest. Phage is propagated in the donor, concentrated, and exposed transiently to recipient at different multiplicity of infection ratios. Transductants are selected for the desired phenotype by culture on selective medium. Here we describe transduction of ermB conferring resistance to erythromycin by the C. difficile phage ϕC2. PMID:27507341

  9. Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome.

    PubMed

    Halmillawewa, Anupama P; Restrepo-Córdoba, Marcela; Perry, Benjamin J; Yost, Christopher K; Hynes, Michael F

    2016-02-01

    Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506 bp in length with an average G+C content of 61.9 %. The genome contains 94 putative open reading frames (ORFs) and 74.5 % of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5 % (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available. PMID:26377943

  10. The SopEPhi phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage.

    PubMed

    Pelludat, Cosima; Mirold, Susanne; Hardt, Wolf-Dietrich

    2003-09-01

    Salmonella spp. are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopEPhi may represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we have analyzed in more detail SopEPhi, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE. We have cloned and characterized the attachment site (att) of SopEPhi and found that its 47-bp core sequence overlaps the 3' terminus of the ssrA gene of serovar Typhimurium. Furthermore, we have demonstrated integration of SopEPhi into the cloned attB site of serovar Typhimurium A36. Sequence analysis of the plasmid-borne prophage revealed that SopEPhi is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome. Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp.

  11. Identification of multiple integration sites for Stx-phage Phi24B in the Escherichia coli genome, description of a novel integrase and evidence for a functional anti-repressor.

    PubMed

    Fogg, Paul C M; Gossage, Sharon M; Smith, Darren L; Saunders, Jon R; McCarthy, Alan J; Allison, Heather E

    2007-12-01

    The key virulence factor in Shiga-toxigenic Escherichia coli is the expression of Shiga toxin (Stx), which is conferred by Stx-encoding temperate lambdoid phages (Stx-phages). It had been assumed that Stx-phages would behave similarly to lambda phage. However, contrary to the lambda superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage Phi24(B). Here, the Phi24(B) integrase gene is identified, and the preferred site of integration defined. Although an E. coli int gene was identified close to the Phi24(B) integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the E. coli genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to E. coli and are therefore unlikely to support phage integration. A Phi24(B) gene, possessing similarity to the well-characterized P22 ant gene, was identified. RT-PCR was used to demonstrate that ant is transcribed in a Phi24(B) E. coli lysogen, and expression of an anti-repressor is the likely explanation for the absence of immunity to superinfection. Demonstration of the ability of Phi24(B) to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the stx gene may lead to an increase in toxin production and consequently increased virulence.

  12. Bacteriophage prevalence in the genus Azospirillum and analysis of the first genome sequence of an Azospirillum brasilense integrative phage.

    PubMed

    Boyer, Mickaël; Haurat, Jacqueline; Samain, Sylvie; Segurens, Béatrice; Gavory, Frédérick; González, Víctor; Mavingui, Patrick; Rohr, René; Bally, René; Wisniewski-Dyé, Florence

    2008-02-01

    The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (phiAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the phiAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of phiAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.

  13. Phage therapy pharmacology: calculating phage dosing.

    PubMed

    Abedon, Stephen

    2011-01-01

    Phage therapy, which can be described as a phage-mediated biocontrol of bacteria (or, simply, biocontrol), is the application of bacterial viruses-also bacteriophages or phages-to reduce densities of nuisance or pathogenic bacteria. Predictive calculations for phage therapy dosing should be useful toward rational development of therapeutic as well as biocontrol products. Here, I consider the theoretical basis of a number of concepts relevant to phage dosing for phage therapy including minimum inhibitory concentration (but also "inundation threshold"), minimum bactericidal concentration (but also "clearance threshold"), decimal reduction time (D value), time until bacterial eradication, threshold bacterial density necessary to support phage population growth ("proliferation threshold"), and bacterial density supporting half-maximal phage population growth rates (K(B)). I also address the concepts of phage killing titers, multiplicity of infection, and phage peak densities. Though many of the presented ideas are not unique to this chapter, I nonetheless provide variations on derivations and resulting formulae, plus as appropriate discuss relative importance. The overriding goal is to present a variety of calculations that are useful toward phage therapy dosing so that they may be found in one location and presented in a manner that allows facile appreciation, comparison, and implementation. The importance of phage density as a key determinant of the phage potential to eradicate bacterial targets is stressed throughout the chapter. PMID:22050820

  14. Engineered phages for electronics.

    PubMed

    Cui, Yue

    2016-11-15

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

  15. N15: the linear phage-plasmid.

    PubMed

    Ravin, Nikolai V

    2011-03-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.

  16. Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by Streptomyces phage phiC31 integrase.

    PubMed

    Bi, Yanzhen; Liu, Ximei; Zhang, Long; Shao, Changwei; Ma, Zhuo; Hua, Zaidong; Zhang, Liping; Li, Li; Hua, Wenjun; Xiao, Hongwei; Wei, Qingxin; Zheng, Xinmin

    2013-09-08

    Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5' and 3' junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position

  17. Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites.

    PubMed Central

    Raya, R R; Fremaux, C; De Antoni, G L; Klaenhammer, T R

    1992-01-01

    The temperate bacteriophage phi adh integrates its genome into the chromosomal DNA of Lactobacillus gasseri ADH by a site-specific recombination process. Southern hybridization analysis of BclI-digested genomic DNA from six relysogenized derivatives of the prophage-cured strain NCK102 displayed phage-chromosomal junction fragments identical to those of the lysogenic parent. The phi adh attachment site sequence, attP, was located within a 365-bp EcoRI-HindIII fragment of phage phi adh. This fragment was cloned and sequenced. DNA sequence analysis revealed striking features common to the attachment sites of other site-specific recombination systems: five direct repeats of the sequence TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived from the sequence of the attP-containing fragment enabled us to amplify predicted junction fragment sequences and thus to identify attL, attR, and attB. The core region was defined as the 16-bp sequence TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific insertion of phage phi adh were located in a 4.5-kb BclI fragment. This fragment was cloned in plasmid pSA34 to generate the insertional vector pTRK182. Plasmid pTRK182 was introduced into L. gasseri NCK102 by electroporation. Hybridization analysis showed that a single copy of pTRK182 had integrated at the attB site of the NCK102 erythromycin-resistant transformants. This is the first site-specific recombination system described in lactobacilli, as well as the first attP-based site-specific integration vector constructed for L. gasseri ADH. Images PMID:1512192

  18. Estimating richness from phage metagenomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages are important drivers of ecosystem functions, yet little is known about the vast majority of phages. Phage metagenomics, or the study of the collective genome of an assemblage of phages, enables the investigation of broad ecological questions in phage communities. One ecological cha...

  19. Phage treatment of human infections

    PubMed Central

    Abedon, Stephen T; Kuhl, Sarah J; Blasdel, Bob G

    2011-01-01

    Phages as bactericidal agents have been employed for 90 years as a means of treating bacterial infections in humans as well as other species, a process known as phage therapy. In this review we explore both the early historical and more modern use of phages to treat human infections. We discuss in particular the little-reviewed French early work, along with the Polish, US, Georgian and Russian historical experiences. We also cover other, more modern examples of phage therapy of humans as differentiated in terms of disease. In addition, we provide discussions of phage safety, other aspects of phage therapy pharmacology, and the idea of phage use as probiotics. PMID:22334863

  20. Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

    PubMed Central

    Davies, Emily V.; James, Chloe E.; Williams, David; O’Brien, Siobhan; Fothergill, Joanne L.; Haldenby, Sam; Paterson, Steve; Winstanley, Craig

    2016-01-01

    Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote within-host evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as free-floating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts. PMID:27382184

  1. Phage on the stage

    PubMed Central

    Temple, Louise; Lewis, Lynn

    2015-01-01

    The resurgence of interest in bacteriophages for use in combating antibiotic resistant bacteria is coincident with an urgent call for more effective science education practices, including hands-on learning opportunities. To address this issue, a number of solutions have been proposed, including a large educational experiment, begun in 2007 by the Howard Hughes Medical Institute and currently involving over 85 colleges and universities, which has students discovering unique phages, obtaining images, and purifying phage DNA. A subset of these phage genomes is sequenced and analyzed using bioinformatics tools. Papers describing individual phage discoveries and comparative genomic studies are being published regularly. The vast majority of students in the program are in their first year of college, a critical time in capturing their interest and retaining them as science majors. This viral discovery model is being adopted and modified by a wide variety of educational institutions using a number of different bacterial hosts. In the opinion of the authors, this program and others like it represent a model accessible to virtually any undergraduate setting. And because of these programs, bacteriophage enthusiasts (academics, health professionals, biotechnology companies) can look forward to more well prepared students entering their ranks and should anticipate many more potentially useful phages discovered and characterized. PMID:26442195

  2. Transposable Phage Mu.

    PubMed

    Harshey, Rasika M

    2014-10-01

    Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a detailed biochemical dissection of the reaction mechanism, as well as of protein and DNA elements that regulate transpososome assembly and function. The deduced phosphotransfer mechanism involves in-line orientation of metal ion-activated hydroxyl groups for nucleophilic attack on reactive diester bonds, a mechanism that appears to be used by all transposable elements examined to date. A crystal structure of the Mu transpososome is available. Mu differs from all other transposable elements in encoding unique adaptations that promote its viral lifestyle. These adaptations include multiple DNA (enhancer, SGS) and protein (MuB, HU, IHF) elements that enable efficient Mu end synapsis, efficient target capture, low target specificity, immunity to transposition near or into itself, and efficient mechanisms for recruiting host repair and replication machineries to resolve transposition intermediates. MuB has multiple functions, including target capture and immunity. The SGS element promotes gyrase-mediated Mu end synapsis, and the enhancer, aided by HU and IHF, participates in directing a unique topological architecture of the Mu synapse. The function of these DNA and protein elements is important during both lysogenic and lytic phases. Enhancer properties have been exploited in the design of mini-Mu vectors for genetic engineering. Mu ends assembled into active transpososomes have been delivered directly into bacterial, yeast, and human genomes, where they integrate efficiently, and may prove useful for gene therapy. PMID:26104374

  3. Satellite phage TLCφ enables toxigenic conversion by CTX phage through dif site alteration.

    PubMed

    Hassan, Faizule; Kamruzzaman, M; Mekalanos, John J; Faruque, Shah M

    2010-10-21

    Bacterial chromosomes often carry integrated genetic elements (for example plasmids, transposons, prophages and islands) whose precise function and contribution to the evolutionary fitness of the host bacterium are unknown. The CTXφ prophage, which encodes cholera toxin in Vibrio cholerae, is known to be adjacent to a chromosomally integrated element of unknown function termed the toxin-linked cryptic (TLC). Here we report the characterization of a TLC-related element that corresponds to the genome of a satellite filamentous phage (TLC-Knφ1), which uses the morphogenesis genes of another filamentous phage (fs2φ) to form infectious TLC-Knφ1 phage particles. The TLC-Knφ1 phage genome carries a sequence similar to the dif recombination sequence, which functions in chromosome dimer resolution using XerC and XerD recombinases. The dif sequence is also exploited by lysogenic filamentous phages (for example CTXφ) for chromosomal integration of their genomes. Bacterial cells defective in the dimer resolution often show an aberrant filamentous cell morphology. We found that acquisition and chromosomal integration of the TLC-Knφ1 genome restored a perfect dif site and normal morphology to V. cholerae wild-type and mutant strains with dif(-) filamentation phenotypes. Furthermore, lysogeny of a dif(-) non-toxigenic V. cholerae with TLC-Knφ1 promoted its subsequent toxigenic conversion through integration of CTXφ into the restored dif site. These results reveal a remarkable level of cooperative interactions between multiple filamentous phages in the emergence of the bacterial pathogen that causes cholera. PMID:20944629

  4. The first phage electron micrographs

    PubMed Central

    Ackermann, Hans-W.

    2011-01-01

    The first phage electron micrographs were published in 1940 in Germany and proved the particulate nature of bacteriophages. Phages and infected bacteria were first examined raw and unstained. US American scientists introduced shadowing and freeze-drying. Phages appeared to be tailed and morphologically heterogeneous. Phage types identified by early electron microscopy include enterobacteriophages T4, T1, T7, T5, 7–11, ViI and Pseudomonas phage PB1. This paper retraces the development of early virus electron microscopy till the introduction of negative staining. PMID:23050215

  5. Generalized transduction Of shigella flexneri by converting phage PE5.

    PubMed

    Financsek, I; Kétyi, I

    1976-01-01

    Phage PE5, responsible for the conversion of type V antigen in Shigella flexneri, has the ability to produce generalized transduction. The correlation between phage multiplicity and the number of transductants, the specific inhibitory activity of anti-PE5 serum, and the lack of transduction in PE5 resistant recipients, indicate the role of phage PE5 in generalized transduction. Transduction of the R100-1 factor resulted in a non-transmissible tetracycline resistance segragation. The characteristics of the tetracycline resistance determinant suggest the possibility of integration.

  6. Phage fitness may help predict phage therapy efficacy

    PubMed Central

    Lindberg, Heather M; McKean, Kurt A; Wang, Ing-Nang

    2014-01-01

    We isolated 6 phages from 2 environmental water sources and assessed their ability to treat Pseudomonas aeruginosa infection of Drosophila melanogaster. We found all 6 phages were able to significantly increase mean survival time (MST) of infected D. melanogaster. Although phage traits, such as adsorption rate, burst size, and lysis time, varied significantly among these phages, none of the traits correlated significantly with MST. Phage growth rate determined in vitro, however, was found to be significantly correlated with MST. Overall, our study shows that infected D. melanogaster can be used as a model system to test the therapeutic efficacy of phages. In addition, a more comprehensive characteristic, like the in vitro growth rate, seems to be a better indicator in predicting therapeutic success than constituent traits like the adsorption rate, burst size, or lysis time. PMID:26713221

  7. Drugs derived from phage display

    PubMed Central

    Nixon, Andrew E; Sexton, Daniel J; Ladner, Robert C

    2014-01-01

    Phage display, one of today’s fundamental drug discovery technologies, allows identification of a broad range of biological drugs, including peptides, antibodies and other proteins, with the ability to tailor critical characteristics such as potency, specificity and cross-species binding. Further, unlike in vivo technologies, generating phage display-derived antibodies is not restricted by immunological tolerance. Although more than 20 phage display-derived antibody and peptides are currently in late-stage clinical trials or approved, there is little literature addressing the specific challenges and successes in the clinical development of phage-derived drugs. This review uses case studies, from candidate identification through clinical development, to illustrate the utility of phage display as a drug discovery tool, and offers a perspective for future developments of phage display technology. PMID:24262785

  8. Inactivation of Burkholderia cepacia Complex Phage KS9 gp41 Identifies the Phage Repressor and Generates Lytic Virions▿ †

    PubMed Central

    Lynch, Karlene H.; Seed, Kimberley D.; Stothard, Paul; Dennis, Jonathan J.

    2010-01-01

    The Burkholderia cepacia complex (BCC) is made up of at least 17 species of Gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3′ end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage φE125 and Burkholderia pseudomallei phage φ1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo. PMID:19939932

  9. Characterization of novel phages isolated in coagulase-negative staphylococci reveals evolutionary relationships with Staphylococcus aureus phages.

    PubMed

    Deghorain, Marie; Bobay, Louis-Marie; Smeesters, Pierre R; Bousbata, Sabrina; Vermeersch, Marjorie; Perez-Morga, David; Drèze, Pierre-Alexandre; Rocha, Eduardo P C; Touchon, Marie; Van Melderen, Laurence

    2012-11-01

    Despite increasing interest in coagulase-negative staphylococci (CoNS), little information is available about their bacteriophages. We isolated and sequenced three novel temperate Siphoviridae phages (StB12, StB27, and StB20) from the CoNS Staphylococcus hominis and S. capitis species. The genome sizes are around 40 kb, and open reading frames (ORFs) are arranged in functional modules encoding lysogeny, DNA metabolism, morphology, and cell lysis. Bioinformatics analysis allowed us to assign a potential function to half of the predicted proteins. Structural elements were further identified by proteomic analysis of phage particles, and DNA-packaging mechanisms were determined. Interestingly, the three phages show identical integration sites within their host genomes. In addition to this experimental characterization, we propose a novel classification based on the analysis of 85 phage and prophage genomes, including 15 originating from CoNS. Our analysis established 9 distinct clusters and revealed close relationships between S. aureus and CoNS phages. Genes involved in DNA metabolism and lysis and potentially in phage-host interaction appear to be widespread, while structural genes tend to be cluster specific. Our findings support the notion of a possible reciprocal exchange of genes between phages originating from S. aureus and CoNS, which may be of crucial importance for pathogenesis in staphylococci.

  10. 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

  11. Highly efficient integration of the viral portal proteins from different types of phages into planar bilayers for the black lipid membrane analysis.

    PubMed

    Jing, Peng; Paraiso, Hallel; Burris, Benjamin

    2016-02-01

    The planar lipid bilayer technology is a technique that yields incredibly useful structural function information about a single channel protein. It is also currently actively utilized as a powerful platform using biological protein nanopores for the development of single-molecule nanopore sensing technology, as well as ultrafast DNA sequencing technology. The portal protein, GP10, from the bacteriophage Φ29 was the first phage portal protein shown to be successfully inserted into planar bilayer membranes, thereby it may inspire more researchers to apply the techniques to portal proteins from the other bacteriophages. However, the technology is far from perfect since the insertion of the channel proteins into planar bilayer membranes is not only technically difficult but also time-consuming. For the fusion of phage portal proteins, vesicles are typically needed to be reconstituted with the portal proteins to form proteoliposomes. However, most of the phage portal proteins have low solubility, and may self-aggregate during the preparation of the proteoliposomes. Furthermore, the fusion of the formed proteoliposomes is sporadic, unpredictable and varied from person to person. Due to the lack of experimental consistency between labs, the results from different methodologies reported for generating fusible proteoliposomes are highly variable. In this research, we propose a new method for the preparation of the fusible proteoliposomes containing portal proteins from bacteriophages, to circumvent the problems aforementioned. Compared to the conventional methods, this method was able to avoid the protein aggregation issues during the vesicle preparation by eliminating the need for detergents and the subsequent time-consuming step for detergent removal. The proteoliposomes prepared by the method were shown to be more efficiently and rapidly inserted into planar bilayer membranes bathed in different conducting buffer solutions including those with nonelectrolytes such as

  12. Clostridium difficile phages: still difficult?

    PubMed Central

    Hargreaves, Katherine R.; Clokie, Martha R. J.

    2014-01-01

    Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however, developing suitable phages is challenging. In this review we summarize the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics. Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage–host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution. No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using “whole-phages” are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem-free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen. PMID:24808893

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

    PubMed Central

    Goerke, Christiane; Köller, Johanna; Wolz, Christiane

    2006-01-01

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

  14. Ecogenomics and genome landscapes of marine Pseudoalteromonas phage H105/1.

    PubMed

    Duhaime, Melissa Beth; Wichels, Antje; Waldmann, Jost; Teeling, Hanno; Glöckner, Frank Oliver

    2011-01-01

    Marine phages have an astounding global abundance and ecological impact. However, little knowledge is derived from phage genomes, as most of the open reading frames in their small genomes are unknown, novel proteins. To infer potential functional and ecological relevance of sequenced marine Pseudoalteromonas phage H105/1, two strategies were used. First, similarity searches were extended to include six viral and bacterial metagenomes paired with their respective environmental contextual data. This approach revealed 'ecogenomic' patterns of Pseudoalteromonas phage H105/1, such as its estuarine origin. Second, intrinsic genome signatures (phylogenetic, codon adaptation and tetranucleotide (tetra) frequencies) were evaluated on a resolved intra-genomic level to shed light on the evolution of phage functional modules. On the basis of differential codon adaptation of Phage H105/1 proteins to the sequenced Pseudoalteromonas spp., regions of the phage genome with the most 'host'-adapted proteins also have the strongest bacterial tetra signature, whereas the least 'host'-adapted proteins have the strongest phage tetra signature. Such a pattern may reflect the evolutionary history of the respective phage proteins and functional modules. Finally, analysis of the structural proteome identified seven proteins that make up the mature virion, four of which were previously unknown. This integrated approach combines both novel and classical strategies and serves as a model to elucidate ecological inferences and evolutionary relationships from phage genomes that typically abound with unknown gene content.

  15. Phage and Yeast Display.

    PubMed

    Sheehan, Jared; Marasco, Wayne A

    2015-02-01

    Despite the availability of antimicrobial drugs, the continued development of microbial resistance--established through escape mutations and the emergence of resistant strains--limits their clinical utility. The discovery of novel, therapeutic, monoclonal antibodies (mAbs) offers viable clinical alternatives in the treatment and prophylaxis of infectious diseases. Human mAb-based therapies are typically nontoxic in patients and demonstrate high specificity for the intended microbial target. This specificity prevents negative impacts on the patient microbiome and avoids driving the resistance of nontarget species. The in vitro selection of human antibody fragment libraries displayed on phage or yeast surfaces represents a group of well-established technologies capable of generating human mAbs. The advantage of these forms of microbial display is the large repertoire of human antibody fragments present during a single selection campaign. Furthermore, the in vitro selection environments of microbial surface display allow for the rapid isolation of antibodies--and their encoding genes--against infectious pathogens and their toxins that are impractical within in vivo systems, such as murine hybridomas. This article focuses on the technologies of phage display and yeast display, as these strategies relate to the discovery of human mAbs for the treatment and vaccine development of infectious diseases. PMID:26104550

  16. Phage therapy of pulmonary infections

    PubMed Central

    Abedon, Stephen T

    2015-01-01

    It is generally agreed that a bacteriophage-associated phenomenon was first unambiguously observed one-hundred years ago with the findings of Twort in 1915. This was independently followed by complementary observations by d'Hérelle in 1917. D'Hérelle's appreciation of the bacteriophage phenomenon appears to have directly led to the development of phages as antibacterial agents within a variety of contexts, including medical and agricultural. Phage use to combat nuisance bacteria appears to be especially useful where targets are sufficiently problematic, suitably bactericidal phages exist, and alternative approaches are lacking in effectiveness, availability, safety, or cost effectiveness, etc. Phage development as antibacterial agents has been strongest particularly when antibiotics have been less available or useful, e.g., such as in the treatment of chronic infections by antibiotic-resistant bacteria. One relatively under-explored or at least not highly reported use of phages as therapeutic agents has been to combat bacterial infections of the lungs and associated tissues. These infections are diverse in terms of their etiologies, manifestations, and also in terms of potential strategies of phage delivery. Here I review the literature considering the phage therapy of pulmonary and pulmonary-related infections, with emphasis on reports of clinical treatment along with experimental treatment of pulmonary infections using animal models. PMID:26442188

  17. Bacteria-phage interactions in natural environments.

    PubMed

    Díaz-Muñoz, Samuel L; Koskella, Britt

    2014-01-01

    Phages are considered the most abundant and diverse biological entities on Earth and are notable not only for their sheer abundance, but also for their influence on bacterial hosts. In nature, bacteria-phage relationships are complex and have far-reaching consequences beyond particular pairwise interactions, influencing everything from bacterial virulence to eukaryotic fitness to the carbon cycle. In this review, we examine bacteria and phage distributions in nature first by highlighting biogeographic patterns and nonhost environmental influences on phage distribution, then by considering the ways in which phages and bacteria interact, emphasizing phage life cycles, bacterial responses to phage infection, and the complex patterns of phage host specificity. Finally, we discuss phage impacts on bacterial abundance, genetics, and physiology, and further aim to clarify distinctions between current theoretical models and point out areas in need of future research. PMID:25131402

  18. The present position in Brucella phage research

    PubMed Central

    Droževkina, M. S.

    1963-01-01

    It is only comparatively recently that efforts to isolate and purify stable Brucella phages have met with success. This success has important implications for the epidemiology and possibly the control of brucellosis, and the author has therefore thought it opportune to summarize and review the present state of research on Brucella phages. After giving an outline of the history of Brucella phage isolation, she goes into the methodology of phage isolation from numerous sources, of phage reinforcement, and of determination of the lytic activity of phage. This is followed by a discussion of the biological properties, morphology and typing of Brucella phage, and of lysogeny in Brucella. Finally, she reviews the promising research that is being done on Brucella typing with specific phage and on the use of antiphage serum in diagnosis, and discusses the possible use of phage in the treatment and prevention of brucellosis. PMID:14043752

  19. Isolation and development of bioluminescent reporter phages for bacterial dysentery.

    PubMed

    Schofield, D A; Wray, D J; Molineux, I J

    2015-02-01

    Shigellosis is a significant cause of morbidity and mortality worldwide, most notably amongst children. Moreover, there is a global increase in the occurrence of multidrug-resistant isolates, including the epidemic and pandemic Shigella dysenteriae type 1 strain. We developed a bioluminescent reporter phage assay to facilitate detection and simultaneously determine antibiotic susceptibility. A Shigella flexneri phage (Shfl25875) was isolated from environmental wastewater and characterized by DNA sequencing. Shfl25875 is T4-like, harbors a 169,062-bp genome, and grows on most (28/29) S. flexneri strains and all 12 S. dysenteriae type 1 strains tested. The genes encoding bacterial luciferase were integrated into the Shfl25875 genome to create a "light-tagged" phage capable of transducing a bioluminescent phenotype to infected cells. Shfl25875::luxAB rapidly detects cultured isolates with high sensitivity. Specificity experiments indicate that the reporter does not respond to Shigella boydii, non-type 1 S. dysenteriae strains, and most non-Shigella Enterobacteriaceae. Shfl25875::luxAB generates ampicillin and ciprofloxacin susceptibility profiles that are similar to the standard Clinical and Laboratory Standards Institute (CLSI) growth microdilution method, but in a significantly shorter time. In addition, the reporter phage detects Shigella in mock-infected stool. This new reporter phage shows promise as a tool for the detection of cultured isolates or complex clinical samples.

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

    PubMed Central

    Cao, Binrui; Yang, Mingying; Mao, Chuanbin

    2016-01-01

    CONSPECTUS Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (~900 nm long and ~8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic–inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their

  1. Membrane fusion during phage lysis

    PubMed Central

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

    2015-01-01

    In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG. PMID:25870259

  2. Phage Specificity of the Freshwater Fish Pathogen Flavobacterium columnare▿

    PubMed Central

    Laanto, Elina; Sundberg, Lotta-Riina; Bamford, Jaana K. H.

    2011-01-01

    Flavobacteria and their phages were isolated from Finnish freshwaters and fish farms. Emphasis was placed on finding phages infecting the fish pathogen Flavobacterium columnare for use as phage therapy agents. The host ranges of the flavobacterial phages varied, phages infecting F. columnare being more host specific than the other phages. PMID:21890667

  3. Complete Genome Sequence of Vibrio anguillarum Phage CHOED Successfully Used for Phage Therapy in Aquaculture

    PubMed Central

    Higuera, Gastón; Gajardo, Felipe; Castillo, Daniel; Middleboe, Mathias; García, Katherine; Ramírez, Carolina; Espejo, Romilio T.

    2014-01-01

    Vibrio anguillarum phage CHOED was isolated from Chilean mussels. It is a virulent phage showing effective inhibition of V. anguillarum. CHOED has potential in phage therapy, because it can protect fish from vibriosis in fish farms. Here, we announce the completely sequenced genome of V. anguillarum phage CHOED. PMID:25013148

  4. Phage Community Dynamics in Hot Springs

    PubMed Central

    Breitbart, Mya; Wegley, Linda; Leeds, Steven; Schoenfeld, Tom; Rohwer, Forest

    2004-01-01

    In extreme thermal environments such as hot springs, phages are the only known microbial predators. Here we present the first study of prokaryotic and phage community dynamics in these environments. Phages were abundant in hot springs, reaching concentrations of a million viruses per milliliter. Hot spring phage particles were resistant to shifts to lower temperatures, possibly facilitating DNA transfer out of these extreme environments. The phages were actively produced, with a population turnover time of 1 to 2 days. Phage-mediated microbial mortality was significant, making phage lysis an important component of hot spring microbial food webs. Together, these results show that phages exert an important influence on microbial community structure and energy flow in extreme thermal environments. PMID:15006788

  5. Insertion of host DNA into PVL-encoding phages of the Staphylococcus aureus lineage ST80 by intra-chromosomal recombination.

    PubMed

    Wirtz, Christiane; Witte, Wolfgang; Wolz, Christiane; Goerke, Christiane

    2010-10-25

    Temperate bacteriophages play a critical role in the pathogenicity of the human pathogen Staphylococcus aureus by mediating positive lysogenic conversion for different virulence factors such as Panton-Valentine leukocidin (PVL) or by interrupting chromosomal virulence genes. PVL-encoding phages are integrated in the S. aureus genome within a conserved ORF which is surrounded by a cluster of tandemly repeated genes. Here we demonstrate that in S. aureus clonal complex ST80 strains PVL-phage induction led to the acquisition of host DNA into the phage genome probably due to a homologous recombination event between direct repeats of the two paralogous genes adjacent to the phage integration site. Phage excision was accompanied by an additional chromosomal deletion in this region. This so far unrecognized mechanism of DNA uptake into the phage genome may play an important role in the co-evolution of phages and bacteria. PMID:20708208

  6. Who went into phage research?

    PubMed Central

    2012-01-01

    A total of 30,000 phage papers, books, or book chapters, published between 1965 and 2010, were analyzed for the ethnic origins of 14,429 first authors. Their names represent 40 linguistic domains or geographic areas and at least 70 languages. British and German names predominate. Results broadly concur with statistics on the frequency of publications by country and show the growing role of Third-World countries in phage research. Irish and Jewish scientists are prominent. Historical and societal factors appear to be very important elements in the advancement of science. PMID:22666657

  7. Synthetic Phage for Tissue Regeneration

    PubMed Central

    Merzlyak, Anna; Lee, Seung-Wuk

    2014-01-01

    Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy. PMID:24991085

  8. On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters.

    PubMed

    Wang, Tao; Kulkarni, Nikita; D'Souza, Gerard G M; Petrenko, Valery A; Torchilin, Vladimir P

    2011-10-01

    The integration of pharmaceutical nanocarriers with phage display techniques is emerging as a new paradigm for targeted cancer nanomedicines. We explored the direct use of landscape phage fusion proteins for the self-assembly of phage-derived binding peptides to liposomes for cancer cell targeting. The primary purpose of this study was to elucidate the targeting mechanism with a particular emphasis on the relative contributions of the two motifs that make up the landscape phage fusion protein (a binding peptide and the phage pVIII coat protein) to the targeting efficiency. Using transmission electron microscopy and dynamic light scattering, we confirmed the formation of phage-liposomes. Using FACS analysis, fluorescence microscopy, and fluorescence photospectrometry, we found that liposomes modified with MCF-7-specific phage fusion proteins (MCF-7 binding peptide, DMPGTVLP, fused to the phage PVIII coat protein) provided a strong and specific association with target MCF-7 cancer cells but not with cocultured, nontarget cells including C166-GFP and NIH3T3. The substitution for the binding peptide fused to phage pVIII coat protein abolished the targeting specificity. The addition of free binding peptide, DMPGTVLP, competitively inhibited the interaction of MCF-7-specific phage-liposomes with target MCF-7 cells but showed no reduction of MCF-7-associated plain liposomes. The proteolysis of the binding peptide reduced MCF-7 cell-associated phage-liposomes in a proteinase K (PK) concentration-dependent manner with no effect on the binding of plain liposomes to MCF-7 cells. Overall, only the binding peptide motif was involved in the targeting specificity of phage-liposomes. The presence of phage pVIII coat protein did not interfere with the targeting efficiency. PMID:21675738

  9. On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters

    PubMed Central

    Wang, Tao; Kulkarni, Nikita; D’Souza, Gerard G.M.; Petrenko, Valery A.; Torchilin, Vladimir P.

    2011-01-01

    The integration of pharmaceutical nanocarriers with phage display techniques is emerging as a new paradigm for targeted cancer nanomedicines. We explored the direct use of landscape phage fusion proteins for the self-assembly of phage-derived binding peptides to liposomes for cancer cell targeting. The primary purpose of this study was to elucidate the targeting mechanism with a particular emphasis on the relative contributions of the two motifs that make up the landscape phage fusion protein (a binding peptide and the phage pVIII coat protein) to the targeting efficiency. Using transmission electron microscopy and dynamic light scattering, we confirmed the formation of phage-liposomes. Using FACS analysis, fluorescence microscopy, and fluorescence photospectrometry, we found that liposomes modified with MCF-7-specific phage fusion proteins (MCF-7 binding peptide, DMPGTVLP, fused to the phage PVIII coat protein) provided a strong and specific association with target MCF-7 cancer cells but not with co-cultured, non-target cells including C166-GFP and NIH3T3. The substitution for the binding peptide fused to phage pVIII coat protein abolished the targeting specificity. The addition of free binding peptide, DMPGTVLP, competitively inhibited the interaction of MCF-7-specific phage-liposomes with target MCF-7 cells but showed no reduction of MCF-7-associated plain liposomes. The proteolysis of the binding peptide reduced MCF-7 cell-associated phage-liposomes in a proteinase K (PK) concentration-dependent manner with no effect on the binding of plain liposomes to MCF-7 cells. Overall, only the binding peptide motif was involved in the targeting specificity of phage-liposomes. The presence of phage pVIII coat protein did not interfere with the targeting efficiency. PMID:21675738

  10. Phage-encoded Serine Integrases and Other Large Serine Recombinases.

    PubMed

    Smith, Margaret C M

    2015-08-01

    The large serine recombinases (LSRs) are a family of enzymes, encoded in temperate phage genomes or on mobile elements, that precisely cut and recombine DNA in a highly controllable and predictable way. In phage integration, the LSRs act at specific sites, the attP site in the phage and the attB site in the host chromosome, where cleavage and strand exchange leads to the integrated prophage flanked by the recombinant sites attL and attR. The prophage can excise by recombination between attL and attR but this requires a phage-encoded accessory protein, the recombination directionality factor (RDF). Although the LSRs can bind specifically to all the recombination sites, only specific integrase-bound sites can pair in a synaptic complex prior to strand exchange. Recent structural information has led to a breakthrough in our understanding of the mechanism of the LSRs, notably how the LSRs bind to their substrates and how LSRs display this site-selectivity. We also understand that the RDFs exercise control over the LSRs by protein-protein interactions. Other recent work with the LSRs have contributed to our understanding of how all serine recombinases undergo strand exchange subunit rotation, facilitated by surfaces that resemble a molecular bearing.

  11. Revenge of the phages: defeating bacterial defences.

    PubMed

    Samson, Julie E; Magadán, Alfonso H; Sabri, Mourad; Moineau, Sylvain

    2013-10-01

    Bacteria and their viral predators (bacteriophages) are locked in a constant battle. In order to proliferate in phage-rich environments, bacteria have an impressive arsenal of defence mechanisms, and in response, phages have evolved counter-strategies to evade these antiviral systems. In this Review, we describe the various tactics that are used by phages to overcome bacterial resistance mechanisms, including adsorption inhibition, restriction-modification, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems and abortive infection. Furthermore, we consider how these observations have enhanced our knowledge of phage biology, evolution and phage-host interactions. PMID:23979432

  12. Phage typing of Staphylococcus saprophyticus.

    PubMed Central

    Torres Pereira, A.; Melo Cristino, J. A.

    1991-01-01

    This study included 502 staphylococcus strains; Staphylococcus saprophyticus (297 strains) S. cohnii (47), S. xylosus (10), S. epidermidis (67) and S. aureus (81). Mitomycin C induction was performed on 100 isolates of S. saprophyticus and all induced strains were reacted with each other. Twenty-six strains proved to be lysogenic. Phages were propagated and titrated. With 12 of the phages there were three frequent associations, named lytic groups A, B and C, which included 75% of all typable strains. Typability of the system was 45% and reproducibility was between 94.2% and 100%. Phages did not lyse S. aureus and S. epidermidis strains, but they lysed S. saprophyticus and only rare strains of other novobiocin resistant species. Effective S. saprophyticus typing serves ecological purposes and tracing the origin of urinary strains from the skin or mucous membranes. Phage typing in association with plasmid profiling previously described, are anticipated as complementary methods with strong discriminatory power for differentiating among S. saprophyticus strains. PMID:1752305

  13. Protein and Antibody Engineering by Phage Display.

    PubMed

    Frei, J C; Lai, J R

    2016-01-01

    Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

  14. The habits of highly effective phages: population dynamics as a framework for identifying therapeutic phages.

    PubMed

    Bull, James J; Gill, Jason J

    2014-01-01

    The use of bacteriophages as antibacterial agents is being actively researched on a global scale. Typically, the phages used are isolated from the wild by plating on the bacteria of interest, and a far larger set of candidate phages is often available than can be used in any application. When an excess of phages is available, how should the best phages be identified? Here we consider phage-bacterial population dynamics as a basis for evaluating and predicting phage success. A central question is whether the innate dynamical properties of phages are the determinants of success, or instead, whether extrinsic, indirect effects can be responsible. We address the dynamical perspective, motivated in part by the absence of dynamics in previously suggested principles of phage therapy. Current mathematical models of bacterial-phage dynamics do not capture the realities of in vivo dynamics, nor is this likely to change, but they do give insight to qualitative properties that may be generalizable. In particular, phage adsorption rate may be critical to treatment success, so understanding the effects of the in vivo environment on host availability may allow prediction of useful phages prior to in vivo experimentation. Principles for predicting efficacy may be derived by developing a greater understanding of the in vivo system, or such principles could be determined empirically by comparing phages with known differences in their dynamic properties. The comparative approach promises to be a powerful method of discovering the key to phage success. We offer five recommendations for future study: (i) compare phages differing in treatment efficacy to identify the phage properties associated with success, (ii) assay dynamics in vivo, (iii) understand mechanisms of bacterial escape from phages, (iv) test phages in model infections that are relevant to the intended clinical applications, and (v) develop new classes of models for phage growth in spatially heterogeneous environments

  15. Recombinant phage probes for Listeria monocytogenes

    NASA Astrophysics Data System (ADS)

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

    2007-10-01

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

  16. 'Big things in small packages: the genetics of filamentous phage and effects on fitness of their host'.

    PubMed

    Mai-Prochnow, Anne; Hui, Janice Gee Kay; Kjelleberg, Staffan; Rakonjac, Jasna; McDougald, Diane; Rice, Scott A

    2015-07-01

    This review synthesizes recent and past observations on filamentous phages and describes how these phages contribute to host phentoypes. For example, the CTXφ phage of Vibrio cholerae encodes the cholera toxin genes, responsible for causing the epidemic disease, cholera. The CTXφ phage can transduce non-toxigenic strains, converting them into toxigenic strains, contributing to the emergence of new pathogenic strains. Other effects of filamentous phage include horizontal gene transfer, biofilm development, motility, metal resistance and the formation of host morphotypic variants, important for the biofilm stress resistance. These phages infect a wide range of Gram-negative bacteria, including deep-sea, pressure-adapted bacteria. Many filamentous phages integrate into the host genome as prophage. In some cases, filamentous phages encode their own integrase genes to facilitate this process, while others rely on host-encoded genes. These differences are mediated by different sets of 'core' and 'accessory' genes, with the latter group accounting for some of the mechanisms that alter the host behaviours in unique ways. It is increasingly clear that despite their relatively small genomes, these phages exert signficant influence on their hosts and ultimately alter the fitness and other behaviours of their hosts.

  17. Effect of preservatives on Shiga toxigenic phages and Shiga toxin of Escherichia coli O157:H7.

    PubMed

    Subils, Tomás; Aquili, Virginia; Ebner, Guillermo; Balagué, Claudia

    2012-05-01

    Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through the induction of the integrated bacteriophages that encode the toxin genes. These phages might be the principal means for the dissemination and release of Shiga toxins. We evaluated the effect of three common food preservatives, potassium sorbate, sodium benzoate, and sodium propionate, on the propagation of the phages and Shiga toxins. We tested each preservative at four concentrations, 1, 1.25, 2.5, and 5 mg/ml, both on free phages and on lysogenic phages in bacteria. We also evaluated the expression of a lambdoid phage, which was exposed to increasing concentrations of preservatives, by measuring β-galactosidase activity from SPC105, a transductant strain. Furthermore, we tested the effect of the preservatives on cytotoxigenic activity of Shiga toxin on Vero cells. We detected an increase of the inhibitory effect of the phage lytic activity, both in lysogenic and free phages, as the preservative concentration increased. However, the inhibition was higher on the lysogenic phages release than on free phages. Sodium benzoate and potassium sorbate were about equal at inhibiting phages; they were more effective than sodium propionate. A significant decrease of lacZ expression, encoded in a lambda phage, was observed. We also found a reduction in Shiga toxin titer caused by exposure of E. coli O157:H7 to 5 mg/ml sodium benzoate or potassium sorbate. These results imply that these three preservatives, used to inhibit microbial spoilage of foods, also act to inhibit lytic activity and dispersion of a phage carrying the gene encoding powerful Shiga cytotoxins. Also notable was the inactivation of Shiga toxin activity, although this effect was detected using concentrations of preservatives greater than those allowed by the Argentine Food Code.

  18. Effect of preservatives on Shiga toxigenic phages and Shiga toxin of Escherichia coli O157:H7.

    PubMed

    Subils, Tomás; Aquili, Virginia; Ebner, Guillermo; Balagué, Claudia

    2012-05-01

    Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through the induction of the integrated bacteriophages that encode the toxin genes. These phages might be the principal means for the dissemination and release of Shiga toxins. We evaluated the effect of three common food preservatives, potassium sorbate, sodium benzoate, and sodium propionate, on the propagation of the phages and Shiga toxins. We tested each preservative at four concentrations, 1, 1.25, 2.5, and 5 mg/ml, both on free phages and on lysogenic phages in bacteria. We also evaluated the expression of a lambdoid phage, which was exposed to increasing concentrations of preservatives, by measuring β-galactosidase activity from SPC105, a transductant strain. Furthermore, we tested the effect of the preservatives on cytotoxigenic activity of Shiga toxin on Vero cells. We detected an increase of the inhibitory effect of the phage lytic activity, both in lysogenic and free phages, as the preservative concentration increased. However, the inhibition was higher on the lysogenic phages release than on free phages. Sodium benzoate and potassium sorbate were about equal at inhibiting phages; they were more effective than sodium propionate. A significant decrease of lacZ expression, encoded in a lambda phage, was observed. We also found a reduction in Shiga toxin titer caused by exposure of E. coli O157:H7 to 5 mg/ml sodium benzoate or potassium sorbate. These results imply that these three preservatives, used to inhibit microbial spoilage of foods, also act to inhibit lytic activity and dispersion of a phage carrying the gene encoding powerful Shiga cytotoxins. Also notable was the inactivation of Shiga toxin activity, although this effect was detected using concentrations of preservatives greater than those allowed by the Argentine Food Code. PMID:22564948

  19. Contribution of phage-derived genomic islands to the virulence of facultative bacterial pathogens.

    PubMed

    Busby, Ben; Kristensen, David M; Koonin, Eugene V

    2013-02-01

    Facultative pathogens have extremely dynamic pan-genomes, to a large extent derived from bacteriophages and other mobile elements. We developed a simple approach to identify phage-derived genomic islands and apply it to show that pathogens from diverse bacterial genera are significantly enriched in clustered phage-derived genes compared with related benign strains. These findings show that genome expansion by integration of prophages containing virulence factors is a major route of evolution of facultative bacterial pathogens.

  20. Development of an engineered bioluminescent reporter phage for detection of bacterial blight of crucifers.

    PubMed

    Schofield, David A; Bull, Carolee T; Rubio, Isael; Wechter, W Patrick; Westwater, Caroline; Molineux, Ian J

    2012-05-01

    Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant "light"-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis. PMID:22427491

  1. Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex

    PubMed Central

    2010-01-01

    Background The Burkholderia cepacia complex (BCC) is comprised of at least seventeen Gram-negative species that cause infections in cystic fibrosis patients. Because BCC bacteria are broadly antibiotic resistant, phage therapy is currently being investigated as a possible alternative treatment for these infections. The purpose of our study was to sequence and characterize three novel BCC-specific phages: KS5 (vB_BceM-KS5 or vB_BmuZ-ATCC 17616), KS14 (vB_BceM-KS14) and KL3 (vB_BamM-KL3 or vB_BceZ-CEP511). Results KS5, KS14 and KL3 are myoviruses with the A1 morphotype. The genomes of these phages are between 32317 and 40555 base pairs in length and are predicted to encode between 44 and 52 proteins. These phages have over 50% of their proteins in common with enterobacteria phage P2 and so can be classified as members of the Peduovirinae subfamily and the "P2-like viruses" genus. The BCC phage proteins similar to those encoded by P2 are predominantly structural components involved in virion morphogenesis. As prophages, KS5 and KL3 integrate into an AMP nucleosidase gene and a threonine tRNA gene, respectively. Unlike other P2-like viruses, the KS14 prophage is maintained as a plasmid. The P2 E+E' translational frameshift site is conserved among these three phages and so they are predicted to use frameshifting for expression of two of their tail proteins. The lysBC genes of KS14 and KL3 are similar to those of P2, but in KS5 the organization of these genes suggests that they may have been acquired via horizontal transfer from a phage similar to λ. KS5 contains two sequence elements that are unique among these three phages: an ISBmu2-like insertion sequence and a reverse transcriptase gene. KL3 encodes an EcoRII-C endonuclease/methylase pair and Vsr endonuclease that are predicted to function during the lytic cycle to cleave non-self DNA, protect the phage genome and repair methylation-induced mutations. Conclusions KS5, KS14 and KL3 are the first BCC-specific phages

  2. Control of Pierce's Disease by Phage

    PubMed Central

    Das, Mayukh; Bhowmick, Tushar Suvra; Ahern, Stephen J.; Young, Ry; Gonzalez, Carlos F.

    2015-01-01

    Pierce’s Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella. PMID:26107261

  3. Control of Pierce's Disease by Phage.

    PubMed

    Das, Mayukh; Bhowmick, Tushar Suvra; Ahern, Stephen J; Young, Ry; Gonzalez, Carlos F

    2015-01-01

    Pierce's Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella. PMID:26107261

  4. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1

    PubMed Central

    Dyson, Zoe A.; Seviour, Robert J.; Tucci, Joseph

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa. The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  5. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

    PubMed

    Dyson, Zoe A; Seviour, Robert J; Tucci, Joseph; Petrovski, Steve

    2016-06-16

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA.

  6. Bacteriophages with potential to inactivate Salmonella Typhimurium: Use of single phage suspensions and phage cocktails.

    PubMed

    Pereira, Carla; Moreirinha, Catarina; Lewicka, Magdalena; Almeida, Paulo; Clemente, Carla; Cunha, Ângela; Delgadillo, Ivonne; Romalde, Jésus L; Nunes, Maria L; Almeida, Adelaide

    2016-07-15

    The aim of this study was to compare the dynamics of three previously isolated bacteriophages (or phages) individually (phSE-1, phSE-2 and phSE-5) or combined in cocktails of two or three phages (phSE-1/phSE-2, phSE-1/phSE-5, phSE-2/phSE-5 and phSE-1/phSE-2/phSE-5) to control Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) in order to evaluate their potential application during depuration. Phages were assigned to the family Siphoviridae and revealed identical restriction digest profiles, although they showed a different phage adsorption, host range, burst size, explosion time and survival in seawater. The three phages were effective against S. Typhimurium (reduction of ∼2.0 log CFU/mL after 4h treatment). The use of cocktails was not significantly more effective than the use of single phages. A big fraction of the remained bacteria are phage-resistant mutants (frequency of phage-resistant mutants 9.19×10(-5)-5.11×10(-4)) but phage- resistant bacterial mutants was lower for the cocktail phages than for the single phage suspensions and the phage phSE-1 presented the highest rate of resistance and phage phSE-5 the lowest one. The spectral changes of S. Typhimurium resistant and phage-sensitive cells were compared and revealed relevant differences for peaks associated to amide I (1620cm(-1)) and amide II (1515cm(-1)) from proteins and from carbohydrates and phosphates region (1080-1000cm(-1)). Despite the similar efficiency of individual phages, the development of lower resistance indicates that phage cocktails might be the most promising choice to be used during the bivalve depuration to control the transmission of salmonellosis.

  7. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

    PubMed

    Dyson, Zoe A; Seviour, Robert J; Tucci, Joseph; Petrovski, Steve

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  8. Enhancing and initiating phage-based therapies

    PubMed Central

    Serwer, Philip; Wright, Elena T; Chang, Juan T; Liu, Xiangan

    2014-01-01

    Drug development has typically been a primary foundation of strategy for systematic, long-range management of pathogenic cells. However, drug development is limited in speed and flexibility when response is needed to changes in pathogenic cells, especially changes that produce drug-resistance. The high replication speed and high diversity of phages are potentially useful for increasing both response speed and response flexibility when changes occur in either drug resistance or other aspects of pathogenic cells. We present strategy, with some empirical details, for (1) using modern molecular biology and biophysics to access these advantages during the phage therapy of bacterial infections, and (2) initiating use of phage capsid-based drug delivery vehicles (DDVs) with procedures that potentially overcome both drug resistance and other present limitations in the use of DDVs for the therapy of neoplasms. The discussion of phage therapy includes (a) historical considerations, (b) changes that appear to be needed in clinical tests if use of phage therapy is to be expanded, (c) recent work on novel phages and its potential use for expanding the capabilities of phage therapy and (d) an outline for a strategy that encompasses both theory and practice for expanding the applications of phage therapy. The discussion of DDVs starts by reviewing current work on DDVs, including work on both liposomal and viral DDVs. The discussion concludes with some details of the potential use of permeability constrained phage capsids as DDVs. PMID:26713220

  9. Experimental Phage Therapy for Burkholderia pseudomallei Infection

    PubMed Central

    Leang-Chung, Choh; Vellasamy, Kumutha Malar; Mariappan, Vanitha; Li-Yen, Chang; Vadivelu, Jamuna

    2016-01-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen intrinsically resistant to a variety of antibiotics. Phages have been developed for use as an alternative treatment therapy, particularly for bacterial infections that do not respond to conventional antibiotics. In this study, we investigated the use of phages to treat cells infected with B. pseudomallei. Phage C34 isolated from seawater was purified and characterised on the basis of its host range and morphology using transmission electron microscopy (TEM). Phage C34 was able to lyse 39.5% of B. pseudomallei clinical strains. Due to the presence of contractile tail, phage C34 is classified as a member of the family Myoviridae, a tailed double-stranded DNA virus. When 2 × 105 A549 cells were exposed to 2 × 107 PFU of phage C34, 24 hours prior to infection with 2 × 106 CFU of B. pseudomallei, it was found that the survivability of the cells increased to 41.6 ± 6.8% as compared to 22.8 ± 6.0% in untreated control. Additionally, application of phage successfully rescued 33.3% of mice infected with B. pseudomallei and significantly reduced the bacterial load in the spleen of the phage-treated mice. These findings indicate that phage can be a potential antimicrobial agent for B. pseudomallei infections. PMID:27387381

  10. Experimental Phage Therapy for Burkholderia pseudomallei Infection.

    PubMed

    Guang-Han, Ong; Leang-Chung, Choh; Vellasamy, Kumutha Malar; Mariappan, Vanitha; Li-Yen, Chang; Vadivelu, Jamuna

    2016-01-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen intrinsically resistant to a variety of antibiotics. Phages have been developed for use as an alternative treatment therapy, particularly for bacterial infections that do not respond to conventional antibiotics. In this study, we investigated the use of phages to treat cells infected with B. pseudomallei. Phage C34 isolated from seawater was purified and characterised on the basis of its host range and morphology using transmission electron microscopy (TEM). Phage C34 was able to lyse 39.5% of B. pseudomallei clinical strains. Due to the presence of contractile tail, phage C34 is classified as a member of the family Myoviridae, a tailed double-stranded DNA virus. When 2 × 105 A549 cells were exposed to 2 × 107 PFU of phage C34, 24 hours prior to infection with 2 × 106 CFU of B. pseudomallei, it was found that the survivability of the cells increased to 41.6 ± 6.8% as compared to 22.8 ± 6.0% in untreated control. Additionally, application of phage successfully rescued 33.3% of mice infected with B. pseudomallei and significantly reduced the bacterial load in the spleen of the phage-treated mice. These findings indicate that phage can be a potential antimicrobial agent for B. pseudomallei infections. PMID:27387381

  11. Use of phages to control Campylobacter spp.

    PubMed

    Janež, Nika; Loc-Carrillo, Catherine

    2013-10-01

    The use of phages to control pathogenic bacteria has been investigated since they were first discovered in the beginning of the 1900s. Over the last century we have slowly gained an in-depth understanding of phage biology including which phage properties are desirable when considering phage as biocontrol agents and which phage characteristics to potentially avoid. Campylobacter infections are amongst the most frequently encountered foodborne bacterial infections around the world. Handling and consumption of raw or undercooked poultry products have been determined to be the main route of transmission. The ability to use phages to target these bacteria has been studied for more than a decade and although we have made progress towards deciphering how best to use phages to control Campylobacter associated with poultry production, there is still much work to be done. This review outlines methods to improve the isolation of these elusive phages, as well as methods to identify desirable characteristics needed for a successful outcome. It also highlights the body of research undertaken so far and what criteria to consider when doing in-vivo studies, especially because some in-vitro studies have not been found to translate into to phage efficacy in-vivo.

  12. A Virulent Phage Infecting Lactococcus garvieae, with Homology to Lactococcus lactis Phages

    PubMed Central

    Eraclio, Giovanni; Tremblay, Denise M.; Lacelle-Côté, Alexia; Labrie, Simon J.; Fortina, Maria Grazia

    2015-01-01

    A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor. PMID:26407890

  13. Mammalian Host-Versus-Phage immune response determines phage fate in vivo.

    PubMed

    Hodyra-Stefaniak, Katarzyna; Miernikiewicz, Paulina; Drapała, Jarosław; Drab, Marek; Jończyk-Matysiak, Ewa; Lecion, Dorota; Kaźmierczak, Zuzanna; Beta, Weronika; Majewska, Joanna; Harhala, Marek; Bubak, Barbara; Kłopot, Anna; Górski, Andrzej; Dąbrowska, Krystyna

    2015-01-01

    Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy. We accommodated the experimental data into a mathematical model. We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations. We found that the mammalian-host response to infecting bacteria causes the concomitant removal of phage from the system. We propose the notion that this effect as an indirect pathway of phage inhibition by bacteria with significant relevance for the clinical outcome of phage therapy. PMID:26440922

  14. Mammalian Host-Versus-Phage immune response determines phage fate in vivo

    PubMed Central

    Hodyra-Stefaniak, Katarzyna; Miernikiewicz, Paulina; Drapała, Jarosław; Drab, Marek; Jończyk-Matysiak, Ewa; Lecion, Dorota; Kaźmierczak, Zuzanna; Beta, Weronika; Majewska, Joanna; Harhala, Marek; Bubak, Barbara; Kłopot, Anna; Górski, Andrzej; Dąbrowska, Krystyna

    2015-01-01

    Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy. We accommodated the experimental data into a mathematical model. We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations. We found that the mammalian-host response to infecting bacteria causes the concomitant removal of phage from the system. We propose the notion that this effect as an indirect pathway of phage inhibition by bacteria with significant relevance for the clinical outcome of phage therapy. PMID:26440922

  15. [Phage therapy for bacterial infection of burn].

    PubMed

    Peng, Y Z; Huang, G T

    2016-09-20

    With the long-term and widespread use of antibiotics, drug resistance of bacteria has become a major problem in the treatment of burn infection. For treating multidrug resistant bacteria, phage therapy has become the focus of attention. Development of phage therapy to fill the blank of this field in China is extremely urgent. PMID:27647065

  16. Preparation and assay of phage lambda.

    PubMed

    Dale, J W; Greenaway, P J

    1985-01-01

    Lambda, a temperate bacteriophage of E. coli, has two alternative modes of replication in sensitive cells, known as the lytic and lysogenic cycles. In the lytic cycle, after the lambda DNA enters the cells, various phage functions are expressed that result in the production of a large number of mature phage particles and cell lysis. In the lysogenic mode, which normally occurs in only a small proportion of the infected cells, the phage forms a more or less stable relationship with the host bacterium; this stable state is known as lysogeny. In a lysogenic cell, phage DNA is normally incorporated into the chromosomal DNA via specific attachment sites on both the phage DNA and the host chromosome. Replication of lambda DNA then occurs only during replication of the host chromosome, and the phage genome is inherited by each daughter cell at cell division. The phage is maintained in this prophage state through the action of a repressor protein, coded for by the phage gene cl. This repressor protein turns off the expression of virtually the whole of the lambda genome. If the repressor is inactivated, the expression of phage genes is initiated. This leads to the excision of lambda DNA from the host chromosome and entry into the lytic cycle. The balance between the lytic and lysogenic modes of replication is a delicate and complex one in which a key factor is the concentration of the cl gene product. Some of the many sources of further information about the basic biology of lambda phage are listed in the references to this chapter.

  17. Holding a grudge: persisting anti-phage CRISPR immunity in multiple human gut microbiomes.

    PubMed

    Mick, Eran; Stern, Adi; Sorek, Rotem

    2013-05-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system of bacteria and archaea constitutes a mechanism of acquired adaptive immunity against phages, which is based on genome-encoded markers of previously infecting phage sequences ("spacers"). As a repository of phage sequences, these spacers make the system particularly suitable for elucidating phage-bacteria interactions in metagenomic studies. Recent metagenomic analyses of CRISPRs associated with the human microbiome intriguingly revealed conserved "memory spacers" shared by bacteria in multiple unrelated, geographically separated individuals. Here, we discuss possible avenues for explaining this phenomenon by integrating insights from CRISPR biology and phage-bacteria ecology, with a special focus on the human gut. We further explore the growing body of evidence for the role of CRISPR/Cas in regulating the interplay between bacteria and lysogenic phages, which may be intimately related to the presence of memory spacers and sheds new light on the multifaceted biological and ecological modes of action of CRISPR/Cas. PMID:23439321

  18. Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display

    PubMed Central

    Tjhung, Katrina F.; Deiss, Frédérique; Tran, Jessica; Chou, Ying; Derda, Ratmir

    2015-01-01

    In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a negatively charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus, as well as positively charged sequences which suppress production of phage when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs) co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc.). PMID:25972845

  19. Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display.

    PubMed

    Tjhung, Katrina F; Deiss, Frédérique; Tran, Jessica; Chou, Ying; Derda, Ratmir

    2015-01-01

    In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a negatively charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus, as well as positively charged sequences which suppress production of phage when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs) co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc.). PMID:25972845

  20. BREX is a novel phage resistance system widespread in microbial genomes.

    PubMed

    Goldfarb, Tamara; Sberro, Hila; Weinstock, Eyal; Cohen, Ofir; Doron, Shany; Charpak-Amikam, Yoav; Afik, Shaked; Ofir, Gal; Sorek, Rotem

    2015-01-13

    The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria.

  1. BREX is a novel phage resistance system widespread in microbial genomes

    PubMed Central

    Goldfarb, Tamara; Sberro, Hila; Weinstock, Eyal; Cohen, Ofir; Doron, Shany; Charpak-Amikam, Yoav; Afik, Shaked; Ofir, Gal; Sorek, Rotem

    2015-01-01

    The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ∼10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria. PMID:25452498

  2. Genomic characterization provides new insight into Salmonella phage diversity

    PubMed Central

    2013-01-01

    Background Salmonella is a widely distributed foodborne pathogen that causes tens of millions of salmonellosis cases globally every year. While the genomic diversity of Salmonella is increasingly well studied, our knowledge of Salmonella phage genomic diversity is still rather limited, despite the contributions of both lysogenic and lytic phages to Salmonella virulence, diversity and ecology (e.g., through horizontal gene transfer and Salmonella lysis). To gain a better understanding of phage diversity in a specific ecological niche, we sequenced 22 Salmonella phages isolated from a number of dairy farms from New York State (United States) and analyzed them using a comparative genomics approach. Results Classification of the 22 phages according to the presence/absence of orthologous genes allowed for classification into 8 well supported clusters. In addition to two phage clusters that represent novel virulent Salmonella phages, we also identified four phage clusters that each contained previously characterized phages from multiple continents. Our analyses also identified two clusters of phages that carry putative virulence (e.g., adhesins) and antimicrobial resistance (tellurite and bicyclomycin) genes as well as virulent and temperate transducing phages. Insights into phage evolution from our analyses include (i) identification of DNA metabolism genes that may facilitate nucleotide synthesis in phages with a G+C % distinct from Salmonella, and (ii) evidence of Salmonella phage tailspike and fiber diversity due to both single nucleotide polymorphisms and major re-arrangements, which may affect the host specificity of Salmonella phages. Conclusions Genomics-based characterization of 22 Salmonella phages isolated from dairy farms allowed for identification of a number of novel Salmonella phages. While the comparative genomics analyses of these phages provide a number of new insights in the evolution and diversity of Salmonella phages, they only represent a first

  3. Bacteriophage 2851 Is a Prototype Phage for Dissemination of the Shiga Toxin Variant Gene 2c in Escherichia coli O157:H7▿ †

    PubMed Central

    Strauch, Eckhard; Hammerl, Jens Andre; Konietzny, Antje; Schneiker-Bekel, Susanne; Arnold, Walter; Goesmann, Alexander; Pühler, Alfred; Beutin, Lothar

    2008-01-01

    The production of Shiga toxin (Stx) (verocytotoxin) is a major virulence factor of Escherichia coli O157:H7 strains (Shiga toxin-producing E. coli [STEC] O157). Two types of Shiga toxins, designated Stx1 and Stx2, are produced in STEC O157. Variants of the Stx2 type (Stx2, Stx2c) are associated with high virulences of these strains for humans. A bacteriophage designated 2851 from a human STEC O157 encoding the Stx2c variant was described previously. Nucleotide sequence analysis of the phage 2851 genome revealed 75 predicted coding sequences and indicated a mosaic structure typical for lambdoid phages. Analyses of free phages and K-12 phage 2851 lysogens revealed that upon excision from the bacterial chromosome, the loss of a phage-encoded IS629 element leads to fusion of phage antA and antB genes, with the generation of a recombined antAB gene encoding a strong antirepressor. In wild-type E. coli O157 as well as in K-12 strains, phage 2851 was found to be integrated in the sbcB locus. Additionally, phage 2851 carries an open reading frame which encodes an OspB-like type III effector similar to that found in Shigella spp. Investigation of 39 stx2c E. coli O157 strains revealed that all except 1 were positive for most phage 2851-specific genes and possessed a prophage with the same border sequences integrated into the sbcB locus. Phage 2851-specific sequences were absent from most stx2c-negative E. coli O157 strains, and we suggest that phage 2851-like phages contributed significantly to the dissemination of the Stx2c variant toxin within this group of E. coli. PMID:18824528

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Inactivation and reactivation of B. megatherium phage.

    PubMed

    NORTHROP, J H

    1955-11-20

    Preparation of Reversibly Inactivated (R.I.) Phage.- If B. megatherium phage (of any type, or in any stage of purification) is suspended in dilute salt solutions at pH 5-6, it is completely inactivated; i.e., it does not form plaques, or give rise to more phage when mixed with a sensitive organism (Northrop, 1954). The inactivation occurs when the phage is added to the dilute salt solution. If a suspension of the inactive phage in pH 7 peptone is titrated to pH 5 and allowed to stand, the activity gradually returns. The inactivation is therefore reversible. Properties of R.I. Phage.- The R.I. phage is adsorbed by sensitive cells at about the same rate as the active phage. It kills the cells, but no active phage is produced. The R.I. phage therefore has the properties of phage "ghosts" (Herriott, 1951) or of colicines (Gratia, 1925), or phage inactivated by ultraviolet light (Luria, 1947). The R.I. phage is sedimented in the centrifuge at the same rate as active phage. It is therefore about the same size as the active phage. The R.I. phage is most stable in pH 7, 5 per cent peptone, and may be kept in this solution for weeks at 0 degrees C. The rate of digestion of R.I. phage by trypsin, chymotrypsin, or desoxyribonuclease is about the same as that of active phage (Northrop, 1955 a). Effect of Various Substances on the Formation of R.I. Phage.- There is an equilibrium between R.I. phage and active phage. The R.I. form is the stable one in dilute salt solution, pH 5 to 6.5 and at low temperature (<20 degrees C.). At pH >6.5, in dilute salt solution, the R.I. phage changes to the active form. The cycle, active right harpoon over left harpoon inactive phage, may be repeated many times at 0 degrees C. by changing the pH of the solution back and forth between pH 7 and pH 6. Irreversible inactivation is caused by distilled water, some heavy metals, concentrated urea or quanidine solutions, and by l-arginine. Reversible inactivation is prevented by all salts tested (except

  6. Rapid enumeration of phage in monodisperse emulsions.

    PubMed

    Tjhung, Katrina F; Burnham, Sean; Anany, Hany; Griffiths, Mansel W; Derda, Ratmir

    2014-06-17

    Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.

  7. Rapid enumeration of phage in monodisperse emulsions.

    PubMed

    Tjhung, Katrina F; Burnham, Sean; Anany, Hany; Griffiths, Mansel W; Derda, Ratmir

    2014-06-17

    Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed. PMID:24892245

  8. The bacteriophage WORiC is the active phage element in wRi of Drosophila simulans and represents a conserved class of WO phages

    PubMed Central

    2011-01-01

    Background The alphaproteobacterium Wolbachia pipientis, the most common endosymbiont in eukaryotes, is found predominantly in insects including many Drosophila species. Although Wolbachia is primarily vertically transmitted, analysis of its genome provides evidence for frequent horizontal transfer, extensive recombination and numerous mobile genetic elements. The genome sequence of Wolbachia in Drosophila simulans Riverside (wRi) is available along with the integrated bacteriophages, enabling a detailed examination of phage genes and the role of these genes in the biology of Wolbachia and its host organisms. Wolbachia is widely known for its ability to modify the reproductive patterns of insects. One particular modification, cytoplasmic incompatibility, has previously been shown to be dependent on Wolbachia density and inversely related to the titer of lytic phage. The wRi genome has four phage regions, two WORiBs, one WORiA and one WORiC. Results In this study specific primers were designed to distinguish between these four prophage types in wRi, and quantitative PCR was used to measure the titer of bacteriophages in testes, ovaries, embryos and adult flies. In all tissues tested, WORiA and WORiB were not found to be present in excess of their integrated prophages; WORiC, however, was found to be present extrachromosomally. WORiC is undergoing extrachromosomal replication in wRi. The density of phage particles was found to be consistent in individual larvae in a laboratory population. The WORiC genome is organized in conserved blocks of genes and aligns most closely with other known lytic WO phages, WOVitA and WOCauB. Conclusions The results presented here suggest that WORiC is the lytic form of WO in D. simulans, is undergoing extrachromosomal replication in wRi, and belongs to a conserved family of phages in Wolbachia. PMID:22085419

  9. Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy.

    PubMed

    Dalmasso, Marion; Strain, Ronan; Neve, Horst; Franz, Charles M A P; Cousin, Fabien J; Ross, R Paul; Hill, Colin

    2016-01-01

    With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI) between 10-3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections. PMID:27280590

  10. Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy

    PubMed Central

    Dalmasso, Marion; Strain, Ronan; Neve, Horst; Franz, Charles M. A. P.; Cousin, Fabien J.; Ross, R. Paul; Hill, Colin

    2016-01-01

    With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI) between 10−3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections. PMID:27280590

  11. Tales from a thousand and one phages

    PubMed Central

    Rodriguez-Valera, Francisco; Mizuno, Carolina Megumi; Ghai, Rohit

    2014-01-01

    The sequencing of marine metagenomic fosmids led to the discovery of several new complete phage genomes. Among the 21 major sequence groups, 10 totally novel groups of marine phages could be identified. Some of these represent the first phages infecting large marine prokaryotic phyla, such as the Verrucomicrobia and the recently described Ca. Actinomarinales. Coming from a single deep photic zone sample the diversity of phages found is astonishing, and the comparison with a metavirome from the same location indicates that only 2% of the real diversity was recovered. In addition to this large macro-diversity, rich micro-diversity was also found, affecting host-recognition modules, mirroring the variation of cell surface components in their host marine microbes. PMID:24616837

  12. PLANET: a phage library analysis expert tool.

    PubMed

    Leplae, R; Tramontano, A

    1995-01-01

    In recent years random peptide libraries displayed on filamentous phage have been widely used and new ideas and techniques are continuously developing in the field (1-5). Notwithstanding this growing interest in the technique and in its promising results, and the enormous increase in usage and scope, very little effort has been devoted to the implementation of software able to handle and analyze the growing number of phage library-derived sequences. In our laboratory, phage libraries are extensively used and peptide sequences are continuously produced, so that the need arose of creating a database (6) to collect all the experimental results in a format compatible with GCG sequence analysis packages (7). We present here the description of an XWindow-based software package named PLANET (Phage Library ANalysis Expert Tool) devoted to the maintenance and statistical analysis of the database.

  13. Supersize me: Cronobacter sakazakii phage GAP32

    SciTech Connect

    Abbasifar, Reza; Griffiths, Mansel W.; Sabour, Parviz M.; Ackermann, Hans-Wolfgang; Vandersteegen, Katrien; Lavigne, Rob; Noben, Jean-Paul; Alanis Villa, Argentina; Abbasifar, Arash; Nash, John H.E.; Kropinski, Andrew M.

    2014-07-15

    Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB{sub C}saM{sub G}AP32 (GAP32), which possesses the second largest sequenced phage genome (358,663 bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily. - Highlights: • Cronobacter sakazakii phage vB{sub C}saM{sub G}AP32 has a genome of 358,663 bp. • It encodes 545 proteins which is more than Mycoplasma genitalium G37. • It is a member of the Myoviridae. • It is peripherally related to coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2. • GAP32 encodes a chromosome condensation protein.

  14. The Staphylococci Phages Family: An Overview

    PubMed Central

    Deghorain, Marie; Van Melderen, Laurence

    2012-01-01

    Due to their crucial role in pathogenesis and virulence, phages of Staphylococcus aureus have been extensively studied. Most of them encode and disseminate potent staphylococcal virulence factors. In addition, their movements contribute to the extraordinary versatility and adaptability of this prominent pathogen by improving genome plasticity. In addition to S. aureus, phages from coagulase-negative Staphylococci (CoNS) are gaining increasing interest. Some of these species, such as S. epidermidis, cause nosocomial infections and are therefore problematic for public health. This review provides an overview of the staphylococcal phages family extended to CoNS phages. At the morphological level, all these phages characterized so far belong to the Caudovirales order and are mainly temperate Siphoviridae. At the molecular level, comparative genomics revealed an extensive mosaicism, with genes organized into functional modules that are frequently exchanged between phages. Evolutionary relationships within this family, as well as with other families, have been highlighted. All these aspects are of crucial importance for our understanding of evolution and emergence of pathogens among bacterial species such as Staphylococci. PMID:23342361

  15. Diversity and censoring of landscape phage libraries

    PubMed Central

    Kuzmicheva, G.A.; Jayanna, P.K.; Sorokulova, I.B.; Petrenko, V.A.

    2009-01-01

    Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd–tet (landscape libraries). They were obtained by replacement of amino acids 2–4 and 2–5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space. PMID:18988692

  16. Landscape Phage Fusion Protein-mediated Targeting of Nanomedicines Enhances their Prostate Tumor Cell Association and Cytotoxic Efficiency

    PubMed Central

    Jayanna, P.K.; Bedi, D; Gillespie, J.W.; DeInnocentes, P.; Wang, T; Torchilin, V.P; Bird, R.C.; Petrenko, V.A.

    2010-01-01

    Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display offers a high-throughput approach to screen for the targeting ligands. We have successfully isolated phage fusion peptides selective and specific for PC3 prostate cancer cells. Also, we have demonstrated a novel approach of targeting liposomes through tumor-specific phage fusion coat proteins, exploiting the intrinsic properties of the phage coat protein as an integral membrane protein. Here we describe the production of Rhodamine-labeled liposomes as well as doxorubicin-loaded long circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides, as an extension of our previous studies. Targeting of labeled liposomes was demonstrated using fluorescence microscopy as well as flow cytometry. Targeting of doxorubicin-loaded liposomes enhanced their cytotoxic effect against PC3 cells in vitro indicating a possible therapeutic advantage. The simplicity of the approach for generating targeted liposomes coupled with the ability to rapidly obtain tumor-specific phage fusion proteins via phage display may contribute to a combinatorial system for the production of targeted liposomal therapeutics for advanced stages of prostate tumor. PMID:20138246

  17. Enhanced Tumor Delivery and Antitumor Activity in Vivo of Liposomal Doxorubicin Modified with MCF-7-Specific Phage Fusion Protein

    PubMed Central

    Wang, Tao; Hartner, William C.; Gillespie, James W.; Praveen, Kulkarni P.; Yang, Shenghong; Mei, Leslie A.; Petrenko, Valery A.; Torchilin, Vladimir P.

    2013-01-01

    A novel strategy to improve the therapeutic index of chemotherapy has been developed by the integration of nanotechnology with phage technique. The objective of this study was to combine phage display, identifying tumor-targeting ligands, with a liposomal nanocarrier for targeted delivery of doxorubicin. Following the proof of concept in cell-based experiments, this study focused on in vivo assessment of antitumor activity and potential side-effects of phage fusion protein-modified liposomal doxorubicin. MCF-7-targeted phage-Doxil treatments led to greater tumor remission and faster onset of antitumor activity than the treatments with non-targeted formulations. The enhanced anticancer effect induced by the targeted phage-Doxil correlated with an improved tumor accumulation of doxorubicin. Tumor sections consistently revealed enhanced apoptosis, reduced proliferation activity and extensive necrosis. Phage-Doxil-treated mice did not show any sign of hepatotoxicity and maintained overall health. Therefore, MCF-7-targeted phage-Doxil seems to be an active and tolerable chemotherapy for breast cancer treatment. PMID:24028893

  18. Scaling Up: Adapting a Phage-Hunting Course to Increase Participation of First-Year Students in Research

    PubMed Central

    Staub, Nancy L.; Poxleitner, Marianne; Braley, Amanda; Smith-Flores, Helen; Pribbenow, Christine M.; Jaworski, Leslie; Lopatto, David; Anders, Kirk R.

    2016-01-01

    Authentic research experiences are valuable components of effective undergraduate education. Research experiences during the first years of college are especially critical to increase persistence in science, technology, engineering, and mathematics fields. The Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) model provides a high-impact research experience to first-year students but is usually available to a limited number of students, and its implementation is costly in faculty time and laboratory space. To offer a research experience to all students taking introductory biology at Gonzaga University (n = 350/yr), we modified the traditional two-semester SEA-PHAGES course by streamlining the first-semester Phage Discovery lab and integrating the second SEA-PHAGES semester into other courses in the biology curriculum. Because most students in the introductory course are not biology majors, the Phage Discovery semester may be their only encounter with research. To discover whether students benefit from the first semester alone, we assessed the effects of the one-semester Phage Discovery course on students’ understanding of course content. Specifically, students showed improvement in knowledge of bacteriophages, lab math skills, and understanding experimental design and interpretation. They also reported learning gains and benefits comparable with other course-based research experiences. Responses to open-ended questions suggest that students experienced this course as a true undergraduate research experience. PMID:27146160

  19. Quantitative Analysis of the Stability of Lysogenic State in Phage lambda

    NASA Astrophysics Data System (ADS)

    Ao, Ping

    2004-03-01

    mutations and parameter fluctuations. This talk is based on work done with L. Hood, C. Kwon, D.J. Thouless, L. Yin and X.-M. Zhu. References: 1. Zhu X-M, Yin L, Hood L, and Ao P; Calculating Biological Behaviors of Epigenetic States in Phage lambda Life Cycle, to appear in Functional and Integrative Genomics. 2. Ao P; Stochastic force defined evolution in dynamical systems. Submitted to Phys. Rev. Lett. (http://it/arXiv.org/find/physics/1/Ao/0/1/0/past/3/0)

  20. [Lytic phages and prophages of Streptococcus suis--a review].

    PubMed

    Tang, Fang; Lu, Chengping

    2015-04-01

    Streptococcus suis (S. suis) is an important zoonosis and pathogen that can carry prophages. In this review, we focus on the recent advances in our understanding of lytic phage and lysogenic phage of S. suis, including the morphology of S. suis lytic phage, the functions of lysin and terminase large subunit encoded by S. suis lytic phage, comparative genomics of S. suis prophages, lysogenic. conversion between S. suis lytic phage and prophage. Furthermore, prospective evolution of interactions between phage and host was discussed. PMID:26211312

  1. Self-assembling Phage-Quantum Dot Nanocomplexes for Quantitative Biodetection

    NASA Astrophysics Data System (ADS)

    Clarke, Matthew; Kang, Hyeonggon; Hwang, Jeeseong

    2010-03-01

    Colloidal quantum dots (QDs) have been used for many biodetection applications because of their brightness and broad spectral coverage in multiplexed approaches. QD surfaces can be functionalized for bio-conjugation to enable self-assembly with other nanomaterials and biomolecules using biological or bio-inspired processes. We demonstrate a model bacterial detection system using phage-QD nanocomplexes. To engineer the nanocomplexes, we genetically modified phage to express lysine residues on the capsid region, resulting in biotin labeling during replication inside the host cell. The biotinylated phages were conjugated with QDs and employed for detection. Bacteriophages have specificity to bacteria, enabling targeted detection of specific strains. Brightness of QDs enables high-throughput optical detection. The properties of nanocomplexes and detection limit/sensitivity were quantitatively evaluated using integrated differential interference contrast and fluorescence microscopy and automated image-based cytometry technique.

  2. Insights into the diversity of φRSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution.

    PubMed

    Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2014-08-01

    The filamentous φRSM phages (φRSM1 and φRSM3) have integration/excision capabilities in the phytopathogenic bacterium Ralstonia solanacearum. In the present study, we further investigated φRSM-like sequences present in the genomes of R. solanacearum strains belonging to the four major evolutionary lineages (phylotypes I-IV). Based on bioinformatics and comparative genomic analyses, we found that φRSM homologs are highly diverse in R. solanacearum complex strains. We detected an open reading frame (ORF)15 located upstream of the gene for φRSM integrase, which exhibited amino acid sequence similarity to phage repressor proteins. ORF15-encoded protein (a putative repressor) was found to encode a 104-residue polypeptide containing a DNA-binding (helix-turn-helix) domain and was expressed in R. solanacearum lysogenic strains. This suggested that φRSM3-ORF15 might be involved in the establishment and maintenance of a lysogenic state, as well as in phage immunity. Comparison of the putative repressor proteins and their binding sites within φRSM-related prophages provides insights into how these regulatory systems of filamentous phages have evolved and diverged in the R. solanacearum complex. In conclusion, φRSM phages represent a unique group of filamentous phages that are equipped with innate integration/excision (ORF14) and regulatory systems (ORF15). PMID:24619102

  3. Insights into the diversity of φRSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution.

    PubMed

    Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2014-08-01

    The filamentous φRSM phages (φRSM1 and φRSM3) have integration/excision capabilities in the phytopathogenic bacterium Ralstonia solanacearum. In the present study, we further investigated φRSM-like sequences present in the genomes of R. solanacearum strains belonging to the four major evolutionary lineages (phylotypes I-IV). Based on bioinformatics and comparative genomic analyses, we found that φRSM homologs are highly diverse in R. solanacearum complex strains. We detected an open reading frame (ORF)15 located upstream of the gene for φRSM integrase, which exhibited amino acid sequence similarity to phage repressor proteins. ORF15-encoded protein (a putative repressor) was found to encode a 104-residue polypeptide containing a DNA-binding (helix-turn-helix) domain and was expressed in R. solanacearum lysogenic strains. This suggested that φRSM3-ORF15 might be involved in the establishment and maintenance of a lysogenic state, as well as in phage immunity. Comparison of the putative repressor proteins and their binding sites within φRSM-related prophages provides insights into how these regulatory systems of filamentous phages have evolved and diverged in the R. solanacearum complex. In conclusion, φRSM phages represent a unique group of filamentous phages that are equipped with innate integration/excision (ORF14) and regulatory systems (ORF15).

  4. Antibody Phage Display Libraries: Contributions to Oncology

    PubMed Central

    Dantas-Barbosa, Carmela; de Macedo Brigido, Marcelo; Maranhao, Andrea Queiroz

    2012-01-01

    Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells’ surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I–III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials. PMID:22754305

  5. Targeting Enterococcus faecalis biofilms with phage therapy.

    PubMed

    Khalifa, Leron; Brosh, Yair; Gelman, Daniel; Coppenhagen-Glazer, Shunit; Beyth, Shaul; Poradosu-Cohen, Ronit; Que, Yok-Ai; Beyth, Nurit; Hazan, Ronen

    2015-04-01

    Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.

  6. Phage display of engineered binding proteins.

    PubMed

    Levisson, Mark; Spruijt, Ruud B; Winkel, Ingrid Nolla; Kengen, Servé W M; van der Oost, John

    2014-01-01

    In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

  7. Network models of phage-bacteria coevolution

    NASA Astrophysics Data System (ADS)

    Rosvall, Martin; Dodd, Ian B.; Krishna, Sandeep; Sneppen, Kim

    2006-12-01

    Bacteria and their bacteriophages are the most abundant, widespread, and diverse groups of biological entities on the planet. In an attempt to understand how the interactions between bacteria, virulent phages, and temperate phages might affect the diversity of these groups, we developed a stochastic network model for examining the coevolution of these ecologies. In our approach, nodes represent whole species or strains of bacteria or phages, rather than individuals, with “speciation” and extinction modeled by duplication and removal of nodes. Phage-bacteria links represent host-parasite relationships and temperate-virulent phage links denote prophage-encoded resistance. The effect of horizontal transfer of genetic information between strains was also included in the dynamical rules. The observed networks evolved in a highly dynamic fashion but the ecosystems were prone to collapse (one or more entire groups going extinct). Diversity could be stably maintained in the model only if the probability of speciation was independent of the diversity. Such an effect could be achieved in real ecosystems if the speciation rate is primarily set by the availability of ecological niches.

  8. The gastrointestinal phage communities of the cultivated freshwater fishes.

    PubMed

    He, Yang; Yang, Hongjiang

    2015-03-01

    The phage communities in the gut of 62 cultivated freshwater fish were investigated by culture-based methods. Using three selective media, 445 pathogenic bacilli strains were isolated and used as indicators for subsequent phage isolations. Totally, 63 phages were detected and the respective host strains were identified with the comparative sequence analysis of 16S rRNA gene, including Aeromonas (29), Vibrio (1), Citrobacter (16), Serratia (4), Enterobacter (2), Proteus (3), Buttiauxella (2), Plesiomonas (2), Kluyvera (1), Morgenella (2) and Providencia (1). The diversity of Aeromonas phages was assessed by discrimination of their host strains with random amplified polymorphic DNA method. Furthermore, the isolated Aeromonas phages were characterized by host range and growth inhibition assay. The results demonstrated that there were abundant and diverse phage populations in the gut environment of the cultivated freshwater fishes. The phages could contribute to the microbiota balance in the gut ecosystem of fishes and provide reliable phage sources for future applications.

  9. Phages of Listeria offer novel tools for diagnostics and biocontrol

    PubMed Central

    Hagens, Steven; Loessner, Martin J.

    2014-01-01

    Historically, bacteriophages infecting their hosts have perhaps been best known and even notorious for being a nuisance in dairy-fermentation processes. However, with the rapid progress in molecular microbiology and microbial ecology, a new dawn has risen for phages. This review will provide an overview on possible uses and applications of Listeria phages, including phage-typing, reporter phage for bacterial diagnostics, and use of phage as biocontrol agents for food safety. The use of phage-encoded enzymes such as endolysins for the detection and as antimicrobial agent will also be addressed. Desirable properties of candidate phages for biocontrol will be discussed. While emphasizing the enormous future potential for applications, we will also consider some of the intrinsic limitations dictated by both phage and bacterial ecology. PMID:24782847

  10. Aerosol Phage Therapy Efficacy in Burkholderia cepacia Complex Respiratory Infections

    PubMed Central

    Semler, Diana D.; Goudie, Amanda D.; Finlay, Warren H.

    2014-01-01

    Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria. PMID:24798268

  11. The gastrointestinal phage communities of the cultivated freshwater fishes.

    PubMed

    He, Yang; Yang, Hongjiang

    2015-03-01

    The phage communities in the gut of 62 cultivated freshwater fish were investigated by culture-based methods. Using three selective media, 445 pathogenic bacilli strains were isolated and used as indicators for subsequent phage isolations. Totally, 63 phages were detected and the respective host strains were identified with the comparative sequence analysis of 16S rRNA gene, including Aeromonas (29), Vibrio (1), Citrobacter (16), Serratia (4), Enterobacter (2), Proteus (3), Buttiauxella (2), Plesiomonas (2), Kluyvera (1), Morgenella (2) and Providencia (1). The diversity of Aeromonas phages was assessed by discrimination of their host strains with random amplified polymorphic DNA method. Furthermore, the isolated Aeromonas phages were characterized by host range and growth inhibition assay. The results demonstrated that there were abundant and diverse phage populations in the gut environment of the cultivated freshwater fishes. The phages could contribute to the microbiota balance in the gut ecosystem of fishes and provide reliable phage sources for future applications. PMID:25743067

  12. Current taxonomy of phages infecting lactic acid bacteria

    PubMed Central

    Mahony, Jennifer; van Sinderen, Douwe

    2013-01-01

    Phages infecting lactic acid bacteria have been the focus of significant research attention over the past three decades. Through the isolation and characterization of hundreds of phage isolates, it has been possible to classify phages of the dairy starter and adjunct bacteria Lactococus lactis, Streptococcus thermophilus, Leuconostoc spp., and Lactobacillus spp. Among these, phages of L. lactis have been most thoroughly scrutinized and serve as an excellent model system to address issues that arise when attempting taxonomic classification of phages infecting other LAB species. Here, we present an overview of the current taxonomy of phages infecting LAB genera of industrial significance, the methods employed in these taxonomic efforts and how these may be employed for the taxonomy of phages of currently underrepresented and emerging phage species. PMID:24478767

  13. Phage therapy in the food industry.

    PubMed

    Endersen, Lorraine; O'Mahony, Jim; Hill, Colin; Ross, R Paul; McAuliffe, Olivia; Coffey, Aidan

    2014-01-01

    Despite advances in modern technologies, the food industry is continuously challenged with the threat of microbial contamination. The overuse of antibiotics has further escalated this problem, resulting in the increasing emergence of antibiotic-resistant foodborne pathogens. Efforts to develop new methods for controlling microbial contamination in food and the food processing environment are extremely important. Accordingly, bacteriophages (phages) and their derivatives have emerged as novel, viable, and safe options for the prevention, treatment, and/or eradication of these contaminants in a range of foods and food processing environments. Whole phages, modified phages, and their derivatives are discussed in terms of current uses and future potential as antimicrobials in the traditional farm-to-fork context, encompassing areas such as primary production, postharvest processing, biosanitation, and biodetection. The review also presents some safety concerns to ensure safe and effective exploitation of bacteriophages in the future.

  14. Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei.

    PubMed

    Mercanti, Diego J; Rousseau, Geneviève M; Capra, María L; Quiberoni, Andrea; Tremblay, Denise M; Labrie, Simon J; Moineau, Sylvain

    2016-01-01

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

  15. Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

    PubMed Central

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

    2015-01-01

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

  16. The Caulobacter crescentus phage phiCbK: genomics of a canonical phage

    PubMed Central

    2012-01-01

    Background The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives. Results Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK) to 280 kb encoding 448 proteins (CcrColossus), and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell’s replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level. Conclusions Due to their lack of any apparent relationship to other described phages, this

  17. Development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study - Cumin seed.

    PubMed

    Siow, Hwee-Leng; Lim, Theam Soon; Gan, Chee-Yuen

    2017-01-01

    The main objective of this study was to develop an efficient workflow to discover α-amylase inhibitory peptides from cumin seed. A total of 56 unknown peptides was initially found in the cumin seed protein hydrolysate. They were subjected to 2 different in silico screenings and 6 peptides were shortlisted. The peptides were then subjected to in vitro selection using phage display technique and 3 clones (CSP3, CSP4 and CSP6) showed high affinity in binding α-amylase. These clones were subjected to the inhibitory test and only CSP4 and CSP6 exhibited high inhibitory activity. Therefore, these peptides were chemically synthesized for validation purposes. CSP4 exhibited inhibition of bacterial and human salivary α-amylases with IC50 values of 0.11 and 0.04μmol, respectively, whereas CSP6 was about 0.10 and 0.15μmol, respectively. Results showed that the strength of each protocol has been successfully combined as deemed fit to enhance the α-amylase inhibitor peptide discovery.

  18. Development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study - Cumin seed.

    PubMed

    Siow, Hwee-Leng; Lim, Theam Soon; Gan, Chee-Yuen

    2017-01-01

    The main objective of this study was to develop an efficient workflow to discover α-amylase inhibitory peptides from cumin seed. A total of 56 unknown peptides was initially found in the cumin seed protein hydrolysate. They were subjected to 2 different in silico screenings and 6 peptides were shortlisted. The peptides were then subjected to in vitro selection using phage display technique and 3 clones (CSP3, CSP4 and CSP6) showed high affinity in binding α-amylase. These clones were subjected to the inhibitory test and only CSP4 and CSP6 exhibited high inhibitory activity. Therefore, these peptides were chemically synthesized for validation purposes. CSP4 exhibited inhibition of bacterial and human salivary α-amylases with IC50 values of 0.11 and 0.04μmol, respectively, whereas CSP6 was about 0.10 and 0.15μmol, respectively. Results showed that the strength of each protocol has been successfully combined as deemed fit to enhance the α-amylase inhibitor peptide discovery. PMID:27507449

  19. HostPhinder: A Phage Host Prediction Tool.

    PubMed

    Villarroel, Julia; Kleinheinz, Kortine Annina; Jurtz, Vanessa Isabell; Zschach, Henrike; Lund, Ole; Nielsen, Morten; Larsen, Mette Voldby

    2016-05-04

    The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within reach. Here, we present HostPhinder, a tool aimed at predicting the bacterial host of phages by examining the phage genome sequence. Using a reference database of 2196 phages with known hosts, HostPhinder predicts the host species of a query phage as the host of the most genomically similar reference phages. As a measure of genomic similarity the number of co-occurring k-mers (DNA sequences of length k) is used. Using an independent evaluation set, HostPhinder was able to correctly predict host genus and species for 81% and 74% of the phages respectively, giving predictions for more phages than BLAST and significantly outperforming BLAST on phages for which both had predictions. HostPhinder predictions on phage draft genomes from the INTESTI phage cocktail corresponded well with the advertised targets of the cocktail. Our study indicates that for most phages genomic similarity correlates well with related bacterial hosts. HostPhinder is available as an interactive web service [1] and as a stand alone download from the Docker registry [2].

  20. Genome Sequences of Gordonia Phages Bowser and Schwabeltier

    PubMed Central

    Montgomery, Matthew T.; Arnold, Zachary M.; Basina, Aleksandra; Iyer, Ankitha M.; Stoner, Ty H.; Kasturiarachi, Naomi S.; Pressimone, Catherine A.; Schiebel, Johnathon G.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    Gordonia phages Bowser and Schwabeltier are newly isolated phages infecting Gordonia terrae 3612. Bowser and Schwabeltier have similar siphoviral morphologies and their genomes are related to each other, but not to other phages. Their lysis cassettes are atypically situated among virion tail genes, and Bowser encodes two tyrosine integrases. PMID:27516498

  1. HostPhinder: A Phage Host Prediction Tool.

    PubMed

    Villarroel, Julia; Kleinheinz, Kortine Annina; Jurtz, Vanessa Isabell; Zschach, Henrike; Lund, Ole; Nielsen, Morten; Larsen, Mette Voldby

    2016-01-01

    The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within reach. Here, we present HostPhinder, a tool aimed at predicting the bacterial host of phages by examining the phage genome sequence. Using a reference database of 2196 phages with known hosts, HostPhinder predicts the host species of a query phage as the host of the most genomically similar reference phages. As a measure of genomic similarity the number of co-occurring k-mers (DNA sequences of length k) is used. Using an independent evaluation set, HostPhinder was able to correctly predict host genus and species for 81% and 74% of the phages respectively, giving predictions for more phages than BLAST and significantly outperforming BLAST on phages for which both had predictions. HostPhinder predictions on phage draft genomes from the INTESTI phage cocktail corresponded well with the advertised targets of the cocktail. Our study indicates that for most phages genomic similarity correlates well with related bacterial hosts. HostPhinder is available as an interactive web service [1] and as a stand alone download from the Docker registry [2]. PMID:27153081

  2. Genome Sequences of Gordonia Phages Bowser and Schwabeltier.

    PubMed

    Montgomery, Matthew T; Pope, Welkin H; Arnold, Zachary M; Basina, Aleksandra; Iyer, Ankitha M; Stoner, Ty H; Kasturiarachi, Naomi S; Pressimone, Catherine A; Schiebel, Johnathon G; Furbee, Emily C; Grubb, Sarah R; Warner, Marcie H; Garlena, Rebecca A; Russell, Daniel A; Jacobs-Sera, Deborah; Hatfull, Graham F

    2016-01-01

    Gordonia phages Bowser and Schwabeltier are newly isolated phages infecting Gordonia terrae 3612. Bowser and Schwabeltier have similar siphoviral morphologies and their genomes are related to each other, but not to other phages. Their lysis cassettes are atypically situated among virion tail genes, and Bowser encodes two tyrosine integrases. PMID:27516498

  3. Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1

    PubMed Central

    Andrade-Domínguez, Andrés

    2016-01-01

    Aspects of the interaction between phages and animals are of interest and importance for medical applications. Here, we report the genome sequence of the lytic Pseudomonas phage AAT-1, isolated from mammalian serum. AAT-1 is a double-stranded DNA phage, with a genome of 57,599 bp, containing 76 predicted open reading frames. PMID:27563032

  4. Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1.

    PubMed

    Andrade-Domínguez, Andrés; Kolter, Roberto

    2016-01-01

    Aspects of the interaction between phages and animals are of interest and importance for medical applications. Here, we report the genome sequence of the lytic Pseudomonas phage AAT-1, isolated from mammalian serum. AAT-1 is a double-stranded DNA phage, with a genome of 57,599 bp, containing 76 predicted open reading frames. PMID:27563032

  5. Vi I typing phage for generalized transduction of Salmonella typhi.

    PubMed

    Cerquetti, M C; Hooke, A M

    1993-08-01

    Salmonella typhi Vi typing phages were used to transduce temperature-sensitive (Ts) mutants of Salmonella typhi. Antibiotic resistance and Ts+ markers were transduced at high frequency (> 10(-4) per virulent phage). Several markers were cotransduced by phage Vi I, suggesting that it may be useful for mapping studies of the S. typhi genome.

  6. HostPhinder: A Phage Host Prediction Tool

    PubMed Central

    Villarroel, Julia; Kleinheinz, Kortine Annina; Jurtz, Vanessa Isabell; Zschach, Henrike; Lund, Ole; Nielsen, Morten; Larsen, Mette Voldby

    2016-01-01

    The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within reach. Here, we present HostPhinder, a tool aimed at predicting the bacterial host of phages by examining the phage genome sequence. Using a reference database of 2196 phages with known hosts, HostPhinder predicts the host species of a query phage as the host of the most genomically similar reference phages. As a measure of genomic similarity the number of co-occurring k-mers (DNA sequences of length k) is used. Using an independent evaluation set, HostPhinder was able to correctly predict host genus and species for 81% and 74% of the phages respectively, giving predictions for more phages than BLAST and significantly outperforming BLAST on phages for which both had predictions. HostPhinder predictions on phage draft genomes from the INTESTI phage cocktail corresponded well with the advertised targets of the cocktail. Our study indicates that for most phages genomic similarity correlates well with related bacterial hosts. HostPhinder is available as an interactive web service [1] and as a stand alone download from the Docker registry [2]. PMID:27153081

  7. Cell biology perspectives in phage biology.

    PubMed

    Ansaldi, Mireille

    2012-01-01

    Cellular biology has long been restricted to large cellular organisms. However, as the resolution of microscopic methods increased, it became possible to study smaller cells, in particular bacterial cells. Bacteriophage biology is one aspect of bacterial cell biology that has recently gained insight from cell biology. Despite their small size, bacteriophages could be successfully labeled and their cycle studied in the host cells. This review aims to put together, although non-extensively, several cell biology studies that recently pushed the elucidation of key mechanisms in phage biology, such as the lysis-lysogeny decision in temperate phages or genome replication and transcription, one step further.

  8. Tolerance of a phage element by Streptococcus pneumoniae leads to a fitness defect during colonization.

    PubMed

    DeBardeleben, Hilary K; Lysenko, Elena S; Dalia, Ankur B; Weiser, Jeffrey N

    2014-07-01

    The pathogenesis of the disease caused by Streptococcus pneumoniae begins with colonization of the upper respiratory tract. Temperate phages have been identified in the genomes of up to 70% of clinical isolates. How these phages affect the bacterial host during colonization is unknown. Here, we examined a clinical isolate that carries a novel prophage element, designated Spn1, which was detected in both integrated and episomal forms. Surprisingly, both lytic and lysogenic Spn1 genes were expressed under routine growth conditions. Using a mouse model of asymptomatic colonization, we demonstrate that the Spn1(-) strain outcompeted the Spn1(+) strain >70-fold. To determine if Spn1 causes a fitness defect through a trans-acting factor, we constructed an Spn1(+) mutant that does not become an episome or express phage genes. This mutant competed equally with the Spn1(-) strain, indicating that expression of phage genes or phage lytic activity is required to confer this fitness defect. In vitro, we demonstrate that the presence of Spn1 correlated with a defect in LytA-mediated autolysis. Furthermore, the Spn1(+) strain displayed increased chain length and resistance to lysis by penicillin compared to the Spn(-) strain, indicating that Spn1 alters the cell wall physiology of its host strain. We posit that these changes in cell wall physiology allow for tolerance of phage gene products and are responsible for the relative defect of the Spn1(+) strain during colonization. This study provides new insight into how bacteria and prophages interact and affect bacterial fitness in vivo. PMID:24816604

  9. Tolerance of a Phage Element by Streptococcus pneumoniae Leads to a Fitness Defect during Colonization

    PubMed Central

    DeBardeleben, Hilary K.; Lysenko, Elena S.; Dalia, Ankur B.

    2014-01-01

    The pathogenesis of the disease caused by Streptococcus pneumoniae begins with colonization of the upper respiratory tract. Temperate phages have been identified in the genomes of up to 70% of clinical isolates. How these phages affect the bacterial host during colonization is unknown. Here, we examined a clinical isolate that carries a novel prophage element, designated Spn1, which was detected in both integrated and episomal forms. Surprisingly, both lytic and lysogenic Spn1 genes were expressed under routine growth conditions. Using a mouse model of asymptomatic colonization, we demonstrate that the Spn1− strain outcompeted the Spn1+ strain >70-fold. To determine if Spn1 causes a fitness defect through a trans-acting factor, we constructed an Spn1+ mutant that does not become an episome or express phage genes. This mutant competed equally with the Spn1− strain, indicating that expression of phage genes or phage lytic activity is required to confer this fitness defect. In vitro, we demonstrate that the presence of Spn1 correlated with a defect in LytA-mediated autolysis. Furthermore, the Spn1+ strain displayed increased chain length and resistance to lysis by penicillin compared to the Spn− strain, indicating that Spn1 alters the cell wall physiology of its host strain. We posit that these changes in cell wall physiology allow for tolerance of phage gene products and are responsible for the relative defect of the Spn1+ strain during colonization. This study provides new insight into how bacteria and prophages interact and affect bacterial fitness in vivo. PMID:24816604

  10. Genomic sequence of temperate phage Smp131 of Stenotrophomonas maltophilia that has similar prophages in xanthomonads

    PubMed Central

    2014-01-01

    Background Stenotrophomonas maltophilia is a ubiquitous Gram-negative bacterium previously named as Xanthomonas maltophilia. This organism is an important nosocomial pathogen associated with infections in immunocompromised patients. Clinical isolates of S. maltophilia are mostly resistant to multiple antibiotics and treatment of its infections is becoming problematic. Several virulent bacteriophages, but not temperate phage, of S. maltophilia have been characterized. Results In this study, a temperate myophage of S. maltophilia (Smp131) was isolated and characterized. Sequence analysis showed that its genome is 33,525-bp long with 47 open reading frames (ORFs). Its similarity to P2-like phages and prophages in S. maltophilia and several Xanthomonas pathovars includes genomic organization, arrangement of several operons, and possession of a slippery sequence T7G for translational frameshifting in tail assembly genes. Smp131 encodes a tyrosine family integrase that shares low degrees of similarity with those of other phages and a lysin belonging to family 19 chitinase that is observed in plants and some bacteria, although not in phages. tRNA are the preferred sites for host integration of Smp131 and the related phages: tRNA-Thr for Smp131 and prophage of S. maltophilia K279a; tRNA-Lys for prophages of X. campestris pv. campestris ATCC33913, X. oryzae pv. oryzae strains MAFF311018, and KACC10331; and tRNA-Asn for prophage of X. oryzae pv. oryzae PXO99A and remnant of X. axonopodis pv. citri 306. Regions flanking the prophages are varied highly in nucleotide sequence and rich in transposase genes, suggesting that frequent insertion/excision had occurred. Conclusions Prevalence of closely related prophages in Stenotrophomonas and Xanthomonads may have contributed to the diversity of these closely related species owing to possible horizontal gene transfer mediated by the phages. PMID:24472137

  11. Vibrio cholerae phage K139: complete genome sequence and comparative genomics of related phages.

    PubMed

    Kapfhammer, Dagmar; Blass, Julia; Evers, Stefan; Reidl, Joachim

    2002-12-01

    In this report, we characterize the complete genome sequence of the temperate phage K139, which morphologically belongs to the Myoviridae phage family (P2 and 186). The prophage genome consists of 33,106 bp, and the overall GC content is 48.9%. Forty-four open reading frames were identified. Homology analysis and motif search were used to assign possible functions for the genes, revealing a close relationship to P2-like phages. By Southern blot screening of a Vibrio cholerae strain collection, two highly K139-related phage sequences were detected in non-O1, non-O139 strains. Combinatorial PCR analysis revealed almost identical genome organizations. One region of variable gene content was identified and sequenced. Additionally, the tail fiber genes were analyzed, leading to the identification of putative host-specific sequence variations. Furthermore, a K139-encoded Dam methyltransferase was characterized.

  12. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies

    PubMed Central

    Mu, Xihui; Tong, Zhaoyang; Huang, Qibin; Liu, Bing; Liu, Zhiwei; Hao, Lanqun; Dong, Hua; Zhang, Jinping; Gao, Chuan

    2016-01-01

    Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL) technique, Staphylococcus protein A (SPA) functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb) as magnetic capturing probes, and Ru(bpy)32+-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD) was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy)32+-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy)32+-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility. PMID:26927130

  13. Phage & phosphatase: a novel phage-based probe for rapid, multi-platform detection of bacteria.

    PubMed

    Alcaine, S D; Pacitto, D; Sela, D A; Nugen, S R

    2015-11-21

    Genetic engineering of bacteriophages allows for the development of rapid, highly specific, and easily manufactured probes for the detection of bacterial pathogens. A challenge for novel probes is the ease of their adoption in real world laboratories. We have engineered the bacteriophage T7, which targets Escherichia coli, to carry the alkaline phosphatase gene, phoA. This inclusion results in phoA overexpression following phage infection of E. coli. Alkaline phosphatase is commonly used in a wide range of diagnostics, and thus a signal produced by our phage-based probe could be detected using common laboratory equipment. Our work demonstrates the successful: (i) modification of T7 phage to carry phoA; (ii) overexpression of alkaline phosphatase in E. coli; and (iii) detection of this T7-induced alkaline phosphatase activity using commercially available colorimetric and chemilumiscent methods. Furthermore, we demonstrate the application of our phage-based probe to rapidly detect low levels of bacteria and discern the antibiotic resistance of E. coli isolates. Using our bioengineered phage-based probe we were able to detect 10(3) CFU per mL of E. coli in 6 hours using a chemiluminescent substrate and 10(4) CFU per mL within 7.5 hours using a colorimetric substrate. We also show the application of this phage-based probe for antibiotic resistance testing. We were able to determine whether an E. coli isolate was resistant to ampicillin within 4.5 hours using chemiluminescent substrate and within 6 hours using a colorimetric substrate. This phage-based scheme could be readily adopted in labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection.

  14. Genome Sequence of Gordonia Phage Emalyn

    PubMed Central

    Guido, Madeline J.; Iyengar, Pragnya; Nigra, Jonathan T.; Serbin, Matthew B.; Kasturiarachi, Naomi S.; Pressimone, Catherine A.; Schiebel, Johnathon G.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Montgomery, Matthew T.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    Emalyn is a newly isolated bacteriophage of Gordonia terrae 3612 and has a double-stranded DNA genome 43,982 bp long with 67 predicted protein-encoding genes, 32 of which we can assign putative functions. Emalyn has a prolate capsid and has extensive nucleotide similarity with several previously sequenced phages. PMID:27516499

  15. Phage therapies for plants and people.

    PubMed

    Gross, Michael

    2014-06-16

    The use of bacteriophages to combat bacterial infections may help to address the current crisis of antibiotic resistance. Fundamental issues arising from the ecological dynamic of host, bacterium and phage can be investigated in trees, offering both a natural approach to treating plant disease, and a chance to avoid creating a new resistance problem. Michael Gross reports. PMID:25075391

  16. Genome Sequence of Gordonia Phage Emalyn.

    PubMed

    Pope, Welkin H; Guido, Madeline J; Iyengar, Pragnya; Nigra, Jonathan T; Serbin, Matthew B; Kasturiarachi, Naomi S; Pressimone, Catherine A; Schiebel, Johnathon G; Furbee, Emily C; Grubb, Sarah R; Warner, Marcie H; Montgomery, Matthew T; Garlena, Rebecca A; Russell, Daniel A; Jacobs-Sera, Deborah; Hatfull, Graham F

    2016-01-01

    Emalyn is a newly isolated bacteriophage of Gordonia terrae 3612 and has a double-stranded DNA genome 43,982 bp long with 67 predicted protein-encoding genes, 32 of which we can assign putative functions. Emalyn has a prolate capsid and has extensive nucleotide similarity with several previously sequenced phages. PMID:27516499

  17. Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages.

    PubMed

    Brüssow, H; Desiere, F

    2001-01-01

    Comparative phage genomics can retrace part of the evolutionary history of phage modules encoding phage-specific functions such as capsid building or establishment of the lysogenic state. The diagnosis of relatedness is not based exclusively on sequence similarity, but includes topological considerations of genome organization. The gene maps from the lambda-, psiM2-, L5-, Sfi21-, Sfi11-, phiC31-, sk1- and TM4-like phages showed a remarkable synteny of their structural genes defining a lambda supergroup within Siphoviridae (Caudovirales with long non-contractile tails). A hierarchy of relatedness within the lambda supergroup suggested elements of vertical evolution in the capsid module of Siphoviridae. Links to P22-like Podoviridae and P2-like Myoviridae were also detected. Numerous cases of horizontal gene transfer were observed, but recent transfers were limited to interbreeding phage populations. We suggest that tailed phages are the result of both vertical and horizontal evolution and are thus a good model system for web-like phylogenies.

  18. Characterization and adsorption of Lactobacillus virulent phage P1.

    PubMed

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

    2016-09-01

    Bacteriophage infection of lactic acid bacteria is considered an important problem worldwide in the food fermentation industry, as it may produce low quality or unsafe foods, cause fermentation failure, and result in economic losses. To increase current knowledge on the properties of Lactobacillus virulent phages, we evaluated the effect of divalent cations, temperature, pH, and chloramphenicol on the adsorption ability of Lactobacillus virulent phage P1. Phage P1 was isolated from the abnormal fermentation liquid of Lactobacillus plantarum IMAU10120. The results showed that this phage belonged to the Siphoviridae family. The latent period of this phage was 45min, and the burst time was 90min. Burst size was 132.88±2.37 phage counts expressed per milliliter per infective center. This phage showed good tolerance at different temperatures, but incubation at 50°C only affected its adsorption. Adsorption rate reached a maximum value between 30 and 42°C. A high adsorption value of phage infectivity was obtained from pH 6 to 8. Moreover, calcium ions promoted and increased the adsorption capacity of phage P1, but magnesium ions had negative effects. Chloramphenicol had no effect on phage adsorption. This study increased current knowledge on the characterization and biological aspects of Lactobacillus virulent phages, and may provide some basic information that can be used to design successful antiphage strategies in the food industry. PMID:27372579

  19. Phage-bacteria interaction network in human oral microbiome.

    PubMed

    Wang, Jinfeng; Gao, Yuan; Zhao, Fangqing

    2016-07-01

    Although increasing knowledge suggests that bacteriophages play important roles in regulating microbial ecosystems, phage-bacteria interaction in human oral cavities remains less understood. Here we performed a metagenomic analysis to explore the composition and variation of oral dsDNA phage populations and potential phage-bacteria interaction. A total of 1,711 contigs assembled with more than 100 Gb shotgun sequencing data were annotated to 104 phages based on their best BLAST matches against the NR database. Bray-Curtis dissimilarities demonstrated that both phage and bacterial composition are highly diverse between periodontally healthy samples but show a trend towards homogenization in diseased gingivae samples. Significantly, according to the CRISPR arrays that record infection relationship between bacteria and phage, we found certain oral phages were able to invade other bacteria besides their putative bacterial hosts. These cross-infective phages were positively correlated with commensal bacteria while were negatively correlated with major periodontal pathogens, suggesting possible connection between these phages and microbial community structure in oral cavities. By characterizing phage-bacteria interaction as networks rather than exclusively pairwise predator-prey relationships, our study provides the first insight into the participation of cross-infective phages in forming human oral microbiota.

  20. Properties and genomic analysis of Lactococcus garvieae lysogenic bacteriophage PLgT-1, a new member of Siphoviridae, with homology to Lactococcus lactis phages.

    PubMed

    Hoai, Truong Dinh; Nishiki, Issei; Yoshida, Terutoyo

    2016-08-15

    The lysogenic phage PLgT-1 is highly prevalent in Lactococcus garvieae, which is a serious bacterial pathogen in marine fish. Therefore, information regarding this phage is one of the key factors to predict the evolution of this bacterium. However, many properties of this phage, its complete genome sequence, and its relationship with other viral communities has not been investigated to date. Here, we demonstrated that the phage PLgT-1 was not only induced by an induction agent (Mitomycin C), but could be released frequently during cell division in a nutrient-rich environment or in natural seawater. Integration of PLgT-1 into non-lysogenic bacteria via transduction changed the genotype, resulting in the diversification of L. garvieae. The complete DNA sequence of PLgT-1 was also determined. This phage has a dsDNA genome of 40,273bp with 66 open reading frames (ORFs). Of these, the biological functions of 24 ORFs could be predicted but those of 42 ORFs are unknown. Thus, PLgT-1 is a novel phage with several novel proteins encoded in its genome. The strict MegaBLAST search program for the PLgT-1 genome revealed that this phage had no similarities with other previously investigated phages specific to L. garvieae (WP-2 and GE1). Notably, PLgT-1 was relatively homologous with several phages of Lactococcus lactis and 17 of the 24 predicted proteins encoded in PLgT-1 were homologous with the deduced proteins of various phages from these dairy bacteria. Comparative genome analysis revealed that the L. garvieae phage PLgT-1 was most closely related to the L. lactis phage TP712. However, they differed from each other in genome size and gene arrangement. The results obtained in this study suggest that the lysogenic phage PLgT-1 is a new member of the family Siphoviridae and has been involved in horizontal gene exchange with microbial communities, especially with L. lactis and its phages. PMID:27234995

  1. Twelve previously unknown phage genera are ubiquitous in global oceans

    SciTech Connect

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

    2013-01-01

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

  2. Twelve previously unknown phage genera are ubiquitous in global oceans.

    PubMed

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

    2013-07-30

    Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing. PMID:23858439

  3. Engineering resistance to phage GVE3 in Geobacillus thermoglucosidasius.

    PubMed

    van Zyl, Leonardo Joaquim; Taylor, Mark Paul; Trindade, Marla

    2016-02-01

    Geobacillus thermoglucosidasius is a promising platform organism for the production of biofuels and other metabolites of interest. G. thermoglucosidasius fermentations could be subject to bacteriophage-related failure and financial loss. We develop two strains resistant to a recently described G. thermoglucosidasius-infecting phage GVE3. The phage-encoded immunity gene, imm, was overexpressed in the host leading to phage resistance. A phage-resistant mutant was isolated following expression of a putative anti-repressor-like protein and phage challenge. A point mutation was identified in the polysaccharide pyruvyl transferase, csaB. A double crossover knockout mutation of csaB confirmed its role in the phage resistance phenotype. These resistance mechanisms appear to prevent phage DNA injection and/or lysogenic conversion rather than just reducing efficiency of plating, as no phage DNA could be detected in resistant bacteria challenged with GVE3 and no plaques observed even at high phage titers. Not only do the strains developed here shed light on the biological relationship between the GVE3 phage and its host, they could be employed by those looking to make use of this organism for metabolite production, with reduced occurrence of GVE3-related failure.

  4. Convergent evolution of pathogenicity islands in helper cos phage interference.

    PubMed

    Carpena, Nuria; Manning, Keith A; Dokland, Terje; Marina, Alberto; Penadés, José R

    2016-11-01

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution.This article is part of the themed issue 'The new bacteriology'.

  5. Engineering resistance to phage GVE3 in Geobacillus thermoglucosidasius.

    PubMed

    van Zyl, Leonardo Joaquim; Taylor, Mark Paul; Trindade, Marla

    2016-02-01

    Geobacillus thermoglucosidasius is a promising platform organism for the production of biofuels and other metabolites of interest. G. thermoglucosidasius fermentations could be subject to bacteriophage-related failure and financial loss. We develop two strains resistant to a recently described G. thermoglucosidasius-infecting phage GVE3. The phage-encoded immunity gene, imm, was overexpressed in the host leading to phage resistance. A phage-resistant mutant was isolated following expression of a putative anti-repressor-like protein and phage challenge. A point mutation was identified in the polysaccharide pyruvyl transferase, csaB. A double crossover knockout mutation of csaB confirmed its role in the phage resistance phenotype. These resistance mechanisms appear to prevent phage DNA injection and/or lysogenic conversion rather than just reducing efficiency of plating, as no phage DNA could be detected in resistant bacteria challenged with GVE3 and no plaques observed even at high phage titers. Not only do the strains developed here shed light on the biological relationship between the GVE3 phage and its host, they could be employed by those looking to make use of this organism for metabolite production, with reduced occurrence of GVE3-related failure. PMID:26536875

  6. Convergent evolution of pathogenicity islands in helper cos phage interference

    PubMed Central

    Manning, Keith A.; Dokland, Terje; Marina, Alberto

    2016-01-01

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution. This article is part of the themed issue ‘The new bacteriology’. PMID:27672154

  7. Convergent evolution of pathogenicity islands in helper cos phage interference.

    PubMed

    Carpena, Nuria; Manning, Keith A; Dokland, Terje; Marina, Alberto; Penadés, José R

    2016-11-01

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution.This article is part of the themed issue 'The new bacteriology'. PMID:27672154

  8. Efficacy of two Staphylococcus aureus phage cocktails in cheese production.

    PubMed

    El Haddad, Lynn; Roy, Jean-Pierre; Khalil, Georges E; St-Gelais, Daniel; Champagne, Claude P; Labrie, Steve; Moineau, Sylvain

    2016-01-18

    Staphylococcus aureus is one of the most prevalent pathogenic bacteria contaminating dairy products. In an effort to reduce food safety risks, virulent phages are investigated as antibacterial agents to control foodborne pathogens. The aim of this study was to compare sets of virulent phages, design phage cocktails, and use them in a cocktail to control pathogenic staphylococci in cheese. Six selected phages belonging to the three Caudovirales families (Myoviridae, Siphoviridae, Podoviridae) were strictly lytic, had a broad host range, and did not carry genes coding for virulence traits in their genomes. However, they were sensitive to pasteurization. At MOI levels of 15, 45, and 150, two anti-S. aureus phage cocktails, each containing three phages, one from each of the three phage families, eradicated a 10(6)CFU/g S. aureus population after 14 days of Cheddar cheese curd ripening at 4°C. The use of these phages did not trigger over-production of S. aureus enterotoxin C. The use of phage cocktails and their rotation may prevent the emergence of phage resistant bacterial strains. PMID:26476571

  9. Efficacy of two Staphylococcus aureus phage cocktails in cheese production.

    PubMed

    El Haddad, Lynn; Roy, Jean-Pierre; Khalil, Georges E; St-Gelais, Daniel; Champagne, Claude P; Labrie, Steve; Moineau, Sylvain

    2016-01-18

    Staphylococcus aureus is one of the most prevalent pathogenic bacteria contaminating dairy products. In an effort to reduce food safety risks, virulent phages are investigated as antibacterial agents to control foodborne pathogens. The aim of this study was to compare sets of virulent phages, design phage cocktails, and use them in a cocktail to control pathogenic staphylococci in cheese. Six selected phages belonging to the three Caudovirales families (Myoviridae, Siphoviridae, Podoviridae) were strictly lytic, had a broad host range, and did not carry genes coding for virulence traits in their genomes. However, they were sensitive to pasteurization. At MOI levels of 15, 45, and 150, two anti-S. aureus phage cocktails, each containing three phages, one from each of the three phage families, eradicated a 10(6)CFU/g S. aureus population after 14 days of Cheddar cheese curd ripening at 4°C. The use of these phages did not trigger over-production of S. aureus enterotoxin C. The use of phage cocktails and their rotation may prevent the emergence of phage resistant bacterial strains.

  10. Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting.

    PubMed

    Goldberg, Gregory W; Jiang, Wenyan; Bikard, David; Marraffini, Luciano A

    2014-10-30

    A fundamental feature of immune systems is the ability to distinguish pathogenic from self and commensal elements, and to attack the former but tolerate the latter. Prokaryotic CRISPR-Cas immune systems defend against phage infection by using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome. Temperate phages include viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity both to lytic and lysogenic infection by temperate phages-compromising the genetic stability of these potentially beneficial elements altogether. Here we show that the Staphylococcus epidermidis CRISPR-Cas system can prevent lytic infection but tolerate lysogenization by temperate phages. Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle. Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity. In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity. PMID:25174707

  11. Therapeutic effect of Pseudomonas aeruginosa phage YH30 on mink hemorrhagic pneumonia.

    PubMed

    Gu, Jingmin; Li, Xinwei; Yang, Mei; Du, Chongtao; Cui, Ziyin; Gong, Pengjuan; Xia, Feifei; Song, Jun; Zhang, Lei; Li, Juecheng; Yu, Chuang; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Han, Wenyu

    2016-07-15

    Hemorrhagic pneumonia caused by Pseudomonas aeruginosa remains one of the most costly infectious diseases among farmed mink and commonly leads to large economic losses during mink production. The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink. A broad-host-range phage from the Podoviridae family, YH30, was isolated using the mink-originating P. aeruginosa (serotype G) D7 strain as a host. The genome of YH30 was 72,192bp (54.92% G+C), contained 86 open reading frames and lacked regions encoding known virulence factors, integration-related proteins or antibiotic resistance determinants. These characteristics make YH30 eligible for use in phage therapy. The results of a curative treatment experiment demonstrated that a single intranasal administration of YH30 was sufficient to cure hemorrhagic pneumonia in mink. The mean colony count of P. aeruginosa in the blood and lung of YH30-protected mink was less than 10(3) CFU/mL (g) within 24h of bacterial challenge and ultimately became undetectable, whereas that in unprotected mink reached more than 10(8) CFU/mL (g). Additionally, YH30 dramatically improved the pathological manifestations of lung injury in mink with hemorrhagic pneumonia. Our work demonstrates the potential of phages to treat P. aeruginosa-caused hemorrhagic pneumonia in mink. PMID:27283850

  12. Twelve previously unknown phage genera are ubiquitous in global oceans

    PubMed Central

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

    2013-01-01

    Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in “unknowns” dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage–host systems for experimental hypothesis testing. PMID:23858439

  13. Indirect Ultraviolet-Reactivation of Phage λ

    PubMed Central

    George, Jacqueline; Devoret, Raymond; Radman, Miroslav

    1974-01-01

    When an F- recipient Escherichia coli K12 bacterium receives Hfr or F-lac+ DNA from an ultraviolet-irradiated donor, its capacity to promote DNA repair and mutagenesis of ultraviolet-damaged phage λ is substantially increased. We call this phenomenon indirect ultraviolet-reactivation, since its features are essentially the same as those of ultraviolet-reactivation; this repair process occurs in pyrimidine dimer excision-deficient strains and produces clear plaque mutations of the restored phage. Moreover, this process is similar to indirect ultraviolet-induction of prophage λ, since it is promoted by conjugation. However, contrarily to indirect induction, it is produced by Hfr donors and occurs in recipients restricting the incoming ultraviolet-damaged donor DNA. The occurrence of indirect ultraviolet-reactivation provides evidence for the existence in E. coli of an inducible error-prone mechanism for the repair of DNA. PMID:4589889

  14. Progress in lactic acid bacterial phage research

    PubMed Central

    2014-01-01

    Research on lactic acid bacteria (LAB) has advanced significantly over the past number of decades and these developments have been driven by the parallel advances in technologies such as genomics, bioinformatics, protein expression systems and structural biology, combined with the ever increasing commercial relevance of this group of microorganisms. Some of the more significant and impressive outputs have been in the domain of bacteriophage-host interactions which provides a prime example of the cutting-edge model systems represented by LAB research. Here, we present a retrospective overview of the key advances in LAB phage research including phage-host interactions and co-evolution. We describe how in many instances this knowledge can be pivotal in creating real improvements in the application of LAB cultures in commercial practice. PMID:25185514

  15. Large scale production of phage antibody libraries using a bioreactor.

    PubMed

    Ferrara, Fortunato; Kim, Chang-Yub; Naranjo, Leslie A; Bradbury, Andrew R M

    2015-01-01

    One of the limitations of the use of phage antibody libraries in high throughput selections is the production of sufficient phage antibody library at the appropriate quality. Here, we successfully adapt a bioreactor-based protocol for the production of phage peptide libraries to the production of phage antibody libraries. The titers obtained in the stirred-tank bioreactor are 4 to 5 times higher than in a standard shake flask procedure, and the quality of the phage antibody library produced is indistinguishable to that produced using standard procedures as assessed by Western blotting and functional selections. Availability of this protocol will facilitate the use of phage antibody libraries in high-throughput scale selections.

  16. A century of the phage: past, present and future.

    PubMed

    Salmond, George P C; Fineran, Peter C

    2015-12-01

    Viruses that infect bacteria (bacteriophages; also known as phages) were discovered 100 years ago. Since then, phage research has transformed fundamental and translational biosciences. For example, phages were crucial in establishing the central dogma of molecular biology - information is sequentially passed from DNA to RNA to proteins - and they have been shown to have major roles in ecosystems, and help drive bacterial evolution and virulence. Furthermore, phage research has provided many techniques and reagents that underpin modern biology - from sequencing and genome engineering to the recent discovery and exploitation of CRISPR-Cas phage resistance systems. In this Timeline, we discuss a century of phage research and its impact on basic and applied biology.

  17. Trivalent Cation Induced Bundle Formation of Filamentous fd Phages.

    PubMed

    Korkmaz Zirpel, Nuriye; Park, Eun Jin

    2015-09-01

    Bacteriophages are filamentous polyelectrolyte viral rods infecting only bacteria. In this study, we investigate the bundle formation of fd phages with trivalent cations having different ionic radii (Al(3+) , La(3+) and Y(3+) ) at various phage and counterion concentrations, and at varying bundling times. Aggregated phage bundles were detected at relatively low trivalent counterion concentrations (1 mM). Although 10 mM and 100 mM Y(3+) and La(3+) treatments formed larger and more intertwined phage bundles, Al(3+) and Fe(3+) treatments lead to the formation of networking filaments. Energy dispersive X-ray spectroscopy (EDX) analyses confirmed the presence of C, N and O peaks on densely packed phage bundles. Immunofluorescence labelling and ELISA analyses with anti-p8 antibodies showed the presence of phage filaments after bundling.

  18. Lysogenic conversion and phage resistance development in phage exposed Escherichia coli biofilms.

    PubMed

    Moons, Pieter; Faster, David; Aertsen, Abram

    2013-01-01

    In this study, three-day old mature biofilms of Escherichia coli were exposed once to either a temperate Shiga-toxin encoding phage (H-19B) or an obligatory lytic phage (T7), after which further dynamics in the biofilm were monitored. As such, it was found that a single dose of H-19B could rapidly lead to a near complete lysogenization of the biofilm, with a subsequent continuous release of infectious H-19B particles. On the other hand, a single dose of T7 rapidly led to resistance development in the biofilm population. Together, our data indicates a profound impact of phages on the dynamics within structured bacterial populations. PMID:23344561

  19. 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

  20. Bacteriophages and phage-derived proteins--application approaches.

    PubMed

    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

  1. Isolation and Characterization of Phages Infecting Bacillus subtilis

    PubMed Central

    Krasowska, Anna; Biegalska, Anna; Augustyniak, Daria; Łoś, Marcin; Richert, Malwina; Łukaszewicz, Marcin

    2015-01-01

    Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages) or noncontractile (ARπ phage) tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0) and alkaline (9.0 and 10.0) pH. PMID:26273592

  2. 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

  3. Quality and safety requirements for sustainable phage therapy products.

    PubMed

    Pirnay, Jean-Paul; Blasdel, Bob G; Bretaudeau, Laurent; Buckling, Angus; Chanishvili, Nina; Clark, Jason R; Corte-Real, Sofia; Debarbieux, Laurent; Dublanchet, Alain; De Vos, Daniel; Gabard, Jérôme; Garcia, Miguel; Goderdzishvili, Marina; Górski, Andrzej; Hardcastle, John; Huys, Isabelle; Kutter, Elizabeth; Lavigne, Rob; Merabishvili, Maia; Olchawa, Ewa; Parikka, Kaarle J; Patey, Olivier; Pouilot, Flavie; Resch, Gregory; Rohde, Christine; Scheres, Jacques; Skurnik, Mikael; Vaneechoutte, Mario; Van Parys, Luc; Verbeken, Gilbert; Zizi, Martin; Van den Eede, Guy

    2015-07-01

    The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.

  4. Ligand-directed profiling of organelles with internalizing phage libraries

    PubMed Central

    Dobroff, Andrey S.; Rangel, Roberto; Guzman-Roja, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Bologa, Cristian G.; Oprea, Tudor I.; Brinker, C. Jeffrey; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, to create vaccines, and to engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents. Recently, our group has developed a new class of internalizing phage (iPhage) for ligand-directed targeting of organelles and/or to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. Here we describe the method for generating and screening the iPhage display system, and explain how to select and validate candidate internalizing homing peptide. PMID:25640897

  5. Distribution of Salmonella enteritidis phage types in Canada.

    PubMed Central

    Khakhria, R.; Duck, D.; Lior, H.

    1991-01-01

    The distribution of Salmonella enteritidis phage types in Canada is described; 606 of 674 strains examined were of human origin. Typable strains of all sources, constituted 99.6% (671/674) of all strains examined, and were representative of 15 different phage types. Five phage types (8, 13, 4, 13a and 1) accounted for 92.4% of the total. Phage type 8 consistently showed the highest incidence in human (69.96%) and non-human (72.05%) sources and appeared to be the most common in North America. Phage type 4, the most prevalent in the UK, is infrequent in Canada (38/674). The distribution of phage types showed regional variation among infrequent phage types, whereas the common type, 8, was observed in different frequencies in all provinces. Examination of 29 outbreaks of S. enteritidis representing 254 isolates for humans revealed 5 different phage types, the highest number of outbreaks (11) were type 8. A study of these outbreaks and the animal-host-associations of the common phage types, 8 and 13, indicated that contaminated poultry appeared to be the most common source of human infection in Canada. PMID:1993451

  6. Identification of Chondrocyte-Binding Peptides by Phage Display

    PubMed Central

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

    2016-01-01

    As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders. PMID:23440926

  7. Complete Genome Sequences of Four Novel Escherichia coli Bacteriophages Belonging to New Phage Groups

    PubMed Central

    Kot, Witold

    2015-01-01

    Here, we describe the sequencing and genome annotations of a set of four Escherichia coli bacteriophages (phages) belonging to newly discovered groups previously consisting of only a single phage and thus expand our knowledge of these phage groups. PMID:26184932

  8. Genome characteristics of a novel phage from Bacillus thuringiensis showing high similarity with phage from Bacillus cereus.

    PubMed

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

    2012-01-01

    Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the "late" region, the "lysogeny-lysis" region and the "early" region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor.

  9. Genome Characteristics of a Novel Phage from Bacillus thuringiensis Showing High Similarity with Phage from Bacillus cereus

    PubMed Central

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

    2012-01-01

    Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the “late” region, the “lysogeny-lysis” region and the “early” region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor. PMID:22649540

  10. Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks

    PubMed Central

    Bonilla, Natasha; Rojas, Maria Isabel; Netto Flores Cruz, Giuliano; Hung, Shr-Hau; Rohwer, Forest

    2016-01-01

    A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 1010–11 PFU·ml−1), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of > 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays. PMID:27547567

  11. Phage on tap-a quick and efficient protocol for the preparation of bacteriophage laboratory stocks.

    PubMed

    Bonilla, Natasha; Rojas, Maria Isabel; Netto Flores Cruz, Giuliano; Hung, Shr-Hau; Rohwer, Forest; Barr, Jeremy J

    2016-01-01

    A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 10(10-11) PFU·ml(-1)), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of > 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays. PMID:27547567

  12. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii

    PubMed Central

    Hamdi, Sana; Rousseau, Geneviève M.; Labrie, Simon J.; Kourda, Rim S.; Tremblay, Denise M.; Moineau, Sylvain; Slama, Karim B.

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus. PMID:27446058

  13. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii.

    PubMed

    Hamdi, Sana; Rousseau, Geneviève M; Labrie, Simon J; Kourda, Rim S; Tremblay, Denise M; Moineau, Sylvain; Slama, Karim B

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus.

  14. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii.

    PubMed

    Hamdi, Sana; Rousseau, Geneviève M; Labrie, Simon J; Kourda, Rim S; Tremblay, Denise M; Moineau, Sylvain; Slama, Karim B

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus. PMID:27446058

  15. Cryptic transposable phages of Pseudomonas aeruginosa

    SciTech Connect

    Krylov, V.N.; Mit`kina, L.N.; Pleteneva, E.A.; Aleshin, V.V.

    1995-11-01

    Frequencies of nucleotide sequences homologous to phage transposons (PT) of two species, D3112 and B3, were assessed in genomes of natural Pseudomonas aeruginosa strains by the dot-blot hybridization method. These strains were incapable of liberating viable phages on a lawn of the PA01 standard indicator strain of P. aeruginosa. It was shown that the homologies detected belong to two groups, high and intermediate, with respect to homology level. Homology patterns were classified as high when they provided signals comparable to those for hybridization in a positive control; patterns were classified as intermediate when the hybridization level was higher than the background level, but lower than in the positive control. Homologous PT sequences were designated as cryptic PT. Intact cryptic PT prophages were shown to exist in genomes of particular natural strains manifesting a higher level of hybridization. However, the growth of these phages was limited by the restriction system of strain PA01. It is possible to isolate strains maintaining the growth of some cryptic PT. These strains differed from P. aeruginosa with respect to the specificity of the restriction and modification system. Nevertheless, in most cases, the attempt to identify a novel host capable of maintaining growth of a cryptic PT failed. Natural strains often carry cryptic PT related to both known PT species, D3112 and B3. The frequency of cryptic PT is extremely high, reaching 30% in strains with a high level of homology only and up to 50% in all strains exhibiting homology. This high PT frequency is assumed to be associated with the considerable variation of P. aeruginosa. 15 refs., 1 fig., 2 tabs.

  16. Electron microscopic studies on intracellular phage development--history and perspectives.

    PubMed

    Kellenberger, E; Wunderli-Allenspach, H

    1995-01-01

    This review is centered on the applications of thin sections to the study of intracellular precursors of bacteriophage heads. Results obtained with other preparation methods are included in so far as they are essential for the comprehension of the biological problems. This type of work was pioneered with phage T4, which contributed much to today's understanding of morphogenesis and form determination. The T4 story is rich in successes, but also in many fallacies. Due to its large size, T4 is obviously prone to preparation artefacts such as emptying, flattening and others. Many of these artefacts were first encountered in T4. Artefacts are mostly found in lysates, however, experience shows that they are not completely absent from thin sections. This can be explained by the fact that permeability changes induced by fixatives occur. The information gained from T4 was profitably used for the study of other phages. They are included in this review as far as electron microscopic studies played a major role in the elucidation of their morphogenetic pathways. Research on phage assembly pathways and form determination is a beautiful illustration for the power of the integrated approach which combines electron microscopy with biochemistry, genetics and biophysics. As a consequence, we did not restrict ourselves to the review of electron microscopic work but tried to integrate pertinent data which contribute to the understanding of the molecular mechanisms acting in determining the form of supramolecular structures.

  17. Electron microscopy of virulent phages for Streptococcus lactis.

    PubMed Central

    Tsaneva, K P

    1976-01-01

    Electron microscopic studies were made on eight virulent Streptococcus lactis bacteriophages. These phages were taken as representative of eight host range groups established in a study of 75 phage isolates and 253 hosts (213 S. lactis, 22 S. cremoris, 18 S. diacetilactis). The phages studied were shown to have an isometric hexagonal head and noncontractile tails, usually several times longer than the head diameter. The virus heads were octahedral. The phages investigated represented three morphological types on the basis of head diameter , tail thickness, and tail length. These dimensions were approximately: for type I phages, 63, 172, and 11 nm, respectively; type II, 73, 200, and 20 nm, respectively; and type III, represented here by a single phage, 98, 551, and 12 nm, respectively. The tail surface revealed a different arrangment of the structural subunits which lent a helical appearance to the tails of type I and II phages and a guaffered tube appearance to the tail of type III phage. The number of turns along the tail axis, turn length, axial pitch, and helix angle were: type I, 32, 12 to 13 nm, 7.14 nm, and 11 degrees 43', respectively; type II, 24, 24, to 28 nm, 40.00 nm, and 32 degrees 30', respectively; and type III, 120, 12 nm, and no visible slope towards the axis. The morphology types showed complete correlation with serological groups, but not with groups based on host range pattern. Images PMID:817668

  18. Phage DNA dynamics in cells with different fates.

    PubMed

    Shao, Qiuyan; Hawkins, Alexander; Zeng, Lanying

    2015-04-21

    Bacteriophage λ begins its infection cycle by ejecting its DNA into its host Escherichia coli cell, after which either a lytic or a lysogenic pathway is followed, resulting in different cell fates. In this study, using a new technique to monitor the spatiotemporal dynamics of the phage DNA in vivo, we found that the phage DNA moves via two distinct modes, localized motion and motion spanning the whole cell. One or the other motion is preferred, depending on where the phage DNA is ejected into the cell. By examining the phage DNA trajectories, we found the motion to be subdiffusive. Moreover, phage DNA motion is the same in the early phase of the infection cycle, irrespective of whether the lytic or lysogenic pathway is followed; hence, cell-fate decision-making appears not to be correlated with the phage DNA motion. However, after the cell commits to one pathway or the other, phage DNA movement slows during the late phase of the lytic cycle, whereas it remains the same during the entire lysogenic cycle. Throughout the infection cycle, phage DNA prefers the regions around the quarter positions of the cell. PMID:25902444

  19. Computational models of populations of bacteria and lytic phage.

    PubMed

    Krysiak-Baltyn, Konrad; Martin, Gregory J O; Stickland, Anthony D; Scales, Peter J; Gras, Sally L

    2016-11-01

    The use of phages to control and reduce numbers of unwanted bacteria can be traced back to the early 1900s, when phages were explored as a tool to treat infections before the wide scale use of antibiotics. Recently, phage therapy has received renewed interest as a method to treat multiresistant bacteria. Phages are also widely used in the food industry to prevent the growth of certain bacteria in foods, and are currently being explored as a tool for use in bioremediation and wastewater treatment. Despite the large body of biological research on phages, relatively little attention has been given to computational modeling of the population dynamics of phage and bacterial interactions. The earliest model was described by Campbell in the 1960s. Subsequent modifications to this model include partial or complete resistance, multiple phage binding sites, and spatial heterogeneity. This review provides a general introduction to modeling of the population dynamics of bacteria and phage. The review introduces the basic model and relevant concepts and evaluates more complex variations of the basic model published to date, including a model of disease epidemics caused by infectious bacteria. Finally, the shortcomings and potential ways to improve the models are discussed.

  20. Detection of TNT-derivatives with recombinant phages

    PubMed Central

    Simonovicá, Mladen; Simonovicá, Branislav

    2012-01-01

    New immunoreagents for detection of TNT-derivatives TNP and TNP-Tris were developed using phage display technique. The monovalent and pentavalent recombinant phages carrying scFv specific for TNT were constructed and compared with each other to define the impact of valency and molecule dimension on antibody binding in immunoassay. Also, the bifunctional phages were generated, which carried TNT-specific scFvs as well as enzyme β-lactamase as a model marker on its surface. The most sensitive recombinant phages were selected and used for detection of TNP-Tris in a competitive ELISA based on immobilized antigen. Preincubation and partial phages saturation with a sample containing antigen allowed competition with immobilized hapten and displacement of free antigen. The phages exposing enzyme were used as immunoreagents for single step detection. The other phages were detected with specific marked antibodies. To date, the results presented in this paper are the first ever published regarding the recombinant phages for the detection of TNT. PMID:23050216

  1. High frequency generalized transduction by miniMu plasmid phage.

    PubMed

    Wang, B M; Liu, L; Groisman, E A; Casadaban, M J; Berg, C M

    1987-06-01

    Deletion derivatives of phage Mu which replicate as multicopy plasmids, and also transpose and package like Mu, have been developed for the in vivo cloning of bacterial genes. We show here that these miniMu plasmid phage are also efficient at generalized transduction and that both in vivo cloning and generalized transduction of a given gene can be accomplished in a single experiment.

  2. Phages preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: past, present and future.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-07-01

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

  3. Phages preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: past, present and future.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-07-01

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

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

    PubMed Central

    Gillis, Annika; Mahillon, Jacques

    2014-01-01

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

  5. Phage Particles as Vaccine Delivery Vehicles: Concepts, Applications and Prospects.

    PubMed

    Jafari, Narjes; Abediankenari, Saeid

    2015-01-01

    The development of new strategies for vaccine delivery for generating protective and long-lasting immune responses has become an expanding field of research. In the last years, it has been recognized that bacteriophages have several potential applications in the biotechnology and medical fields because of their intrinsic advantages, such as ease of manipulation and large-scale production. Over the past two decades, bacteriophages have gained special attention as vehicles for protein/peptide or DNA vaccine delivery. In fact, whole phage particles are used as vaccine delivery vehicles to achieve the aim of enhanced immunization. In this strategy, the carried vaccine is protected from environmental damage by phage particles. In this review, phage-based vaccine categories and their development are presented in detail, with discussion of the potential of phage-based vaccines for protection against microbial diseases and cancer treatment. Also reviewed are some recent advances in the field of phage- based vaccines.

  6. Phages in the global fruit and vegetable industry.

    PubMed

    Żaczek, M; Weber-Dąbrowska, B; Górski, A

    2015-03-01

    From recent articles, we have learned that phages can constitute a promising alternative in the food industry to eliminate bacterial pathogens from seedlings in greenhouse and field environments, as well as from fresh-cut food products. The fruit and vegetable industry requires quite a different approach than the meat or dairy industry. Several factors can inhibit efficacy of phage treatment such as plant watering or washing ready-to-eat products (water may dilute therapeutic doses), UV irradiation or extensive spreading of phytopathogens by wind, insects or even humans. Spontaneously occurring anomalous weather conditions in different parts of the world also may have an enormous impact on phage persistence in cultivations and on yields. Despite that, some phage preparations are commercially available and, without doubt, are much safer than chemical treatments. Along with increasing worldwide fruit and vegetable consumption, plant diseases and human foodborne illnesses are becoming a serious economic problem, resulting in a focus on optimization of phage treatment.

  7. Phage Particles as Vaccine Delivery Vehicles: Concepts, Applications and Prospects.

    PubMed

    Jafari, Narjes; Abediankenari, Saeid

    2015-01-01

    The development of new strategies for vaccine delivery for generating protective and long-lasting immune responses has become an expanding field of research. In the last years, it has been recognized that bacteriophages have several potential applications in the biotechnology and medical fields because of their intrinsic advantages, such as ease of manipulation and large-scale production. Over the past two decades, bacteriophages have gained special attention as vehicles for protein/peptide or DNA vaccine delivery. In fact, whole phage particles are used as vaccine delivery vehicles to achieve the aim of enhanced immunization. In this strategy, the carried vaccine is protected from environmental damage by phage particles. In this review, phage-based vaccine categories and their development are presented in detail, with discussion of the potential of phage-based vaccines for protection against microbial diseases and cancer treatment. Also reviewed are some recent advances in the field of phage- based vaccines. PMID:26745034

  8. Exclusion of polyvalent T7-like phages by prophage elements.

    PubMed

    Faidiuk, I V; Tovkach, E I

    2014-01-01

    The study presents new insights into the process of interaction of T7-like bacteriophages FE44 and BA14 with lysogenic cells. It was demonstrated that single and double lysogens possess Abiphenotype regardless of genera, species and strain of bacteria that initially had normal phage sensitivity. Efficiency of plating of these phages is reduced by two orders of magnitude on monolysogens, whereas it decreases by 4-6 orders on bilysogens. In the latter case, phage infection leads to formation of more than 60% of aberrant capsids in phage progeny. Abortive phage infection is suggested to be associated with defects in general dynamics of the bacterial chromosome in single and double lysogens of Erwinia "horticola" and Escherichia coli. PMID:25434214

  9. Possible association between phages, Hoc protein, and the immune system.

    PubMed

    Dabrowska, K; Switała-Jeleń, K; Opolski, A; Górski, A

    2006-02-01

    Mammals have become "an environment" for enterobacterial phage life cycles. Therefore it could be expected that bacteriophages adapt to them. This adaptation must comprise bacteriophage proteins. Gp Hoc seems to have significance neither for phage particle structure nor for phage antibacterial activity. It is evidently not necessary for the "typical" antibacterial actions of bacteriophages. But the rules of evolution make it improbable that gp Hoc really has no function, and non-essential genes of T4-type phages are probably important for phages' adaptation to their particular lifestyle. More interesting is the eukaryotic origin of gp Hoc: a resemblance to immunoglobulin-like proteins that reflects their evolutionary relation. Substantial differences in biological activity between T4 and a mutant that lacks gp Hoc were observed in a mammalian system. Hoc protein seems to be one of the molecules predicted to interact with mammalian organisms and/or modulate these interactions. PMID:16195787

  10. Methods for identification of recombinants of phage lambda.

    PubMed

    Sanzey, B; Mercereau, O; Ternynck, T; Kourilsky, P

    1976-10-01

    Two methods are described which allow the screening of a large number of phage plaques for a specific DNA sequence carried by the phage or a specific antigen produced within the phage plaque. These methods were set up with lambda and lambdalac phages. Phage plaques were transferred onto nitrocellulose filters by desiccation in 0.1 M NaOH, and the lac sequence was detected by hybridization to radioactive lac mRNA. Beta-Galactosidase was detected by reaction with anti-beta-galactosidase immune serum included in the soft agar of the titration plates; the precipitate thus formed was revealed by means of enzyme-coupled antibodies and in situ coloration. These methods are potentially useful for the identification of lambda transducers, including those which are generated by in vitro recombination with eukaryotic DNA.

  11. Chemical strategies for the covalent modification of filamentous phage

    PubMed Central

    Bernard, Jenna M. L.; Francis, Matthew B.

    2014-01-01

    Historically filamentous bacteriophage have been known to be the workhorse of phage display due to their ability to link genotype to phenotype. More recently, the filamentous phage scaffold has proven to be powerful outside the realm of phage display technology in fields such as molecular imaging, cancer research and materials, and vaccine development. The ability of the virion to serve as a platform for a variety of applications heavily relies on the functionalization of the phage coat proteins with a wide variety of functionalities. Genetic modification of the coat proteins has been the most widely used strategy for functionalizing the virion; however, complementary chemical modification strategies can help to diversify the range of materials that can be developed. This review emphasizes the recent advances that have been made in the chemical modification of filamentous phage as well as some of the challenges that are involved in functionalizing the virion. PMID:25566240

  12. Nucleotide sequence of Bacillus phage Nf terminal protein gene.

    PubMed Central

    Leavitt, M C; Ito, J

    1987-01-01

    The nucleotide sequence of Bacillus phage Nf gene E has been determined. Gene E codes for phage terminal protein which is the primer necessary for the initiation of DNA replication. The deduced amino acid sequence of Nf terminal protein is approximately 66% homologous with the terminal proteins of Bacillus phages PZA and luminal diameter 29, and shows similar hydropathy and secondary structure predictions. A serine which has been identified as the residue which covalently links the protein to the 5' end of the genome in luminal diameter 29, is conserved in all three phages. The hydropathic and secondary structural environment of this serine is similar in these phage terminal proteins and also similar to the linking serine of adenovirus terminal protein. PMID:3601672

  13. Phage abortive infection in lactococci: variations on a theme.

    PubMed

    Chopin, Marie-Christine; Chopin, Alain; Bidnenko, Elena

    2005-08-01

    Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance.

  14. Filamentous Phages As a Model System in Soft Matter Physics.

    PubMed

    Dogic, Zvonimir

    2016-01-01

    Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science. PMID:27446051

  15. Filamentous Phages As a Model System in Soft Matter Physics.

    PubMed

    Dogic, Zvonimir

    2016-01-01

    Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science.

  16. Filamentous Phages As a Model System in Soft Matter Physics

    PubMed Central

    Dogic, Zvonimir

    2016-01-01

    Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science. PMID:27446051

  17. Challenges in predicting the evolutionary maintenance of a phage transgene

    PubMed Central

    2014-01-01

    Background In prior work, a phage engineered with a biofilm-degrading enzyme (dispersin B) cleared artificial, short-term biofilms more fully than the phage lacking the enzyme. An unresolved question is whether the transgene will be lost or maintained during phage growth – its loss would limit the utility of the engineering. Broadly supported evolutionary theory suggests that transgenes will be lost through a ‘tragedy of the commons’ mechanism unless the ecology of growth in biofilms meets specific requirements. We test that theory here. Results Functional properties of the transgenic phage were identified. Consistent with the previous study, the dispersin phage was superior to unmodified phage at clearing short term biofilms grown in broth, shown here to be an effect attributable to free enzyme. However, the dispersin phage was only marginally better than control phages on short term biofilms in minimal media and was no better than control phages in clearing long term biofilms. There was little empirical support for the tragedy of the commons framework despite a strong theoretical foundation for its supposed relevance. The framework requires that the transgene imposes an intrinsic cost, yet the transgene was intrinsically neutral or beneficial when expressed from one part of the phage genome. Expressed from a different part of the genome, the transgene did behave as if intrinsically costly, but its maintenance did not benefit from spatially structured growth per se – violating the tragedy framework. Conclusions Overall, the transgene was beneficial under many conditions, but no insight to its maintenance was attributable to the established evolutionary framework. The failure likely resides in system details that would be used to parameterize the models. Our study cautions against naive applications of evolutionary theory to synthetic biology, even qualitatively. PMID:25126112

  18. Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

    PubMed Central

    Holguín, Angela V.; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C.; Barrios, Andrés Fernando González; Vives, Martha J.

    2015-01-01

    Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization. PMID:26274971

  19. Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays.

    PubMed

    Holguín, Angela V; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C; Barrios, Andrés Fernando González; Vives, Martha J

    2015-08-01

    Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization.

  20. The XXIIIrd Phage/Virus Assembly Meeting

    PubMed Central

    Serwer, Philip

    2014-01-01

    The XXIIIrd Phage/Virus Assembly (PVA) meeting returned to its birthplace in Lake Arrowhead, CA on September 8–13, 2013 (Fig. 1). The original meeting occurred in 1968, organized by Bob Edgar (Caltech, Pasadena, CA USA), Fred Eiserling (University of California, Los Angeles, Los Angeles, CA USA) and Bill Wood (Caltech, Pasadena, CA USA). The organizers of the 2013 meeting were Bill Gelbart (University of California, Los Angeles, Los Angeles, CA USA) and Jack Johnson (Scripps Research Institute, La Jolla, CA USA). This meeting specializes in an egalitarian format. Students are distinguished from senior faculty primarily by the signs of age. With the exception of historically based introductory talks, all talks were allotted the same time and freedom. This tradition began when the meeting was phage-only and has been continued now that all viruses are included. Many were the animated conversations about basic questions. New and international participants were present, a sign that the field has significant attraction, as it should, based on details below. The meeting was also characterized by a sense of humor and generally good times, a chance to both enjoy the science and forget the funding malaise to which many participants are exposed. I will present some of the meeting content, without attempting to be comprehensive. PMID:24498537

  1. Vibrio vulnificus Phage PV94 Is Closely Related to Temperate Phages of V. cholerae and Other Vibrio Species

    PubMed Central

    Reetz, Jochen; Strauch, Eckhard; Hertwig, Stefan

    2014-01-01

    Background Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1) infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species. Results In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5′-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218. Conclusion We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species. PMID:24732980

  2. Biofilm control with natural and genetically-modified phages.

    PubMed

    Motlagh, Amir Mohaghegh; Bhattacharjee, Ananda Shankar; Goel, Ramesh

    2016-04-01

    Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended.

  3. Coevolution of CRISPR bacteria and phage in 2 dimensions

    NASA Astrophysics Data System (ADS)

    Han, Pu; Deem, Michael

    2014-03-01

    CRISPR (cluster regularly interspaced short palindromic repeats) is a newly discovered adaptive, heritable immune system of prokaryotes. It can prevent infection of prokaryotes by phage. Most bacteria and almost all archae have CRISPR. The CRISPR system incorporates short nucleotide sequences from viruses. These incorporated sequences provide a historical record of the host and predator coevolution. We simulate the coevolution of bacteria and phage in 2 dimensions. Each phage has multiple proto-spacers that the bacteria can incorporate. Each bacterium can store multiple spacers in its CRISPR. Phages can escape recognition by the CRISPR system via point mutation or recombination. We will discuss the different evolutionary consequences of point mutation or recombination on the coevolution of bacteria and phage. We will also discuss an intriguing ``dynamic phase transition'' in the number of phage as a function of time and mutation rate. We will show that due to the arm race between phages and bacteria, the frequency of spacers and proto-spacers in a population can oscillate quite rapidly.

  4. Phage Therapy – Everything Old is New Again

    PubMed Central

    Kropinski, Andrew M

    2006-01-01

    The study of bacterial viruses (bacteriophages or phages) proved pivotal in the nascence of the disciplines of molecular biology and microbial genetics, providing important information on the central processes of the bacterial cell (DNA replication, transcription and translation) and on how DNA can be transferred from one cell to another. As a result of the pioneering genetics studies and modern genomics, it is now known that phages have contributed to the evolution of the microbial cell and to its pathogenic potential. Because of their ability to transmit genes, phages have been exploited to develop cloning vector systems. They also provide a plethora of enzymes for the modern molecular biologist. Until the introduction of antibiotics, phages were used to treat bacterial infections (with variable success). Western science is now having to re-evaluate the application of phage therapy – a therapeutic modality that never went out of vogue in Eastern Europe – because of the emergence of an alarming number of antibiotic-resistant bacteria. The present article introduces the reader to phage biology, and the benefits and pitfalls of phage therapy in humans and animals. PMID:18382643

  5. Tumor cell-targeting by phage-displayed peptides.

    PubMed

    Rasmussen, Ulla B; Schreiber, Valerie; Schultz, Huguette; Mischler, Fabienne; Schughart, Klaus

    2002-07-01

    We isolated cancer cell-specific phages by subtracting and selecting complex peptide display phage libraries on cultured human cancer cells. The best candidate was selected by performing three rounds of subtraction before each of five selections on the human colorectal WiDr cell line. The phage showed more than 1000-fold higher binding efficiency for WiDr cells when compared to five other human cancer cell lines, including two of colorectal origin, and when compared to wild-type M13 phage. Fifty-fold higher binding efficiency was also seen for a human breast cancer cell line. We show that the WiDr cell binding of the selected phage was efficiently competed by the synthetic peptide HEWSYLAPYPWF, predicted from the phage sequence. This confirms that the specificity of the peptide is independent of the display by the phage coat proteins. The identified peptide may target biomarkers linked to colorectal cancer, and thus be useful for designing gene transfer vectors as well as diagnostic and prognostic tools for this disease. PMID:12082461

  6. Chemical and Genetic Wrappers for Improved Phage and RNA Display

    PubMed Central

    Lamboy, Jorge A.; Tam, Phillip Y.; Lee, Lucie S.; Jackson, Pilgrim J.; Avrantinis, Sara K.; Lee, Hye Jin; Corn, Robert M.

    2009-01-01

    An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high non-specificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. We report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. First, a genetic strategy introduced a short charge neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7+) reduced or eliminated non-specific binding to the problematic high pI proteins. In the second, chemical approach, oligolysine wrappers for phage and total RNA blocked non-specific interactions. For phage display applications, the peptides Lysn (where n = 16 to 24) emerged as optimal for wrapping the phage. Lys8, however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked non-specific binding to allow successful selections, screens, and assays with five previously unworkable protein targets. PMID:18973165

  7. Phage-bacteria infection networks: From nestedness to modularity

    NASA Astrophysics Data System (ADS)

    Flores, Cesar O.; Valverde, Sergi; Weitz, Joshua S.

    2013-03-01

    Bacteriophages (viruses that infect bacteria) are the most abundant biological life-forms on Earth. However, very little is known regarding the structure of phage-bacteria infections. In a recent study we re-evaluated 38 prior studies and demonstrated that phage-bacteria infection networks tend to be statistically nested in small scale communities (Flores et al 2011). Nestedness is consistent with a hierarchy of infection and resistance within phages and bacteria, respectively. However, we predicted that at large scales, phage-bacteria infection networks should be typified by a modular structure. We evaluate and confirm this hypothesis using the most extensive study of phage-bacteria infections (Moebus and Nattkemper 1981). In this study, cross-infections were evaluated between 215 marine phages and 286 marine bacteria. We develop a novel multi-scale network analysis and find that the Moebus and Nattkemper (1981) study, is highly modular (at the whole network scale), yet also exhibits nestedness and modularity at the within-module scale. We examine the role of geography in driving these modular patterns and find evidence that phage-bacteria interactions can exhibit strong similarity despite large distances between sites. CFG acknowledges the support of CONACyT Foundation. JSW holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund and acknowledges the support of the James S. McDonnell Foundation

  8. Biofilm control with natural and genetically-modified phages.

    PubMed

    Motlagh, Amir Mohaghegh; Bhattacharjee, Ananda Shankar; Goel, Ramesh

    2016-04-01

    Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended. PMID:26931607

  9. A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students

    PubMed Central

    Jordan, Tuajuanda C.; Burnett, Sandra H.; Carson, Susan; Caruso, Steven M.; Clase, Kari; DeJong, Randall J.; Dennehy, John J.; Denver, Dee R.; Dunbar, David; Elgin, Sarah C. R.; Findley, Ann M.; Gissendanner, Chris R.; Golebiewska, Urszula P.; Guild, Nancy; Hartzog, Grant A.; Grillo, Wendy H.; Hollowell, Gail P.; Hughes, Lee E.; Johnson, Allison; King, Rodney A.; Lewis, Lynn O.; Li, Wei; Rosenzweig, Frank; Rubin, Michael R.; Saha, Margaret S.; Sandoz, James; Shaffer, Christopher D.; Taylor, Barbara; Temple, Louise; Vazquez, Edwin; Ware, Vassie C.; Barker, Lucia P.; Bradley, Kevin W.; Jacobs-Sera, Deborah; Pope, Welkin H.; Russell, Daniel A.; Cresawn, Steven G.; Lopatto, David; Bailey, Cheryl P.; Hatfull, Graham F.

    2014-01-01

    ABSTRACT Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students’ interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. PMID:24496795

  10. Characterization of Streptomyces plasmid-phage pFP4 and its evolutionary implications.

    PubMed

    Chen, Zhenhua; Zhong, Li; Shen, Meijuan; Fang, Ping; Qin, Zhongjun

    2012-11-01

    Autonomous-replicating plasmid pFP4 of Streptomyces sp. FR1 isolated from a heavy metal-contaminated land was cloned and sequenced. Surprisingly, the 40,949-bp pFP4 contains a cluster of 20 genes, resembling these chromosome-integrated prophages of Streptomyces sp. SPB78 and Streptomyces scabiei 87.22. Plasmid pFP4 could transfer by conjugation and a replication locus, iteron/repA/repB, was identified. The filtered FR1 culture could infect both FR1 and FR1 cured of pFP4 to form plaques, and also six out of 13 strains from the same land, but failed to form plaques on other seven strains from same source and all ten Streptomyces species from different sources. pFP4 phage particles were observed by transmission electron microscopy. Major structural proteins (capsid, portal and tail, etc.) of pFP4 virions were encoded by twelve pFP4 genes. pFP4 phage DNA contained 3' protruding cohesive ends of 9-nt. Streptomyces pFP4 represents a novel plasmid-phage.

  11. Review: phage therapy: a modern tool to control bacterial infections.

    PubMed

    Qadir, Muhammad Imran

    2015-01-01

    The evolution of antibiotic-resistant in bacteria has aggravated curiosity in development of alternative therapy to conventional drugs. One of the emerging drugs that can be used alternative to antibiotics is bacteriophage therapy. The use of living phages in the cure of lethal infectious life threatening diseases caused by Gram positive and Gram negative bacteria has been reported. Another development in the field of bacteriophage therapy is the use of genetically modified and non replicating phages in the treatment of bacterial infection. Genetically engineered bacteriophages can be used as adjuvant along with antibiotic therapy. Phages encoded with lysosomal enzymes are also effectual in the treatment of infectious diseases.

  12. The factors affecting effectiveness of treatment in phages therapy

    PubMed Central

    Ly-Chatain, Mai Huong

    2014-01-01

    In recent years, the use of lytic bacteriophages as antimicrobial agents controlling pathogenic bacteria has appeared as a promising new alternative strategy in the face of growing antibiotic resistance which has caused problems in many fields including medicine, veterinary medicine, and aquaculture. The use of bacteriophages has numerous advantages over traditional antimicrobials. The effectiveness of phage applications in fighting against pathogenic bacteria depends on several factors such as the bacteriophages/target bacteria ratio, the mode and moment of treatment, environmental conditions (pH, temperature...), the neutralization of phage and accessibility to target bacteria, amongst others. This report presents these factors and the challenges involved in developing phage therapy applications. PMID:24600439

  13. The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family.

    PubMed

    Shen, Xiaona; Zhang, Jingyun; Xu, Jialiang; Du, Pengcheng; Pang, Bo; Li, Jie; Kan, Biao

    2016-01-01

    Bacteriophage 919TP is a temperate phage of Vibrio cholerae serogroup O1 El Tor and is used as a subtyping phage in the phage-biotyping scheme in cholera surveillance in China. In this study, sequencing of the 919TP genome showed that it belonged to the Vibrio phage K139 family. The mechanisms conferring resistance to 919TP infection of El Tor strains were explored to help understand the subtyping basis of phage 919TP and mutations related to 919TP resistance. Among the test strains resistant to phage 919TP, most contained the temperate 919TP phage genome, which facilitated superinfection exclusion to 919TP. Our data suggested that this immunity to Vibrio phage 919TP occurred after absorption of the phage onto the bacteria. Other strains contained LPS receptor synthesis gene mutations that disable adsorption of phage 919TP. Several strains resistant to 919TP infection possessed unknown resistance mechanisms, since they did not contain LPS receptor mutations or temperate K139 phage genome. Further research is required to elucidate the phage infection steps involved in the resistance of these strains to phage infection. PMID:27242744

  14. The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family

    PubMed Central

    Shen, Xiaona; Zhang, Jingyun; Xu, Jialiang; Du, Pengcheng; Pang, Bo; Li, Jie; Kan, Biao

    2016-01-01

    Bacteriophage 919TP is a temperate phage of Vibrio cholerae serogroup O1 El Tor and is used as a subtyping phage in the phage-biotyping scheme in cholera surveillance in China. In this study, sequencing of the 919TP genome showed that it belonged to the Vibrio phage K139 family. The mechanisms conferring resistance to 919TP infection of El Tor strains were explored to help understand the subtyping basis of phage 919TP and mutations related to 919TP resistance. Among the test strains resistant to phage 919TP, most contained the temperate 919TP phage genome, which facilitated superinfection exclusion to 919TP. Our data suggested that this immunity to Vibrio phage 919TP occurred after absorption of the phage onto the bacteria. Other strains contained LPS receptor synthesis gene mutations that disable adsorption of phage 919TP. Several strains resistant to 919TP infection possessed unknown resistance mechanisms, since they did not contain LPS receptor mutations or temperate K139 phage genome. Further research is required to elucidate the phage infection steps involved in the resistance of these strains to phage infection. PMID:27242744

  15. CRISPR/Cas9-mediated phage resistance is not impeded by the DNA modifications of phage T4.

    PubMed

    Yaung, Stephanie J; Esvelt, Kevin M; Church, George M

    2014-01-01

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

  16. Hydroxyapatite chromatography of phage-display virions.

    PubMed

    Smith, George P; Gingrich, Todd R

    2005-12-01

    Hydroxyapatite column chromatography can be used to purify filamentous bacteriophage--the phage most commonly used for phage display. Virions that have been partially purified from culture supernatant by two cycles of precipitation in 2% polyethylene glycol are adsorbed onto the matrix at a density of at least 7.6 x 10(13) virions (about 3 mg) per milliliter of packed bed volume in phosphate-buffered saline (PBS; 0.15 M NaCl, 5 mM NaH2PO4, pH-adjusted to 7.0 with NaOH). The matrix is washed successively with wash buffer I(150 mM NaCl, 125 mM phosphate, pH 7.0), wash buffer II (2.55 M NaCl, 125 mM phosphate, pH 7.0), and wash buffer I; after which virions are desorbed in desorption buffer (150 mM NaCl, 200 mM phosphate, pH 7.0), and the matrix is stripped with stripping buffer (150 mM NaCl, 1 Mphosphate, pH 7.0). About half of the applied virions are recovered in desorption buffer. Western blot analysis shows that they have undetectable levels of host-derived protein contaminants that are present in the input virions and in virions purified by CsCl equilibrium density gradient centrifugation--the method most commonly used to prepare virions in high purity. Hydroxyapatite chromatography is thus an attractive alternative method for purifying filamentous virions, particularly when the scale is too large for ultracentrifugation to be practical. PMID:16382907

  17. Plasmids and packaging cell lines for use in phage display

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  18. Complete Genome Sequence of the Streptomyces Phage Nanodon

    PubMed Central

    2016-01-01

    Streptomyces phage Nanodon is a temperate double-stranded DNA Siphoviridae belonging to cluster BD1. It was isolated from soil collected in Kilauea, HI, using Streptomyces griseus subsp. griseus as a host. PMID:27795236

  19. Sequence analysis of the Lactobacillus temperate phage Sha1.

    PubMed

    Yoon, Bo Hyun; Jang, Se Hwan; Chang, Hyo-Ihl

    2011-09-01

    Bacteriophage Sha1, a newly isolated temperate phage from a mitomycin-C-induced lysate of Lactobacillus plantarum isolated from Kimchi, has an isometric head (58 nm × 60 nm) and a long tail (259 nm × 11 nm). The double-strand DNA genome of the phage Sha1 was 41,726 base pairs (bp) long, with a G+C content of 40.61%. Bioinformatic analysis of Sha1 shows that this phage contains 58 putative open reading frames (ORFs). Sha1 can be classified as a member of the large family Siphoviridae by genomic structure and morphology. To our knowledge, this is the first report of genomic sequencing and characterization of temperate phage Sha1 from wild-type L. plantarum isolated from kimchi in Korea. PMID:21701917

  20. [Inactivation of T4 phage in water environment using proteinase].

    PubMed

    Lü, Wen-zhou; Yang, Qing-xiang; Zhang, Yu; Yang, Min; Zhu, Chun-fang

    2004-09-01

    The inactivation effectiveness of proteinase to viruses was investigated by using T4 phage as a model virus. The results showed that the inactivation effectiveness of proteinase to T4 phage was obvious. In the optimum conditions and 67.5 u/mL concentration, the inactivation rate of proteinase K to T4 phage in sterilized water and in sewage achieved 99.4% and 49.4% respectively in an hour, and achieved >99.9% and 81.1% in three hours. The inactivation rate of the industrial proteinase 1398 to T4 phage in sterilized water achieved 74.4% in an hour. The effects of pH and temperature on the inactivation effectiveness was not evident.

  1. Phage display creates innovative applications to combat hepatitis B virus

    PubMed Central

    Tan, Wen Siang; Ho, Kok Lian

    2014-01-01

    Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20th century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases. PMID:25206271

  2. Salmonella phages and prophages--genomics and practical aspects.

    PubMed

    Kropinski, Andrew M; Sulakvelidze, Alexander; Konczy, Paulina; Poppe, Cornelius

    2007-01-01

    Numerous bacteriophages specific to Salmonella have been isolated or identified as part of host genome sequencing projects. Phylogenetic analysis of the sequenced phages, based on related protein content using CoreGenes, reveals that these viruses fall into five groupings (P27-like, P2-like, lambdoid, P22-like, and T7-like) and three outliers (epsilon15, KS7, and Felix O1). The P27 group is only represented by ST64B; the P2 group contains Fels-2, SopEphi, and PSP3; the lambdoid Salmonella phages include Gifsy-1, Gifsy-2, and Fels-1. The P22-like viruses include epsilon34, ES18, P22, ST104, and ST64T. The only member of the T7-like group is SP6. The properties of each of these phages are discussed, along with their role as agents of genetic exchange and as therapeutic agents and their involvement in phage typing.

  3. Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells)

    PubMed Central

    Serwer, Philip

    2011-01-01

    I present a hypothesis that begins with the proposal that abiotic ancestors of phage RNA and DNA packaging systems (and cells) include mobile shells with an internal, molecule-transporting cavity. The foundations of this hypothesis include the conjecture that current nucleic acid packaging systems have imprints from abiotic ancestors. The abiotic shells (1) initially imbibe and later also bind and transport organic molecules, thereby providing a means for producing molecular interactions that are links in the chain of events that produces ancestors to the first molecules that are both information carrying and enzymatically active, and (2) are subsequently scaffolds on which proteins assemble to form ancestors common to both shells of viral capsids and cell membranes. Emergence of cells occurs via aggregation and merger of shells and internal contents. The hypothesis continues by using proposed imprints of abiotic and biotic ancestors to deduce an ancestral thermal ratchet-based DNA packaging motor that subsequently evolves to integrate a DNA packaging ATPase that provides a power stroke. PMID:21994778

  4. Neisseria gonorrhoeae filamentous phage NgoΦ6 is capable of infecting a variety of Gram-negative bacteria.

    PubMed

    Piekarowicz, Andrzej; Kłyż, Aneta; Majchrzak, Michał; Szczêsna, Ewa; Piechucki, Marcin; Kwiatek, Agnieszka; Maugel, Timothy K; Stein, Daniel C

    2014-01-01

    We constructed a phagemid consisting of the whole genome of the Neisseria gonorrhoeae bacteriophage NgoΦ6 cloned into a pBluescript plasmid derivative lacking the f1 origin of replication (named pBS::Φ6). Escherichia coli cells harboring pBS::Φ6 were able to produce a biologically active phagemid, NgoΦ6fm, capable of infecting, integrating its DNA into the chromosome of, and producing progeny phagemids in, a variety of taxonomically distant Gram-negative bacteria, including E. coli, Haemophilus influenzae, Neisseria sicca, Pseudomonas sp., and Paracoccus methylutens. A derivative of pBS::Φ6 lacking the phage orf7 gene, a positional homolog of filamentous phage proteins that mediate the interaction between the phage and the bacterial pilus, was capable of producing phagemid particles that were able to infect E. coli, Haemophilus influenzae, N. sicca, Pseudomonas sp., and Paracoccus methylutens, indicating that NgoΦ6 infects cells of these species using a mechanism that does not involve the Orf7 gene product and that NgoΦ6 initiates infection through a novel process in these species. We further demonstrate that the establishment of the lysogenic state does not require an active phage integrase. Since phagemid particles were capable of infecting diverse hosts, this indicates that NgoΦ6 is the first broad-host-range filamentous bacteriophage described. PMID:24198404

  5. Deep sequencing analysis of phage libraries using Illumina platform.

    PubMed

    Matochko, Wadim L; Chu, Kiki; Jin, Bingjie; Lee, Sam W; Whitesides, George M; Derda, Ratmir

    2012-09-01

    This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve

  6. Properties of Klebsiella phage P13 and associated exopolysaccharide depolymerase

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Li, Guiyang; Mo, Zhaolan; Chai, Zihan; Shang, Anqi; Mou, Haijin

    2013-11-01

    The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide (EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70°C for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60°C and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a doublestrand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.

  7. Molecular architecture of tailed double-stranded DNA phages

    PubMed Central

    Fokine, Andrei; Rossmann, Michael G

    2014-01-01

    The tailed double-stranded DNA bacteriophages, or Caudovirales, constitute ~96% of all the known phages. Although these phages come in a great variety of sizes and morphology, their virions are mainly constructed of similar molecular building blocks via similar assembly pathways. Here we review the structure of tailed double-stranded DNA bacteriophages at a molecular level, emphasizing the structural similarity and common evolutionary origin of proteins that constitute these virions. PMID:24616838

  8. Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

    PubMed Central

    Sørensen, Martine C. Holst; Gencay, Yilmaz Emre; Birk, Tina; Baldvinsson, Signe Berg; Jäckel, Claudia; Hammerl, Jens A.; Vegge, Christina S.; Neve, Horst; Brøndsted, Lone

    2015-01-01

    In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages. PMID:25585385

  9. Shiga toxins and stx phages: highly diverse entities.

    PubMed

    Krüger, Alejandra; Lucchesi, Paula M A

    2015-03-01

    Shiga toxins are the main virulence factors of a group of Escherichia coli strains [Shiga toxin-producing E. coli (STEC)] that cause severe human diseases, such as haemorrhagic colitis and haemolytic-uraemic syndrome. The Shiga toxin family comprises several toxin subtypes, which have been differentially related to clinical manifestations. In addition, the phages that carry the Shiga toxin genes (stx phages) are also diverse. These phages play an important role not only in the dissemination of Shiga toxin genes and the emergence of new STEC strains, but also in the regulation of Shiga toxin production. Consequently, differences in stx phages may affect the dissemination of stx genes as well as the virulence of STEC strains. In addition to presenting an overview of Shiga toxins and stx phages, in this review we highlight current knowledge about the diversity of stx phages, with emphasis on its impact on STEC virulence. We consider that this diversity should be taken into account when developing STEC infection treatments and diagnostic approaches, and when conducting STEC control in reservoirs. PMID:25479836

  10. The Legacy of 20th Century Phage Research.

    PubMed

    Campbell, Allan M

    2010-09-01

    The Golden Age of Phage Research, where phage was the favored material for attacking many basic questions in molecular biology, lasted from about 1940 to 1970. The era was initiated by Ellis and Delbrück, whose analysis defined the relevant parameters to measure in studying phage growth, and depended on the fact that the contents of a plaque can comprise descendants of a single infecting particle. It ended around 1970 because definitive methods had then become available for answering the same questions in other systems. Some of the accomplishments of phage research were the demonstration by Hershey and Chase that the genetic material of phage T2 is largely composed of DNA, the construction of linkage maps of T2 and T4 by Hershey and Rotman and their extension to very short molecular distances by Benzer, and the isolation of conditionally lethal mutants in T4 by Epstein et al. and in λ by Campbell. The dissection of the phage life cycle into causal chains was explored by Edgar and Wood for T4 assembly and later in the regulation of lysogeny by Kaiser, extended to the molecular level by Ptashne and others. Restriction/modification was discovered in λ by Bertani and Weigle, and the biochemical mechanism was elucidated by Arber and by Smith.

  11. Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

    PubMed Central

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; Edwards, Robert A.; Burgin, Alex B.; Segall, Anca M.; Rohwer, Forest

    2015-01-01

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented. PMID:26132888

  12. Phage therapy against Enterococcus faecalis in dental root canals.

    PubMed

    Khalifa, Leron; Shlezinger, Mor; Beyth, Shaul; Houri-Haddad, Yael; Coppenhagen-Glazer, Shunit; Beyth, Nurit; Hazan, Ronen

    2016-01-01

    Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE) in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages). Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals. PMID:27640530

  13. Experimental phage therapy of burn wound infection: difficult first steps

    PubMed Central

    Rose, Thomas; Verbeken, Gilbert; Vos, Daniel De; Merabishvili, Maya; Vaneechoutte, Mario; Lavigne, Rob; Jennes, Serge; Zizi, Martin; Pirnay, Jean-Paul

    2014-01-01

    Antibiotic resistance has become a major public health problem and the antibiotics pipeline is running dry. Bacteriophages (phages) may offer an ‘innovative’ means of infection treatment, which can be combined or alternated with antibiotic therapy and may enhance our abilities to treat bacterial infections successfully. Today, in the Queen Astrid Military Hospital, phage therapy is increasingly considered as part of a salvage therapy for patients in therapeutic dead end, particularly those with multidrug resistant infections. We describe the application of a well-defined and quality controlled phage cocktail, active against Pseudomonas aeruginosa and Staphylococcus aureus, on colonized burn wounds within a modest clinical trial (nine patients, 10 applications), which was approved by a leading Belgian Medical Ethical Committee. No adverse events, clinical abnormalities or changes in laboratory test results that could be related to the application of phages were observed. Unfortunately, this very prudent ‘clinical trial’ did not allow for an adequate evaluation of the efficacy of the phage cocktail. Nevertheless, this first ‘baby step’ revealed several pitfalls and lessons for future experimental phage therapy and helped overcome the psychological hurdles that existed to the use of viruses in the treatment of patients in our burn unit. PMID:25356373

  14. Conserved termini and adjacent variable region of Twortlikevirus Staphylococcus phages.

    PubMed

    Zhang, Xianglilan; Kang, Huaixing; Li, Yuyuan; Liu, Xiaodong; Yang, Yu; Li, Shasha; Pei, Guangqian; Sun, Qiang; Shu, Peng; Mi, Zhiqiang; Huang, Yong; Zhang, Zhiyi; Liu, Yannan; An, Xiaoping; Xu, Xiaolu; Tong, Yigang

    2015-12-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing cause of serious infection, both in the community and hospital settings. Despite sophisticated strategies and efforts, the antibiotic options for treating MRSA infection are narrowing because of the limited number of newly developed antimicrobials. Here, four newly-isolated MRSA-virulent phages, IME-SA1, IMESA2, IME-SA118 and IME-SA119, were sequenced and analyzed. Their genome termini were identified using our previously proposed "termini analysis theory". We provide evidence that remarkable conserved terminus sequences are found in IME-SA1/2/118/119, and, moreover, are widespread throughout Twortlikevirus Staphylococcus phage G1 and K species. Results also suggested that each phage of the two species has conserved 5' terminus while the 3' terminus is variable. More importantly, a variable region with a specific pattern was found to be present near the conserved terminus of Twortlikevirus S. phage G1 species. The clone with the longest variable region had variable terminus lengths in successive generations, while the clones with the shortest variable region and with the average length variable region maintained the same terminal length as themselves during successive generations. IME-SA1 bacterial infection experiments showed that the variation is not derived from adaptation of the phage to different host strains. This is the first study of the conserved terminus and variable region of Twortlikevirus S. phages.

  15. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa.

    PubMed

    Chan, Benjamin K; Sistrom, Mark; Wertz, John E; Kortright, Kaitlyn E; Narayan, Deepak; Turner, Paul E

    2016-01-01

    Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections. PMID:27225966

  16. Phage therapy against Enterococcus faecalis in dental root canals

    PubMed Central

    Khalifa, Leron; Shlezinger, Mor; Beyth, Shaul; Houri-Haddad, Yael; Coppenhagen-Glazer, Shunit; Beyth, Nurit; Hazan, Ronen

    2016-01-01

    Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE) in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages). Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals. PMID:27640530

  17. Shiga toxins and stx phages: highly diverse entities.

    PubMed

    Krüger, Alejandra; Lucchesi, Paula M A

    2015-03-01

    Shiga toxins are the main virulence factors of a group of Escherichia coli strains [Shiga toxin-producing E. coli (STEC)] that cause severe human diseases, such as haemorrhagic colitis and haemolytic-uraemic syndrome. The Shiga toxin family comprises several toxin subtypes, which have been differentially related to clinical manifestations. In addition, the phages that carry the Shiga toxin genes (stx phages) are also diverse. These phages play an important role not only in the dissemination of Shiga toxin genes and the emergence of new STEC strains, but also in the regulation of Shiga toxin production. Consequently, differences in stx phages may affect the dissemination of stx genes as well as the virulence of STEC strains. In addition to presenting an overview of Shiga toxins and stx phages, in this review we highlight current knowledge about the diversity of stx phages, with emphasis on its impact on STEC virulence. We consider that this diversity should be taken into account when developing STEC infection treatments and diagnostic approaches, and when conducting STEC control in reservoirs.

  18. Phage phenomics: Physiological approaches to characterize novel viral proteins

    DOE PAGES

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; et al

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

  19. Phage phenomics: Physiological approaches to characterize novel viral proteins

    SciTech Connect

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; Edwards, Robert A.; Burgin, Alex B.; Segall, Anca M.; Rohwer, Forest

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

  20. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa

    PubMed Central

    Chan, Benjamin K.; Sistrom, Mark; Wertz, John E.; Kortright, Kaitlyn E.; Narayan, Deepak; Turner, Paul E.

    2016-01-01

    Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections. PMID:27225966

  1. Evolutionary consequences of intra-patient phage predation on microbial populations.

    PubMed

    Seed, Kimberley D; Yen, Minmin; Shapiro, B Jesse; Hilaire, Isabelle J; Charles, Richelle C; Teng, Jessica E; Ivers, Louise C; Boncy, Jacques; Harris, Jason B; Camilli, Andrew

    2014-08-26

    The impact of phage predation on bacterial pathogens in the context of human disease is not currently appreciated. Here, we show that predatory interactions of a phage with an important environmentally transmitted pathogen, Vibrio cholerae, can modulate the evolutionary trajectory of this pathogen during the natural course of infection within individual patients. We analyzed geographically and temporally disparate cholera patient stool samples from Haiti and Bangladesh and found that phage predation can drive the genomic diversity of intra-patient V. cholerae populations. Intra-patient phage-sensitive and phage-resistant isolates were isogenic except for mutations conferring phage resistance, and moreover, phage-resistant V. cholerae populations were composed of a heterogeneous mix of many unique mutants. We also observed that phage predation can significantly alter the virulence potential of V. cholerae shed from cholera patients. We provide the first molecular evidence for predatory phage shaping microbial community structure during the natural course of infection in humans.

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

    PubMed Central

    Khan Mirzaei, Mohammadali; Nilsson, Anders S.

    2015-01-01

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

  3. An Unusual Phage Repressor Encoded by Mycobacteriophage BPs.

    PubMed

    Villanueva, Valerie M; Oldfield, Lauren M; Hatfull, Graham F

    2015-01-01

    Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection-lysogenic or lytic growth-as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33-34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome. PMID:26332854

  4. 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.

  5. 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

  6. Bacteriophage exploitation of bacterial biofilms: phage preference for less mature targets?

    PubMed

    Abedon, Stephen T

    2016-02-01

    Robust evidence is somewhat lacking for biofilm susceptibility to bacteriophages in nature, contrasting often substantial laboratory biofilm vulnerability to phages. To help bridge this divide, I review a two-part scenario for 'heterogeneous' phage interaction even with phage-permissive single-species biofilms. First, through various mechanisms, those bacteria which are both more newly formed and located at biofilm surfaces may be particularly vulnerable to phage adsorption, rather than biofilm matrix being homogeneously resistant to phage penetration. Second, though phage infection of older, less metabolically active bacteria may still be virion productive, nevertheless the majority of phage population growth in association with biofilm bacteria could involve infection particularly of those bacteria which are more metabolically active and thereby better able to support larger phage bursts, versus clonally related biofilm bacteria equivalently supporting phage production. To the extent that biofilms are physiologically or structurally heterogeneous, with phages exploiting particularly relatively newly divided biofilm-surface bacteria, then even effective phage predation of natural biofilms could result in less than complete overall biofilm clearance. Phage tendencies toward only partial exploitation of even single-species biofilms could be consistent with observations that chronic bacterial infections in the clinic can require more aggressive or extensive phage therapy to eradicate.

  7. Proteomic Analysis of a Novel Bacillus Jumbo Phage Revealing Glycoside Hydrolase As Structural Component.

    PubMed

    Yuan, Yihui; Gao, Meiying

    2016-01-01

    Tailed phages with genomes of larger than 200 kbp are classified as Jumbo phages and exhibited extremely high uncharted diversity. The genomic annotation of Jumbo phage is often disappointing because most of the predicted proteins, including structural proteins, failed to make good hits to the sequences in the databases. In this study, 23 proteins of a novel Bacillus Jumbo phage, vB_BpuM_BpSp, were identified as phage structural proteins by the structural proteome analysis, including 14 proteins of unknown function, 5 proteins with predicted function as structural proteins, a glycoside hydrolase, a Holliday junction resolvase, a RNA-polymerase β-subunit, and a host-coding portal protein, which might be hijacked from the host strain during phage virion assembly. The glycoside hydrolase (Gp255) was identified as phage virion component and was found to interact with the phage baseplate protein. Gp255 shows specific lytic activity against the phage host strain GR8 and has high temperature tolerance. In situ peptidoglycan-hydrolyzing activities analysis revealed that the expressed Gp255 and phage structural proteome exhibited glycoside hydrolysis activity against the tested GR8 cell extracts. This study identified the first functional individual structural glycoside hydrolase in phage virion. The presence of activated glycoside hydrolase in phage virions might facilitate the injection of the phage genome during infection by forming pores on the bacterial cell wall. PMID:27242758

  8. Bacteriophage exploitation of bacterial biofilms: phage preference for less mature targets?

    PubMed

    Abedon, Stephen T

    2016-02-01

    Robust evidence is somewhat lacking for biofilm susceptibility to bacteriophages in nature, contrasting often substantial laboratory biofilm vulnerability to phages. To help bridge this divide, I review a two-part scenario for 'heterogeneous' phage interaction even with phage-permissive single-species biofilms. First, through various mechanisms, those bacteria which are both more newly formed and located at biofilm surfaces may be particularly vulnerable to phage adsorption, rather than biofilm matrix being homogeneously resistant to phage penetration. Second, though phage infection of older, less metabolically active bacteria may still be virion productive, nevertheless the majority of phage population growth in association with biofilm bacteria could involve infection particularly of those bacteria which are more metabolically active and thereby better able to support larger phage bursts, versus clonally related biofilm bacteria equivalently supporting phage production. To the extent that biofilms are physiologically or structurally heterogeneous, with phages exploiting particularly relatively newly divided biofilm-surface bacteria, then even effective phage predation of natural biofilms could result in less than complete overall biofilm clearance. Phage tendencies toward only partial exploitation of even single-species biofilms could be consistent with observations that chronic bacterial infections in the clinic can require more aggressive or extensive phage therapy to eradicate. PMID:26738755

  9. Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

    PubMed Central

    Denes, Thomas; den Bakker, Henk C.; Tokman, Jeffrey I.; Guldimann, Claudia

    2015-01-01

    Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA. PMID:25888172

  10. Topoisomerase Involvement in Multiplicity Reactivation of Phage T4

    PubMed Central

    Miskimins, Robin; Schneider, Silke; Johns, Virginia; Bernstein, Harris

    1982-01-01

    The products of phage T4 genes 39, 52 and probably 60 have been previously characterized as forming a type II DNA topoisomerase. Other evidence suggested that this topoisomerase promotes normal initiation of DNA replication, and that when it is defective its loss is partially compensated for by the host gyrase. We present evidence here that mutants defective in genes 39, 52 and 60 have reduced ability to carry out multiplicity reactivation (MR, a form of recombinational repair) of phage DNA damaged either by mitomycin C (MMC) or psoralen plus near-UV light (PUVA). We also observed that there is not extensive superhelicity in the intracellular phage DNA either in the presence or absence of the phage topoisomerase. This tends to rule out the possibility that the topoisomerase influences MR by controlling the general superhelicity of the phage DNA. The dependence of MR on topoisomerase could occur in several possible ways. However, we favor the explanation that the lesions are bypassed by a postreplication recombinational repair process that is influenced by the topoisomerase through its role in initiating replication. PMID:6293912

  11. Discovery of internalizing antibodies to tumor antigens from phage libraries

    PubMed Central

    Zhou, Yu; Marks, James D

    2014-01-01

    Phage antibody technology can be used to generate human antibodies to essentially any antigen. Many therapeutic target antigens are cell surface receptors, which can be challenging targets for antibody generation. In addition, for many therapeutic applications, one needs antibodies that not only bind the cell surface receptor but that also are internalized into the cell upon binding. This allows use of the antibody to deliver a range of payloads into the cell to achieve a therapeutic effect. In this chapter we describe how human phage antibody libraries can be selected directly on tumor cell lines to generate antibodies that bind cell surface receptors and which upon binding are rapidly internalized into the cell. Specific protocols show how to: 1) directly select cell binding and internalizing antibodies from human phage antibody libraries; 2) screen the phage antibodies in a high throughput flow cytometry assay for binding to the tumor cell line used for selection; 3) identify the antigen bound by the phage antibody using immunoprecipitation and mass spectrometry; and 4) direct cell binding and internalizing selections to a specific tumor antigen by sequential selection on a tumor cell line followed by selection on yeast displaying the target tumor antigen on the yeast surface. PMID:22208981

  12. Abortive infection mechanisms and prophage sequences significantly influence the genetic makeup of emerging lytic lactococcal phages.

    PubMed

    Labrie, Simon J; Moineau, Sylvain

    2007-02-01

    In this study, we demonstrated the remarkable genome plasticity of lytic lactococcal phages that allows them to rapidly adapt to the dynamic dairy environment. The lytic double-stranded DNA phage ul36 was used to sequentially infect a wild-type strain of Lactococcus lactis and two isogenic derivatives with genes encoding two phage resistance mechanisms, AbiK and AbiT. Four phage mutants resistant to one or both Abi mechanisms were isolated. Comparative analysis of their complete genomes, as well as morphological observations, revealed that phage ul36 extensively evolved by large-scale homologous and nonhomologous recombination events with the inducible prophage present in the host strain. One phage mutant exchanged as much as 79% of its genome compared to the core genome of ul36. Thus, natural phage defense mechanisms and prophage elements found in bacterial chromosomes contribute significantly to the evolution of the lytic phage population.

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

    PubMed Central

    Grose, Julianne H.; Casjens, Sherwood R.

    2014-01-01

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

  14. Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage.

    PubMed

    Hendrix, R W; Smith, M C; Burns, R N; Ford, M E; Hatfull, G F

    1999-03-01

    We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage phiC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, phiflu, in the Haemophilus influenzae genome, and two small prophage-like elements, phiRv1 and phiRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage.

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

    PubMed

    Grose, Julianne H; Casjens, Sherwood R

    2014-11-01

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

  16. 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

  17. A comparative study and phage typing of silage-making Lactobacillus bacteriophages.

    PubMed

    Doi, Katsumi; Zhang, Ye; Nishizaki, Yousuke; Umeda, Akiko; Ohmomo, Sadahiro; Ogata, Seiya

    2003-01-01

    To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus. Phage phiPY4 (group III) infected both L. casei and Lactobacillus rhamnosus. Phage phiPY5 (group IV) specifically infected Lactobacillus casei. Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G + C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli. PMID:16233449

  18. Molecular imaging of T4 phage in mammalian tissues and cells

    PubMed Central

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

    2014-01-01

    Advances in phage therapy encourage scientific interest in interactions of phages with human and animal organisms. This has created a need for developing tools that facilitate studies of phage circulation and deposition in tissues and cells. Here we propose a new green fluorescent protein (GFP)-based method for T4 phage molecular imaging in living systems. The method employs decoration of a phage capsid with GFP fused to the N-terminus of Hoc protein by in vivo phage display. Fluorescent phages were positively assessed as regards their applicability for detection inside living mammalian cells (by phagocytosis) and tissues (filtering and retention by lymph nodes and spleen). Molecular imaging provides innovative techniques that have brought substantial progress in life sciences. We propose it as a useful tool for studies of phage biology. PMID:24653943

  19. Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus.

    PubMed

    Wei, Yunzhou; Chesne, Megan T; Terns, Rebecca M; Terns, Michael P

    2015-02-18

    CRISPR-Cas systems are RNA-based immune systems that protect prokaryotes from invaders such as phages and plasmids. In adaptation, the initial phase of the immune response, short foreign DNA fragments are captured and integrated into host CRISPR loci to provide heritable defense against encountered foreign nucleic acids. Each CRISPR contains a ∼100-500 bp leader element that typically includes a transcription promoter, followed by an array of captured ∼35 bp sequences (spacers) sandwiched between copies of an identical ∼35 bp direct repeat sequence. New spacers are added immediately downstream of the leader. Here, we have analyzed adaptation to phage infection in Streptococcus thermophilus at the CRISPR1 locus to identify cis-acting elements essential for the process. We show that the leader and a single repeat of the CRISPR locus are sufficient for adaptation in this system. Moreover, we identified a leader sequence element capable of stimulating adaptation at a dormant repeat. We found that sequences within 10 bp of the site of integration, in both the leader and repeat of the CRISPR, are required for the process. Our results indicate that information at the CRISPR leader-repeat junction is critical for adaptation in this Type II-A system and likely other CRISPR-Cas systems.

  20. Recent findings about the Yersinia enterocolitica phage shock protein response.

    PubMed

    Yamaguchi, Saori; Darwin, Andrew J

    2012-02-01

    The phage shock protein (Psp) system is a conserved extracytoplasmic stress response in bacteria that is essential for virulence of the human pathogen Yersinia enterocolitica. This article summarizes some recent findings about Y. enterocolitica Psp system function. Increased psp gene expression requires the transcription factor PspF, but under non-inducing conditions PspF is inhibited by an interaction with another protein, PspA, in the cytoplasm. A Psp-inducing stimulus causes PspA to relocate to the cytoplasmic membrane, freeing PspF to induce psp gene expression. This PspA relocation requires the integral cytoplasmic membrane proteins, PspB and PspC, which might sense an inducing trigger and sequester PspA by direct interaction. The subsequent induction of psp gene expression increases the PspA concentration, which also allows it to contact the membrane directly, perhaps for its physiological function. Mutational analysis of the PspB and PspC proteins has revealed that they both positively and negatively regulate psp gene expression and has also identified PspC domains associated with each function. We also compare the contrasting physiological roles of the Psp system in the virulence of Y. enterocolitica and Salmonella enterica sv. Typhimurium (S. Typhimurium). In S. Typhimurium, PspA maintains the proton motive force, which provides the energy needed to drive ion importers required for survival within macrophages. In contrast, in the extracellular pathogen Y. enterocolitica, PspB and PspC, but not PspA, are the Psp components needed for virulence. PspBC protect Y. enterocolitica from damage caused by the secretin component of its type 3 secretion system, an essential virulence factor.

  1. Influence of Environmental Factors on Phage-Bacteria Interaction and on the Efficacy and Infectivity of Phage P100.

    PubMed

    Fister, Susanne; Robben, Christian; Witte, Anna K; Schoder, Dagmar; Wagner, Martin; Rossmanith, Peter

    2016-01-01

    When using bacteriophages to control food-borne bacteria in food production plants and processed food, it is crucial to consider that environmental conditions influence their stability. These conditions can also affect the physiological state of bacteria and consequently host-virus interaction and the effectiveness of the phage ability to reduce bacteria numbers. In this study we investigated the stability, binding, and replication capability of phage P100 and its efficacy to control Listeria monocytogenes under conditions typically encountered in dairy plants. The influences of SDS, Lutensol AO 7, salt, smear water, and different temperatures were investigated. Results indicate that phage P100 is stable and able to bind to the host under most conditions tested. Replication was dependent upon the growth of L. monocytogenes and efficacy was higher when bacterial growth was reduced by certain environmental conditions. In long-term experiments at different temperatures phages were initially able to reduce bacteria up to seven log10 units after 2 weeks at 4°C. However, thereafter, re-growth and development of phage-resistant L. monocytogenes isolates were encountered. PMID:27516757

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

    PubMed Central

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

    2015-01-01

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

  3. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    NASA Astrophysics Data System (ADS)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  4. Biochemical functionalization of peptide nanotubes with phage displayed peptides.

    PubMed

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering. PMID:27479451

  5. A simple method for the isolation of phages from Listeria monocytogenes.

    PubMed

    Durst, J; Rau, E; Kemenes, F; Berencsi, G

    1980-02-01

    By application of a combined mitomycin-/heat treatment after freezing, 7 out of 29 Listeria monocytogenes strains which were found to be non-phage carriers by UV irradiation could release phages. Propagation of the obtained phages was promoted by storage at 4 degrees C. Apart from TNSA plates the application of chocolate plates appears to be necessary in order to study these phages.

  6. Phage cluster relationships identified through single gene analysis

    PubMed Central

    2013-01-01

    Background Phylogenetic comparison of bacteriophages requires whole genome approaches such as dotplot analysis, genome pairwise maps, and gene content analysis. Currently mycobacteriophages, a highly studied phage group, are categorized into related clusters based on the comparative analysis of whole genome sequences. With the recent explosion of phage isolation, a simple method for phage cluster prediction would facilitate analysis of crude or complex samples without whole genome isolation and sequencing. The hypothesis of this study was that mycobacteriophage-cluster prediction is possible using comparison of a single, ubiquitous, semi-conserved gene. Tape Measure Protein (TMP) was selected to test the hypothesis because it is typically the longest gene in mycobacteriophage genomes and because regions within the TMP gene are conserved. Results A single gene, TMP, identified the known Mycobacteriophage clusters and subclusters using a Gepard dotplot comparison or a phylogenetic tree constructed from global alignment and maximum likelihood comparisons. Gepard analysis of 247 mycobacteriophage TMP sequences appropriately recovered 98.8% of the subcluster assignments that were made by whole-genome comparison. Subcluster-specific primers within TMP allow for PCR determination of the mycobacteriophage subcluster from DNA samples. Using the single-gene comparison approach for siphovirus coliphages, phage groupings by TMP comparison reflected relationships observed in a whole genome dotplot comparison and confirm the potential utility of this approach to another widely studied group of phages. Conclusions TMP sequence comparison and PCR results support the hypothesis that a single gene can be used for distinguishing phage cluster and subcluster assignments. TMP single-gene analysis can quickly and accurately aid in mycobacteriophage classification. PMID:23777341

  7. The diverse genetic switch of enterobacterial and marine telomere phages.

    PubMed

    Hammerl, Jens A; Jäckel, Claudia; Funk, Eugenia; Pinnau, Sabrina; Mache, Christin; Hertwig, Stefan

    2016-01-01

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the enterobacterial telomere phages N15, PY54 and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator, similar to CI, Cro and Q of lambda, respectively. Moreover, N15 and ϕKO2 contain 3 related operator (OR) sites between cI and cro, while only one site (OR3) has been detected in PY54. Marine telomere phages possess a putative cI gene but not a cro-like gene. Instead, a gene is located at the position of cro, whose product shows some similarity to the PY54 ORF42 product, the function of which is unknown. We have determined the transcription start sites of the predicted repressor genes of N15, PY54, ϕKO2 and of the marine telomere phage VP58.5. The influence of the genes on phage propagation was analyzed in E. coli, Y. enterocolitica and V.parahaemolyticus. We show that the repressors and antiterminators of N15, ϕKO2 and PY54 exerted their predicted activities. However, while the proteins of both N15 and ϕKO2 affected lysis and lysogeny by N15, they did not affect PY54 propagation. On the other hand, the respective PY54 proteins exclusively influenced the propagation of this phage. The immB region of VP58.5 contains 2 genes that revealed prophage repressor activity, while a lytic repressor gene could not be identified. The results indicate an unexpected diversity of the growth regulation mechanisms in these temperate phages.

  8. The diverse genetic switch of enterobacterial and marine telomere phages.

    PubMed

    Hammerl, Jens A; Jäckel, Claudia; Funk, Eugenia; Pinnau, Sabrina; Mache, Christin; Hertwig, Stefan

    2016-01-01

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the enterobacterial telomere phages N15, PY54 and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator, similar to CI, Cro and Q of lambda, respectively. Moreover, N15 and ϕKO2 contain 3 related operator (OR) sites between cI and cro, while only one site (OR3) has been detected in PY54. Marine telomere phages possess a putative cI gene but not a cro-like gene. Instead, a gene is located at the position of cro, whose product shows some similarity to the PY54 ORF42 product, the function of which is unknown. We have determined the transcription start sites of the predicted repressor genes of N15, PY54, ϕKO2 and of the marine telomere phage VP58.5. The influence of the genes on phage propagation was analyzed in E. coli, Y. enterocolitica and V.parahaemolyticus. We show that the repressors and antiterminators of N15, ϕKO2 and PY54 exerted their predicted activities. However, while the proteins of both N15 and ϕKO2 affected lysis and lysogeny by N15, they did not affect PY54 propagation. On the other hand, the respective PY54 proteins exclusively influenced the propagation of this phage. The immB region of VP58.5 contains 2 genes that revealed prophage repressor activity, while a lytic repressor gene could not be identified. The results indicate an unexpected diversity of the growth regulation mechanisms in these temperate phages. PMID:27607141

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

    PubMed Central

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

    2011-01-01

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

  10. Decoupling of Host–Symbiont–Phage Coadaptations Following Transfer Between Insect Species

    PubMed Central

    Chafee, Meghan E.; Zecher, Courtney N.; Gourley, Michelle L.; Schmidt, Victor T.; Chen, John H.; Bordenstein, Sarah R.; Clark, Michael E.; Bordenstein, Seth R.

    2011-01-01

    Transferring endosymbiotic bacteria between different host species can perturb the coordinated regulation of the host and bacterial genomes. Here we use the most common maternally transmitted bacteria, Wolbachia pipientis, to test the consequences of host genetic background on infection densities and the processes underlying those changes in the parasitoid wasp genus Nasonia. Introgressing the genome of Nasonia giraulti into the infected cytoplasm of N. vitripennis causes a two-order-of-magnitude increase in bacterial loads in adults and a proliferation of the infection to somatic tissues. The host effect on W. pipientis distribution and densities is associated with a twofold decrease in densities of the temperate phage WO-B. Returning the bacteria from the new host species back to the resident host species restores the bacteria and phage to their native densities. To our knowledge, this is the first study to report a host–microbe genetic interaction that affects the densities of both W. pipientis and bacteriophage WO-B. The consequences of the increased bacterial density include a reduction in fecundity, an increase in levels of cytoplasmic incompatibility (CI), and unexpectedly, male-to-female transfer of the bacteria to uninfected females and an increased acceptance of densely infected females to interspecific mates. While paternal inheritance of the W. pipientis was not observed, the high incidence of male-to-female transfer in the introgressed background raises the possibility that paternal transmission could be more likely in hybrids where paternal leakage of other cytoplasmic elements is also known to occur. Taken together, these results establish a major change in W. pipientis densities and tissue tropism between closely related species and support a model in which phage WO, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis. PMID:20944019

  11. Genome Sequence of Bacillus cereus Phage vB_BceS-MY192.

    PubMed

    Yang, Yong; Zhan, Li; Chen, Jiancai; Zhang, Yunyi; Sun, Yi; Yang, Zhangnv; Jiang, Liping; Zhu, Hanping; Zhang, Yanjun; Lu, Yiyu; Mei, Lingling

    2016-01-01

    ITALIC! Bacillus cereusis an opportunistic foodborne pathogen. The phage vB_BceS-MY192 was isolated from ITALIC! B. cereus192 in a cooked rice sample. The temperate phage belongs to the ITALIC! Siphoviridaefamily, ITALIC! Caudoviralesorder. Here we announce the phage genome sequence and its annotation, which may expand the understanding of ITALIC! B. cereussiphophages.

  12. Genome Sequences of Gordonia Phages BaxterFox, Kita, Nymphadora, and Yeezy

    PubMed Central

    Bandla, Sharanya; Colbert, Alexandra K.; Eichinger, Fiona G.; Gamburg, Michelle B.; Horiates, Stavroula G.; Jamison, Jerrica M.; Julian, Dana R.; Moore, Whitney A.; Murthy, Pranav; Powell, Meghan C.; Smith, Sydney V.; Mezghani, Nadia; Milliken, Katherine A.; Thompson, Paige K.; Toner, Chelsea L.; Ulbrich, Megan C.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Montgomery, Matthew T.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae, isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717 bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%. PMID:27516501

  13. Genome Sequence of Bacillus cereus Phage vB_BceS-MY192

    PubMed Central

    Yang, Yong; Zhan, Li; Chen, Jiancai; Zhang, Yunyi; Sun, Yi; Yang, Zhangnv; Jiang, Liping; Zhu, Hanping; Zhang, Yanjun; Lu, Yiyu

    2016-01-01

    Bacillus cereus is an opportunistic foodborne pathogen. The phage vB_BceS-MY192 was isolated from B. cereus 192 in a cooked rice sample. The temperate phage belongs to the Siphoviridae family, Caudovirales order. Here we announce the phage genome sequence and its annotation, which may expand the understanding of B. cereus siphophages. PMID:27103733

  14. Phage mutations in response to CRISPR diversification in a bacterial population.

    PubMed

    Sun, Christine L; Barrangou, Rodolphe; Thomas, Brian C; Horvath, Philippe; Fremaux, Christophe; Banfield, Jillian F

    2013-02-01

    Interactions between bacteria and their coexisting phage populations impact evolution and can strongly influence biogeochemical processes in natural ecosystems. Periodically, mutation or migration results in exposure of a host to a phage to which it has no immunity; alternatively, a phage may be exposed to a host it cannot infect. To explore the processes by which coexisting, co-evolving hosts and phage populations establish, we cultured Streptococcus thermophilus DGCC7710 with phage 2972 and tracked CRISPR (clustered regularly interspaced short palindromic repeats) diversification and host-phage co-evolution in a population derived from a colony that acquired initial CRISPR-encoded immunity. After 1 week of co-culturing, the coexisting host-phage populations were metagenomically characterized using 454 FLX Titanium sequencing. The evolved genomes were compared with reference genomes to identify newly incorporated spacers in S. thermophilus DGCC7710 and recently acquired single-nucleotide polymorphisms (SNPs) in phage 2972. Following phage exposure, acquisition of immune elements (spacers) led to a genetically diverse population with multiple subdominant strain lineages. Phage mutations that circumvented three early immunization events were localized in the proto-spacer adjacent motif (PAM) or near the PAM end of the proto-spacer, suggesting a strong selective advantage for the phage that mutated in this region. The sequential fixation or near fixation of these single mutations indicates selection events so severe that single phage genotypes ultimately gave rise to all surviving lineages and potentially carried traits unrelated to immunity to fixation.

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

    DOE PAGES

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

    2015-05-08

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

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

    SciTech Connect

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

    2015-05-08

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

  17. Phage mutations in response to CRISPR diversification in a bacterial population.

    PubMed

    Sun, Christine L; Barrangou, Rodolphe; Thomas, Brian C; Horvath, Philippe; Fremaux, Christophe; Banfield, Jillian F

    2013-02-01

    Interactions between bacteria and their coexisting phage populations impact evolution and can strongly influence biogeochemical processes in natural ecosystems. Periodically, mutation or migration results in exposure of a host to a phage to which it has no immunity; alternatively, a phage may be exposed to a host it cannot infect. To explore the processes by which coexisting, co-evolving hosts and phage populations establish, we cultured Streptococcus thermophilus DGCC7710 with phage 2972 and tracked CRISPR (clustered regularly interspaced short palindromic repeats) diversification and host-phage co-evolution in a population derived from a colony that acquired initial CRISPR-encoded immunity. After 1 week of co-culturing, the coexisting host-phage populations were metagenomically characterized using 454 FLX Titanium sequencing. The evolved genomes were compared with reference genomes to identify newly incorporated spacers in S. thermophilus DGCC7710 and recently acquired single-nucleotide polymorphisms (SNPs) in phage 2972. Following phage exposure, acquisition of immune elements (spacers) led to a genetically diverse population with multiple subdominant strain lineages. Phage mutations that circumvented three early immunization events were localized in the proto-spacer adjacent motif (PAM) or near the PAM end of the proto-spacer, suggesting a strong selective advantage for the phage that mutated in this region. The sequential fixation or near fixation of these single mutations indicates selection events so severe that single phage genotypes ultimately gave rise to all surviving lineages and potentially carried traits unrelated to immunity to fixation. PMID:23057534

  18. Genome Sequences of Gordonia Phages BaxterFox, Kita, Nymphadora, and Yeezy.

    PubMed

    Pope, Welkin H; Bandla, Sharanya; Colbert, Alexandra K; Eichinger, Fiona G; Gamburg, Michelle B; Horiates, Stavroula G; Jamison, Jerrica M; Julian, Dana R; Moore, Whitney A; Murthy, Pranav; Powell, Meghan C; Smith, Sydney V; Mezghani, Nadia; Milliken, Katherine A; Thompson, Paige K; Toner, Chelsea L; Ulbrich, Megan C; Furbee, Emily C; Grubb, Sarah R; Warner, Marcie H; Montgomery, Matthew T; Garlena, Rebecca A; Russell, Daniel A; Jacobs-Sera, Deborah; Hatfull, Graham F

    2016-01-01

    Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae, isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717 bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%. PMID:27516501

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

    PubMed

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

    2015-01-01

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

  20. Selective posttranslational modification of phage-displayed polypeptides

    DOEpatents

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-02-05

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2]cycloaddition reactions and Staudinger modifications.

  1. Selective posttranslational modification of phage-displayed polypeptides

    DOEpatents

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-11-19

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2] cycloaddition reactions and Staudinger modifications.

  2. Novel strategy for the selection of human recombinant Fab fragments to membrane proteins from a phage-display library.

    PubMed

    Labrijn, Aran F; Koppelman, Marco H G M; Verhagen, Janneke; Brouwer, Mieke C; Schuitemaker, Hanneke; Hack, C Erik; Huisman, Han G

    2002-03-01

    Traditionally, the selection of phage-display libraries is performed on purified antigens (Ags), immobilized to a solid substrate. However, this approach may not be applicable for some Ags, such as membrane proteins, which for structural integrity strongly rely on their native environment. Here we describe an approach for the selection of phage-libraries against membrane proteins. The envelope glycoproteins (Env) of the Human Immunodeficiency Virus type-1 (HIV-1) were used as a model for a type-1 integral membrane protein. HIV-1IHI Env, expressed on the surface of Rabbit Kidney cells (RK13) with a recombinant vaccinia virus (rVV), was solubilized using the non-ionic detergent n-Octyl beta-D-glucopyranoside (OG). Membrane associated Env was reconstituted into vesicles by the simultaneous removal of detergent and free monomeric Env subunits by gel-filtration. The resulting antigen preparation, termed OG-P1IHI, was captured on microtiter plates coated with Galanthus nivalis agglutinin (GNA) and used for rounds of selection (panning) of a well-characterized phage-display library derived from an HIV-1 seropositive donor. Simultaneously, an identical experiment was performed with OG-P1IHI vesicles disrupted by Nonidet P-40 (NP-P1IHI). Both membrane-associated and soluble Ags were selected for vaccinia-specific clones (OG-P1IHI: 59/75 and NP-P1IHI: 1/75) and HIV-1-specific clones (OG-P1IHI: 11/75 and NP-P1IHI: 65/75) using our approach. Hence, the novel panning strategy described here may be applicable for selection of phage-libraries against membrane proteins.

  3. Elevated lytic phage production as a consequence of particle colonization by a marine Flavobacterium (Cellulophaga sp.).

    PubMed

    Riemann, Lasse; Grossart, Hans-Peter

    2008-10-01

    Bacteria growing on marine particles generally have higher densities and cell-specific activities than free-living bacteria. Since rapidity of phage adsorption is dependent on host density, while infection productivity is a function of host physiological status, we hypothesized that marine particles are sites of elevated phage production. In the present study, organic-matter-rich agarose beads and a marine phage-host pair (Cellulophaga sp., PhiS(M)) were used as a model system to examine whether bacterial colonization of particles increases phage production. While no production of phages was observed in plain seawater, the presence of beads enhanced attachment and growth of bacteria, as well as phage production. This was observed because of extensive lysis of bacteria in the presence of beads and a subsequent increase in phage abundance both on beads and in the surrounding water. After 12 h, extensive phage lysis reduced the density of attached bacteria; however, after 32 h, bacterial abundance increased again. Reexposure to phages and analyses of bacterial isolates suggested that this regrowth on particles was by phage-resistant clones. The present demonstration of elevated lytic phage production associated with model particles illustrates not only that a marine phage has the ability to successfully infect and lyse surface-attached bacteria but also that acquisition of resistance may affect temporal phage-host dynamics on particles. These findings from a model system may have relevance to the distribution of phage production in environments rich in particulate matter (e.g., in coastal areas or during phytoplankton blooms) where a significant part of phage production may be directly linked to these nutrient-rich "hot spots."

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  5. The filamentous phage XacF1 causes loss of virulence in Xanthomonas axonopodis pv. citri, the causative agent of citrus canker disease.

    PubMed

    Ahmad, Abdelmonim Ali; Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2014-01-01

    In this study, filamentous phage XacF1, which can infect Xanthomonas axonopodis pv. citri (Xac) strains, was isolated and characterized. Electron microscopy showed that XacF1 is a member of the family Inoviridae and is about 600 nm long. The genome of XacF1 is 7325 nucleotides in size, containing 13 predicted open reading frames (ORFs), some of which showed significant homology to Ff-like phage proteins such as ORF1 (pII), ORF2 (pV), ORF6 (pIII), and ORF8 (pVI). XacF1 showed a relatively wide host range, infecting seven out of 11 strains tested in this study. Frequently, XacF1 was found to be integrated into the genome of Xac strains. This integration occurred at the host dif site (attB) and was mediated by the host XerC/D recombination system. The attP sequence was identical to that of Xanthomonas phage Cf1c. Interestingly, infection by XacF1 phage caused several physiological changes to the bacterial host cells, including lower levels of extracellular polysaccharide production, reduced motility, slower growth rate, and a dramatic reduction in virulence. In particular, the reduction in virulence suggested possible utilization of XacF1 as a biological control agent against citrus canker disease. PMID:25071734

  6. The filamentous phage XacF1 causes loss of virulence in Xanthomonas axonopodis pv. citri, the causative agent of citrus canker disease

    PubMed Central

    Ahmad, Abdelmonim Ali; Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2014-01-01

    In this study, filamentous phage XacF1, which can infect Xanthomonas axonopodis pv. citri (Xac) strains, was isolated and characterized. Electron microscopy showed that XacF1 is a member of the family Inoviridae and is about 600 nm long. The genome of XacF1 is 7325 nucleotides in size, containing 13 predicted open reading frames (ORFs), some of which showed significant homology to Ff-like phage proteins such as ORF1 (pII), ORF2 (pV), ORF6 (pIII), and ORF8 (pVI). XacF1 showed a relatively wide host range, infecting seven out of 11 strains tested in this study. Frequently, XacF1 was found to be integrated into the genome of Xac strains. This integration occurred at the host dif site (attB) and was mediated by the host XerC/D recombination system. The attP sequence was identical to that of Xanthomonas phage Cf1c. Interestingly, infection by XacF1 phage caused several physiological changes to the bacterial host cells, including lower levels of extracellular polysaccharide production, reduced motility, slower growth rate, and a dramatic reduction in virulence. In particular, the reduction in virulence suggested possible utilization of XacF1 as a biological control agent against citrus canker disease. PMID:25071734

  7. Prevalence of Stx phages in environments of a pig farm and lysogenic infection of the field E. coli O157 isolates with a recombinant converting Phage.

    PubMed

    Yan, Yaxian; Shi, Yibo; Cao, Dongmei; Meng, Xiangpeng; Xia, Luming; Sun, Jianhe

    2011-02-01

    The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx. PMID:20697714

  8. Prevalence of Stx phages in environments of a pig farm and lysogenic infection of the field E. coli O157 isolates with a recombinant converting Phage.

    PubMed

    Yan, Yaxian; Shi, Yibo; Cao, Dongmei; Meng, Xiangpeng; Xia, Luming; Sun, Jianhe

    2011-02-01

    The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.

  9. Transmission of phage by glassy-winged sharpshooters, a vector of Xylella fastidiosa

    PubMed Central

    Bhowmick, Tushar Suvra; Das, Mayukh; Heinz, Kevin M.; Krauter, Peter C.; Gonzalez, Carlos F.

    2016-01-01

    ABSTRACT Xylella fastidiosa subsp. fastidiosa (Xff) is the causal agent of Pierce's Disease (PD) of grapevines and is vectored by the glassy-winged sharpshooter (GWSS, Homalodisca vitripennis). Previously we have reported the development of a bacteriophage (phage) based biocontrol system for PD, but no information on insect transmission of phages has been reported. Here we communicate that laboratory reared GWSSs fed on cowpea plants (Vigna unguiculata subsp. unguiculata) harboring the virulent phage Paz were able to uptake of phage efficiently when the phage was present in high concentration, but were inefficient in transfer to plants. PMID:27738554

  10. Genome Sequence of Bacillus cereus Group Phage SalinJah.

    PubMed

    Erill, Ivan; Caruso, Steven M

    2016-01-01

    The double-stranded DNA (dsDNA) Myoviridae Bacillus cereus group bacteriophage SalinJah was isolated from soil collected in Gyeonggi-do, South Korea. SalinJah, a cluster C phage with a broad host range, suggests the need to create a new subcluster with SalinJah and Helga as founding members.

  11. Genome Sequence of Gordonia Phage BetterKatz

    PubMed Central

    Berryman, Emily N.; Forrest, Kaitlyn M.; McHale, Lilliana; Wertz, Anthony T.; Zhuang, Zenas; Kasturiarachi, Naomi S.; Pressimone, Catherine A.; Schiebel, Johnathon G.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Montgomery, Matthew T.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    BetterKatz is a bacteriophage isolated from a soil sample collected in Pittsburgh, Pennsylvania using the host Gordonia terrae 3612. BetterKatz’s genome is 50,636 bp long and contains 75 predicted protein-coding genes, 35 of which have been assigned putative functions. BetterKatz is not closely related to other sequenced Gordonia phages. PMID:27516497

  12. Genome Sequence of Bacillus cereus Group Phage SalinJah

    PubMed Central

    2016-01-01

    The double-stranded DNA (dsDNA) Myoviridae Bacillus cereus group bacteriophage SalinJah was isolated from soil collected in Gyeonggi-do, South Korea. SalinJah, a cluster C phage with a broad host range, suggests the need to create a new subcluster with SalinJah and Helga as founding members. PMID:27688335

  13. CRISPR: new horizons in phage resistance and strain identification.

    PubMed

    Barrangou, Rodolphe; Horvath, Philippe

    2012-01-01

    Bacteria have been widely used as starter cultures in the food industry, notably for the fermentation of milk into dairy products such as cheese and yogurt. Lactic acid bacteria used in food manufacturing, such as lactobacilli, lactococci, streptococci, Leuconostoc, pediococci, and bifidobacteria, are selectively formulated based on functional characteristics that provide idiosyncratic flavor and texture attributes, as well as their ability to withstand processing and manufacturing conditions. Unfortunately, given frequent viral exposure in industrial environments, starter culture selection and development rely on defense systems that provide resistance against bacteriophage predation, including restriction-modification, abortive infection, and recently discovered CRISPRs (clustered regularly interspaced short palindromic repeats). CRISPRs, together with CRISPR-associated genes (cas), form the CRISPR/Cas immune system, which provides adaptive immunity against phages and invasive genetic elements. The immunization process is based on the incorporation of short DNA sequences from virulent phages into the CRISPR locus. Subsequently, CRISPR transcripts are processed into small interfering RNAs that guide a multifunctional protein complex to recognize and cleave matching foreign DNA. Hypervariable CRISPR loci provide insights into the phage and host population dynamics, and new avenues for enhanced phage resistance and genetic typing and tagging of industrial strains.

  14. Prospective identification of parasitic sequences in phage display screens

    PubMed Central

    Matochko, Wadim L.; Cory Li, S.; Tang, Sindy K.Y.; Derda, Ratmir

    2014-01-01

    Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones. PMID:24217917

  15. Identification of Soft Matter Binding Peptide Ligands Using Phage Display.

    PubMed

    Günay, Kemal Arda; Klok, Harm-Anton

    2015-10-21

    Phage display is a powerful tool for the selection of highly affine, short peptide ligands. While originally primarily used for the identification of ligands to proteins, the scope of this technique has significantly expanded over the past two decades. Phage display nowadays is also increasingly applied to identify ligands that selectively bind with high affinity to a broad range of other substrates including natural and biological polymers as well as a variety of low-molecular-weight organic molecules. Such peptides are of interest for various reasons. The ability to selectively and with high affinity bind to the substrate of interest allows the conjugation or immobilization of, e.g., nanoparticles or biomolecules, or generally, facilitates interactions at materials interfaces. On the other hand, presentation of peptide ligands that selectively bind to low-molecular-weight organic materials is of interest for the development of sensor surfaces. The aim of this article is to highlight the opportunities provided by phage display for the identification of peptide ligands that bind to synthetic or natural polymer substrates or to small organic molecules. The article will first provide an overview of the different peptide ligands that have been identified by phage display that bind to these "soft matter" targets. The second part of the article will discuss the different characterization techniques that allow the determination of the affinity of the identified ligands to the respective substrates. PMID:26275106

  16. Phage selection for bacterial cheats leads to population decline.

    PubMed

    Vasse, Marie; Torres-Barceló, Clara; Hochberg, Michael E

    2015-11-01

    While predators and parasites are known for their effects on bacterial population biology, their impact on the dynamics of bacterial social evolution remains largely unclear. Siderophores are iron-chelating molecules that are key to the survival of certain bacterial species in iron-limited environments, but their production can be subject to cheating by non-producing genotypes. In a selection experiment conducted over approximately 20 bacterial generations and involving 140 populations of the pathogenic bacterium Pseudomonas aeruginosa PAO1, we assessed the impact of a lytic phage on competition between siderophore producers and non-producers. We show that the presence of lytic phages favours the non-producing genotype in competition, regardless of whether iron use relies on siderophores. Interestingly, phage pressure resulted in higher siderophore production, which constitutes a cost to the producers and may explain why they were outcompeted by non-producers. By the end of the experiment, however, cheating load reduced the fitness of mixed populations relative to producer monocultures, and only monocultures of producers managed to grow in the presence of phage in situations where siderophores were necessary to access iron. These results suggest that public goods production may be modulated in the presence of natural enemies with consequences for the evolution of social strategies. PMID:26538598

  17. Genome Sequence of Bacillus cereus Group Phage SalinJah.

    PubMed

    Erill, Ivan; Caruso, Steven M

    2016-01-01

    The double-stranded DNA (dsDNA) Myoviridae Bacillus cereus group bacteriophage SalinJah was isolated from soil collected in Gyeonggi-do, South Korea. SalinJah, a cluster C phage with a broad host range, suggests the need to create a new subcluster with SalinJah and Helga as founding members. PMID:27688335

  18. The phage-driven microbial loop in petroleum bioremediation.

    PubMed

    Rosenberg, Eugene; Bittan-Banin, Gili; Sharon, Gil; Shon, Avital; Hershko, Galit; Levy, Itzik; Ron, Eliora Z

    2010-07-01

    During the drilling process and transport of crude oil, water mixes with the petroleum. At oil terminals, the water settles to the bottom of storage tanks. This drainage water is contaminated with emulsified oil and water-soluble hydrocarbons and must be treated before it can be released into the environment. In this study, we tested the efficiency of a continuous flow, two-stage bioreactor for treating drainage water from an Israeli oil terminal. The bioreactor removed all of the ammonia, 93% of the sulfide and converted 90% of the total organic carbon (TOC) into carbon dioxide. SYBR Gold staining indicated that reactor 1 contained 1.7 × 10(8) bacteria and 3.7 × 10(8) phages per millilitre, and reactor 2 contained 1.3 × 10(8) bacteria and 1.7 × 10(9) phages per millilitre. The unexpectedly high mineralization of TOC and high concentration of phage in reactor 2 support the concept of a phage-driven microbial loop in the bioremediation of the drainage water. In general, application of this concept in bioremediation of contaminated water has the potential to increase the efficiency of processes. PMID:21255344

  19. Nitrous acid induced damage in T7 DNA and phage

    SciTech Connect

    Scearce, L.M.; Masker, W.E.

    1986-05-01

    The response of bacteriophage T7 to nitrous acid damage was investigated. The T7 system allows in vitro mimicry of most aspects of in vivo DNA metabolism. Nitrous acid is of special interest since it has been previously shown to induce deletions and point mutations as well as novel adducts in DNA. T7 phage was exposed to 56 mM nitrous acid at pH 4.6 in vivo, causing a time dependent 98% decrease in survival for each 10 min duration of exposure to nitrous acid. These studies were extended to include examination of pure T7 DNA exposed in vitro to nitrous acid conditions identical to those used in the in vivo survival studies. The treated DNA was dialyzed to remove the nitrous acid and the DNA was encapsulated into empty phage heads. These in vitro packaged phage showed a survival curve analogous to the in vivo system. There was no change in survival when either in vitro or in vivo exposed phage were grown on wild type E. coli or on E. coli strains deficient in DNA repair due to mutations in DNA polymerase I, exonuclease III or a uvrA mutation. Survival was not increased when nitrous acid treated T7 were grown on E. coli induced for SOS repair. In vitro replication of nitrous acid treated DNA showed a time dependent decrease in the total amount of DNA synthesized.

  20. Genome Sequence of Gordonia Phage BetterKatz.

    PubMed

    Pope, Welkin H; Berryman, Emily N; Forrest, Kaitlyn M; McHale, Lilliana; Wertz, Anthony T; Zhuang, Zenas; Kasturiarachi, Naomi S; Pressimone, Catherine A; Schiebel, Johnathon G; Furbee, Emily C; Grubb, Sarah R; Warner, Marcie H; Montgomery, Matthew T; Garlena, Rebecca A; Russell, Daniel A; Jacobs-Sera, Deborah; Hatfull, Graham F

    2016-01-01

    BetterKatz is a bacteriophage isolated from a soil sample collected in Pittsburgh, Pennsylvania using the host Gordonia terrae 3612. BetterKatz's genome is 50,636 bp long and contains 75 predicted protein-coding genes, 35 of which have been assigned putative functions. BetterKatz is not closely related to other sequenced Gordonia phages. PMID:27516497

  1. Genome Sequences of Gordonia terrae Phages Benczkowski14 and Katyusha.

    PubMed

    Pope, Welkin H; Benczkowski, Matthew S; Green, Daryn E; Hwang, Melina; Kennedy, Bryan; Kocak, Bradley; Kruczek, Ellen; Lin, Leon; Moretti, Matthew L; Onelangsy, Faith L; Mezghani, Nadia; Milliken, Katherine A; Toner, Chelsea L; Thompson, Paige K; Ulbrich, Megan C; Furbee, Emily C; Grubb, Sarah R; Warner, Marcie H; Montgomery, Matthew T; Garlena, Rebecca A; Russell, Daniel A; Jacobs-Sera, Deborah; Hatfull, Graham F

    2016-01-01

    Bacteriophages Katyusha and Benczkowski14 are newly isolated phages that infect Gordonia terrae 3612. Both have siphoviral morphologies with isometric heads and long tails (500 nm). The genomes are 75,380 bp long and closely related, and the tape measure genes (9 kbp) are among the largest to be identified. PMID:27340062

  2. Genome Sequences of Gordonia terrae Phages Benczkowski14 and Katyusha

    PubMed Central

    Benczkowski, Matthew S.; Green, Daryn E.; Hwang, Melina; Kennedy, Bryan; Kocak, Bradley; Kruczek, Ellen; Lin, Leon; Moretti, Matthew L.; Onelangsy, Faith L.; Mezghani, Nadia; Milliken, Katherine A.; Toner, Chelsea L.; Thompson, Paige K.; Ulbrich, Megan C.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Montgomery, Matthew T.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    Bacteriophages Katyusha and Benczkowski14 are newly isolated phages that infect Gordonia terrae 3612. Both have siphoviral morphologies with isometric heads and long tails (500 nm). The genomes are 75,380 bp long and closely related, and the tape measure genes (9 kbp) are among the largest to be identified. PMID:27340062

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

    PubMed

    Hobbs, Zack; Abedon, Stephen T

    2016-04-01

    Bacteriophages, or phages, are viruses of members of domain Bacteria. These viruses play numerous roles in shaping the diversity of microbial communities, with impact differing depending on what infection strategies specific phages employ. From an applied perspective, these especially are communities containing undesired or pathogenic bacteria that can be modified through phage-mediated bacterial biocontrol, that is, through phage therapy. Here we seek to categorize phages in terms of their infection strategies as well as review or suggest more descriptive, accurate or distinguishing terminology. Categories can be differentiated in terms of (1) whether or not virion release occurs (productive infections versus lysogeny, pseudolysogeny and/or the phage carrier state), (2) the means of virion release (lytic versus chronic release) and (3) the degree to which phages are genetically equipped to display lysogenic cycles (temperate versus non-temperate phages). We address in particular the use or overuse of what can be a somewhat equivocal phrase, 'Lytic or lysogenic', especially when employed as a means of distinguishing among phages types. We suggest that the implied dichotomy is inconsistent with both modern as well as historical understanding of phage biology. We consider, therefore, less ambiguous terminology for distinguishing between 'Lytic' versus 'Lysogenic' phage types. PMID:26925588

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

    PubMed

    Hobbs, Zack; Abedon, Stephen T

    2016-04-01

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

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

    PubMed

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

    2016-08-15

    Bacteriophage (phage) amplification is an attractive method for the detection of bacteria due to a narrow phage-host specificity, short amplification times, and the phages' ability to differentiate between viable and non-viable bacterial cells. The next step in phage-based bacteria detection is leveraging bioengineered phages to create low-cost, rapid, and easy-to-use detection platforms such as lateral flow assays. Our work establishes the proof-of-concept for the use of bioengineered T7 phage strains to increase the sensitivity of phage amplification-based lateral flow assays. We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 10(3)CFU/mL of Escherichia coli in broth after 7h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100CFU/100mL in inoculated river water after 9h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use. PMID:27031186

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

    PubMed

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

    2016-08-15

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

  7. The use of phage FCL-2 as an alternative to chemotherapy against columnaris disease in aquaculture.

    PubMed

    Laanto, Elina; Bamford, Jaana K H; Ravantti, Janne J; Sundberg, Lotta-Riina

    2015-01-01

    Flavobacterium columnare, the causative agent of columnaris disease in fish, causes millions of dollars of losses in the US channel catfish industry alone, not to mention aquaculture industry worldwide. Novel methods are needed for the control and treatment of bacterial diseases in aquaculture to replace traditionally used chemotherapies. A potential solution could be the use of phages, i.e., bacterial viruses, host-specific and self-enriching particles that can be can easily distributed via water flow. We examined the efficacy of phages to combat columnaris disease. A previously isolated phage, FCL-2, infecting F. columnare, was characterized by sequencing. The 47 142 bp genome of the phage had G + C content of 30.2%, and the closest similarities regarding the structural proteins were found in Cellulophaga phage phiSM. Under controlled experimental conditions, two host fish species, rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), were used to study the success of phage therapy to prevent F. columnare infections. The survival of both fish species was significantly higher in the presence of the phage. Hundred percent of the zebrafish and 50% of the rainbow trout survived in the phage treatment (survival without phage 0 and 8.3%, respectively). Most importantly, the rainbow trout population was rescued from infection by a single addition of the phage into the water in a flow-through fish tank system. Thus, F. columnare could be used as a model system to test the benefits and risks of phage therapy on a larger scale.

  8. Genome organization of temperate phage 11143 from emetic Bacillus cereus NCTC11143.

    PubMed

    Lee, Young-Duck; Park, Jong-Hyun

    2012-05-01

    A temperate phage was isolated from emetic Bacillus cereus NCTC 11143 by mitomycin C and characterized by transmission electron microscopy and DNA and protein analyses. Whole genome sequencing of Bacillus phage 11143 was performed by GS-FLX. The phage has a dsDNA genome of 39,077 bp and a 35% G+C content. Bioinformatic analysis of the phage genome revealed 49 putative ORFs involved in replication, morphogenesis, DNA packaging, lysogeny, and host lysis. Bacillus phage 11143 could be classified as a member of the Siphoviridae family by morphology and genome structure. Genomic comparisons at the DNA and protein levels revealed homologous genetic modules with patterns and morphogenesis proteins similar to those of other Bacillus phages. Thus, Bacillus phages might have a mosaic genetic relationship.

  9. Phage-based platforms for the clinical detection of human bacterial pathogens

    PubMed Central

    Schofield, David A.; Sharp, Natasha J.; Westwater, Caroline

    2012-01-01

    Bacteriophages (phages) have been utilized for decades as a means for uniquely identifying their target bacteria. Due to their inherent natural specificity, ease of use, and straightforward production, phage possess a number of desirable attributes which makes them particularly suited as bacterial detectors. As a result, extensive research has been conducted into the development of phage, or phage-derived products to expedite the detection of human pathogens. However, very few phage-based diagnostics have transitioned from the research lab into a clinical diagnostic tool. Herein we review the phage-based platforms that are currently used for the detection of Mycobacterium tuberculosis, Yersinia pestis, Bacillus anthracis and Staphylococcus aureus in the clinical field. We briefly describe the disease, the current diagnostic options, and the role phage diagnostics play in identifying the cause of infection, and determining antibiotic susceptibility. PMID:23050221

  10. The phage-related chromosomal islands of Gram-positive bacteria

    PubMed Central

    Novick, Richard P.; Christie, Gail E.; Penadés, Jose R.

    2012-01-01

    The phage-related chromosomal islands (PRCIs) were first identified in Staphylococcus aureus as highly mobile, superantigen-encoding genetic elements known as the S. aureus pathogenicity islands (SaPIs). These elements are characterized by a specific set of phage-related functions that enable them to use the phage reproduction cycle for their own transduction and inhibit phage reproduction in the process. SaPIs produce many phage-like infectious particles; their streptococcal counterparts have a role in gene regulation but may not be infectious. These elements therefore represent phage satellites or parasites, not defective phages. In this Review, we discuss the shared genetic content of PRCIs, their life cycle and their ability to be transferred across large phylogenetic distances. PMID:20634809

  11. CTXϕ: Exploring new alternatives in host factor-mediated filamentous phage replications.

    PubMed

    Martínez, Eriel; Campos-Gómez, Javier; Barre, François-Xavier

    2016-01-01

    For a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXϕ, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXϕ and VGJϕ replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages. PMID:27607139

  12. Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

    PubMed

    Rossant, Christine J; Carroll, Danielle; Huang, Ling; Elvin, John; Neal, Frances; Walker, Edward; Benschop, Joris J; Kim, Eldar E; Barry, Simon T; Vaughan, Tristan J

    2014-01-01

    Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.

  13. The expression of Streptomyces and Escherichia coli drug-resistance determinants cloned into the Streptomyces phage phi C31.

    PubMed

    Chater, K F; Bruton, C J; King, A A; Suarez, J E

    1982-01-01

    Lysogens obtained by infecting Streptomyces albus G with a phi C31-pBR322 chimaeric prophage or its delta W12 deletion derivative had increased tetracycline resistance. The ability of the delta W12 derivative to transduce tetracycline resistance was inactivated by inserting a viomycin resistance determinant (vph) into the BamHI site of the pBR322 tet gene, and restored by excising the vph gene. Another deletion mutant (delta W17) of the chimaera, carrying an intact tet gene, was normally unable to transduce tetracycline resistance. This inability was correlated with the finding, by Southern hybridisation analysis, that the att site required for insertion of phi C31 prophage into the host chromosome was located within the delta W17 deletion. Use of phi C31 lysogenic recipient permitted the integration of the att-deleted phage, presumably by homologous recombination, giving tetracycline-resistant double lysogens. This technique was extended to S. coelicolor A3(2) in the detection of derivatives of the att-deleted phage into which a thiostrepton-resistance determinant (tsr) had been inserted in vitro. Phage released from double lysogens were mainly recombinants. One such recombinant is a PstI vector for DNA cloning, able to accommodate up to 6 kb of introduced DNA.

  14. Development of a highly sensitive noncompetitive electrochemical immunosensor for the detection of atrazine by phage anti-immunocomplex assay.

    PubMed

    González-Techera, Andrés; Zon, María Alicia; Molina, Patricia Gabriela; Fernández, Héctor; González-Sapienza, Gualberto; Arévalo, Fernando Javier

    2015-02-15

    The development of immunosensors for the detection of small molecules is of great interest because of their simplicity, high sensitivity and extended analytical range. Due to their size, small compounds cannot be simultaneously recognized by two antibodies impeding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. In this work, we combine the advantages of magneto-electrochemical immunosensors with the improved sensitivity and direct proportional signal of noncompetitive immunoassays to develop a new Phage Anti-Immunocomplex Electrochemical Immunosensor (PhAIEI) for the detection of the herbicide atrazine. The noncompetitive assay is based on the use of recombinant M13 phage particles bearing a peptide that specifically recognizes the immunocomplex of atrazine with an anti-atrazine monoclonal antibody. The PhAIEI performed with a limit of detection (LOD) of 0.2 pg mL(-1), which is 200-fold better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA, with a large increase in working range. The developed PhAIEI was successfully used to assay undiluted river water samples with no pretreatment and excellent recoveries. Apart from the first demonstration of the benefits of integrating phage anti-immunocomplex particles into electrochemical immunosensors, the extremely low and environmentally relevant detection limits of atrazine attained with the PhAIEIS may have direct applicability to fast and sensitive detection of this herbicide in the environment.

  15. Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China

    PubMed Central

    Zhao, Huanqiang; Hu, Fupin; Jin, Shu; Xu, Xiaogang; Zou, Yuhan; Ding, Baixing; He, Chunyan; Gong, Fang; Liu, Qingzhong

    2016-01-01

    Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages. PMID:27536288

  16. Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China.

    PubMed

    Zhao, Huanqiang; Hu, Fupin; Jin, Shu; Xu, Xiaogang; Zou, Yuhan; Ding, Baixing; He, Chunyan; Gong, Fang; Liu, Qingzhong

    2016-01-01

    Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages. PMID:27536288

  17. Antibacterial phage ORFans of Pseudomonas aeruginosa phage LUZ24 reveal a novel MvaT inhibiting protein.

    PubMed

    Wagemans, Jeroen; Delattre, Anne-Sophie; Uytterhoeven, Birgit; De Smet, Jeroen; Cenens, William; Aertsen, Abram; Ceyssens, Pieter-Jan; Lavigne, Rob

    2015-01-01

    The functional elucidation of small unknown phage proteins ('ORFans') presents itself as one of the major challenges of bacteriophage molecular biology. In this work, we mined the Pseudomonas aeruginosa-infecting phage LUZ24 proteome for antibacterial and antibiofilm proteins against its host. Subsequently, their putative host target was identified. In one example, we observed an interaction between LUZ24 gp4 and the host transcriptional regulator MvaT. The polymerization of MvaT across AT-rich DNA strands permits gene silencing of foreign DNA, thereby limiting any potentially adverse effects of such DNA. Gel shift assays proved the inhibitory effect of LUZ24 gp4 on MvaT DNA binding activity. Therefore, we termed this gene product as Mip, the MvaT inhibiting protein. We hypothesize Mip prevents the AT-rich LUZ24 DNA from being physically blocked by MvaT oligomers right after its injection in the host cell, thereby allowing phage transcription and thus completion of the phage infection cycle. PMID:26594207

  18. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library.

    PubMed

    Han, Lei; Liu, Pei; Petrenko, Valery A; Liu, Aihua

    2016-02-24

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 10(2) - 2.0 × 10(8) cells mL(-1)), a low limit of detection (79 cells mL(-1), S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  19. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    NASA Astrophysics Data System (ADS)

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-02-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 ‑ 2.0 × 108 cells mL‑1), a low limit of detection (79 cells mL‑1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  20. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    PubMed Central

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-01-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 − 2.0 × 108 cells mL−1), a low limit of detection (79 cells mL−1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis. PMID:26908277

  1. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library.

    PubMed

    Han, Lei; Liu, Pei; Petrenko, Valery A; Liu, Aihua

    2016-01-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 10(2) - 2.0 × 10(8) cells mL(-1)), a low limit of detection (79 cells mL(-1), S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis. PMID:26908277

  2. Tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage N15.

    PubMed

    Mardanov, Andrey V; Strakhova, Taisia S; Smagin, Vladimir A; Ravin, Nikolai V

    2007-06-15

    A new Escherichia coli host/vector system has been developed to allow a dual regulation of both the plasmid copy number and gene expression. The new pN15E vectors are low copy number plasmids based on the replicon of temperate phage N15, comprising the repA replicase gene and cB repressor gene, controlling the plasmid copy number. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the PBAD promoter. The host strain also carried phage N15 partition operon, sop, allowing stable inheritance of pN15E vectors in the absence of selection pressure. In the first vector, pN15E4, the same PBAD promoter controls expression of a cloned gene. The second vector, pN15E6, carries the phage T5 promoter with a double lac operator repression module thus allowing independent regulation of promoter activity and copy number. Using the lacZ gene to monitor expression in these vectors, we show that the ratio of induction/repression can be about 7600-fold for pN15E4 and more than 15,000-fold for pN15E6. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products that was demonstrated by the stable maintenance of a gene encoding a restriction endonuclease in pN15E4. The tight control of transcription and the potential to regulate gene activities quantitatively over wide ranges will open up new approaches in the study of gene function in vivo and controlled expression of heterologous genes.

  3. Salmonella phages isolated from dairy farms in Thailand show wider host range than a comparable set of phages isolated from U.S. dairy farms.

    PubMed

    Wongsuntornpoj, Sarach; Moreno Switt, Andrea I; Bergholz, Peter; Wiedmann, Martin; Chaturongakul, Soraya

    2014-08-01

    Salmonella is a zoonotic pathogen with globally distributed serovars as well as serovars predominantly found in certain regions; for example, serovar Weltevreden is rarely isolated in the U.S., but is common in Thailand. Relative to our understanding of Salmonella diversity, our understanding of the global diversity of Salmonella phages is limited. We hypothesized that the serovar diversity in a given environment and farming system will affect the Salmonella phage diversity associated with animal hosts. We thus isolated and characterized Salmonella phages from 15 small-scale dairy farms in Thailand and compared the host ranges of the 62 Salmonella phage isolates obtained with host range diversity for 129 phage isolates obtained from dairy farms in the U.S. The 62 phage isolates from Thailand represented genome sizes ranging from 40 to 200 kb and showed lysis of 6-25 of the 26 host strains tested (mean number of strain lysed=19). By comparison, phage isolates previously obtained in a survey of 15 U.S. dairy farms showed a narrow host range (lysis of 1-17; mean number of strains lysed=4); principal coordinate analysis also confirmed U.S. and Thai phages had distinct host lysis profiles. Our data indicate that dairy farms that differ in management practices and are located on different continents can yield phage isolates that differ in their host ranges, providing an avenue for isolation of phages with desirable host range characteristics for commercial applications. Farming systems characterized by coexistence of different animals may facilitate presence of Salmonella phages with wide host ranges.

  4. Listeria phage and phage tail induction triggered by components of bacterial growth media (phosphate, LiCl, nalidixic acid, and acriflavine).

    PubMed

    Lemaître, Jean-Paul; Duroux, Amandine; Pimpie, Romain; Duez, Jean-Marie; Milat, Marie-Louise

    2015-03-01

    The detection of Listeria monocytogenes from food is currently carried out using a double enrichment. For the ISO methodology, this double enrichment is performed using half-Fraser and Fraser broths, in which the overgrowth of L. innocua can occur in samples where both species are present. In this study, we analyzed the induction of phages and phage tails of Listeria spp. in these media and in two brain heart infusion (BHI) broths (BHIM [bioMérieux] and BHIK [Biokar]) to identify putative effectors. It appears that Na2HPO4 at concentrations ranging from 1 to 40 g/liter with an initial pH of 7.5 can induce phage or phage tail production of Listeria spp., especially with 10 g/liter of Na2HPO4 and a pH of 7.5, conditions present in half-Fraser and Fraser broths. Exposure to LiCl in BHIM (18 to 21 g/liter) can also induce phage and phage tail release, but in half-Fraser and Fraser broths, the concentration of LiCl is much lower (3 g/liter). Low phage titers were induced by acriflavine and/or nalidixic acid. We also show that the production of phages and phage tails can occur in half-Fraser and Fraser broths. This study points out that induction of phages and phage tails could be triggered by compounds present in enrichment media. This could lead to a false-negative result for the detection of L. monocytogenes in food products.

  5. Oligopeptidase A is required for normal phage P22 development.

    PubMed Central

    Conlin, C A; Vimr, E R; Miller, C G

    1992-01-01

    The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing. Images PMID:1522065

  6. Multi-scale structure and geographic drivers of cross-infection within marine bacteria and phages

    PubMed Central

    Flores, Cesar O; Valverde, Sergi; Weitz, Joshua S

    2013-01-01

    Bacteriophages are the most abundant biological life forms on Earth. However, relatively little is known regarding which bacteriophages infect and exploit which bacteria. A recent meta-analysis showed that empirically measured phage-bacteria infection networks are often significantly nested, on average, and not modular. A perfectly nested network is one in which phages can be ordered from specialist to generalist such that the host range of a given phage is a subset of the host range of the subsequent phage in the ordering. The same meta-analysis hypothesized that modularity, in which groups of phages specialize on distinct groups of hosts, should emerge at larger geographic and/or taxonomic scales. In this paper, we evaluate the largest known phage-bacteria interaction data set, representing the interaction of 215 phage types with 286 host types sampled from geographically separated sites in the Atlantic Ocean. We find that this interaction network is highly modular. In addition, some of the modules identified in this data set are nested or contain submodules, indicating the presence of multi-scale structure, as hypothesized in the earlier meta-analysis. We examine the role of geography in driving these patterns and find evidence that the host range of phages and the phage permissibility of bacteria is driven, in part, by geographic separation. We conclude by discussing approaches to disentangle the roles of ecology and evolution in driving complex patterns of interaction between phages and bacteria. PMID:23178671

  7. A polystyrene binding target-unrelated peptide isolated in the screening of phage display library.

    PubMed

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2016-11-01

    Phage display is a powerful methodology for the identification of peptide ligands binding to any desired target. However, the selection of target-unrelated peptides (TUPs) appears as a huge problem in the screening of phage display libraries through biopanning. The phage-displayed peptide TLHPAAD has been isolated both in our laboratory and by another reserach group on completely different screening targets prompting us to hypothesize that it may be a potential TUP. In the current study, we analyzed the binding characteristics and propagation rate of phage clone displaying TLHPAAD peptide (SW-TUP clone). The results of ELISA experiment and phage recovery assay provided strong support for the notion that SW-TUP phage binds to polystyrene with a significantly higher affinity than control phage clones. Furthermore, this polystyrene binding was demonstrated to occur in a concentration- and pH-dependent mode. Characterization of the propagation profile of phage clones within a specified time course revealed no statistically significant difference between the amplification rate of SW-TUP and control phages. Our findings lead us to the conclusion that SW-TUP phage clone with the displayed peptide TLHPAAD is not a true target binder and its selection in biopanning experiments results from its bidning affinity to the polystyrene surface of the solid phase.

  8. Comparative genomic and morphological analyses of Listeria phages isolated from farm environments.

    PubMed

    Denes, Thomas; Vongkamjan, Kitiya; Ackermann, Hans-Wolfgang; Moreno Switt, Andrea I; Wiedmann, Martin; den Bakker, Henk C

    2014-08-01

    The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large genome (~135-kb) myoviruses belonging to the genus “Twort-like viruses,” three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (~66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (~56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.

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

    PubMed Central

    Abedon, Stephen T.

    2016-01-01

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

  10. Single-stranded DNA phages: from early molecular biology tools to recent revolutions in environmental microbiology.

    PubMed

    Székely, Anna J; Breitbart, Mya

    2016-03-01

    Single-stranded DNA (ssDNA) phages are profoundly different from tailed phages in many aspects including the nature and size of their genome, virion size and morphology, mutation rate, involvement in horizontal gene transfer, infection dynamics and cell lysis mechanisms. Despite the importance of ssDNA phages as molecular biology tools and model systems, the environmental distribution and ecological roles of these phages have been largely unexplored. Viral metagenomics and other culture-independent viral diversity studies have recently challenged the perspective of tailed, double-stranded DNA (dsDNA) phages, dominance by demonstrating the prevalence of ssDNA phages in diverse habitats. However, the differences between ssDNA and dsDNA phages also substantially limit the efficacy of simultaneously assessing the abundance and diversity of these two phage groups. Here we provide an overview of the major differences between ssDNA and tailed dsDNA phages that may influence their effects on bacterial communities. Furthermore, through the analysis of 181 published metaviromes we demonstrate the environmental distribution of ssDNA phages and present an analysis of the methodological biases that distort their study through metagenomics. PMID:26850442

  11. Inactivation of Escherichia coli phage by pulsed electric field treatment and analysis of inactivation mechanism

    NASA Astrophysics Data System (ADS)

    Tanino, Takanori; Yoshida, Tomoki; Sakai, Kazuki; Ohshima, Takayuki

    2013-03-01

    Inactivation of bacteriophage by pulsed electric field (PEF) treatment, one of the effective procedures for bacteria nonthermal inactivation, was studied. Model phage particles Escherichia coli bacteriophages M13mp18 and λ phage, were successfully inactivated by PEF treatment. The survival ratios of both bacteriophages decreased depending on the PEF treatment time when applied peak voltage was 5 or 7 kV, and the survival ratios after 12 min PEF treatment were 10-4 - 10-5. Electrophoresis analyses of biological molecules of inactivated λ phage detected no degradation of total protein and genomic DNA. These results suggested that the factor of phage inactivation by PEF treatment was not based on the degradation of protein or DNA, but on the destruction of phage particle structure. Sensitivity of E. coli phage to PEF treatment was compared with that of E. coli cell. Phage and MV1184 cell were treated with same condition PEF at 5 kV, respectively. After 12 min treatment, the survival ration of λ phage and MV1184 were 4.0 × 10-5 and 1.7 × 10-3, respectively. The survival ratio of phage was lower than that of MV1184. E. coli cell is more tolerant to inactivation with PEF treatment than coli phage.

  12. Comparative Genomic and Morphological Analyses of Listeria Phages Isolated from Farm Environments

    PubMed Central

    Denes, Thomas; Ackermann, Hans-Wolfgang; Moreno Switt, Andrea I.; Wiedmann, Martin; den Bakker, Henk C.

    2014-01-01

    The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large-genome (∼135-kb) myoviruses belonging to the genus “Twort-like viruses,” three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (∼66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (∼56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes. PMID:24837381

  13. A polystyrene binding target-unrelated peptide isolated in the screening of phage display library.

    PubMed

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2016-11-01

    Phage display is a powerful methodology for the identification of peptide ligands binding to any desired target. However, the selection of target-unrelated peptides (TUPs) appears as a huge problem in the screening of phage display libraries through biopanning. The phage-displayed peptide TLHPAAD has been isolated both in our laboratory and by another reserach group on completely different screening targets prompting us to hypothesize that it may be a potential TUP. In the current study, we analyzed the binding characteristics and propagation rate of phage clone displaying TLHPAAD peptide (SW-TUP clone). The results of ELISA experiment and phage recovery assay provided strong support for the notion that SW-TUP phage binds to polystyrene with a significantly higher affinity than control phage clones. Furthermore, this polystyrene binding was demonstrated to occur in a concentration- and pH-dependent mode. Characterization of the propagation profile of phage clones within a specified time course revealed no statistically significant difference between the amplification rate of SW-TUP and control phages. Our findings lead us to the conclusion that SW-TUP phage clone with the displayed peptide TLHPAAD is not a true target binder and its selection in biopanning experiments results from its bidning affinity to the polystyrene surface of the solid phase. PMID:27555439

  14. Phage sensitivity and prophage carriage in Staphylococcus aureus isolated from foods in Spain and New Zealand.

    PubMed

    Gutiérrez, Diana; Rodríguez-Rubio, Lorena; García, Pilar; Billington, Craig; Premarante, Aruni; Rodríguez, Ana; Martínez, Beatriz

    2016-08-01

    Bacteriophages (phages) are a promising tool for the biocontrol of pathogenic bacteria, including those contaminating food products and causing infectious diseases. However, the success of phage preparations is limited by the host ranges of their constituent phages. The phage resistance/sensitivity profile of eighty seven Staphylococcus aureus strains isolated in Spain and New Zealand from dairy, meat and seafood sources was determined for six phages (Φ11, K, ΦH5, ΦA72, CAPSa1 and CAPSa3). Most of the S. aureus strains were sensitive to phage K (Myoviridae) and CAPSa1 (Siphoviridae) regardless of their origin. There was a higher sensitivity of New Zealand S. aureus strains to phages isolated from both Spain (ΦH5 and ΦA72) and New Zealand (CAPSa1 and CAPSa3). Spanish phages had a higher infectivity on S. aureus strains of Spanish dairy origin, while Spanish strains isolated from other environments were more sensitive to New Zealand phages. Lysogeny was more prevalent in Spanish S. aureus compared to New Zealand strains. A multiplex PCR reaction, which detected ΦH5 and ΦA72 sequences, indicated a high prevalence of these prophages in Spanish S. aureus strains, but were infrequently detected in New Zealand strains. Overall, the correlation between phage resistance and lysogeny in S. aureus strains was found to be weak.

  15. Methicillin-resistant Staphylococcus aureus phage plaque size enhancement using sublethal concentrations of antibiotics.

    PubMed

    Kaur, Sandeep; Harjai, Kusum; Chhibber, Sanjay

    2012-12-01

    Phage therapy presents an alternative approach against the emerging methicillin-resistant Staphylococcus aureus (MRSA) threat. Some of the problems encountered during isolation of MRSA phages include the high prevalence of enteric phages in natural sources, nonspecific absorption of viable phage, and the formation of pinpoint or tiny plaques. The phage isolated in this study, MR-5, also formed tiny plaques against its host S. aureus ATCC 43300 (MRSA), making its detection and enumeration difficult. An improved method of increasing the plaque size of MRSA phage by incorporating sublethal concentrations of three different classes of antibiotics (inhibitors of protein synthesis) in the classical double-layer agar (DLA) method was investigated. The β-lactam and quinolone antibiotics commonly employed in earlier studies for increasing the plaque size did not show any significant effect on the plaque size of isolated MR-5 phage. Linezolid (oxazolidinone class), tetracycline, and ketolide antibiotics brought significant enhancements (3 times the original size) in the plaque size of MR-5 phage. Prior treatment with these antibiotics resulted in significant reductions in the time of adsorption and the latent period of MR-5 phage. To rule out whether the action of linezolid (which brought the maximum increase in plaque size) was specific for a single phage only, its effect on the plaque size of seven other S. aureus-specific phages was also assessed. Significant enhancements in the plaque size of these phages were observed. These results indicate that this modification can therefore safely be incorporated in the traditional DLA overlay method to search for new MRSA-virulent phages. PMID:23001655

  16. Genomics of staphylococcal Twort-like phages--potential therapeutics of the post-antibiotic era.

    PubMed

    Łobocka, Małgorzata; Hejnowicz, Monika S; Dąbrowski, Kamil; Gozdek, Agnieszka; Kosakowski, Jarosław; Witkowska, Magdalena; Ulatowska, Magdalena I; Weber-Dąbrowska, Beata; Kwiatek, Magdalena; Parasion, Sylwia; Gawor, Jan; Kosowska, Helena; Głowacka, Aleksandra

    2012-01-01

    Polyvalent bacteriophages of the genus Twort-like that infect clinically relevant Staphylococcus strains may be among the most promising phages with potential therapeutic applications. They are obligatorily lytic, infect the majority of Staphylococcus strains in clinical strain collections, propagate efficiently and do not transfer foreign DNA by transduction. Comparative genomic analysis of 11 S. aureus/S. epidermidis Twort-like phages, as presented in this chapter, emphasizes their strikingly high similarity and clear divergence from phage Twort of the same genus, which might have evolved in hosts of a different species group. Genetically, these phages form a relatively isolated group, which minimizes the risk of acquiring potentially harmful genes. The order of genes in core parts of their 127 to 140-kb genomes is conserved and resembles that found in related representatives of the Spounavirinae subfamily of myoviruses. Functions of certain conserved genes can be predicted based on their homology to prototypical genes of model spounavirus SPO1. Deletions in the genomes of certain phages mark genes that are dispensable for phage development. Nearly half of the genes of these phages have no known homologues. Unique genes are mostly located near termini of the virion DNA molecule and are expressed early in phage development as implied by analysis of their potential transcriptional signals. Thus, many of them are likely to play a role in host takeover. Single genes encode homologues of bacterial virulence-associated proteins. They were apparently acquired by a common ancestor of these phages by horizontal gene transfer but presumably evolved towards gaining functions that increase phage infectivity for bacteria or facilitate mature phage release. Major differences between the genomes of S. aureus/S. epidermidis Twort-like phages consist of single nucleotide polymorphisms and insertions/deletions of short stretches of nucleotides, single genes, or introns of group I

  17. Effect of Bacteriophages on the Growth of Flavobacterium psychrophilum and Development of Phage-Resistant Strains.

    PubMed

    Christiansen, Rói Hammershaimb; Madsen, Lone; Dalsgaard, Inger; Castillo, Daniel; Kalatzis, Panos G; Middelboe, Mathias

    2016-05-01

    The controlling effect of single and multiple phages on the density of Flavobacterium psychrophilum at different initial multiplicity of infection (MOI) was assessed in batch cultures to explore the potential for phage-based treatment of this important fish pathogen. A high initial phage concentration (MOI = 0.3-4) was crucial for efficient viral lysis, resulting in a 10(4)-10(5)-fold reduction of phage-sensitive cells (both single phages and phage cocktails), which was maintained throughout the incubation (>10 days). Following cell lysis, regrowth of phage-resistant strains was examined and resistant strains were isolated for further characterization. The application of a mathematical model allowed simulation of phage-host interactions and resistance development, confirming indications from strain isolations that phage-sensitive strains dominated the regrowing population (>99.8%) at low MOI and phage-resistant strains (>87.8%) dominated at high MOI. A cross-infectivity test covering 68 isolated strains and 22 phages resulted in 23 different host susceptibility patterns, with 20 of the isolates being resistant to all the applied phages. Eleven isolated strains with different susceptibility patterns had lower growth rates (0.093 to 0.31 h(-1)) than the host strain (0.33 h(-1)), while 10 of 14 examined strains had lost the ability to take up specific substrates as shown by BIOLOG profiles. Despite increased selection for phage resistance at high MOI, the results emphasize that high initial MOI is essential for fast and effective control of F. psychrophilum infection and suggest that the small populations of resistant clones had reduced competitive abilities relative to the sensitive ancestral strain.

  18. Differential Codon Adaptation between dsDNA and ssDNA Phages in Escherichia coli

    PubMed Central

    Chithambaram, Shivapriya; Prabhakaran, Ramanandan; Xia, Xuhua

    2014-01-01

    Because phages use their host translation machinery, their codon usage should evolve toward that of highly expressed host genes. We used two indices to measure codon adaptation of phages to their host, rRSCU (the correlation in relative synonymous codon usage [RSCU] between phages and their host) and Codon Adaptation Index (CAI) computed with highly expressed host genes as the reference set (because phage translation depends on host translation machinery). These indices used for this purpose are appropriate only when hosts exhibit little mutation bias, so only phages parasitizing Escherichia coli were included in the analysis. For double-stranded DNA (dsDNA) phages, both rRSCU and CAI decrease with increasing number of transfer RNA genes encoded by the phage genome. rRSCU is greater for dsDNA phages than for single-stranded DNA (ssDNA) phages, and the low rRSCU values are mainly due to poor concordance in RSCU values for Y-ending codons between ssDNA phages and the E. coli host, consistent with the predicted effect of C→T mutation bias in the ssDNA phages. Strong C→T mutation bias would improve codon adaptation in codon families (e.g., Gly) where U-ending codons are favored over C-ending codons (“U-friendly” codon families) by highly expressed host genes but decrease codon adaptation in other codon families where highly expressed host genes favor C-ending codons against U-ending codons (“U-hostile” codon families). It is remarkable that ssDNA phages with increasing C→T mutation bias also increased the usage of codons in the “U-friendly” codon families, thereby achieving CAI values almost as large as those of dsDNA phages. This represents a new type of codon adaptation. PMID:24586046

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

    PubMed

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

    2011-08-01

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

  20. Analyses of the Distribution Patterns of Burkholderia pseudomallei and Associated Phages in Soil Samples in Thailand Suggest That Phage Presence Reduces the Frequency of Bacterial Isolation

    PubMed Central

    Withatanung, Patoo; Chantratita, Narisara; Muangsombut, Veerachat; Saiprom, Natnaree; Lertmemongkolchai, Ganjana; Klumpp, Jochen; Clokie, Martha R. J.; Galyov, Edouard E.

    2016-01-01

    Background Burkholderia pseudomallei is a soil saprophytic bacterium that causes melioidosis. The infection occurs through cutaneous inoculation, inhalation or ingestion. Bacteriophages (phages) in the same ecosystem may significantly impact the biology of this bacterium in the environment, and in their culturability in the laboratory. Methods/Principal Findings The soil samples were analysed for the presence of bacteria using culture methods, and for phages using plaque assays on B. pseudomallei strain 1106a lawns. Of the 86 soil samples collected from northeastern Thailand, B. pseudomallei was cultured from 23 (26.7%) samples; no phage capable of infecting B. pseudomallei was detected in these samples. In contrast, phages capable of infecting B. pseudomallei, but no bacteria, were present in 10 (11.6%) samples. B. pseudomallei and their phages were co-isolated from only 3 (3.5%) of soil samples. Since phage capable of infecting B. pseudomallei could not have appeared in the samples without the prior presence of bacteria, or exposure to bacteria nearby, our data suggest that all phage-positive/bacteria-negative samples have had B. pseudomallei in or in a close proximity to them. Taken together, these findings indicate that the presence of phages may influence the success of B. pseudomallei isolation. Transmission electron microscopy revealed that the isolated phages are podoviruses. The temperate phages residing in soil-isolated strains of B. pseudomallei that were resistant to the dominant soil borne phages could be induced by mitomycin C. These induced-temperate phages were closely related, but not identical, to the more dominant soil-isolated phage type. Conclusion/Significance The presence of podoviruses capable of infecting B. pseudomallei may affect the success of the pathogen isolation from the soil. The currently used culture-based methods of B. pseudomallei isolation appear to under-estimate the bacterial abundance. The detection of phage capable of

  1. In vitro packaging of phage DNA with defined lesions

    SciTech Connect

    Lee, E.; Hays, J.B.

    1986-05-01

    In vitro packaging of undamaged phage lambda DNA at efficiencies in excess of 2% has been routinely achieved using slightly modified standard techniques. In order to test the ability of nicks and gaps to stimulate genetic recombination during homoimmune and lytic infections, DNA was randomly nicked by treatment with methidium-propyl-EDTA-Fe(II) (MPE), and the nicks expanded to gaps with exonuclease III. DNA with about 15 MPE nicks per lambda duplex or the same number of 30-nucleotide gaps was packaged with slightly reduced efficiency. Surprisingly, DNA irradiated at 254 nm, containing 50 cyclobutane pyrimidine dimers per lambda duplex (plus other photoproducts) was packaged as efficiently as unirradiated DNA. Thus, phage containing only minor 254 nm photoproducts can be prepared efficiently by irradiation, enzymatic photoreactivation, and packaging.

  2. Clear Plaque Mutants of Lactococcal Phage TP901-1

    PubMed Central

    Kot, Witold; Kilstrup, Mogens; Vogensen, Finn K.; Hammer, Karin

    2016-01-01

    We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1. PMID:27258092

  3. Targeting membrane proteins for antibody discovery using phage display.

    PubMed

    Jones, Martina L; Alfaleh, Mohamed A; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B; Chin, David Y; Mahler, Stephen M

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  4. Sad State of Phage Electron Microscopy. Please Shoot the Messenger

    PubMed Central

    Ackermann, Hans-W.

    2013-01-01

    Two hundred and sixty publications from 2007 to 2012 were classified according to the quality of electron micrographs; namely as good (71); mediocre (21); or poor (168). Publications were from 37 countries; appeared in 77 journals; and included micrographs produced with about 60 models of electron microscopes. The quality of the micrographs was not linked to any country; journal; or electron microscope. Main problems were poor contrast; positive staining; low magnification; and small image size. Unsharp images were frequent. Many phage descriptions were silent on virus purification; magnification control; even the type of electron microscope and stain used. The deterioration in phage electron microscopy can be attributed to the absence of working instructions and electron microscopy courses; incompetent authors and reviewers; and lenient journals. All these factors are able to cause a gradual lowering of standards.

  5. A generalized transducing phage for the murine pathogen Citrobacter rodentium

    PubMed Central

    Petty, Nicola K.; Toribio, Ana L.; Goulding, David; Foulds, Ian; Thomson, Nicholas; Dougan, Gordon; Salmond, George P. C.

    2008-01-01

    A virulent phage (φCR1) capable of generalized transduction in Citrobacter rodentium was isolated from the environment and characterized. C. rodentium is a natural pathogen of mice, causing transmissible murine colonic hyperplasia. Sequencing of its genome has recently been completed and will soon be fully annotated and published. C. rodentium is an important model organism for infections caused by the human pathogens enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC). φCR1 uses a lipopolysaccharide receptor, has a genome size of approximately 300 kb, and is able to transduce a variety of markers. φCR1 is the first reported transducing phage for C. rodentium and will be a useful tool for functional genomic analysis of this important natural murine pathogen. PMID:17768241

  6. Targeting membrane proteins for antibody discovery using phage display

    PubMed Central

    Jones, Martina L.; Alfaleh, Mohamed A.; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W.; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B.; Chin, David Y.; Mahler, Stephen M.

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  7. Genome Sequences of Gordonia Phages Hotorobo, Woes, and Monty

    PubMed Central

    Davis, Jameson P.; O’Shea, Sirinya; Pfeiffer, Anastasia C.; Rich, Alexandra N.; Xue, Jason C.; Shedlock, Kathleen A.; Stanton, Ann-Catherine J.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Montgomery, Matthew T.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    Hotorobo, Woes, and Monty are newly isolated bacteriophages of Gordonia terrae 3612. The three phages are related, and their genomes are similarly sized (76,972 bp, 73,752 bp, and 75,680 bp for Hotorobo, Woes, and Monty, respectively) and organized. They have extremely long tails and among the longest tape measure protein genes described to date. PMID:27516500

  8. Genome Sequences of Gordonia Phages Hotorobo, Woes, and Monty.

    PubMed

    Pope, Welkin H; Davis, Jameson P; O'Shea, Sirinya; Pfeiffer, Anastasia C; Rich, Alexandra N; Xue, Jason C; Shedlock, Kathleen A; Stanton, Ann-Catherine J; Furbee, Emily C; Grubb, Sarah R; Warner, Marcie H; Montgomery, Matthew T; Garlena, Rebecca A; Russell, Daniel A; Jacobs-Sera, Deborah; Hatfull, Graham F

    2016-01-01

    Hotorobo, Woes, and Monty are newly isolated bacteriophages of Gordonia terrae 3612. The three phages are related, and their genomes are similarly sized (76,972 bp, 73,752 bp, and 75,680 bp for Hotorobo, Woes, and Monty, respectively) and organized. They have extremely long tails and among the longest tape measure protein genes described to date. PMID:27516500

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

    PubMed

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

    2013-09-01

    Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb, including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure. PMID:23755967

  10. Phage therapy treatment of the coral pathogen Vibrio coralliilyticus

    PubMed Central

    Cohen, Yossi; Joseph Pollock, F; Rosenberg, Eugene; Bourne, David G

    2013-01-01

    Vibrio coralliilyticus is an important coral pathogen demonstrated to cause disease outbreaks worldwide. This study investigated the feasibility of applying bacteriophage therapy to treat the coral pathogen V. coralliilyticus. A specific bacteriophage for V. coralliilyticus strain P1 (LMG23696), referred to here as bacteriophage YC, was isolated from the seawater above corals at Nelly Bay, Magnetic Island, central Great Barrier Reef (GBR), the same location where the bacterium was first isolated. Bacteriophage YC was shown to be a lytic phage belonging to the Myoviridae family, with a rapid replication rate, high burst size, and high affinity to its host. By infecting its host bacterium, bacteriophage YC was able to prevent bacterial-induced photosystem inhibition in pure cultures of Symbiodinium, the photosymbiont partner of coral and a target for virulence factors produced by the bacterial pathogen. Phage therapy experiments using coral juveniles in microtiter plates as a model system revealed that bacteriophage YC was able to prevent V. coralliilyticus-induced photoinactivation and tissue lysis. These results demonstrate that bacteriophage YC has the potential to treat coral disease outbreaks caused by the bacterial pathogen V. coralliilyticus, making it a good candidate for phage therapy treatment of coral disease. PMID:23239510

  11. Communication: Origin of the contributions to DNA structure in phages.

    PubMed

    Myers, Christopher G; Pettitt, B Montgomery

    2013-02-21

    Cryo electron microscopy (cryo-EM) data of the interior of phages show ordering of the interior DNA that has been interpreted as a nearly perfectly ordered polymer. We show surface-induced correlations, excluded volume, and electrostatic forces are sufficient to predict most of the major features of the current structural data for DNA packaged within viral capsids without additional ordering due to elastic bending forces for the polymer. Current models assume highly-ordered, even spooled, hexagonally packed conformations based on interpretation of cryo-EM density maps. We show herein that the surface induced packing of short (6mer), unconnected DNA polymer segments is the only necessary ingredient in creating ringed densities consistent with experimental density maps. This implies the ensemble of possible conformations of polymeric DNA within the capsid that are consistent with cryo-EM data may be much larger than implied by traditional interpretations where such rings can only result from highly-ordered spool-like conformations. This opens the possibility of a more disordered, entropically-driven view of phage packaging thermodynamics. We also show the electrostatics of the DNA contributes a large portion of the internal hydrostatic and osmotic pressures of a phage virion, suggesting that nonlinear elastic anomalies might reduce the overall elastic bending enthalpy of more disordered conformations to have allowable free energies.

  12. T4 bacteriophage as a phage display platform.

    PubMed

    Gamkrelidze, Mariam; Dąbrowska, Krystyna

    2014-07-01

    Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems.

  13. T4 bacteriophage as a phage display platform.

    PubMed

    Gamkrelidze, Mariam; Dąbrowska, Krystyna

    2014-07-01

    Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems. PMID:24828789

  14. Archaeal Viruses, Not Archaeal Phages: An Archaeological Dig

    PubMed Central

    Abedon, Stephen T.; Murray, Kelly L.

    2013-01-01

    Viruses infect members of domains Bacteria, Eukarya, and Archaea. While those infecting domain Eukarya are nearly universally described as “Viruses”, those of domain Bacteria, to a substantial extent, instead are called “Bacteriophages,” or “Phages.” Should the viruses of domain Archaea therefore be dubbed “Archaeal phages,” “Archaeal viruses,” or some other construct? Here we provide documentation of published, general descriptors of the viruses of domain Archaea. Though at first the term “Phage” or equivalent was used almost exclusively in the archaeal virus literature, there has been a nearly 30-year trend away from this usage, with some persistence of “Phage” to describe “Head-and-tail” archaeal viruses, “Halophage” to describe viruses of halophilic Archaea, use of “Prophage” rather than “Provirus,” and so forth. We speculate on the root of the early 1980's transition from “Phage” to “Virus” to describe these infectious agents, consider the timing of introduction of “Archaeal virus” (which can be viewed as analogous to “Bacterial virus”), identify numerous proposed alternatives to “Archaeal virus,” and also provide discussion of the general merits of the term, “Phage.” Altogether we identify in excess of one dozen variations on how the viruses of domain Archaea are described, and document the timing of both their introduction and use. PMID:23653528

  15. Probing ADAMTS13 Substrate Specificity using Phage Display

    PubMed Central

    Desch, Karl C.; Kretz, Colin; Yee, Andrew; Gildersleeve, Robert; Metzger, Kristin; Agrawal, Nidhi; Cheng, Jane; Ginsburg, David

    2015-01-01

    Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73. PMID:25849793

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

    PubMed Central

    Majewska, Joanna; Beta, Weronika; Lecion, Dorota; Hodyra-Stefaniak, Katarzyna; Kłopot, Anna; Kaźmierczak, Zuzanna; Miernikiewicz, Paulina; Piotrowicz, Agnieszka; Ciekot, Jarosław; Owczarek, Barbara; Kopciuch, Agnieszka; Wojtyna, Karolina; Harhala, Marek; Mąkosa, Mateusz; Dąbrowska, Krystyna

    2015-01-01

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

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

    PubMed

    Majewska, Joanna; Beta, Weronika; Lecion, Dorota; Hodyra-Stefaniak, Katarzyna; Kłopot, Anna; Kaźmierczak, Zuzanna; Miernikiewicz, Paulina; Piotrowicz, Agnieszka; Ciekot, Jarosław; Owczarek, Barbara; Kopciuch, Agnieszka; Wojtyna, Karolina; Harhala, Marek; Mąkosa, Mateusz; Dąbrowska, Krystyna

    2015-08-01

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

  18. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.

    PubMed

    Pope, Welkin H; Bowman, Charles A; Russell, Daniel A; Jacobs-Sera, Deborah; Asai, David J; Cresawn, Steven G; Jacobs, William R; Hendrix, Roger W; Lawrence, Jeffrey G; Hatfull, Graham F

    2015-01-01

    The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery. PMID:25919952

  19. The phage-host arms race: Shaping the evolution of microbes

    SciTech Connect

    Stern, Adi; Sorek, Rotem

    2010-10-26

    Bacteria, the most abundant organisms on the planet, are outnumbered by a factor of 10 to 1 by phages that infect them. Faced with the rapid evolution and turnover of phage particles, bacteria have evolved various mechanisms to evade phage infection and killing, leading to an evolutionary arms race. The extensive co-evolution of both phage and host has resulted in considerable diversity on the part of both bacterial and phage defensive and offensive strategies. In this paper, we discuss the unique and common features of phage resistance mechanisms and their role in global biodiversity. Finally, the commonalities between defense mechanisms suggest avenues for the discovery of novel forms of these mechanisms based on their evolutionary traits.

  20. Advancement and applications of peptide phage display technology in biomedical science.

    PubMed

    Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

    2016-01-01

    Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

  1. P087, a lactococcal phage with a morphogenesis module similar to an Enterococcus faecalis prophage.

    PubMed

    Villion, Manuela; Chopin, Marie-Christine; Deveau, Hélène; Ehrlich, S Dusko; Moineau, Sylvain; Chopin, Alain

    2009-05-25

    The virulent lactococcal phage P087 was isolated from a dairy environment in 1978. This phage was then recognized as the reference member for one of the ten phage groups currently known to infect Lactococcus lactis strains. The double-stranded DNA genome of this Siphoviridae phage is composed of 60,074 bp and is circularly permuted. Five tRNA and 88 orfs were found within an uncommon genome architecture. Eleven structural proteins were also identified through SDS-PAGE and LC-MS/MS analyses. Of note, 11 translated orfs from the structural module of phage P087 have identities to gene products found in a prophage located in the genome of Enterococcus faecalis V583. The alignment of both genomic sequences suggests that DNA exchanges could occur between these two phages which are infecting low G+C bacteria found in similar ecological niches.

  2. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.

    PubMed

    Pope, Welkin H; Bowman, Charles A; Russell, Daniel A; Jacobs-Sera, Deborah; Asai, David J; Cresawn, Steven G; Jacobs, William R; Hendrix, Roger W; Lawrence, Jeffrey G; Hatfull, Graham F

    2015-04-28

    The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.

  3. 60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage.

    PubMed

    Yamamoto, Tatsuo; Kojio, Seiichi; Taneike, Ikue; Nakagawa, Saori; Iwakura, Nobuhiro; Wakisaka-Saito, Noriko

    2003-05-16

    Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher. However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1). although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2). irradiation differentially inactivates some activities of Stx.

  4. Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection.

    PubMed

    Reyes-Cortés, Ruth; Arguijo-Hernández, Emma S; Carballo-Ontiveros, Marco A; Martínez-Peñafiel, Eva; Kameyama, Luis

    2016-01-01

    In order to identify host components involved in the infective process of bacteriophages, we developed a wide-range strategy to obtain cell envelope mutants, using Escherichia coli W3110 and its specific phage mEp213. The strategy consisted in four steps: (1) random mutagenesis using transposon miniTn10Km(r); (2) selection of phage-resistant mutants by replica-plating; (3) electroporation of the phage-resistant mutants with mEp213 genome, followed by selection of those allowing phage development; and (4) sequencing of the transposon-disrupted genes. This strategy allowed us to distinguish the host factors related to phage development or multiplication within the cell, from those involved in phage infection at the level of the cell envelope. PMID:27311665

  5. Intragenus generalized transduction in Staphylococcus spp. by a novel giant phage.

    PubMed

    Uchiyama, Jumpei; Takemura-Uchiyama, Iyo; Sakaguchi, Yoshihiko; Gamoh, Keiji; Kato, Shin-ichiro; Daibata, Masanori; Ujihara, Takako; Misawa, Naoaki; Matsuzaki, Shigenobu

    2014-09-01

    Bacteriophage (phage)-mediated generalized transduction is expected to contribute to the emergence of drug-resistant staphylococcal clones in various environments. In this study, novel phage S6 was isolated from sewage and used to test generalized transduction in human- and animal-derived staphylococci. Phage S6 was a novel type of giant myophage, which possessed a DNA genome that contained uracil instead of thymine, and it could infect all of the tested staphylococcal species. The phage S6 appeared to be similar to the transducing phage PBS1, which infects Bacillus spp. Moreover, phage S6 facilitated the transduction of a plasmid in Staphylococcus aureus and from S. aureus to non-aureus staphylococcal species, as well as vice versa. Transduction of methicillin resistance also occurred in S. aureus. This is the first report of successful intragenus generalized transduction among staphylococci.

  6. The complete genome sequence of PE3-1, a novel E. coli O153 phage.

    PubMed

    Liu, Honghui; Liu, Xinchun; Yi, Xin; Liu, Ruyin; Huang, Jing

    2016-11-01

    A novel virulent phage PE3-1 against E. coli O153 was isolated from an aeration tank in a wastewater treatment plant. Transmission electron microscopy images showed that phage PE3-1 had an icosahedral head and a short tail, which revealed that it was a member of the family Podoviridae of the order Caudovirales. The complete PE3-1 genome consisted of 39,093 bp and was a linear double-stranded DNA with an average GC content of 49.93 %. Phage PE3-1 showed homology to the T7-like phages in 48 open reading frames (ORFs), but it differed from previously reported E .coli phages in morphology and bioinformatics analysis. This indicated that phage PE3-1 is a new member of the genus T7 virus. PMID:27541819

  7. Salmonella typhimurium phage type 141 infections in Sheffield during 1984 and 1985: association with hens' eggs.

    PubMed Central

    Chapman, P. A.; Rhodes, P.; Rylands, W.

    1988-01-01

    Food poisoning due to Salmonella typhimurium phage type 141 was unusual in the Sheffield area before 1984. The sudden increase in incidence of this phage type during 1984 and 1985, and its causative role in several small outbreaks in this period have been investigated. Epidemiological and laboratory investigations suggested that hens' eggs were the most likely source of S. typhimurium phage type 141. PMID:3042440

  8. The Population and Evolutionary Dynamics of Phage and Bacteria with CRISPR–Mediated Immunity

    PubMed Central

    Levin, Bruce R.; Moineau, Sylvain; Bushman, Mary; Barrangou, Rodolphe

    2013-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with associated genes (cas), form the CRISPR–cas adaptive immune system, which can provide resistance to viruses and plasmids in bacteria and archaea. Here, we use mathematical models, population dynamic experiments, and DNA sequence analyses to investigate the host–phage interactions in a model CRISPR–cas system, Streptococcus thermophilus DGCC7710 and its virulent phage 2972. At the molecular level, the bacteriophage-immune mutant bacteria (BIMs) and CRISPR–escape mutant phage (CEMs) obtained in this study are consistent with those anticipated from an iterative model of this adaptive immune system: resistance by the addition of novel spacers and phage evasion of resistance by mutation in matching sequences or flanking motifs. While CRISPR BIMs were readily isolated and CEMs generated at high rates (frequencies in excess of 10−6), our population studies indicate that there is more to the dynamics of phage–host interactions and the establishment of a BIM–CEM arms race than predicted from existing assumptions about phage infection and CRISPR–cas immunity. Among the unanticipated observations are: (i) the invasion of phage into populations of BIMs resistant by the acquisition of one (but not two) spacers, (ii) the survival of sensitive bacteria despite the presence of high densities of phage, and (iii) the maintenance of phage-limited communities due to the failure of even two-spacer BIMs to become established in populations with wild-type bacteria and phage. We attribute (i) to incomplete resistance of single-spacer BIMs. Based on the results of additional modeling and experiments, we postulate that (ii) and (iii) can be attributed to the phage infection-associated production of enzymes or other compounds that induce phenotypic phage resistance in sensitive bacteria and kill resistant BIMs. We present evidence in support of these hypotheses and discuss the implications of

  9. What are the limitations on the wider therapeutic use of phage?

    PubMed Central

    Henein, Alexandra

    2013-01-01

    Bacterial resistance to antibiotics poses a serious health threat. Since research into new antibiotics is not progressing at the same rate as the development of bacterial resistance, widespread calls for alternatives to antibiotics have been made. Phage therapy is an ideal alternative candidate to be investigated. However the success of phage therapy may be hampered by a lack of investment support from large pharmaceutical companies, due to their narrow spectrum of activity in antibiotics, very large costs associated with clinical trials of the variety of phages needed, and regulatory requirements remaining unclear. Intellectual property is difficult to secure for therapeutic phage products for a variety of reasons, and patenting procedures vary widely between the US and the EU. Consequently, companies are more likely to invest in phage products for decontamination or veterinary use, rather than clinical use in humans. Some still raise questions as to the safety of phage therapy overall, suggesting the possibility of cytotoxicity and immunogenicity, depending on the phage preparation and route. On the other hand, with patients dying because of infections untreatable with conventional antibiotics, the question arises as to whether it is ethical not to pursue phage therapy more diligently. A paradigm shift about how phage therapy is perceived is required, as well as more rigorous proof of efficacy in the form of clinical trials of existing medicinal phage products. Phage therapy potential may be fulfilled in the meantime by allowing individual preparations to be used on a named-patient basis, with extensive monitoring and multidisciplinary team input. The National Health Service and academia have a role in carrying out clinical phage research, which would be beneficial to public health, but not necessarily financially rewarding. PMID:24228220

  10. In vitro management of hospital Pseudomonas aeruginosa biofilm using indigenous T7-like lytic phage.

    PubMed

    Ahiwale, Sangeeta; Tamboli, Nilofer; Thorat, Kiran; Kulkarni, Rajendra; Ackermann, Hans; Kapadnis, Balasaheb

    2011-02-01

    Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1-HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.

  11. Control of directionality in Streptomyces phage φBT1 integrase-mediated site-specific recombination.

    PubMed

    Zhang, Lin; Zhu, Binyan; Dai, Ruixue; Zhao, Guoping; Ding, Xiaoming

    2013-01-01

    Streptomyces phage φBT1 integrates its genome into the attB site of the host chromosome with the attP site to generate attL and attR. The φBT1 integrase belongs to the large serine recombinase subfamily which directly binds to target sites to initiate double strand breakage and exchange. A recombination directionality factor (RDF) is commonly required for switching integration to excision. Here we report the characterization of the RDF protein for φBT1 recombination. The RDF, is a phage-encoded gp3 gene product (28 KDa), which allows efficient active excision between attL and attR, and inhibits integration between attB and attP; Gp3 can also catalyze topological relaxation with the integrase of supercoiled plasmids containing a single excision site. Further study showed that Gp3 could form a dimer and interact with the integrase whether it bound to the substrate or not. The synapse formation of attL or attR alone with integrase and Gp3 showed that synapsis did not discriminate between the two sites, indicating that complementarity of central dinucleotides is the sole determinant of outcome in correct excision synapses. Furthermore, both in vitro and in vivo evidence support that the RDFs of φBT1 and φC31 were fully exchangeable, despite the low amino acid sequence identity of the two integrases.

  12. Comparative genomics of the T4-Like Escherichia coli phage JS98: implications for the evolution of T4 phages.

    PubMed

    Chibani-Chennoufi, Sandra; Canchaya, Carlos; Bruttin, Anne; Brüssow, Harald

    2004-12-01

    About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh. The DNA shared up to 81% base pair identity with phage T4. The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform. The head genes showed the highest sequence conservation, followed by the tail, baseplate, and tail fiber genes. Many tail fiber genes shared only protein sequence identity. Except for the insertion of endonuclease genes in T4 and gene 24 duplication in JS98, the structural gene maps of the two phages were colinear. The receptor-recognizing tail fiber proteins gp37 and gp38 were only distantly related to T4, but shared up to 83% amino acid identity to other T6-like phages, suggesting lateral gene transfer. A greater degree of variability was seen between JS98 and T4 over DNA replication and DNA transaction genes. While most of these genes came in the same order and shared up to 76% protein sequence identity, a few rearrangements, insertions, and replacements of genes were observed. Many putative gene insertions in the DNA replication module of T4 were flanked by intron-related endonuclease genes, suggesting mobile DNA elements. A hotspot of genome diversification was located downstream of the DNA polymerase gene 43 and the DNA binding gene 32. Comparative genomics of 100-kb genome sequence revealed that T4-like phages diversify more by the accumulation of point mutations and occasional gene duplication events than by modular exchanges. PMID:15576776

  13. The production of generalized transducing phage by bacteriophage lambda.

    PubMed

    Sternberg, N

    1986-01-01

    Generalized transduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage lambda to assess some of the factors that affect that process. The results of those experiments indicate: the production of generalized transducing particles by bacteriophage lambda is inhibited by the phage lambda exonuclease (Exo). Also inhibited by lambda Exo is the production of lambda docR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of lambda docL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by lambda Exo; lambda-generalized transducing particles are not detected in induced lysis-defective (S-) lambda lysogens until about 60-90 min after prophage induction. Since wild-type lambda would normally lyse cells by 60 min, the production of lambda-generalized transducing particles depends on the phage being lysis-defective; if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10-20-fold range. In contrast, if transducing lysates are prepared by the induction of a lambda lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers; if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the

  14. Comparative genomics and functional analysis of the 936 group of lactococcal Siphoviridae phages

    PubMed Central

    Murphy, James; Bottacini, Francesca; Mahony, Jennifer; Kelleher, Philip; Neve, Horst; Zomer, Aldert; Nauta, Arjen; van Sinderen, Douwe

    2016-01-01

    Genome sequencing and comparative analysis of bacteriophage collections has greatly enhanced our understanding regarding their prevalence, phage-host interactions as well as the overall biodiversity of their genomes. This knowledge is very relevant to phages infecting Lactococcus lactis, since they constitute a significant risk factor for dairy fermentations. Of the eighty four lactococcal phage genomes currently available, fifty five belong to the so-called 936 group, the most prevalent of the ten currently recognized lactococcal phage groups. Here, we report the genetic characteristics of a new collection of 936 group phages. By combining these genomes to those sequenced previously we determined the core and variable elements of the 936 genome. Genomic variation occurs across the 936 phage genome, such as genetic elements that (i) lead to a +1 translational frameshift resulting in the formation of additional structures on the phage tail, (ii) specify a double neck passage structure, and (iii) encode packaging module-associated methylases. Hierarchical clustering of the gene complement of the 936 group phages and nucleotide alignments allowed grouping of the ninety 936 group phages into distinct clusters, which in general appear to correspond with their geographical origin. PMID:26892066

  15. Engineered phage films as scaffolds for CaCO3 biomineralization.

    PubMed

    Tom, Steven; Jin, Hyo-Eon; Heo, Kwang; Lee, Seung-Wuk

    2016-08-25

    M13 bacteriophages (phage) were exploited as CaCO3 mineralization scaffolds for hard tissue engineering applications. M13 phage was first self-assembled into biomimetic fibrous scaffolds, followed by CaCO3 biomineralization via the polymer-induced liquid precursor process. The phage scaffolds successfully incorporated calcium carbonate, facilitating nucleation and growth of spherulitically textured calcite. The Young's modulus of the scaffolds increased by an order of magnitude after mineralization while also supporting the growth of mouse fibroblasts. These findings demonstrate that phage-based biomaterials are a feasible platform for creating biomineralized hard tissue constructs, in support of future studies in hard tissue engineering and biomedical applications.

  16. Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

    PubMed

    Regeimbal, James M; Jacobs, Anna C; Corey, Brendan W; Henry, Matthew S; Thompson, Mitchell G; Pavlicek, Rebecca L; Quinones, Javier; Hannah, Ryan M; Ghebremedhin, Meron; Crane, Nicole J; Zurawski, Daniel V; Teneza-Mora, Nimfa C; Biswas, Biswajit; Hall, Eric R

    2016-10-01

    Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe.

  17. Directed synthesis of bio-inorganic vanadium oxide composites using genetically modified filamentous phage

    NASA Astrophysics Data System (ADS)

    Mueller, Michael; Baik, Seungyun; Jeon, Hojeong; Kim, Yuchan; Kim, Jungtae; Kim, Young Jun

    2015-05-01

    The growth of crystalline vanadium oxide using a filamentous bacteriophage template was investigated using sequential incubation in a V2O5 precursor. Using the genetic modification of the bacteriophage, we displayed two cysteines that constrained the RSTB-1 peptide on the major coat protein P8, resulting in vanadium oxide crystallization. The phage-driven vanadium oxide crystals with different topologies, microstructures, photodegradation and vanadium oxide composites were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), quartz microbalance and dissipation (QCM-D) and X-ray photoelectron spectroscopy (XPS). Non-specific electrostatic attraction between a wild-type phage (wt-phage) and vanadium cations in the V2O5 precursor caused phage agglomeration and fiber formation along the length of the viral scaffold. As a result, the addition of recombinant phage (re-phage) in V2O5 precursors formed heterogeneous structures, which led to efficient condensation of vanadium oxide crystal formation in lines, shown by QCM-D analysis. Furthermore, re-phage/VxOx composites showed significantly enhanced photodegradation activities compared with the synthesized wt-phage-V2O5 composite under illumination. This study demonstrates that peptide-mediated vanadium oxide mineralization is governed by a complicated interplay of peptide sequence, local structure, kinetics and the presence of a mineralizing aid, such as the two cysteine-constrained peptides on the phage surface, and has potential for use in nanotechnology applications.

  18. On-Demand Isolation of Bacteriophages Against Drug-Resistant Bacteria for Personalized Phage Therapy

    PubMed Central

    Mattila, Sari; Ruotsalainen, Pilvi; Jalasvuori, Matti

    2015-01-01

    Bacteriophages are bacterial viruses, capable of killing even multi-drug resistant bacterial cells. For this reason, therapeutic use of phages is considered as a possible alternative to conventional antibiotics. However, phages are very host specific in comparison to wide-spectrum antibiotics and thus preparation of phage-cocktails beforehand against pathogens can be difficult. In this study, we evaluate whether it may be possible to isolate phages on-demand from environmental reservoir. We attempted to enrich infectious bacteriophages from sewage against nosocomial drug-resistant bacterial strains of different medically important species in order to evaluate the probability of discovering novel therapeutic phages. Stability and host-range were determined for the acquired phages. Our results suggest that on-demand isolation of phages is possible against Pseudomonas aeruginosa, Salmonella and extended spectrum beta-lactamase Escherichia coli and Klebsiella pneumoniae. The probability of finding suitable phages was less than 40% against vancomycin resistant Enterococcus and Acinetobacter baumannii strains. Furthermore, isolation of new phages against methicillin resistant Staphylococcus aureus strains was found to be very difficult. PMID:26617601

  19. On-Demand Isolation of Bacteriophages Against Drug-Resistant Bacteria for Personalized Phage Therapy.

    PubMed

    Mattila, Sari; Ruotsalainen, Pilvi; Jalasvuori, Matti

    2015-01-01

    Bacteriophages are bacterial viruses, capable of killing even multi-drug resistant bacterial cells. For this reason, therapeutic use of phages is considered as a possible alternative to conventional antibiotics. However, phages are very host specific in comparison to wide-spectrum antibiotics and thus preparation of phage-cocktails beforehand against pathogens can be difficult. In this study, we evaluate whether it may be possible to isolate phages on-demand from environmental reservoir. We attempted to enrich infectious bacteriophages from sewage against nosocomial drug-resistant bacterial strains of different medically important species in order to evaluate the probability of discovering novel therapeutic phages. Stability and host-range were determined for the acquired phages. Our results suggest that on-demand isolation of phages is possible against Pseudomonas aeruginosa, Salmonella and extended spectrum beta-lactamase Escherichia coli and Klebsiella pneumoniae. The probability of finding suitable phages was less than 40% against vancomycin resistant Enterococcus and Acinetobacter baumannii strains. Furthermore, isolation of new phages against methicillin resistant Staphylococcus aureus strains was found to be very difficult.

  20. Improving the safety of Staphylococcus aureus polyvalent phages by their production on a Staphylococcus xylosus strain.

    PubMed

    El Haddad, Lynn; Ben Abdallah, Nour; Plante, Pier-Luc; Dumaresq, Jeannot; Katsarava, Ramaz; Labrie, Steve; Corbeil, Jacques; St-Gelais, Daniel; Moineau, Sylvain

    2014-01-01

    Team1 (vB_SauM_Team1) is a polyvalent staphylococcal phage belonging to the Myoviridae family. Phage Team1 was propagated on a Staphylococcus aureus strain and a non-pathogenic Staphylococcus xylosus strain used in industrial meat fermentation. The two Team1 preparations were compared with respect to their microbiological and genomic properties. The burst sizes, latent periods, and host ranges of the two derivatives were identical as were their genome sequences. Phage Team1 has 140,903 bp of double stranded DNA encoding for 217 open reading frames and 4 tRNAs. Comparative genomic analysis revealed similarities to staphylococcal phages ISP (97%) and G1 (97%). The host range of Team1 was compared to the well-known polyvalent staphylococcal phages phi812 and K using a panel of 57 S. aureus strains collected from various sources. These bacterial strains were found to represent 18 sequence types (MLST) and 14 clonal complexes (eBURST). Altogether, the three phages propagated on S. xylosus lysed 52 out of 57 distinct strains of S. aureus. The identification of phage-insensitive strains underlines the importance of designing phage cocktails with broadly varying and overlapping host ranges. Taken altogether, our study suggests that some staphylococcal phages can be propagated on food-grade bacteria for biocontrol and safety purposes. PMID:25061757

  1. Improving the Safety of Staphylococcus aureus Polyvalent Phages by Their Production on a Staphylococcus xylosus Strain

    PubMed Central

    El Haddad, Lynn; Ben Abdallah, Nour; Plante, Pier-Luc; Dumaresq, Jeannot; Katsarava, Ramaz; Labrie, Steve; Corbeil, Jacques; St-Gelais, Daniel; Moineau, Sylvain

    2014-01-01

    Team1 (vB_SauM_Team1) is a polyvalent staphylococcal phage belonging to the Myoviridae family. Phage Team1 was propagated on a Staphylococcus aureus strain and a non-pathogenic Staphylococcus xylosus strain used in industrial meat fermentation. The two Team1 preparations were compared with respect to their microbiological and genomic properties. The burst sizes, latent periods, and host ranges of the two derivatives were identical as were their genome sequences. Phage Team1 has 140,903 bp of double stranded DNA encoding for 217 open reading frames and 4 tRNAs. Comparative genomic analysis revealed similarities to staphylococcal phages ISP (97%) and G1 (97%). The host range of Team1 was compared to the well-known polyvalent staphylococcal phages phi812 and K using a panel of 57 S. aureus strains collected from various sources. These bacterial strains were found to represent 18 sequence types (MLST) and 14 clonal complexes (eBURST). Altogether, the three phages propagated on S. xylosus lysed 52 out of 57 distinct strains of S. aureus. The identification of phage-insensitive strains underlines the importance of designing phage cocktails with broadly varying and overlapping host ranges. Taken altogether, our study suggests that some staphylococcal phages can be propagated on food-grade bacteria for biocontrol and safety purposes. PMID:25061757

  2. Going viral: next generation sequencing applied to human gut phage populations

    PubMed Central

    Reyes, Alejandro; Semenkovich, Nicholas P.; Whiteson, Katrine; Rohwer, Forest; Gordon, Jeffrey I.

    2013-01-01

    Over the past decade researchers have begun to characterize viral diversity using metagenomic methods. These studies have shown that viruses, the majority of which infect bacteria (bacteriophages), are likely the most genetically diverse components of the biosphere. Here we briefly review the incipient rise of a phage biology renaissance catalyzed by recent advances in next generation sequencing. We explore how work characterizing phage diversity and their lifestyles in the gut is changing our view of ourselves as supra-organisms. Finally, we discuss how a new appreciation of phage dynamics may yield new applications for phage therapies designed to manipulate the structure and functions of our gut microbiomes. PMID:22864264

  3. Novel phage-based bio-processing of pathogenic Escherichia coli and its biofilms.

    PubMed

    Jassim, S A A; Abdulamir, A S; Abu Bakar, F

    2012-01-01

    To explore new approaches of phage-based bio-process of specifically pathogenic Escherichia coli bacteria in food products within a short period. One hundred and forty highly lytic designed coliphages were used. Escherichia coli naturally contaminated and Enterohemorrhagic Escherichia coli experimentally inoculated samples of lettuce, cabbage, meat, and egg were used. In addition, experimentally produced biofilms of E. coli were tested. A phage concentration of 10(3) PFU/ml was used for food products immersion, and for spraying of food products, 10(5) PFU/ml of a phage cocktail was used by applying a 20-s optimal dipping time in a phage cocktail. Food samples were cut into pieces and were either sprayed with or held in a bag immersed in lambda buffer containing a cocktail of 140 phages. Phage bio-processing was successful in eliminating completely E. coli in all processed samples after 48 h storage at 4°C. Partial elimination of E. coli was observed in earlier storage periods (7 and 18 h) at 24° and 37°C. Moreover, E. coli biofilms were reduced >3 log cycles upon using the current phage bio-processing. The use of a phage cocktail of 140 highly lytic designed phages proved highly effective in suppressing E. coli contaminating food products. Proper decontamination/prevention methods of pathogenic E. coli achieved in this study can replace the current chemically less effective decontamination methods.

  4. Phage Morphology Recapitulates Phylogeny: The Comparative Genomics of a New Group of Myoviruses

    PubMed Central

    Comeau, André M.; Tremblay, Denise; Moineau, Sylvain; Rattei, Thomas; Kushkina, Alla I.; Tovkach, Fedor I.; Krisch, Henry M.; Ackermann, Hans-Wolfgang

    2012-01-01

    Among dsDNA tailed bacteriophages (Caudovirales), members of the Myoviridae family have the most sophisticated virion design that includes a complex contractile tail structure. The Myoviridae generally have larger genomes than the other phage families. Relatively few “dwarf” myoviruses, those with a genome size of less than 50 kb such as those of the Mu group, have been analyzed in extenso. Here we report on the genome sequencing and morphological characterization of a new group of such phages that infect a diverse range of Proteobacteria, namely Aeromonas salmonicida phage 56, Vibrio cholerae phages 138 and CP-T1, Bdellovibrio phage φ1422, and Pectobacterium carotovorum phage ZF40. This group of dwarf myoviruses shares an identical virion morphology, characterized by usually short contractile tails, and have genome sizes of approximately 45 kb. Although their genome sequences are variable in their lysogeny, replication, and host adaption modules, presumably reflecting differing lifestyles and hosts, their structural and morphogenesis modules have been evolutionarily constrained by their virion morphology. Comparative genomic analysis reveals that these phages, along with related prophage genomes, form a new coherent group within the Myoviridae. The results presented in this communication support the hypothesis that the diversity of phages may be more structured than generally believed and that the innumerable phages in the biosphere all belong to discrete lineages or families. PMID:22792219

  5. Tests of the Double-Strand-Break Repair Model for Red-Mediated Recombination of Phage λ and Plasmid λdv

    PubMed Central

    Thaler, David S.; Stahl, Mary M.; Stahl, Franklin W.

    1987-01-01

    The double-strand-break repair (DSBR) model was formulated to account for various aspects of yeast mitotic and meiotic recombination. In this study three features of the DSBR model are tested for Red-mediated recombination between phage λ and λdv, a plasmid that is perfectly homologous to about 10% of λ. The results support the applicability of the DSBR model to λ's Red system: (1) Creating a double-strand-break (DSB) within the region of homology shared by phage and plasmid increases their genetic interaction by about 20-fold. A DSB outside the region of shared homology has no such effect. (2) Both patches, i.e., simple marker rescue, and splices, i.e., co-integration of the phage and plasmid, are stimulated by a DSB in the region of shared homology. (3) Co-integrants harbor a duplication of the region of shared homology. Among co-integrants that were formed by the creation of a DSB, there is a preferential loss of whichever allele was in cis to a utilized cut site. The DSBR model as originally formulated involves the isomerization and cleavage of Holliday junctions to resolve the canonical intermediate. We propose as an alternative mechanism that a topoisomerase can resolve the canonical DSBR intermediate. PMID:2957271

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

    PubMed

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

    2015-11-01

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

  7. Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000.

    PubMed

    Fujiwara, Akiko; Kawasaki, Takeru; Usami, Shoji; Fujie, Makoto; Yamada, Takashi

    2008-01-01

    PhiRSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the phiRSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5'-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the phiRSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A phiRSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these phiRSA1 genes as 5' TGTTGT-(X)13-ACAACA. The genomic sequence similarity between phiRSA1 and related phages phi52237 and phiCTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. phiRSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3' portion of the arginine tRNA(CCG) gene. In the light of the phiRSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a phiRSA1-related prophage (designated phiRSX). phiRSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. phiRSX ORFs shared very high amino acid identity with their phiRSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.

  8. The use of phage FCL-2 as an alternative to chemotherapy against columnaris disease in aquaculture.

    PubMed

    Laanto, Elina; Bamford, Jaana K H; Ravantti, Janne J; Sundberg, Lotta-Riina

    2015-01-01

    Flavobacterium columnare, the causative agent of columnaris disease in fish, causes millions of dollars of losses in the US channel catfish industry alone, not to mention aquaculture industry worldwide. Novel methods are needed for the control and treatment of bacterial diseases in aquaculture to replace traditionally used chemotherapies. A potential solution could be the use of phages, i.e., bacterial viruses, host-specific and self-enriching particles that can be can easily distributed via water flow. We examined the efficacy of phages to combat columnaris disease. A previously isolated phage, FCL-2, infecting F. columnare, was characterized by sequencing. The 47 142 bp genome of the phage had G + C content of 30.2%, and the closest similarities regarding the structural proteins were found in Cellulophaga phage phiSM. Under controlled experimental conditions, two host fish species, rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), were used to study the success of phage therapy to prevent F. columnare infections. The survival of both fish species was significantly higher in the presence of the phage. Hundred percent of the zebrafish and 50% of the rainbow trout survived in the phage treatment (survival without phage 0 and 8.3%, respectively). Most importantly, the rainbow trout population was rescued from infection by a single addition of the phage into the water in a flow-through fish tank system. Thus, F. columnare could be used as a model system to test the benefits and risks of phage therapy on a larger scale. PMID:26347722

  9. Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp.

    PubMed

    Colom, Joan; Cano-Sarabia, Mary; Otero, Jennifer; Cortés, Pilar; Maspoch, Daniel; Llagostera, Montserrat

    2015-07-01

    Bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals.

  10. Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

    PubMed Central

    Filippov, Andrey A.; Sergueev, Kirill V.; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T.; Mueller, Allen J.; Fernandez-Prada, Carmen M.; Nikolich, Mikeljon P.

    2011-01-01

    Background Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis. PMID:21980477

  11. Development of phoH as a novel signature gene for assessing marine phage diversity.

    PubMed

    Goldsmith, Dawn B; Crosti, Giuseppe; Dwivedi, Bhakti; McDaniel, Lauren D; Varsani, Arvind; Suttle, Curtis A; Weinbauer, Markus G; Sandaa, Ruth-Anne; Breitbart, Mya

    2011-11-01

    Phages play a key role in the marine environment by regulating the transfer of energy between trophic levels and influencing global carbon and nutrient cycles. The diversity of marine phage communities remains difficult to characterize because of the lack of a signature gene common to all phages. Recent studies have demonstrated the presence of host-derived auxiliary metabolic genes in phage genomes, such as those belonging to the Pho regulon, which regulates phosphate uptake and metabolism under low-phosphate conditions. Among the completely sequenced phage genomes in GenBank, this study identified Pho regulon genes in nearly 40% of the marine phage genomes, while only 4% of nonmarine phage genomes contained these genes. While several Pho regulon genes were identified, phoH was the most prevalent, appearing in 42 out of 602 completely sequenced phage genomes. Phylogenetic analysis demonstrated that phage phoH sequences formed a cluster distinct from those of their bacterial hosts. PCR primers designed to amplify a region of the phoH gene were used to determine the diversity of phage phoH sequences throughout a depth profile in the Sargasso Sea and at six locations worldwide. phoH was present at all sites examined, and a high diversity of phoH sequences was recovered. Most phoH sequences belonged to clusters without any cultured representatives. Each depth and geographic location had a distinct phoH composition, although most phoH clusters were recovered from multiple sites. Overall, phoH is an effective signature gene for examining phage diversity in the marine environment. PMID:21926220

  12. Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean.

    PubMed

    Tucker, Kimberly P; Parsons, Rachel; Symonds, Erin M; Breitbart, Mya

    2011-05-01

    Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem. PMID:21124487

  13. Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages.

    PubMed

    Berg, Jordan A; Merrill, Bryan D; Crockett, Justin T; Esplin, Kyle P; Evans, Marlee R; Heaton, Karli E; Hilton, Jared A; Hyde, Jonathan R; McBride, Morgan S; Schouten, Jordan T; Simister, Austin R; Thurgood, Trever L; Ward, Andrew T; Breakwell, Donald P; Hope, Sandra; Grose, Julianne H

    2016-01-01

    Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared.

  14. Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp.

    PubMed Central

    Colom, Joan; Cano-Sarabia, Mary; Otero, Jennifer; Cortés, Pilar

    2015-01-01

    Bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals. PMID:25956778

  15. Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages

    PubMed Central

    Berg, Jordan A.; Merrill, Bryan D.; Crockett, Justin T.; Esplin, Kyle P.; Evans, Marlee R.; Heaton, Karli E.; Hilton, Jared A.; Hyde, Jonathan R.; McBride, Morgan S.; Schouten, Jordan T.; Simister, Austin R.; Thurgood, Trever L.; Ward, Andrew T.; Breakwell, Donald P.; Hope, Sandra; Grose, Julianne H.

    2016-01-01

    Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared. PMID:27304881

  16. Selective and Sensitive Sensing of Flame Retardant Chemicals Through Phage Display Discovered Recognition Peptide.

    PubMed

    Jin, Hyo-Eon; Zueger, Chris; Chung, Woo-Jae; Wong, Winnie; Lee, Byung Yang; Lee, Seung-Wuk

    2015-11-11

    We report a highly selective and sensitive biosensor for the detection of an environmentally toxic molecule, decabrominated diphenyl ether (DBDE), one of the most common congeners of the polybrominated frame retardants (polybrominated diphenyl ether (PBDE)), using newly discovered DBDE peptide receptors integrated with carbon nanotube field-effect transistors (CNT-FET). The specific DBDE peptide receptor was identified using a high-throughput screening process of phage library display. The resulting binding peptide carries an interesting consensus binding pocket with two Trp-His/Asn-Trp repeats, which binds to the DBDE in a multivalent manner. We integrated the novel DBDE binding peptide onto the CNT-FET using polydiacetylene coating materials linked through cysteine-maleimide click chemistry. The resulting biosensor could detect the desired DBDE selectively with a 1 fM detection limit. Our combined approaches of selective receptor discovery, material nanocoating through click chemistry, and integration onto a sensitive CNT-FET electronic sensor for desired target chemicals will pave the way toward the rapid development of portable and easy-to-use biosensors for desired chemicals to protect our health and environment. PMID:26455834

  17. Selective and Sensitive Sensing of Flame Retardant Chemicals Through Phage Display Discovered Recognition Peptide.

    PubMed

    Jin, Hyo-Eon; Zueger, Chris; Chung, Woo-Jae; Wong, Winnie; Lee, Byung Yang; Lee, Seung-Wuk

    2015-11-11

    We report a highly selective and sensitive biosensor for the detection of an environmentally toxic molecule, decabrominated diphenyl ether (DBDE), one of the most common congeners of the polybrominated frame retardants (polybrominated diphenyl ether (PBDE)), using newly discovered DBDE peptide receptors integrated with carbon nanotube field-effect transistors (CNT-FET). The specific DBDE peptide receptor was identified using a high-throughput screening process of phage library display. The resulting binding peptide carries an interesting consensus binding pocket with two Trp-His/Asn-Trp repeats, which binds to the DBDE in a multivalent manner. We integrated the novel DBDE binding peptide onto the CNT-FET using polydiacetylene coating materials linked through cysteine-maleimide click chemistry. The resulting biosensor could detect the desired DBDE selectively with a 1 fM detection limit. Our combined approaches of selective receptor discovery, material nanocoating through click chemistry, and integration onto a sensitive CNT-FET electronic sensor for desired target chemicals will pave the way toward the rapid development of portable and easy-to-use biosensors for desired chemicals to protect our health and environment.

  18. Bacteriophage module reshuffling results in adaptive host range as exemplified by the baseplate model of listerial phage A118.

    PubMed

    Cambillau, Christian

    2015-10-01

    Each phage infects its specific bacterial host strain through highly specific interactions between the baseplate-associated receptor binding protein (RBP) at the tip of the phage tail and the receptor at the host surface. Baseplates incorporate structural core modules, Dit and Tal, largely conserved among phages, and peripheral modules anchoring the RBPs. Exploiting structural information from the HHpred program and EM data from the Bielmann et al. (2015) paper, a molecular model of the A118 phage baseplate was generated from different building blocks. This model implies the occurrence of baseplate module reshuffling and suggests that listerial phage A118 may have been derived from lactococcal phage TP901-1 through host species exchange. With the increase of available viral module structures, modelling phage baseplates will become easier and more reliant, and will provide insightful information on the nature of the phage host receptor and its mode of recognition.

  19. Bacteriophage module reshuffling results in adaptive host range as exemplified by the baseplate model of listerial phage A118.

    PubMed

    Cambillau, Christian

    2015-10-01

    Each phage infects its specific bacterial host strain through highly specific interactions between the baseplate-associated receptor binding protein (RBP) at the tip of the phage tail and the receptor at the host surface. Baseplates incorporate structural core modules, Dit and Tal, largely conserved among phages, and peripheral modules anchoring the RBPs. Exploiting structural information from the HHpred program and EM data from the Bielmann et al. (2015) paper, a molecular model of the A118 phage baseplate was generated from different building blocks. This model implies the occurrence of baseplate module reshuffling and suggests that listerial phage A118 may have been derived from lactococcal phage TP901-1 through host species exchange. With the increase of available viral module structures, modelling phage baseplates will become easier and more reliant, and will provide insightful information on the nature of the phage host receptor and its mode of recognition. PMID:26074066

  20. Characteristics of defective phage particles of Pectobacterium carotovorum ZM1.

    PubMed

    Tovkach, F I; Ivanytsia, T V; Kushkina, A I

    2012-01-01

    It is shown for the first time that the expression products of defective prophages are typical of defective lysogenic systems of phytopathogenic Pectobacterium carotovorum. It is established that virus-like particles (LP) such as phage capsids are packing bacterial DNA which size is determined by pulse field gel electrophoresis separation. Based on data about capsid structures which are formed by the virulent mutant ZF40/421, there is made a suggestion about the forming mechanism of defective virions of P carotovorum. PMID:22545442

  1. Specific electrochemical phage sensing for Bacillus cereus and Mycobacterium smegmatis.

    PubMed

    Yemini, Miri; Levi, Yaron; Yagil, Ezra; Rishpon, Judith

    2007-01-01

    The rapid and reliable detection of pathogenic microorganisms is an important issue for the safety and security of our society. Here we describe the use of a sensitive, inexpensive, amperometric, phage-based biosensor for the detection of extremely low concentrations of Bacillus cereus and Mycobacterium smegmatis as models for Bacillus anthracis (the causative agent of anthrax) and for Mycobacterium tuberculosis (the causative agent of tuberculosis), respectively. The detection procedure developed here enabled the determination of bacteria at a low concentration of 10 viable cells/mL within 8 h. This experimental setup allows the simultaneous analysis of up to eight independent samples, using disposable screen-printed electrodes.

  2. Phage Peptide Libraries As a Source of Targeted Ligands

    PubMed Central

    Nemudraya, A. A.; Richter, V. A.; Kuligina, E. V.

    2016-01-01

    One of the dominant trends in modern pharmacology is the creation of drugs that act directly on the lesion focus and have minimal toxicity on healthy tissues and organs. This problem is particularly acute in relation to oncologic diseases. Short tissue- and organ-specific peptides capable of delivering drugs to the affected organ or tissue are considered promising targeted agents that can be used in the diagnosis and therapy of diseases, including cancer. The review discusses in detail the technology of phage display as a method for obtaining specific targeted peptide agents and offers examples of their use in diagnostic and clinical practice. PMID:27099784

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

    NASA Astrophysics Data System (ADS)

    Moebus, K.

    1983-12-01

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

  4. Role of phages in the control of bacterial pathogens in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages (phages) are viruses of bacteria and were independently discovered by English bacteriologist Twort in 1915 and Franco-Canadian microbiologist d’Hérelle in 1917 (d'Hérelle, 1919). Since then, phages have been used to treat infectious disease in humans, animals, and plants, but in the ...

  5. Lytic phages obscure the cost of antibiotic resistance in Escherichia coli.

    PubMed

    Tazzyman, Samuel J; Hall, Alex R

    2015-04-01

    The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods.

  6. Three proposed new bacteriophage genera of staphylococcal phages: "3alikevirus", "77likevirus" and "Phietalikevirus".

    PubMed

    Gutiérrez, Diana; Adriaenssens, Evelien M; Martínez, Beatriz; Rodríguez, Ana; Lavigne, Rob; Kropinski, Andrew M; García, Pilar

    2014-02-01

    To date, most members of the Siphoviridae family of bacteriophages remain unclassified, including the 46 staphylococcal phages for which the complete genome sequences have been deposited in public databases. Comparative nucleotide and protein sequence analysis, in addition to available data on phage morphology, allowed us to propose three new phage genera within the family Siphoviridae: "3alikevirus", "77likevirus" and "Phietalikevirus", which include related phages infecting Staphylococcus aureus and Staphylococcus epidermidis. However, six phages infecting S. aureus, Staphylococcus pasteuri, Staphylococcus hominis and Staphylococcus capitis strains remain to be classified (orphan phages). Overall, the former phages share morphological features and genome organization. The three groups have conserved domains containing peptidoglycan hydrolytic activities clearly identified as part of tape measure proteins ("3alikevirus" and "77likevirus") or as individual virionassociated proteins ("Phietalikevirus"). In addition, bacteriophages belonging to the genus "3alikevirus" share closely related DNA-processing and packaging proteins, while bacteriophages included in the genus "Phietalikevirus" encode specific tail proteins for host interaction. These properties are considered distinctive for these genera. Orphan phages seem to have a more divergent organization, but they share some properties with members of these proposed genera.

  7. Analysis of Lactobacillus Products for Phages and Bacteriocins That Inhibit Vaginal Lactobacilli

    PubMed Central

    Pavlova, Sylvia I.; Mou, Susan M.; Ma, Wen-ge; Kiliç, Ali O.

    1997-01-01

    Objective: Bacterial vaginosis is associated with an unexplained loss of vaginal lactobacilli. Previously, we have identified certain vaginal lactobacilli-released phages that can inhibit in vitro other vaginal lactobacilli. However, there is no apparent route for phages to be transmitted among women. The purpose of this study was to identify whether certain Lactobacillus products commonly used by women release phages or bacteriocins that can inhibit vaginal lactobacilli. Methods: From 26 Lactobacillus products (2 acidophilus milks, 20 yogurts, 3 Lactobacillus pills, and 1 vaginal douche mix), lactobacilli were isolated with Rogosa SL agar (Difco, Detroit, MI). From these lactobacilli, phages and bacteriocins were induced with mitomycin C and tested against a collection of vaginal Lactobacillus strains. Results: From the 26 products, 43 Lactobacillus strains were isolated. Strains from 11 yogurts released phages, among which 7 inhibited vaginal lactobacilli. Eleven strains released bacteriocins that inhibited vaginal lactobacilli. While about one-half of the vaginal strains were lysed by bacteriocins, less than 20% were lysed by phages. Conclusions: Some vaginal lactobacilli were inhibited in vitro by phages or bacteriocins released from Lactobacillus products used by women, implying that vaginal lactobacilli may be reduced naturally due to phages or bacteriocins from the environment. PMID:18476145

  8. Sewage as a rich source of phage study against Pseudomonas aeruginosa PAO.

    PubMed

    Azizian, Reza; Nasser, Ahmad; Askari, Hasan; Taheri Kalani, Morovat; Sadeghifard, Nourkhoda; Pakzad, Iraj; Amini, Razieh; Mozaffari Nejad, Amir Sasan; Azizi Jalilian, Farid

    2015-07-01

    Pseudomonas aeruginosa is a ubiquitous organism which has emerged as a major public health threat in hospital environments. Overuse of antibiotics has significantly exacerbated the emergence of multi-drug resistant bacteria such as P. aeruginosa. Phages are currently being utilized successfully for aquaculture, agriculture and veterinary applications. The aim of this study was to isolate and characterize of lytic P. aeruginosa phage from sewage of Ilam, Iran. Phage was isolated from sewage that was added to the enrichment along with the host and subsequently filtered. Plaque assay was done by using an overlay method (also called the double agar layer method). Purified plaques were then amplified for characterization. Finally, RAPD-PCR method was conducted for genotyping and Transition electron micrograph (TEM) recruited to determine the morphology and phage family. The phage had high concentration and tremendous effects against a variety of clinical and general laboratory strains (ATCC15693) of P. aeruginosa. Among a set of primers in RAPD panel, only P2 and RAPD5 primers, were useful in differentiating the phages. TEM images revealed that the isolated phages were members of the Siphoviridae family. The phage effectiveness and specificity towards target bacteria and potential to control biofilm formations will be investigate in our further studies.

  9. N-Terminal Labeling Of Filamentous Phage To Create Cancer Marker Imaging Agents

    PubMed Central

    Carrico, Zachary M.; Farkas, Michelle E.; Zhou, Yu; Hsiao, Sonny C.; Marks, James D.; Chokhawala, Harshal; Clark, Douglas S.; Francis, Matthew B.

    2012-01-01

    We report a convenient new technique for the labeling of filamentous phage capsid proteins. Previous reports have shown that phage coat protein residues can be modified, but the lack of chemically distinct amino acids in the coat protein sequences makes it difficult to attach high levels of synthetic molecules without altering the binding capabilities of the phage. To modify the phage with polymer chains, imaging groups, and other molecules, we have developed chemistry to convert the N-terminal amines of the ~4,200 coat proteins into ketone groups. These sites can then serve as chemospecific handles for the attachment of alkoxyamine groups through oxime formation. Specifically, we demonstrate the attachment of fluorophores and up to 3,000 molecules of 2 kD poly(ethylene glycol) (PEG2k) to each of the phage capsids without significantly affecting the binding of phage-displayed antibody fragments to EGFR and HER2 (two important epidermal growth factor receptors). We also demonstrate the utility of the modified phage for the characterization of breast cancer cells using multicolor fluorescence microscopy. Due to the widespread use of filamentous phage as display platforms for peptide and protein evolution, we envision that the ability to attach large numbers of synthetic functional groups to their coat proteins will be of significant value to the biological and materials communities. PMID:22830952

  10. Phospholipid vesicles encapsulated bacteriophage: A novel approach to enhance phage biodistribution.

    PubMed

    Singla, S; Harjai, K; Raza, K; Wadhwa, S; Katare, O P; Chhibber, S

    2016-10-01

    Phage therapy has been at the centre of attraction for combating multi-drug resistant strains. However, less stability and rapid clearance of phage by mononuclear phagocytic system (MPS) restricts its use in humans. In the present study, aim was to develop a liposomal delivery system for bacteriophage that can assure efficient phage delivery and retention at the site of infection. Different ratios of cholesterol, lipids and surfactant along with different charge inducers were employed to prepare liposomes. Phage was then entrapped in the liposomes and characterized on the basis of morphology, size, entrapment efficiency and stability. Further, in vivo biodistribution of free phage and liposome entrapped phage was compared in different organs of mice. A cationic liposomal formulation showed maximum encapsulation efficiency of 92%. Transmission electron microscopy (TEM) confirmed the entrapment of phages in liposomes. Liposome preparation was found to be most stable at 4°C during storage. Liposome entrapped bacteriophage was retained for longer duration in different organs i.e. upto day 4 in blood, day 6 in liver, lungs and kidney, 14days in spleen of mice as compared to free phage that became undetectable by 36th h in blood as well as lungs and by 48th h in all other organs. PMID:27393682

  11. Development of a Phage Cocktail to Control Proteus mirabilis Catheter-associated Urinary Tract Infections.

    PubMed

    Melo, Luís D R; Veiga, Patrícia; Cerca, Nuno; Kropinski, Andrew M; Almeida, Carina; Azeredo, Joana; Sillankorva, Sanna

    2016-01-01

    Proteus mirabilis is an enterobacterium that causes catheter-associated urinary tract infections (CAUTIs) due to its ability to colonize and form crystalline biofilms on the catheters surface. CAUTIs are very difficult to treat, since biofilm structures are highly tolerant to antibiotics. Phages have been used widely to control a diversity of bacterial species, however, a limited number of phages for P. mirabilis have been isolated and studied. Here we report the isolation of two novel virulent phages, the podovirus vB_PmiP_5460 and the myovirus vB_PmiM_5461, which are able to target, respectively, 16 of the 26 and all the Proteus strains tested in this study. Both phages have been characterized thoroughly and sequencing data revealed no traces of genes associated with lysogeny. To further evaluate the phages' ability to prevent catheter's colonization by Proteus, the phages adherence to silicone surfaces was assessed. Further tests in phage-coated catheters using a dynamic biofilm model simulating CAUTIs, have shown a significant reduction of P. mirabilis biofilm formation up to 168 h of catheterization. These results highlight the potential usefulness of the two isolated phages for the prevention of surface colonization by this bacterium. PMID:27446059

  12. Filamentous phages prevalent in Pseudoalteromonas spp. confer properties advantageous to host survival in Arctic sea ice.

    PubMed

    Yu, Zi-Chao; Chen, Xiu-Lan; Shen, Qing-Tao; Zhao, Dian-Li; Tang, Bai-Lu; Su, Hai-Nan; Wu, Zhao-Yu; Qin, Qi-Long; Xie, Bin-Bin; Zhang, Xi-Ying; Yu, Yong; Zhou, Bai-Cheng; Chen, Bo; Zhang, Yu-Zhong

    2015-03-17

    Sea ice is one of the most frigid environments for marine microbes. In contrast to other ocean ecosystems, microbes in permanent sea ice are space confined and subject to many extreme conditions, which change on a seasonal basis. How these microbial communities are regulated to survive the extreme sea ice environment is largely unknown. Here, we show that filamentous phages regulate the host bacterial community to improve survival of the host in permanent Arctic sea ice. We isolated a filamentous phage, f327, from an Arctic sea ice Pseudoalteromonas strain, and we demonstrated that this type of phage is widely distributed in Arctic sea ice. Growth experiments and transcriptome analysis indicated that this phage decreases the host growth rate, cell density and tolerance to NaCl and H2O2, but enhances its motility and chemotaxis. Our results suggest that the presence of the filamentous phage may be beneficial for survival of the host community in sea ice in winter, which is characterized by polar night, nutrient deficiency and high salinity, and that the filamentous phage may help avoid over blooming of the host in sea ice in summer, which is characterized by polar day, rich nutrient availability, intense radiation and high concentration of H2O2. Thus, while they cannot kill the host cells by lysing them, filamentous phages confer properties advantageous to host survival in the Arctic sea ice environment. Our study provides a foremost insight into the ecological role of filamentous phages in the Arctic sea ice ecosystem.

  13. Filamentous phages prevalent in Pseudoalteromonas spp. confer properties advantageous to host survival in Arctic sea ice

    PubMed Central

    Yu, Zi-Chao; Chen, Xiu-Lan; Shen, Qing-Tao; Zhao, Dian-Li; Tang, Bai-Lu; Su, Hai-Nan; Wu, Zhao-Yu; Qin, Qi-Long; Xie, Bin-Bin; Zhang, Xi-Ying; Yu, Yong; Zhou, Bai-Cheng; Chen, Bo; Zhang, Yu-Zhong

    2015-01-01

    Sea ice is one of the most frigid environments for marine microbes. In contrast to other ocean ecosystems, microbes in permanent sea ice are space confined and subject to many extreme conditions, which change on a seasonal basis. How these microbial communities are regulated to survive the extreme sea ice environment is largely unknown. Here, we show that filamentous phages regulate the host bacterial community to improve survival of the host in permanent Arctic sea ice. We isolated a filamentous phage, f327, from an Arctic sea ice Pseudoalteromonas strain, and we demonstrated that this type of phage is widely distributed in Arctic sea ice. Growth experiments and transcriptome analysis indicated that this phage decreases the host growth rate, cell density and tolerance to NaCl and H2O2, but enhances its motility and chemotaxis. Our results suggest that the presence of the filamentous phage may be beneficial for survival of the host community in sea ice in winter, which is characterized by polar night, nutrient deficiency and high salinity, and that the filamentous phage may help avoid over blooming of the host in sea ice in summer, which is characterized by polar day, rich nutrient availability, intense radiation and high concentration of H2O2. Thus, while they cannot kill the host cells by lysing them, filamentous phages confer properties advantageous to host survival in the Arctic sea ice environment. Our study provides a foremost insight into the ecological role of filamentous phages in the Arctic sea ice ecosystem. PMID:25303713

  14. Bacteriophage phi11 lysin: physicochemical characterization and comparison with phage phi80a lysin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phage lytic enzymes are promising antimicrobial agents. Lysins of phage phi11 (LysPhi11) and phi80a (LysPhi80a) can lyse (destroy) biofilms and cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The obj...

  15. Complete Genome Sequence of Pseudomonas aeruginosa Phage-Resistant Variant PA1RG

    PubMed Central

    Li, Gang; Lu, Shuguang; Shen, Mengyu; Le, Shuai; Tan, Yinling; Li, Ming; Zhao, Xia; Wang, Jing; Shen, Wei; Guo, Keke; Yang, Yuhui; Zhu, Hongbin; Li, Shu; Zhu, Junmin; Rao, Xiancai

    2016-01-01

    Bacteria have evolved several defense systems against phage predation. Here, we report the 6,500,439-bp complete genome sequence of the Pseudomonas aeruginosa phage-resistant variant PA1RG. Single-molecule real-time (SMRT) sequencing and de novo assembly revealed a single contig with 320-fold sequence coverage. PMID:26893434

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

    PubMed Central

    Yang, Yongheng

    2013-01-01

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

  17. RIFAMYCIN. XXXIII. ISOLATION OF ACTINOPHAGES ACTIVE ON STREPTOMYCES MEDITERRANEI AND CHARACTERISTICS OF PHAGE-RESISTANT STRAINS.

    PubMed

    THIEMANN, J E; HENGELLER, C; VIRGILIO, A; BUELLI, O; LICCIARDELLO, G

    1964-05-01

    Five actinophages highly specific for Streptomyces mediterranei were isolated from lysed broth cultures. Studies were performed on the effect of plating conditions on plaque formation. The development of phage-resistant strains of S. mediterranei not only eliminated the phage but also significantly increased rifamycin yields. The phage-resistant cultures proved to be more unstable than the original sensitive strain. Maintenance of the cultures as frozen vegetative mycelium assured culture stability and reproducibility of the results. Strict aseptic precautions throughout the laboratories and fermentation areas did not eliminate the danger of phage infection; effective control was obtained only with the introduction of resistant strains. S. mediterranei phages proved to be highly specific for calcium as an adsorption cofactor; addition of calcium-sequestering agents to sensitive mycelium completely prevented its lysis by the phage. The resistant strains developed were capable of adsorbing the phage and of releasing it without multiplication upon aging of the mycelium. No marked morphological, cultural, or biochemical differences were found among the various phage-resistant strains.

  18. Location of the Coat Cistron on the RNA of Phage

    PubMed Central

    Hindley, J.; Staples, D. H.; Billeter, M. A.; Weissmann, C.

    1970-01-01

    A new approach for the localization of a cistron on a phage RNA has been developed. The coat protein cistron of phage Qβ was found to begin between the 1100th and 1400th nucleotide from the 5′ terminus of Qβ RNA and therefore lies in the middle of the genome. Images PMID:5274447

  19. Complete Genome Sequence of Pseudomonas aeruginosa Phage-Resistant Variant PA1RG.

    PubMed

    Li, Gang; Lu, Shuguang; Shen, Mengyu; Le, Shuai; Tan, Yinling; Li, Ming; Zhao, Xia; Wang, Jing; Shen, Wei; Guo, Keke; Yang, Yuhui; Zhu, Hongbin; Li, Shu; Zhu, Junmin; Rao, Xiancai; Hu, Fuquan

    2016-01-01

    Bacteria have evolved several defense systems against phage predation. Here, we report the 6,500,439-bp complete genome sequence of the Pseudomonas aeruginosa phage-resistant variant PA1RG. Single-molecule real-time (SMRT) sequencing and de novo assembly revealed a single contig with 320-fold sequence coverage. PMID:26893434

  20. Complete Genome Sequence of Bacillus subtilis Strain CU1050, Which Is Sensitive to Phage SPβ

    PubMed Central

    2016-01-01

    The Gram-positive bacterium Bacillus subtilis is used as a model organism to study cellular and molecular processes. Here, we announce the complete genomic sequence of B. subtilis strain CU1050, derived from B. subtilis strain 168. CU1050 has historically been used to study suppressor mutations and phage biology, especially the lysogenic phage SPβ. PMID:27056236

  1. Isolation and Characterization of a Novel Virulent Phage of Lactobacillus casei ATCC 393.

    PubMed

    Zhang, Xi; Lan, Yu; Jiao, Wenchao; Li, Yijing; Tang, Lijie; Jiang, Yanping; Cui, Wen; Qiao, Xinyuan

    2015-12-01

    A new virulent phage (Lcb) of Lactobacillus casei ATCC 393 was isolated from Chinese sauerkraut. It was specific to L. casei ATCC 393. Electron micrograph revealed that it had an icosahedral head (60.2 ± 0.8 nm in diameter) and a long tail (251 ± 2.6 nm). It belonged to the Siphoviridae family. The genome of phage Lcb was estimated to be approximately 40 kb and did not contain cohesive ends. One-step growth kinetics of its lytic development revealed latent and burst periods of 75 and 45 min, respectively, with a burst size of 16 PFU per infected cell. The phage was able to survive in a pH range between 4 and 11. However, a treatment of 70 °C for 30 min and 75% ethanol or isopropanol for 20 min was observed to inactivate phage Lcb thoroughly. The presence of both Ca(2+) and Mg(2+) showed a little influence on phage adsorption, but they were indispensable to gain complete lysis and improve plaque formation. The adsorption kinetics were similar on viable or nonviable cells, and high adsorption rates maintained between 10 and 37 °C. The highest adsorption rate was at 30 °C. This study increased the knowledge on phages of L. casei. The characterization of phage Lcb is helpful to establish a basis for adopting effective strategies to control phage attack in industry. PMID:26123178

  2. Intravenous Infusion of Phage-displayed Antibody Library in Human Cancer Patients: Enrichment and Cancer-Specificity of Tumor-Homing Phage-Antibodies

    PubMed Central

    Shukla, Girja S.; Krag, David N.; Peletskaya, Elena N.; Pero, Stephanie C.; Sun, Yu-Jing; Carman, Chelsea L.; McCahill, Laurence E.; Roland, Thomas A.

    2013-01-01

    Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage antibodies and a Stage IV cancer status. All patients were intravenously administered 1×1011 TUs/kg of an scFv library 1 to 4 hours before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0 to 5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2355) that was found to share a 12-amino acid long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis. PMID:23736951

  3. Phage based green chemistry for gold ion reduction and gold retrieval.

    PubMed

    Setyawati, Magdiel I; Xie, Jianping; Leong, David T

    2014-01-22

    The gold mining industry has taken its toll on the environment, triggering the development of more environmentally benign processes to alleviate the waste load release. Here, we demonstrate the use of bacteriophages (phages) for biosorption and bioreduction of gold ions from aqueous solution, which potentially can be applied to remediate gold ions from gold mining waste effluent. Phage has shown a remarkably efficient sorption of gold ions with a maximum gold adsorption capacity of 571 mg gold/g dry weight phage. The product of this phage mediated process is gold nanocrystals with the size of 30-630 nm. Biosorption and bioreduction processes are mediated by the ionic and covalent interaction between gold ions and the reducing groups on the phage protein coat. The strategy offers a simple, ecofriendly and feasible option to recover of gold ions to form readily recoverable products of gold nanoparticles within 24 h. PMID:24359519

  4. Phage therapy of the white plague-like disease of Favia favus in the Red Sea

    NASA Astrophysics Data System (ADS)

    Atad, I.; Zvuloni, A.; Loya, Y.; Rosenberg, E.

    2012-09-01

    Coral disease is a major factor in the global decline of coral reefs. At present, there are no known procedures for preventing or treating infectious diseases of corals. Immunization is not possible because corals have a restricted adaptive immune system and antibiotics are neither ecologically safe nor practical in an open system. Thus, we tested phage therapy as an alternative therapeutic method for treating diseased corals. Phage BA3, specific to the coral pathogen Thalassomonas loyana, inhibited the progression of the white plague-like disease and transmission to healthy corals in the Gulf of Aqaba, Red Sea. Only one out of 19 (5 %) of the healthy corals became infected when placed near phage-treated diseased corals, whereas 11 out of 18 (61 %) healthy corals were infected in the no-phage control. This is the first successful treatment for a coral disease in the sea. We posit that phage therapy of certain coral diseases is achievable in situ.

  5. Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

    PubMed

    Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

    2015-05-15

    The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.

  6. DNA fusion product of phage P2 with plasmid pBR322 - A new phasmid

    NASA Technical Reports Server (NTRS)

    Nicoletti, M.; Bertani, G.

    1983-01-01

    The chromosome of the temperate bacteriophage P2 and that of the plasmid pBR322 have been joined in vitro after treatment with restriction endonuclease EcoRI. The fusion product - a phasmid - can behave as a plasmid, as a phage and as a prophage. It can replicate its DNA under the control of either the specific replication mechanism of the parent phage in a polA mutant or that of the parent plasmid in a rep mutant. Several interesting interactions between the two replication modes are indicated. In particular, phage particles may be produced even when the phage mode of DNA replication is blocked, and this throws new light on the involvement of the early gene A in the regulation of late gene expression in phage P2.

  7. Involvement of the htpR gene product of Escherichia coli in phage lambda development.

    PubMed

    Waghorne, C; Fuerst, C R

    1985-02-01

    Growth of phage lambda at high temperature requires a functional htpR host gene. The stages of the phage growth cycle shown to be dependent on htpR gene function include prophage excision and particle morphogenesis. Two types of morphogenetic abnormalities have been detected. One is a defect in phage tail assembly that results from a deficiency in tail fibers even though gpJ is produced. The severity of this defect is phage-strain specific. The second morphogenetic defect is less clearly defined, but results in formation of aberrant phage head structures. These abnormalities in lambda reproduction are presumed to be caused by the absence in htpR mutant host cells at high temperature of one or more of the heat-shock proteins of Escherichia coli whose synthesis is known to be regulated by the htpR gene.

  8. Phage based green chemistry for gold ion reduction and gold retrieval.

    PubMed

    Setyawati, Magdiel I; Xie, Jianping; Leong, David T

    2014-01-22

    The gold mining industry has taken its toll on the environment, triggering the development of more environmentally benign processes to alleviate the waste load release. Here, we demonstrate the use of bacteriophages (phages) for biosorption and bioreduction of gold ions from aqueous solution, which potentially can be applied to remediate gold ions from gold mining waste effluent. Phage has shown a remarkably efficient sorption of gold ions with a maximum gold adsorption capacity of 571 mg gold/g dry weight phage. The product of this phage mediated process is gold nanocrystals with the size of 30-630 nm. Biosorption and bioreduction processes are mediated by the ionic and covalent interaction between gold ions and the reducing groups on the phage protein coat. The strategy offers a simple, ecofriendly and feasible option to recover of gold ions to form readily recoverable products of gold nanoparticles within 24 h.

  9. Detection of E. coli O157:H7 with a reporter phage containing the luxCDABE cassette

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage and reporter phage are used for typing and/or detection of pathogens. The temperate tailed phage fV10 has been utilized for phage-typing E. coli O157:H7. By modifying fV10 to transduce kanamycin resistance and the a luxCDABE cassette, we developed a reporter bacteriophage (fV10-lux) p...

  10. Complete Genome Sequences of Nine Phages Capable of Infecting Paenibacillus larvae, the Causative Agent of American Foulbrood Disease in Honeybees.

    PubMed

    Tsourkas, Philippos K; Yost, Diane G; Krohn, Andrew; LeBlanc, Lucy; Zhang, Anna; Stamereilers, Casey; Amy, Penny S

    2015-01-01

    We present here the complete genome sequences of nine phages that infect Paenibacillus larvae, the causative agent of American foulbrood disease in honeybees. The phages were isolated from soil, propolis, and infected bees from three U.S. states. This is the largest number of P. larvae phage genomes sequenced in a single publication to date.

  11. Coverage of diarrhoea-associated Escherichia coli isolates from different origins with two types of phage cocktails

    PubMed Central

    Bourdin, Gilles; Navarro, Armando; Sarker, Shafiqul A; Pittet, Anne-C; Qadri, Firdausi; Sultana, Shamima; Cravioto, Alejandro; Talukder, Kaisar A; Reuteler, Gloria; Brüssow, Harald

    2014-01-01

    Eighty-nine T4-like phages from our phage collection were tested against four collections of childhood diarrhoea-associated Escherichia coli isolates representing different geographical origins (Mexico versus Bangladesh), serotypes (69 O, 27 H serotypes), pathotypes (ETEC, EPEC, EIEC, EAEC, VTEC, Shigella), epidemiological settings (community and hospitalized diarrhoea) and years of isolation. With a cocktail consisting of 3 to 14 T4-like phages, we achieved 54% to 69% coverage against predominantly EPEC isolates from Mexico, 30% to 53% against mostly ETEC isolates from a prospective survey in Bangladesh, 24% to 61% against a mixture of pathotypes isolated from hospitalized children in Bangladesh, and 60% coverage against Shigella isolates. In comparison a commercial Russian phage cocktail containing a complex mixture of many different genera of coliphages showed 19%, 33%, 50% and 90% coverage, respectively, against the four above-mentioned collections. Few O serotype-specific phages and no broad-host range phages were detected in our T4-like phage collection. Interference phenomena between the phage isolates were observed when constituting larger phage cocktails. Since the coverage of a given T4-like phage cocktail differed with geographical area and epidemiological setting, a phage composition adapted to a local situation is needed for phage therapy approaches against E. coli pathogens. PMID:24528873

  12. Complete Genome Sequences of Nine Phages Capable of Infecting Paenibacillus larvae, the Causative Agent of American Foulbrood Disease in Honeybees.

    PubMed

    Tsourkas, Philippos K; Yost, Diane G; Krohn, Andrew; LeBlanc, Lucy; Zhang, Anna; Stamereilers, Casey; Amy, Penny S

    2015-01-01

    We present here the complete genome sequences of nine phages that infect Paenibacillus larvae, the causative agent of American foulbrood disease in honeybees. The phages were isolated from soil, propolis, and infected bees from three U.S. states. This is the largest number of P. larvae phage genomes sequenced in a single publication to date. PMID:26472825

  13. Complete Genome Sequences of Nine Phages Capable of Infecting Paenibacillus larvae, the Causative Agent of American Foulbrood Disease in Honeybees

    PubMed Central

    Yost, Diane G.; Krohn, Andrew; LeBlanc, Lucy; Zhang, Anna; Stamereilers, Casey; Amy, Penny S.

    2015-01-01

    We present here the complete genome sequences of nine phages that infect Paenibacillus larvae, the causative agent of American foulbrood disease in honeybees. The phages were isolated from soil, propolis, and infected bees from three U.S. states. This is the largest number of P. larvae phage genomes sequenced in a single publication to date. PMID:26472825

  14. Sequence of Shiga Toxin 2 Phage 933W from Escherichia coli O157:H7: Shiga Toxin as a Phage Late-Gene Product†

    PubMed Central

    Plunkett, Guy; Rose, Debra J.; Durfee, Timothy J.; Blattner, Frederick R.

    1999-01-01

    Lysogenic bacteriophages are major vehicles for the transfer of genetic information between bacteria, including pathogenicity and/or virulence determinants. In the enteric pathogen Escherichia coli O157:H7, which causes hemorrhagic colitis and hemolytic-uremic syndrome, Shiga toxins 1 and 2 (Stx1 and Stx2) are phage encoded. The sequence and analysis of the Stx2 phage 933W is presented here. We find evidence that the toxin genes are part of a late-phage transcript, suggesting that toxin production may be coupled with, if not dependent upon, phage release during lytic growth. Another phage gene, stk, encodes a product resembling eukaryotic serine/threonine protein kinases. Based on its position in the sequence, Stk may be produced by the prophage in the lysogenic state, and, like the YpkA protein of Yersinia species, it may interfere with the signal transduction pathway of the mammalian host. Three novel tRNA genes present in the phage genome may serve to increase the availability of rare tRNA species associated with efficient expression of pathogenicity determinants: both the Shiga toxin and serine/threonine kinase genes contain rare isoleucine and arginine codons. 933W also has homology to lom, encoding a member of a family of outer membrane proteins associated with virulence by conferring the ability to survive in macrophages, and bor, implicated in serum resistance. PMID:10074068

  15. The tail-associated depolymerase of Erwinia amylovora phage L1 mediates host cell adsorption and enzymatic capsule removal, which can enhance infection by other phage.

    PubMed

    Born, Yannick; Fieseler, Lars; Klumpp, Jochen; Eugster, Marcel R; Zurfluh, Katrin; Duffy, Brion; Loessner, Martin J

    2014-07-01

    The depolymerase enzyme (DpoL1) encoded by the T7-like phage L1 efficiently degrades amylovoran, an important virulence factor and major component of the extracellular polysaccharide (EPS) of its host, the plant pathogen Erwinia amylovora. Mass spectrometry analysis of hydrolysed EPS revealed that DpoL1 cleaves the galactose-containing backbone of amylovoran. The enzyme is most active at pH 6 and 50°C, and features a modular architecture. Removal of 180 N-terminal amino acids was shown not to affect enzyme activity. The C-terminus harbours the hydrolase activity, while the N-terminal domain links the enzyme to the phage particle. Electron microscopy demonstrated that DpoL1-specific antibodies cross-link phage particles at their tails, either lateral or frontal, and immunogold staining confirmed that DpoL1 is located at the tail spikes. Exposure of high-level EPS-producing Er. amylovora strain CFBP1430 to recombinant DpoL1 dramatically increased sensitivity to the Dpo-negative phage Y2, which was not the case for EPS-negative mutants or low-level EPS-producing Er. amylovora. Our findings indicate that enhanced phage susceptibility is based on enzymatic removal of the EPS capsule, normally a physical barrier to Y2 infection, and that use of DpoL1 together with the broad host range, virulent phage Y2 represents an attractive combination for biocontrol of fire blight.

  16. Genome Sequences of Two Closely Related Vibrio parahaemolyticus Phages, VP16T and VP16C†

    PubMed Central

    Seguritan, Victor; Feng, I-Wei; Rohwer, Forest; Swift, Mark; Segall, Anca M.

    2003-01-01

    Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced. The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes. Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database. Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit). Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon. In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins. Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus. Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other. We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V. parahaemolyticus strain 16 host. Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V. parahaemolyticus. The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages. PMID:14563879

  17. Characterization of temperate phages infecting Clostridium difficile isolates of human and animal origins.

    PubMed

    Sekulovic, Ognjen; Garneau, Julian R; Néron, Audrey; Fortier, Louis-Charles

    2014-04-01

    Clostridium difficile is a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs of C. difficile that could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. In C. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and human C. difficile isolates. Prophages were induced by UV light from 58 C. difficile isolates of animal and human origins. Using soft agar overlays with 27 different C. difficile test strains, we isolated and further propagated nine temperate phages: two from horse isolates (ΦCD481-1 and ΦCD481-2), three from dog isolates (ΦCD505, ΦCD506, and ΦCD508), and four from human isolates (ΦCD24-2, ΦCD111, ΦCD146, and ΦCD526). Two phages are members of the Siphoviridae family (ΦCD111 and ΦCD146), while the others are Myoviridae phages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from human C. difficile isolates, especially the members of the Siphoviridae family, had a broader host range than phages from animal C. difficile isolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported in C. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and human C. difficile isolates.

  18. Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

    PubMed Central

    Ahern, Stephen J.; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry

    2014-01-01

    The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ∼4 × 10−12 ml cell−1 min−1 for X. fastidiosa strain Temecula 1 and ∼5 × 10−10 to 7 × 10−10 ml cell−1 min−1 for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa. PMID:24214944

  19. Chemically linked phage idiotype vaccination in the murine B cell lymphoma 1 model

    PubMed Central

    2013-01-01

    Background B cell malignancies are characterized by clonal expansion of B cells expressing tumor-specific idiotypes on their surface. These idiotypes are ideal target antigens for an individualized immunotherapy. However, previous idiotype vaccines mostly lacked efficiency due to a low immunogenicity of the idiotype. The objective of the present study was the determination of the feasibility, safety and immunogenicity of a novel chemically linked phage idiotype vaccine. Methods In the murine B cell lymphoma 1 model, tumor idiotypes were chemically linked to phage particles used as immunological carriers. For comparison, the idiotype was genetically expressed on the major phage coat protein g8 or linked to keyhole limpet hemocynanin. After intradermal immunizations with idiotype vaccines, tolerability and humoral immune responses were assessed. Results Feasibility and tolerability of the chemically linked phage idiotype vaccine was demonstrated. Vaccination with B cell lymphoma 1 idiotype expressing phage resulted in a significant survival benefit in the murine B cell lymphoma 1 protection model (60.2 ± 23.8 days vs. 41.8 ± 1.6 days and 39.8 ± 3.8 days after vaccination with wild type phage or phosphate buffered saline, respectively). Superior immunogenicity of the chemically linked phage idiotype vaccine compared to the genetically engineered phage idiotype and keyhole limpet hemocynanin-coupled idiotype vaccine was demonstrated by significantly higher B cell lymphoma 1 idiotype-specific IgG levels after vaccination with chemically linked phage idiotype. Conclusion We present a novel, simple, time- and cost-efficient phage idiotype vaccination strategy, which represents a safe and feasible therapy and may produce a superior immune response compared to previously employed idiotype vaccination strategies. PMID:24152874

  20. Temperature Significantly Affects the Plaquing and Adsorption Efficiencies of Listeria Phages

    PubMed Central

    Tokman, Jeffrey I.; Kent, David J.; Wiedmann, Martin; Denes, Thomas

    2016-01-01

    Listeria-infecting phages are currently being used to control and detect the important foodborne pathogen Listeria monocytogenes; however, the influence of environmental conditions on the interactions between L. monocytogenes and its phages has not been explored in depth. Here, we examined the infective potential of four Listeria phages (two each from the P70-like and P100-like phages of Listeria) against five strains of L. monocytogenes (representing serotypes 1/2a, 1/2b, 4a, and 4b) grown under a range of temperatures (7–37°C). We show that the plaquing efficiencies for all four phages were significantly affected by temperature. Interestingly, no plaques were observed for any of the four phages at 37°C. Adsorption assays performed with the P100-like phages, LP-048 and LP-125, showed that LP-048 had a severely reduced adsorption efficiency against susceptible strains at 37°C as compared to 30°C, suggesting that there is considerably less accessible rhamnose (LP-048’s putative phage receptor) on the host at 37°C than at 30°C. LP-125 adsorbed to host cells at 37°C, indicating that the inability for LP-125 to plaque at 37°C is not due to adsorption inhibition. LP-048 showed significantly higher adsorption efficiency against a mutant strain lacking N-acetylglucosamine in its wall teichoic acids (WTA) than the parental strain at both 30 and 37°C, suggesting that N-acetylglucosamine competes with rhamnose for glycosylation sites on the WTA. The data presented here clearly shows that L. monocytogenes can gain physiological refuge from phage infection, which should be carefully considered for both the design and implementation of phage-based control and detection applications. PMID:27199957

  1. The filamentous phages fd and IF1 use different mechanisms to infect Escherichia coli.

    PubMed

    Lorenz, Stefan H; Jakob, Roman P; Weininger, Ulrich; Balbach, Jochen; Dobbek, Holger; Schmid, Franz X

    2011-01-28

    The filamentous phage fd uses its gene 3 protein (G3P) to target Escherichia coli cells in a two-step process. First, the N2 domain of G3P attaches to an F pilus, and then the N1 domain binds to TolA-C. N1 and N2 are tightly associated, rendering the phage robust but noninfectious because the binding site for TolA-C is buried at the domain interface. Binding of N2 to the F pilus initiates partial unfolding, domain disassembly, and prolyl cis-to-trans isomerization in the hinge between N1 and N2. This activates the phage, and trans-Pro213 maintains this state long enough for N1 to reach TolA-C. Phage IF1 targets I pili, and its G3P contains also an N1 domain and an N2 domain. The pilus-binding N2 domains of the phages IF1 and fd are unrelated, and the N1 domains share a 31% sequence identity. We show that N2 of phage IF1 mediates binding to the I pilus, and that N1 targets TolA. Crystallographic and NMR analyses of the complex between N1 and TolA-C indicate that phage IF1 interacts with the same site on TolA-C as phage fd. In IF1-G3P, N1 and N2 are independently folding units, however, and the TolA binding site on N1 is permanently accessible. Activation by unfolding and prolyl isomerization, as in the case of phage fd, is not observed. In IF1-G3P, the absence of stabilizing domain interactions is compensated for by a strong increase in the stabilities of the individual domains. Apparently, these closely related filamentous phages evolved different mechanisms to reconcile robustness with high infectivity.

  2. Genome sequences of two closely related Vibrio parahaemolyticus phages, VP16T and VP16C.

    PubMed

    Seguritan, Victor; Feng, I-Wei; Rohwer, Forest; Swift, Mark; Segall, Anca M

    2003-11-01

    Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced. The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes. Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database. Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit). Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon. In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins. Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus. Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other. We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V. parahaemolyticus strain 16 host. Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V. parahaemolyticus. The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages.

  3. Phylogenetic structure and evolution of regulatory genes and integrases of P2-like phages.

    PubMed

    Nilsson, Hanna; Cardoso-Palacios, Carlos; Haggård-Ljungquist, Elisabeth; Nilsson, Anders S

    2011-07-01

    The phylogenetic relationships and structural similarities of the proteins encoded within the regulatory region (containing the integrase gene and the lytic-lysogenic transcriptional switch genes) of P2-like phages were analyzed, and compared with the phylogenetic relationship of P2-like phages inferred from four structural genes. P2-like phages are thought to be one of the most genetically homogenous phage groups but the regulatory region nevertheless varies extensively between different phage genomes.   The analyses showed that there are many types of regulatory regions, but two types can be clearly distinguished; regions similar either to the phage P2 or to the phage 186 regulatory regions. These regions were also found to be most frequent among the sequenced P2-like phage or prophage genomes, and common in phages using Escherichia coli as a host. Both the phylogenetic and the structural analyses showed that these two regions are related. The integrases as well as the cox/apl genes show a common monophyletic origin but the immunity repressor genes, the type P2 C gene and the type 186 cI gene, are likely of different origin. There was no indication of recombination between the P2-186 types of regulatory genes but the comparison of the phylogenies of the regulatory region with the phylogeny based on four structural genes revealed recombinational events between the regulatory region and the structural genes. Less common regulatory regions were phylogenetically heterogeneous and typically contained a fusion of genes from distantly related or unknown phages and P2-like genes.

  4. Phylogenetic structure and evolution of regulatory genes and integrases of P2-like phages

    PubMed Central

    Nilsson, Hanna; Cardoso-Palacios, Carlos; Haggård-Ljungquist, Elisabeth; Nilsson, Anders S.

    2011-01-01

    The phylogenetic relationships and structural similarities of the proteins encoded within the regulatory region (containing the integrase gene and the lytic–lysogenic transcriptional switch genes) of P2-like phages were analyzed, and compared with the phylogenetic relationship of P2-like phages inferred from four structural genes. P2-like phages are thought to be one of the most genetically homogenous phage groups but the regulatory region nevertheless varies extensively between different phage genomes.   The analyses showed that there are many types of regulatory regions, but two types can be clearly distinguished; regions similar either to the phage P2 or to the phage 186 regulatory regions. These regions were also found to be most frequent among the sequenced P2-like phage or prophage genomes, and common in phages using Escherichia coli as a host. Both the phylogenetic and the structural analyses showed that these two regions are related. The integrases as well as the cox/apl genes show a common monophyletic origin but the immunity repressor genes, the type P2 C gene and the type 186 cI gene, are likely of different origin. There was no indication of recombination between the P2–186 types of regulatory genes but the comparison of the phylogenies of the regulatory region with the phylogeny based on four structural genes revealed recombinational events between the regulatory region and the structural genes. Less common regulatory regions were phylogenetically heterogeneous and typically contained a fusion of genes from distantly related or unknown phages and P2-like genes. PMID:23050214

  5. Dualities in the analysis of phage DNA packaging motors

    PubMed Central

    Serwer, Philip; Jiang, Wen

    2012-01-01

    The DNA packaging motors of double-stranded DNA phages are models for analysis of all multi-molecular motors and for analysis of several fundamental aspects of biology, including early evolution, relationship of in vivo to in vitro biochemistry and targets for anti-virals. Work on phage DNA packaging motors both has produced and is producing dualities in the interpretation of data obtained by use of both traditional techniques and the more recently developed procedures of single-molecule analysis. The dualities include (1) reductive vs. accretive evolution, (2) rotation vs. stasis of sub-assemblies of the motor, (3) thermal ratcheting vs. power stroking in generating force, (4) complete motor vs. spark plug role for the packaging ATPase, (5) use of previously isolated vs. new intermediates for analysis of the intermediate states of the motor and (6) a motor with one cycle vs. a motor with two cycles. We provide background for these dualities, some of which are under-emphasized in the literature. We suggest directions for future research. PMID:23532204

  6. Diversity-generating Retroelements in Phage and Bacterial Genomes.

    PubMed

    Guo, Huatao; Arambula, Diego; Ghosh, Partho; Miller, Jeff F

    2014-12-01

    Diversity-generating retroelements (DGRs) are DNA diversification machines found in diverse bacterial and bacteriophage genomes that accelerate the evolution of ligand-receptor interactions. Diversification results from a unidirectional transfer of sequence information from an invariant template repeat (TR) to a variable repeat (VR) located in a protein-encoding gene. Information transfer is coupled to site-specific mutagenesis in a process called mutagenic homing, which occurs through an RNA intermediate and is catalyzed by a unique, DGR-encoded reverse transcriptase that converts adenine residues in the TR into random nucleotides in the VR. In the prototype DGR found in the Bordetella bacteriophage BPP-1, the variable protein Mtd is responsible for phage receptor recognition. VR diversification enables progeny phage to switch tropism, accelerating their adaptation to changes in sequence or availability of host cell-surface molecules for infection. Since their discovery, hundreds of DGRs have been identified, and their functions are just beginning to be understood. VR-encoded residues of many DGR-diversified proteins are displayed in the context of a C-type lectin fold, although other scaffolds, including the immunoglobulin fold, may also be used. DGR homing is postulated to occur through a specialized target DNA-primed reverse transcription mechanism that allows repeated rounds of diversification and selection, and the ability to engineer DGRs to target heterologous genes suggests applications for bioengineering. This chapter provides a comprehensive review of our current understanding of this newly discovered family of beneficial retroelements.

  7. Ionic switch controls the DNA state in phage λ

    SciTech Connect

    Li, Dong; Liu, Ting; Zuo, Xiaobing; Li, Tao; Qiu, Xiangyun; Evilevitch, Alex

    2015-06-19

    We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. The results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.

  8. Ionic switch controls the DNA state in phage λ

    DOE PAGES

    Li, Dong; Liu, Ting; Zuo, Xiaobing; Li, Tao; Qiu, Xiangyun; Evilevitch, Alex

    2015-06-19

    We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid bymore » changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. The results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.« less

  9. Ionic switch controls the DNA state in phage λ.

    PubMed

    Li, Dong; Liu, Ting; Zuo, Xiaobing; Li, Tao; Qiu, Xiangyun; Evilevitch, Alex

    2015-07-27

    We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is 'switched on' at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. These results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.

  10. Dynamics of adaptive immunity against phage in bacterial populations

    NASA Astrophysics Data System (ADS)

    Bradde, Serena; Vucelja, Marija; Tesileanu, Tiberiu; Balasubramanian, Vijay

    The CRISPR (clustered regularly interspaced short palindromic repeats) mechanism allows bacteria to adaptively defend against phages by acquiring short genomic sequences (spacers) that target specific sequences in the viral genome. We propose a population dynamical model where immunity can be both acquired and lost. The model predicts regimes where bacterial and phage populations can co-exist, others where the populations oscillate, and still others where one population is driven to extinction. Our model considers two key parameters: (1) ease of acquisition and (2) spacer effectiveness in conferring immunity. Analytical calculations and numerical simulations show that if spacers differ mainly in ease of acquisition, or if the probability of acquiring them is sufficiently high, bacteria develop a diverse population of spacers. On the other hand, if spacers differ mainly in their effectiveness, their final distribution will be highly peaked, akin to a ``winner-take-all'' scenario, leading to a specialized spacer distribution. Bacteria can interpolate between these limiting behaviors by actively tuning their overall acquisition rate.

  11. Structural Conservation of the Myoviridae Phage Tail Sheath Protein Fold

    SciTech Connect

    Aksyuk, Anastasia A.; Kurochkina, Lidia P.; Fokine, Andrei; Forouhar, Farhad; Mesyanzhinov, Vadim V.; Tong, Liang; Rossmann, Michael G.

    2012-02-21

    Bacteriophage phiKZ is a giant phage that infects Pseudomonas aeruginosa, a human pathogen. The phiKZ virion consists of a 1450 {angstrom} diameter icosahedral head and a 2000 {angstrom}-long contractile tail. The structure of the whole virus was previously reported, showing that its tail organization in the extended state is similar to the well-studied Myovirus bacteriophage T4 tail. The crystal structure of a tail sheath protein fragment of phiKZ was determined to 2.4 {angstrom} resolution. Furthermore, crystal structures of two prophage tail sheath proteins were determined to 1.9 and 3.3 {angstrom} resolution. Despite low sequence identity between these proteins, all of these structures have a similar fold. The crystal structure of the phiKZ tail sheath protein has been fitted into cryo-electron-microscopy reconstructions of the extended tail sheath and of a polysheath. The structural rearrangement of the phiKZ tail sheath contraction was found to be similar to that of phage T4.

  12. Biopanning of endotoxin-specific phage displayed peptides.

    PubMed

    Thomas, Celestine J; Sharma, Shilpi; Kumar, Gyanendra; Visweswariah, Sandhya S; Surolia, Avadhesha

    2003-07-18

    Systemic bacterial infections frequently lead to a plethora of symptoms termed "endotoxic shock" or "sepsis." Characterized by hypotension, coagulation abnormalities, and multiple organ failure, treatment of sepsis still remains mostly supportive. Of the various experimental therapeutic interventional strategies, neutralization of endotoxin by peptides or proteins is becoming popular recently. Hence, design of endotoxin binding peptides is gaining currency as their structural complexity and mode of recognition of endotoxin precludes mounting of resistance against them by the susceptible bacteria by genetic recombination, mutation, etc. Earlier work from our laboratory had shown that the amphiphilic cationic peptides are good ligands for endotoxin binding. In this study, we report the results of studies with the 12 selected lipid A binding phage displayed peptides by biopanning of a repertoire of a random pentadecapeptide library displayed on the filamentous M-13 phage. A comparison of the sequences revealed no consensus sequence between the 12 selected peptides suggesting that the lipid A binding motif is not sequence specific which is in accord with the sequence variation seen with the naturally occurring anti-microbial and/or endotoxin binding peptides. Thus, the flexibility of the peptides coupled with their plasticity in recognizing the lipid A moiety, explains their tight binding to endotoxin. At a structural level, asymmetric distribution of the charged polar residues on one face of the helix and non-polar residues on the opposite face appears to correlate with their activity.

  13. Comparative Genomics and Extensive Recombinations in Phage Communities

    NASA Astrophysics Data System (ADS)

    Poisson, Guylaine; Belcaid, Mahdi; Bergeron, Anne

    Comparing the genomes of two closely related viruses often produces mosaics where nearly identical sequences alternate with sequences that are unique to each genome. When several closely related genomes are compared, the unique sequences are likely to be shared with third genomes, leading to virus mosaic communities. Here we present comparative analysis of sets of Staphylococcus aureus phages that share large identical sequences with up to three other genomes, and with different partners along their genomes. We introduce mosaic graphs to represent these complex recombination events, and use them to illustrate the breath and depth of sequence sharing: some genomes are almost completely made up of shared sequences, while genomes that share very large identical sequences can adopt alternate functional modules. Mosaic graphs also allow us to identify breakpoints that could eventually be used for the construction of recombination networks. These findings have several implications on phage metagenomics assembly, on the horizontal gene transfer paradigm, and more generally on the understanding of the composition and evolutionary dynamics of virus communities.

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

    PubMed

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

    2012-01-01

    Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥6log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions. PMID:22024504

  15. Magnetically-assisted impedimetric detection of bacteria using phage-modified carbon microarrays.

    PubMed

    Shabani, Arghavan; Marquette, Christophe A; Mandeville, Rosemonde; Lawrence, Marcus F

    2013-11-15

    This study presents an investigation on the possibility of improving the detection limit of bacteria with an inexpensive electrochemical, impedimetric sensor platform, by integrating the sensor with magnetic manipulation. The approach uses T4 bacteriophage coated Dynabeads to selectively capture and concentrate E. coli K12 cells from samples, to increase the sensitivity of detection at the surface of functionalized screen-printed carbon microarrays. Fluorescence and flow cytometry measurements indicate that the surface modification of the magnetic beads, with phages, and binding with the bacteria, were successful. Integration of the screen-printed carbon-based impedimetric sensor, with a magnetic manipulation system, was found to improve the sensitivity of the device, decreasing the limit of detection of E. coli K12 from 10(4) to 10(3) cfu/mL. We have also demonstrated that this approach provides for more specific detection of bacteria, enabling the operator to account for non-specific adsorption, and detection of bacteria in more complex (real) samples (milk).

  16. Following cell-fate in E. coli after infection by phage lambda.

    PubMed

    Zeng, Lanying; Golding, Ido

    2011-01-01

    The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant). We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells. Here, we describe the full procedure for performing the infection experiments described in our earlier work. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome

  17. A tail of two phages: genomic and functional analysis of Listeria monocytogenes phages vB_LmoS_188 and vB_LmoS_293 reveal the receptor-binding proteins involved in host specificity

    PubMed Central

    Casey, Aidan; Jordan, Kieran; Neve, Horst; Coffey, Aidan; McAuliffe, Olivia

    2015-01-01

    The physical characteristics of bacteriophages establish them as viable candidates for downstream development of pathogen detection assays and biocontrol measures. To utilize phages for such purposes, a detailed knowledge of their host interaction mechanisms is a prerequisite. There is currently a wealth of knowledge available concerning Gram-negative phage-host interaction, but little by comparison for Gram-positive phages and Listeria phages in particular. In this research, the lytic spectrum of two recently isolated Listeria monocytogenes phages (vB_LmoS_188 and vB_LmoS_293) was determined, and the genomic basis for their observed serotype 4b/4e host-specificity was investigated using comparative genomics. The late tail genes of these phages were identified to be highly conserved when compared to other serovar 4-specific Listeria phages. Spontaneous mutants of each of these phages with broadened host specificities were generated. Their late tail gene sequences were compared with their wild-type counterparts resulting in the putative identification of the products of ORF 19 of vB_LmoS_188 and ORF 20 of vB_LmoS_293 as the receptor binding proteins of these phages. The research findings also indicate that conserved baseplate architectures and host interaction mechanisms exist for Listeria siphoviruses with differing host-specificities, and further contribute to the current knowledge of phage-host interactions with regard to Listeria phages. PMID:26500641

  18. Fingerprint-like Analysis of “Nanoantibody” Selection by Phage Display Using Two Helper Phage Variants

    PubMed Central

    Tillib, S.V.; Ivanova, T.I.; Vasilev, L.A.

    2010-01-01

    This paper discusses the selection of mini-antibody (nanoantibody, nanobody® or single domain antibody) sequences of desired specificity by phage display-based method using a generated library of antigen-binding domains of special heavy-chain only antibodies (single-stranded antibodies) of immunized camel. A comprehensive comparison of the efficiency of parallel selection procedures was performed by using the traditional (M13KO7) and modified (with N-terminal deletion in the surface gIII protein) helper phages. These two methods are partly complementary, and by using them in parallel one can significantly improve the selection efficiency. Parallel restriction analysis (fingerprinting) of PCR-amplified cloned sequences coding for mini-antibodies (HMR-analysis) is proposed for identifying individual clones, as a replacement to sequencing (to a certain extent). Using this method, unique data were collected on the selection of mini-antibody variants with the required specificity at various stages of a multi-stage selection procedure. It has been shown that different sequences coding for mini-antibodies are selected in different ways, and that, if this feature is not taken into account, some mini-antibody variants may be lost. PMID:22649655

  19. Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. Establishment of lysogeny and lytic growth efficiency

    SciTech Connect

    Gorbunova, S.A.; Yanenko, A.S.; Akhverdyan, V.Z.; Reulets, M.A.; Krylov, V.N.

    1986-03-01

    Expression of the genomes of Pseudomonas aeruginosa transposable phages (TP) in the cells of a heterologous host, P. putida PpGl, was studied. A high efficiency of TP lytic growth in PpGl cells was obtained both after zygotic induction following RP4::TP plasmid transfer and after thermoinduction of PpGl cells lysogenic for thermoinducible prophage D3112cts15. Characteristic for PpGl cells was a high TP yield (20-25 phage D3112cts15 particles per cell), which was evidence of a high level of TP transposition in cells of this species. The frequency of RP4::TP transfer into PpGl and PA01 cells was equal, but the lysogeny detection rat was somewhat lower in PpGl. Pseudomonas aeruginosa TP can integrate into the PpGl chromosome, producing inducible lysogens. The presence of RP4 is not necessary for the expression of the TP genome in PpGl cells. The D3112cts15 TP may be used for interspecific transduction of plasmids and chromosomal markers.

  20. Generation and Validation of a Shewanella oneidensis MR-1 Clone Set for Protein Expression and Phage Display

    PubMed Central

    Wang, Xiaohu; Petrosino, Joseph; Hemphill, Lisa; Wan, Xiufeng; Leaphart, Adam B.; Weinstock, George M.; Palzkill, Timothy; Zhou, Jizhong

    2008-01-01

    A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST- tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray) and other proteomic tools (e.g., mass spectrometry-based proteomic analysis), and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro. PMID:18714347

  1. A Phage Tail-Derived Element with Wide Distribution among Both Prokaryotic Domains: A Comparative Genomic and Phylogenetic Study

    PubMed Central

    Sarris, Panagiotis F.; Ladoukakis, Emmanuel D.; Panopoulos, Nickolas J.; Scoulica, Effie V.

    2014-01-01

    Prophage sequences became an integral part of bacterial genomes as a consequence of coevolution, encoding fitness or virulence factors. Such roles have been attributed to phage-derived elements identified in several Gram-negative species: The type VI secretion system (T6SS), the R- and F-type pyocins, and the newly discovered Serratia entomophila antifeeding prophage (Afp), and the Photorhabdus luminescens virulence cassette (PVC). In this study, we provide evidence that remarkably conserved gene clusters, homologous to Afp/PVC, are not restricted to Gram-negative bacteria but are widespread throughout all prokaryotes including the Archaea. Even though they are phylogenetically closer to pyocins, they share key characteristics in common with the T6SS, such as the use of a chaperon-type AAA+ ATPase and the lack of a host cell lysis mechanism. We thus suggest that Afp/PVC-like elements could be classified as phage-like-protein-translocation structures (PLTSs) rather than as pyocins. The reconstruction of phylogeny and the conserved gene content suggest that the diversification of prophage sequences to PLTS occurred in bacteria early in evolution and only once, but PLTS clusters have been horizontally transferred to some of the bacterial lineages and to the Archaea. The adaptation of this element in such a wide host range is suggestive of its versatile use in prokaryotes. PMID:25015235

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

    SciTech Connect

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

    2013-09-01

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

  3. Cytoplasmic Delivery of Liposomes into MCF-7 Breast Cancer Cells Mediated by Cell-Specific Phage Fusion Coat Protein

    PubMed Central

    Wang, Tao; Yang, Shenghong; Petrenko, Valery A; Torchilin, Vladimir P

    2010-01-01

    Earlier, we have shown that doxorubicin-loaded liposomes (Doxil) modified with a chimeric phage fusion coat protein specific towards MCF-7 breast cancer cells identified from a phage landscape library demonstrated a significantly enhanced association with target cells and an increased cytotoxicity. Based on some structural similarities between the N-terminus of the phage potein and known fusogenic peptides, we hypothesized that, in addition to the specific targeting, the phage protein may possess endosome-escaping potential and an increased cytotoxicity of drug-loaded phage protein-targeted liposomes may be explained by an advantageous combination of both, cell targeting and endosomal escape of drug-loaded nanocarrier. The use of the fluorescence resonance energy transfer (FRET) technique allowed us to clearly demonstrate the pH-dependent membrane fusion activity of the phage protein. Endosomal escape and cytosolic delivery of phage-liposomes was visualized with fluorescence microscopy. Endosome acidification inhibition by bafilomycin A 1 resulted in decreased cytotoxicity of the phage-Doxil, while the endosome disruption by chloroquine had a negligible effect on efficacy of phage-Doxil, confirming its endosomal escape. Our results demonstrated an endosome-escaping property of the phage protein and provided an insight on mechanism of the enhanced cytotoxicity of phage-Doxil. PMID:20438086

  4. Evolved Populations of Shigella flexneri Phage Sf6 Acquire Large Deletions, Altered Genomic Architecture, and Faster Life Cycles.

    PubMed

    Dover, John A; Burmeister, Alita R; Molineux, Ian J; Parent, Kristin N

    2016-01-01

    Genomic architecture is the framework within which genes and regulatory elements evolve and where specific constructs may constrain or potentiate particular adaptations. One such construct is evident in phages that use a headful packaging strategy that results in progeny phage heads packaged with DNA until full rather than encapsidating a simple unit-length genome. Here, we investigate the evolution of the headful packaging phage Sf6 in response to barriers that impede efficient phage adsorption to the host cell. Ten replicate populations evolved faster Sf6 life cycles by parallel mutations found in a phage lysis gene and/or by large, 1.2- to 4.0-kb deletions that remove a mobile genetic IS911 element present in the ancestral phage genome. The fastest life cycles were found in phages that acquired both mutations. No mutations were found in genes encoding phage structural proteins, which were a priori expected from the experimental design that imposed a challenge for phage adsorption by using a Shigella flexneri host lacking receptors preferred by Sf6. We used DNA sequencing, molecular approaches, and physiological experiments on 82 clonal isolates taken from all 10 populations to reveal the genetic basis of the faster Sf6 life cycle. The majority of our isolates acquired deletions in the phage genome. Our results suggest that deletions are adaptive and can influence the duration of the phage life cycle while acting in conjunction with other lysis time-determining point mutations. PMID:27497318

  5. Bacillus cereus phage typing as an epidemiological tool in outbreaks of food poisoning.

    PubMed

    Ahmed, R; Sankar-Mistry, P; Jackson, S; Ackermann, H W; Kasatiya, S S

    1995-03-01

    Bacillus cereus is responsible for an increasing number of food poisoning cases. By using 12 bacteriophages isolated from sewage, a typing scheme for B. cereus isolates from outbreaks or sporadic cases of food poisoning was developed. The phages belonged to three morphotypes. Ten phages with contractile tails and icosahedral heads were members of the Myoviridae family, and two phages with noncontractile tails belonged to the Siphoviridae family. Phage 11 represented a new species. It had an isometric head and a very long contractile tail with long wavy tail fibers and was one of the largest viruses known. The vast majority of 166 B. cereus strains (161, or 97%) isolated from food poisoning cases were typeable. Of 146 strains isolated from 18 outbreaks, 142 (97%) could be divided into 17 phage types. A good correlation, on the order of 80 to 100%, between phage types of strains isolated from suspected foods and those of strains isolated from stools of symptomatic patients was observed. Most Bacillus thuringiensis strains were also typeable, providing further evidence of the close relatedness of B. cereus and B. thuringiensis. This phage typing scheme can be a valuable epidemiological tool in tracing the origins of food poisoning caused by B. cereus.

  6. Genetic Diversity and Evolution of Salmonella enterica Serovar Enteritidis Strains with Different Phage Types

    PubMed Central

    Pettengill, James; Strain, Errol; Allard, Marc W.; Ahmed, Rafiq; Zhao, Shaohua; Brown, Eric W.

    2014-01-01

    Phage typing has been used for the epidemiological surveillance of Salmonella enterica serovar Enteritidis for over 2 decades. However, knowledge of the genetic and evolutionary relationships between phage types is very limited, making differences difficult to interpret. Here, single nucleotide polymorphisms (SNPs) identified from whole-genome comparisons were used to determine the relationships between some S. Enteritidis phage types (PTs) commonly associated with food-borne outbreaks in the United States. Emphasis was placed on the predominant phage types PT8, PT13a, and PT13 in North America. With >89,400 bp surveyed across 98 S. Enteritidis isolates representing 14 distinct phage types, 55 informative SNPs were discovered within 23 chromosomally anchored loci. To maximize the discriminatory and evolutionary partitioning of these highly homogeneous strains, sequences comprising informative SNPs were concatenated into a single combined data matrix and subjected to phylogenetic analysis. The resultant phylogeny allocated most S. Enteritidis isolates into two distinct clades (clades I and II) and four subclades. Synapomorphic (shared and derived) sets of SNPs capable of distinguishing individual clades/subclades were identified. However, individual phage types appeared to be evolutionarily disjunct when mapped to this phylogeny, suggesting that phage typing may not be valid for making phylogenetic inferences. Furthermore, the set of SNPs identified here represents useful genetic markers for strain differentiation of more clonal S. Enteritidis strains and provides core genotypic markers for future development of a SNP typing scheme with S. Enteritidis. PMID:24574287

  7. Absorption Kinetics of Phage Lambda on Its Host Under Shear Flow

    NASA Astrophysics Data System (ADS)

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

    2000-03-01

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

  8. A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents

    PubMed Central

    Zhan, Yuanchao; Huang, Sijun; Voget, Sonja; Simon, Meinhard; Chen, Feng

    2016-01-01

    Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205–279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics. PMID:27460944

  9. The use of specialised transducing phages in the amplification of enzyme production.

    PubMed

    Moir, A; Brammar, W J

    1976-11-24

    Two types of lambdatrp phages have been used as model systems to investigate ways of optimising the expression of bacterial genes from transducing phage genomes. Excellent yields of trp enzymes were achieved by infecting a trpR- host with Q- or Q-S- derivatives of lambdatrpAM1, which expresses its trp genese exclusively from the trp promoter. The five trp geneproducts constituted more than 50% of the total soluble protein of infected cells under these conditions, and an even higher proportion of the protein synthesized after infection. In a trpR+ host, phage DNA replication was easily able to override tryptophan-mediated repression by titration of the trp promoter were equally productive, while having the advantage of being much simpler to construct and propagate. lambdatrp phages lacking the trp promoter were used to investigate ways of optimising gene expression initiated at the phage promoter, PL. Though very powerful, the latter promoter is more difficult to harness then the trp promoter. Derepression of transcription from PL by the use of cro- mutations is accompanied by poor replication of transducing phage DNA. Attempts to circumvent this difficulty using virulent of cro,cII double mutants have not been successful. Nevertheless, cells infected with a lambdatrp phage expressing its trp genes exclusively from PL made up to 16 per cent of their protein as trp gene-products.

  10. A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents

    NASA Astrophysics Data System (ADS)

    Zhan, Yuanchao; Huang, Sijun; Voget, Sonja; Simon, Meinhard; Chen, Feng

    2016-07-01

    Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205–279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics.

  11. Phage Therapy is Effective in Protecting Honeybee Larvae from American Foulbrood Disease

    PubMed Central

    Ghorbani-Nezami, Sara; LeBlanc, Lucy; Yost, Diane G.; Amy, Penny S.

    2015-01-01

    American foulbrood disease has a major impact on honeybees (Apis melifera) worldwide. It is caused by a Gram-positive, spore-forming bacterium, Paenibacillus larvae. The disease can only affect larval honeybees, and the bacterial endospores are the infective unit of the disease. Antibiotics are not sufficient to combat the disease due to increasing resistance among P. larvae strains. Because of the durability and virulence of P. larvae endospores, infections spread rapidly, and beekeepers are often forced to burn beehives and equipment. To date, very little information is available on the use of bacteriophage therapy in rescuing and preventing American foulbrood disease, therefore the goal of this study was to test the efficacy of phage therapy against P. larvae infection. Out of 32 previously isolated P. larvae phages, three designated F, WA, and XIII were tested on artificially reared honeybee larvae infected with P. larvae strain NRRL B-3650 spores. The presence of P. larvae DNA in dead larvae was confirmed by 16S rRNA gene-specific polymerase chain reaction amplification. Survival rates for phage-treated larvae were approximately the same as for larvae never infected with spores (84%), i.e., the phages had no deleterious effect on the larvae. Additionally, prophylactic treatment of larvae with phages before spore infection was more effective than administering phages after infection, although survival in both cases was higher than spores alone (45%). Further testing to determine the optimal combination and concentration of phages, and testing in actual hive conditions are needed. PMID:26136497

  12. Phage Therapy is Effective in Protecting Honeybee Larvae from American Foulbrood Disease.

    PubMed

    Ghorbani-Nezami, Sara; LeBlanc, Lucy; Yost, Diane G; Amy, Penny S

    2015-01-01

    American foulbrood disease has a major impact on honeybees (Apis melifera) worldwide. It is caused by a Gram-positive, spore-forming bacterium, Paenibacillus larvae. The disease can only affect larval honeybees, and the bacterial endospores are the infective unit of the disease. Antibiotics are not sufficient to combat the disease due to increasing resistance among P. larvae strains. Because of the durability and virulence of P. larvae endospores, infections spread rapidly, and beekeepers are often forced to burn beehives and equipment. To date, very little information is available on the use of bacteriophage therapy in rescuing and preventing American foulbrood disease, therefore the goal of this study was to test the efficacy of phage therapy against P. larvae infection. Out of 32 previously isolated P. larvae phages, three designated F, WA, and XIII were tested on artificially reared honeybee larvae infected with P. larvae strain NRRL B-3650 spores. The presence of P. larvae DNA in dead larvae was confirmed by 16S rRNA gene-specific polymerase chain reaction amplification. Survival rates for phage-treated larvae were approximately the same as for larvae never infected with spores (84%), i.e., the phages had no deleterious effect on the larvae. Additionally, prophylactic treatment of larvae with phages before spore infection was more effective than administering phages after infection, although survival in both cases was higher than spores alone (45%). Further testing to determine the optimal combination and concentration of phages, and testing in actual hive conditions are needed.

  13. Oral T4-like phage cocktail application to healthy adult volunteers from Bangladesh

    SciTech Connect

    Sarker, Shafiqul Alam; McCallin, Shawna; Barretto, Caroline; Berger, Bernard; Pittet, Anne-Cecile; Sultana, Shamima; Krause, Lutz; Huq, Sayeda; Bibiloni, Rodrigo; Bruttin, Anne; Reuteler, Gloria; Bruessow, Harald

    2012-12-20

    The genomic diversity of 99 T4-like coliphages was investigated by sequencing an equimolar mixture with Illumina technology and screening them against different databases for horizontal gene transfer and undesired genes. A 9-phage cocktail was given to 15 healthy adults from Bangladesh at a dose of 3 Multiplication-Sign 10{sup 9} and 3 Multiplication-Sign 10{sup 7} plaque-forming units and placebo respectively. Phages were detected in 64% of the stool samples when subjects were treated with higher titer phage, compared to 30% and 28% with lower-titer phage and placebo, respectively. No Escherichia coli was present in initial stool samples, and no amplification of phage was observed. One percent of the administered oral phage was recovered from the feces. No adverse events were observed by self-report, clinical examination, or from laboratory tests for liver, kidney, and hematology function. No impact of oral phage was seen on the fecal microbiota composition with respect to bacterial 16S rRNA from stool.

  14. The temperate phages RP2 and RP3 of Streptomyces rimosus.

    PubMed

    Rausch, H; Vesligaj, M; Pocta, D; Biuković, G; Pigac, J; Cullum, J; Schmieger, H; Hranueli, D

    1993-10-01

    The oxytetracycline-producing Streptomyces rimosus strains R6-65 and R7 (ATCC 10970) are lysogenic for the two narrow-host-range phages RP2 and RP3. Both phages are released at low frequency from the lysogenic strains and form plaques on 'cured' S. rimosus strains. RP2 and RP3 are of similar shape with flexible tails and contain double-stranded DNA of about 70% G+C with cohesive ends (group B1 of bacteriophage classification). The two phages also have identical, very slow, growth kinetics in S. rimosus, with a latent phase of about 6 h and a rise period of about 4 h. RP2 and RP3 are heteroimmune and they differ slightly in their size of phage particles and length of DNA (64.7 and 62.4 kb for RP2 and RP3, respectively). The restriction maps of the two phages are completely different, and hybridization experiments showed only one short region of sequence similarity (less than 430 bp); the two phages are thus essentially unrelated. Both phages lysogenize their hosts by recombination via defined attachment (att) sites. The positions of the attP sites have been localized on the restriction maps of RP2 and RP3 to restriction fragments of 800 and 300 bp, respectively. The prophages did not affect the level of oxytetracycline production or the genetic instability of this trait.

  15. A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents.

    PubMed

    Zhan, Yuanchao; Huang, Sijun; Voget, Sonja; Simon, Meinhard; Chen, Feng

    2016-01-01

    Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205-279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics. PMID:27460944

  16. Nanodiscs allow phage display selection for ligands to non-linear epitopes on membrane proteins.

    PubMed

    Pavlidou, Marina; Hänel, Karen; Möckel, Luis; Willbold, Dieter

    2013-01-01

    In this work, we exploited a method that uses polytopic membrane proteins as targets for phage display selections. Membrane proteins represent the largest class of drug targets and drug discovery is mostly based on the identification of ligands binding to target molecules. The screening of a phage display library for ligands against membrane proteins is typically hindered by the requirement of these proteins for a membrane environment, which is necessary to retain correct folding and epitope formation. Especially in proteins with multiple transmembrane domains, epitopes often are non-linear and consist of a combination of loops between transmembrane stretches of the proteins. Here, we have used bacteriorhodopsin (bR) as a model of polytopic membrane protein, assembled into nanoscale phospholipid bilayers, so called nanodiscs, to screen a phage display library for potential ligands. Nanodiscs provide a native-like environment to membrane proteins and thus selection of ligands can take place in a near physiological state. Screening a 12-mer phage display peptide library against bR nanodiscs led to the isolation of phage clones binding specifically to bR. We were further able to identify the binding site of selected phage clones proving that the clones bind to extramembranous, non-linear epitopes of bR. Thus, nanodiscs provide a suitable and general tool that allows screening of a phage display library against membrane proteins in a near native environment.

  17. On size-dependent stability and infectivity of λ bacterial phages

    NASA Astrophysics Data System (ADS)

    Li, Long; Wang, Jizeng

    2015-02-01

    The elastic icosahedral capsid of λ phages plays an important role in the life cycle of these phages, such as holding the viral genome and releasing confinement for DNA ejection. Understanding how a nanosized elastic capsid guarantees the stability and infectivity of λ phages is challenging. In this article, we propose a combined nonlinear continuum and statistical mechanics model by considering the effects of DNA bending deformation, electrostatic repulsion between DNA-DNA strands, and elastic deformation of phage capsid to investigate the coupled process between capsid and DNA in packaging and ejection. Based on this model, we show that packaging DNA into immature λ phage capsid uses less force than packaging DNA into mature λ phage because of the deformability and softness of the former. Consequently, resistance to DNA packaging inside capsid decreases compared with mature ones. We also observe relationships between phage capsid size and the maximum shear stress on the inner surface of capsid and required osmotic pressure for the complete inhibition of DNA ejection. An optimized radius of capsid, i.e., around 30 nm, is found for both stable DNA packaging and effective viral infection from mechanical standpoints, which may result from physical evolution. All these findings may be interesting to toxicologists, nanotechnologists, and virologists.

  18. Development of a Phage Cocktail to Control Proteus mirabilis Catheter-associated Urinary Tract Infections

    PubMed Central

    Melo, Luís D. R.; Veiga, Patrícia; Cerca, Nuno; Kropinski, Andrew M.; Almeida, Carina; Azeredo, Joana; Sillankorva, Sanna

    2016-01-01

    Proteus mirabilis is an enterobacterium that causes catheter-associated urinary tract infections (CAUTIs) due to its ability to colonize and form crystalline biofilms on the catheters surface. CAUTIs are very difficult to treat, since biofilm structures are highly tolerant to antibiotics. Phages have been used widely to control a diversity of bacterial species, however, a limited number of phages for P. mirabilis have been isolated and studied. Here we report the isolation of two novel virulent phages, the podovirus vB_PmiP_5460 and the myovirus vB_PmiM_5461, which are able to target, respectively, 16 of the 26 and all the Proteus strains tested in this study. Both phages have been characterized thoroughly and sequencing data revealed no traces of genes associated with lysogeny. To further evaluate the phages’ ability to prevent catheter’s colonization by Proteus, the phages adherence to silicone surfaces was assessed. Further tests in phage-coated catheters using a dynamic biofilm model simulating CAUTIs, have shown a significant reduction of P. mirabilis biofilm formation up to 168 h of catheterization. These results highlight the potential usefulness of the two isolated phages for the prevention of surface colonization by this bacterium. PMID:27446059

  19. 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. PMID:11853411

  20. Phage passage after extended processing in small-virus-retentive filters.

    PubMed

    Lute, Scott; Bailey, Mark; Combs, Jessica; Sukumar, Muppalla; Brorson, Kurt

    2007-07-01

    Retention of a two small phages (PhiX-174 and pp7) by direct-flow small-virus-retentive filters [Viresolve NFP (normal-flow parvovirus), Virosart CPV (canine parvovirus), Ultipor DV20 and Planova 20N] was studied using a commercial-process fluid. Phage passage occurred in each filter type, particularly when overloaded with phage. Clearances of pp7 and PhiX-174 were similar for any given filter brand, arguing that the two phages are equivalent for testing small-virus-retentive filters. The patterns of flux under constant pressure and instantaneous LRV (log reduction value) in relationship to cumulative phage load differed between brands, consistent with the current industry understanding that each brand possesses specific performance attributes. Phages are a powerful and universal tool for evaluating filter performance. Validation of filter performance with phages such as pp7 or PhiX-174 as models for small mammalian viruses represents an attractive alternative to the current practice.

  1. A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents.

    PubMed

    Zhan, Yuanchao; Huang, Sijun; Voget, Sonja; Simon, Meinhard; Chen, Feng

    2016-01-01

    Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205-279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics.

  2. Genomic characterization of a temperate phage of the psychrotolerant deep-sea bacterium Aurantimonas sp.

    PubMed

    Yoshida, Mitsuhiro; Yoshida-Takashima, Yukari; Nunoura, Takuro; Takai, Ken

    2015-01-01

    A temperate phage (termed AmM-1) was identified from the psychrotolerant Rhizobiales bacterium Aurantimonas sp. C5-1, which was isolated from bathypelagic water (water depth = 1,500 m) in the northwest Pacific. The AmM-1 genome is 47,800 bp in length and contains 67 coding sequences. Although phage AmM-1 morphologically belongs to the family Myoviridae, its genomic structure, particularly modular genome organization, is similar to that of lambda-type phages of Siphoviridae. Genetic and phylogenetic analyses of the structural core genes also revealed that AmM-1 has a mosaic genomic structure that includes a lambda-like head (Siphoviridae) and P2-like tail (Myoviridae). The sequences of the structural core genes of AmM-1 are distinct from those of previously characterized phage groups but similar to those of recently identified one prophage element and one phage of marine Rhizobiales bacteria: a potential prophage element in the marine psychrotolerant Aureimonas ureilytica DSM 18598 genome and the temperate phage RR-1A infecting Rhizobium radiobacter P007 isolated from deep subseafloor sediment. The mosaic genome structure of AmM-1 suggests the occurrence of genetic exchange between distinct temperate phages in marine Rhizobiales populations.

  3. Genomic and Proteomic Analyses of the Terminally Redundant Genome of the Pseudomonas aeruginosa Phage PaP1: Establishment of Genus PaP1-Like Phages

    PubMed Central

    Lu, Shuguang; Le, Shuai; Tan, Yinling; Zhu, Junmin; Li, Ming; Rao, Xiancai; Zou, Lingyun; Li, Shu; Wang, Jing; Jin, Xiaolin; Huang, Guangtao; Zhang, Lin; Zhao, Xia; Hu, Fuquan

    2013-01-01

    We isolated and characterized a new Pseudomonas aeruginosa myovirus named PaP1. The morphology of this phage was visualized by electron microscopy and its genome sequence and ends were determined. Finally, genomic and proteomic analyses were performed. PaP1 has an icosahedral head with an apex diameter of 68–70 nm and a contractile tail with a length of 138–140 nm. The PaP1 genome is a linear dsDNA molecule containing 91,715 base pairs (bp) with a G+C content of 49.36% and 12 tRNA genes. A strategy to identify the genome ends of PaP1 was designed. The genome has a 1190 bp terminal redundancy. PaP1 has 157 open reading frames (ORFs). Of these, 143 proteins are homologs of known proteins, but only 38 could be functionally identified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-mass spectrometry allowed identification of 12 ORFs as structural protein coding genes within the PaP1 genome. Comparative genomic analysis indicated that the Pseudomonas aeruginosa phage PaP1, JG004, PAK_P1 and vB_PaeM_C2-10_Ab1 share great similarity. Besides their similar biological characteristics, the phages contain 123 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore propose that these four phages be classified as PaP1-like phages, a new phage genus of Myoviridae that infects Pseudomonas aeruginosa. PMID:23675441

  4. Development of phage/antibody immobilized magnetostrictive biosensors

    NASA Astrophysics Data System (ADS)

    Fu, Liling

    There is an urgent need for biosensors that are able to detect and quantify the presence of a small amount of pathogens in a real-time manner accurately and quickly to guide prevention efforts and assay food and water quality. Acoustic wave (AW) devices, whose performance is defined by mass sensitivity (Sm) and quality factor (Q value), have been extensively studied as high performance biosensor platforms. However, current AW devices still face some challenges such as the difficulty to be employed in liquid and low Q value in practical applications. The objective of this research is to develop magnetostrictive sensors which include milli/microcantilever type (MSMC) and particle type (MSP). Compared to other AW devices, MSMC exhibits the following advantages: (1) wireless/remote driving and sensing; (2) easy to fabricate; (3) works well in liquid; (4) exhibits a high Q value (> 500 in air). The fundamental study of the damping effect on MSMCs from the surrounding media including air and liquids were conducted to improve the Q value of MSMCs. The experiment results show that the Q value is dependent on the properties of surrounding media (e.g. viscosity, density), the geometry of the MSMCs, and the harmonic mode on the resonance behavior of MSMCs, etc. The phage-coated MSMC has high specificity and sensitivity even while used in water with a low concentration of targeted bacteria. Two currently developed phages, JRB7 and E2, respectively respond to Bacillus anthracis spores and Salmonella typhimurium, were employed as bio-recognition elements in this research. The phage-immobilized MSMC biosensors exhibited high performance and detection of limit was 5 x 104 cfu/ml for the MSMC in size of 1.4 x 0.8 x 0.035 mm. The MSMC-based biosensors were indicated as a very potential method for in-situ monitoring of the biological quality in water. The MSP combine antibody was used to detect Staphylococcus aureus in this experiment. The interface between MSPs and antibody was

  5. Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections

    PubMed Central

    Regeimbal, James M.; Jacobs, Anna C.; Corey, Brendan W.; Henry, Matthew S.; Thompson, Mitchell G.; Pavlicek, Rebecca L.; Quinones, Javier; Hannah, Ryan M.; Ghebremedhin, Meron; Crane, Nicole J.; Zurawski, Daniel V.; Teneza-Mora, Nimfa C.; Hall, Eric R.

    2016-01-01

    Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe. PMID:27431214

  6. Complete genome sequence analysis of two Pseudomonas plecoglossicida phages, potential therapeutic agents.

    PubMed

    Kawato, Yasuhiko; Yasuike, Motoshige; Nakamura, Yoji; Shigenobu, Yuya; Fujiwara, Atushi; Sano, Motohiko; Nakai, Toshihiro

    2015-02-01

    Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416-1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33-39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ϕIBB-PF7A and Pseudomonas putida phage ϕ15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents. PMID:25416766

  7. Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

    PubMed

    Regeimbal, James M; Jacobs, Anna C; Corey, Brendan W; Henry, Matthew S; Thompson, Mitchell G; Pavlicek, Rebecca L; Quinones, Javier; Hannah, Ryan M; Ghebremedhin, Meron; Crane, Nicole J; Zurawski, Daniel V; Teneza-Mora, Nimfa C; Biswas, Biswajit; Hall, Eric R

    2016-10-01

    Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The