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

PubMed Central

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

2010-01-01

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

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

PubMed

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

2017-06-27

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

High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum.

PubMed

Christiansen, Anders; Kringelum, Jens V; Hansen, Christian S; Bøgh, Katrine L; Sullivan, Eric; Patel, Jigar; Rigby, Neil M; Eiwegger, Thomas; Szépfalusi, Zsolt; de Masi, Federico; Nielsen, Morten; Lund, Ole; Dufva, Martin

2015-08-06

Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds.

Molecular characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages.

PubMed

Casey, Eoghan; Mahony, Jennifer; O'Connell-Motherway, Mary; Bottacini, Francesca; Cornelissen, Anneleen; Neve, Horst; Heller, Knut J; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2014-09-01

In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Comparative (Meta)genomic Analysis and Ecological Profiling of Human Gut-Specific Bacteriophage φB124-14

PubMed Central

Ogilvie, Lesley A.; Caplin, Jonathan; Dedi, Cinzia; Diston, David; Cheek, Elizabeth; Bowler, Lucas; Taylor, Huw; Ebdon, James; Jones, Brian V.

2012-01-01

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome. PMID:22558115

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

PubMed

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

2014-01-27

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

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

PubMed

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

2015-02-01

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

A Novel Bacteriophage Targeting Cronobacter sakazakii Is a Potential Biocontrol Agent in Foods

PubMed Central

Lee, Ju-Hoon; Bai, Jaewoo; Shin, Hakdong; Kim, Yeran; Park, Bookyung; Heu, Sunggi

2015-01-01

Cronobacter sakazakii is an important pathogen that causes high mortality in infants. Due to its occasional antibiotic resistance, a bacteriophage approach might be an alternative effective method for the control of this pathogen. To develop a novel biocontrol agent using bacteriophages, the C. sakazakii-infecting phage CR5 was newly isolated and characterized. Interestingly, this phage exhibited efficient and relatively durable host lysis activity. In addition, a specific gene knockout study and subsequent complementation experiment revealed that this phage infected the host strain using the bacterial flagella. The complete genome sequence analysis of phage CR5 showed that its genome contains 223,989 bp of DNA, including 231 predicted open reading frames (ORFs), and it has a G+C content of 50.06%. The annotated ORFs were classified into six functional groups (structure, packaging, host lysis, DNA manipulation, transcription, and additional functions); no gene was found to be related to virulence or toxin or lysogen formation, but >80% of the predicted ORFs are unknown. In addition, a phage proteomic analysis using SDS-PAGE and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) revealed that seven phage structural proteins are indeed present, supporting the ORF predictions. To verify the potential of this phage as a biocontrol agent against C. sakazakii, it was added to infant formula milk contaminated with a C. sakazakii clinical isolate or food isolate, revealing complete growth inhibition of the isolates by the addition of phage CR5 when the multiplicity of infection (MOI) was 105. PMID:26497465

Energy-efficient growth of phage Q Beta in Escherichia coli.

PubMed

Kim, Hwijin; Yin, John

2004-10-20

The role of natural selection in the optimal design of organisms is controversial. Optimal forms, functions, or behaviors of organisms have long been claimed without knowledge of how genotype contributes to phenotype, delineation of design constraints, or reference to alternative designs. Moreover, arguments for optimal designs have been often based on models that were difficult, if not impossible, to test. Here, we begin to address these issues by developing and probing a kinetic model for the intracellular growth of bacteriophage Q beta in Escherichia coli. The model accounts for the energetic costs of all template-dependent polymerization reactions, in ATP equivalents, including RNA-dependent RNA elongation by the phage replicase and synthesis of all phage proteins by the translation machinery of the E. coli host cell. We found that translation dominated phage growth, requiring 85% of the total energy expenditure. Only 10% of the total energy was applied to activities other than the direct synthesis of progeny phage components, reflecting primarily the cost of making the negative-strand RNA template that is needed for replication of phage genomic RNA. Further, we defined an energy efficiency of phage growth and showed its direct relationship to the yield of phage progeny. Finally, we performed a sensitivity analysis and found that the growth of wild-type phage was optimized for progeny yield or energy efficiency, suggesting that phage Q beta has evolved to optimally utilize the finite resources of its host cells.

Genomic analysis of Staphylococcus phage Stau2 isolated from medical specimen.

PubMed

Hsieh, Sue-Er; Tseng, Yi-Hsiung; Lo, Hsueh-Hsia; Chen, Shui-Tu; Wu, Cheng-Nan

2016-02-01

Stau2 is a lytic myophage of Staphylococcus aureus isolated from medical specimen. Exhibiting a broad host range against S. aureus clinical isolates, Stau2 is potentially useful for topical phage therapy or as an additive in food preservation. In this study, Stau2 was firstly revealed to possess a circularly permuted linear genome of 133,798 bp, with low G + C content, containing 146 open reading frames, but encoding no tRNA. The genome is organized into several modules containing genes for packaging, structural proteins, replication/transcription and host-cell-lysis, with the structural proteins and DNA polymerase modules being organized similarly to that in Twort-like phages of Staphylococcus. With the encoded DNA replication genes, Stau2 can possibly use its own system for replication. In addition, analysis in silico found several introns in seven genes, including those involved in DNA metabolism, packaging, and structure, while one of them (helicase gene) is experimentally confirmed to undergo splicing. Furthermore, phylogenetic analysis suggested Stau2 to be most closely related to Staphylococcus phages SA11 and Remus, members of Twort-like phages. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 14 structural proteins of Stau2 and N-terminal sequencing identified three of them. Importantly, this phage does not encode any proteins which are known or suspected to be involved in toxicity, pathogenicity, or antibiotic resistance. Therefore, further investigations of feasible therapeutic application of Stau2 are needed.

Advance in phage display technology for bioanalysis.

PubMed

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

2016-06-01

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

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

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

PubMed Central

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

2017-01-01

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

Genome Sequences of Three Cluster AU Arthrobacter Phages, Caterpillar, Nightmare, and Teacup

PubMed Central

Adair, Tamarah L.; Stowe, Emily; Pizzorno, Marie C.; Krukonis, Gregory; Harrison, Melinda; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah

2017-01-01

ABSTRACT Caterpillar, Nightmare, and Teacup are cluster AU siphoviral phages isolated from enriched soil on Arthrobacter sp. strain ATCC 21022. These genomes are 58 kbp long with an average G+C content of 50%. Sequence analysis predicts 86 to 92 protein-coding genes, including a large number of small proteins with predicted transmembrane domains. PMID:29122860

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

PubMed Central

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

2014-01-01

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

Characterization of Sinorhizobium sp. LM21 Prophages and Virus-Encoded DNA Methyltransferases in the Light of Comparative Genomic Analyses of the Sinorhizobial Virome

PubMed Central

Decewicz, Przemyslaw; Radlinska, Monika; Dziewit, Lukasz

2017-01-01

The genus Sinorhizobium/Ensifer mostly groups nitrogen-fixing bacteria that create root or stem nodules on leguminous plants and transform atmospheric nitrogen into ammonia, which improves the productivity of the plants. Although these biotechnologically-important bacteria are commonly found in various soil environments, little is known about their phages. In this study, the genome of Sinorhizobium sp. LM21 isolated from a heavy-metal-contaminated copper mine in Poland was investigated for the presence of prophages and DNA methyltransferase-encoding genes. In addition to the previously identified temperate phage, ΦLM21, and the phage-plasmid, pLM21S1, the analysis revealed the presence of three prophage regions. Moreover, four novel phage-encoded DNA methyltransferase (MTase) genes were identified and the enzymes were characterized. It was shown that two of the identified viral MTases methylated the same target sequence (GANTC) as cell cycle-regulated methyltransferase (CcrM) of the bacterial host strain, LM21. This discovery was recognized as an example of the evolutionary convergence between enzymes of sinorhizobial viruses and their host, which may play an important role in virus cycle. In the last part of the study, thorough comparative analyses of 31 sinorhizobial (pro)phages (including active sinorhizobial phages and novel putative prophages retrieved and manually re-annotated from Sinorhizobium spp. genomes) were performed. The networking analysis revealed the presence of highly conserved proteins (e.g., holins and endolysins) and a high diversity of viral integrases. The analysis also revealed a large number of viral DNA MTases, whose genes were frequently located within the predicted replication modules of analyzed prophages, which may suggest their important regulatory role. Summarizing, complex analysis of the phage protein similarity network enabled a new insight into overall sinorhizobial virome diversity. PMID:28672885

T4-Like Genome Organization of the Escherichia coli O157:H7 Lytic Phage AR1▿†

PubMed Central

Liao, Wei-Chao; Ng, Wailap Victor; Lin, I-Hsuan; Syu, Wan-Jr; Liu, Tze-Tze; Chang, Chuan-Hsiung

2011-01-01

We report the genome organization and analysis of the first completely sequenced T4-like phage, AR1, of Escherichia coli O157:H7. Unlike most of the other sequenced phages of O157:H7, which belong to the temperate Podoviridae and Siphoviridae families, AR1 is a T4-like phage known to efficiently infect this pathogenic bacterial strain. The 167,435-bp AR1 genome is currently the largest among all the sequenced E. coli O157:H7 phages. It carries a total of 281 potential open reading frames (ORFs) and 10 putative tRNA genes. Of these, 126 predicted proteins could be classified into six viral orthologous group categories, with at least 18 proteins of the structural protein category having been detected by tandem mass spectrometry. Comparative genomic analysis of AR1 and four other completely sequenced T4-like genomes (RB32, RB69, T4, and JS98) indicated that they share a well-organized and highly conserved core genome, particularly in the regions encoding DNA replication and virion structural proteins. The major diverse features between these phages include the modules of distal tail fibers and the types and numbers of internal proteins, tRNA genes, and mobile elements. Codon usage analysis suggested that the presence of AR1-encoded tRNAs may be relevant to the codon usage of structural proteins. Furthermore, protein sequence analysis of AR1 gp37, a potential receptor binding protein, indicated that eight residues in the C terminus are unique to O157:H7 T4-like phages AR1 and PP01. These residues are known to be located in the T4 receptor recognition domain, and they may contribute to specificity for adsorption to the O157:H7 strain. PMID:21507986

Bacteriophages BCP1-1 and BCP8-2 require divalent cations for efficient control of Bacillus cereus in fermented foods.

PubMed

Bandara, Nadeeka; Jo, Junhee; Ryu, Sangryeol; Kim, Kwang-Pyo

2012-08-01

Bacillus cereus is a foodborne bacterial pathogen that causes diarrhea and vomiting. In this study, the usefulness of bacteriophages to eradicate B. cereus from fermented foods was investigated. A total of 13 phages were isolated from Korean fermented food products, and 2 (BCP1-1 and BCP8-2) were further characterized. Transmission electron microscopy (TEM), restriction enzyme digestion pattern analysis, and SDS-PAGE of the structural proteins suggest that both phages belong to the family Myoviridae, containing approximately 150 kbp-long genomes. The host ranges of both phages were limited to B. cereus group species (12/13), as they were not able to lyse other Gram-positive or negative strains including Bacillus subtilis. Purified phages were used to inhibit B. cereus growth in a model fermented food system, cheonggukjang, a fast-fermented soybean paste product. BCP1-1 and BCP8-2 were able to effectively eradicate B. cereus from the food only if divalent cations (Ca²⁺, Mg²⁺, or Mn²⁺) were added to the medium. Further studies reveal that divalent cations are essential for phage adsorption, while a monovalent cation (Na⁺) is required for the post-adsorption phase of phage infection. Taken together, our findings imply that a phage could be an ideal anti-bacterial agent for use in fermented food products that require the presence of beneficial microflora and, during phage application, optimization of phage reaction conditions is critical for the successful utilization of phage biocontrol. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

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

1997-02-01

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

Targeting Enterococcus faecalis Biofilms with Phage Therapy

PubMed Central

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

2015-01-01

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. PMID:25662974

Phylogenetic and Complementation Analysis of a Single-Stranded DNA Binding Protein Family from Lactococcal Phages Indicates a Non-Bacterial Origin

PubMed Central

Mariadassou, Mahendra; Bardowski, Jacek K.; Bidnenko, Elena

2011-01-01

Background The single-stranded-nucleic acid binding (SSB) protein superfamily includes proteins encoded by different organisms from Bacteria and their phages to Eukaryotes. SSB proteins share common structural characteristics and have been suggested to descend from an ancestor polypeptide. However, as other proteins involved in DNA replication, bacterial SSB proteins are clearly different from those found in Archaea and Eukaryotes. It was proposed that the corresponding genes in the phage genomes were transferred from the bacterial hosts. Recently new SSB proteins encoded by the virulent lactococcal bacteriophages (Orf14bIL67-like proteins) have been identified and characterized structurally and biochemically. Methodology/Principal Findings This study focused on the determination of phylogenetic relationships between Orf14bIL67-like proteins and other SSBs. We have performed a large scale phylogenetic analysis and pairwise sequence comparisons of SSB proteins from different phyla. The results show that, in remarkable contrast to other phage SSBs, the Orf14bIL67–like proteins form a distinct, self-contained and well supported phylogenetic group connected to the archaeal SSBs. Functional studies demonstrated that, despite the structural and amino acid sequence differences from bacterial SSBs, Orf14bIL67 protein complements the conditional lethal ssb-1 mutation of Escherichia coli. Conclusions/Significance Here we identified for the first time a group of phages encoded SSBs which are clearly distinct from their bacterial counterparts. All methods supported the recognition of these phage proteins as a new family within the SSB superfamily. Our findings suggest that unlike other phages, the virulent lactococcal phages carry ssb genes that were not acquired from their hosts, but transferred from an archaeal genome. This represents a unique example of a horizontal gene transfer between Archaea and bacterial phages. PMID:22073223

The Effect of Bacteriophage Preparations on Intracellular Killing of Bacteria by Phagocytes

PubMed Central

Jończyk-Matysiak, Ewa; Łusiak-Szelachowska, Marzanna; Kłak, Marlena; Bubak, Barbara; Międzybrodzki, Ryszard; Weber-Dąbrowska, Beata; Żaczek, Maciej; Fortuna, Wojciech; Rogóż, Paweł; Letkiewicz, Sławomir; Szufnarowski, Krzysztof; Górski, Andrzej

2015-01-01

Intracellular killing of bacteria is one of the fundamental mechanisms against invading pathogens. Impaired intracellular killing of bacteria by phagocytes may be the reason of chronic infections and may be caused by antibiotics or substances that can be produced by some bacteria. Therefore, it was of great practical importance to examine whether phage preparations may influence the process of phagocyte intracellular killing of bacteria. It may be important especially in the case of patients qualified for experimental phage therapy (approximately half of the patients with chronic bacterial infections have their immunity impaired). Our analysis included 51 patients with chronic Gram-negative and Gram-positive bacterial infections treated with phage preparations at the Phage Therapy Unit in Wroclaw. The aim of the study was to investigate the effect of experimental phage therapy on intracellular killing of bacteria by patients' peripheral blood monocytes and polymorphonuclear neutrophils. We observed that phage therapy does not reduce patients' phagocytes' ability to kill bacteria, and it does not affect the activity of phagocytes in patients with initially reduced ability to kill bacteria intracellularly. Our results suggest that experimental phage therapy has no significant adverse effects on the bactericidal properties of phagocytes, which confirms the safety of the therapy. PMID:26783541

Broad-range lytic bacteriophages that kill Staphylococcus aureus local field strains

PubMed Central

Boncompain, Carina A.; Amadio, Ariel A.; Carrasco, Soledad; Suárez, Cristian A.

2017-01-01

Staphylococcus aureus is a very successful opportunistic pathogen capable of causing a variety of diseases ranging from mild skin infections to life-threatening sepsis, meningitis and pneumonia. Its ability to display numerous virulence mechanisms matches its skill to display resistance to several antibiotics, including β-lactams, underscoring the fact that new anti-S. aureus drugs are urgently required. In this scenario, the utilization of lytic bacteriophages that kill bacteria in a genus -or even species- specific way, has become an attractive field of study. In this report, we describe the isolation, characterization and sequencing of phages capable of killing S. aureus including methicillin resistant (MRSA) and multi-drug resistant S. aureus local strains from environmental, animal and human origin. Genome sequencing and bio-informatics analysis showed the absence of genes encoding virulence factors, toxins or antibiotic resistance determinants. Of note, there was a high similarity between our set of phages to others described in the literature such as phage K. Considering that reported phages were obtained in different continents, it seems plausible that there is a commonality of genetic features that are needed for optimum, broad host range anti-staphylococcal activity of these related phages. Importantly, the high activity and broad host range of one of our phages underscores its promising value to control the presence of S. aureus in fomites, industry and hospital environments and eventually on animal and human skin. The development of a cocktail of the reported lytic phages active against S. aureus–currently under way- is thus, a sensible strategy against this pathogen. PMID:28742812

Broad-range lytic bacteriophages that kill Staphylococcus aureus local field strains.

PubMed

Abatángelo, Virginia; Peressutti Bacci, Natalia; Boncompain, Carina A; Amadio, Ariel F; Carrasco, Soledad; Suárez, Cristian A; Morbidoni, Héctor R

2017-01-01

Staphylococcus aureus is a very successful opportunistic pathogen capable of causing a variety of diseases ranging from mild skin infections to life-threatening sepsis, meningitis and pneumonia. Its ability to display numerous virulence mechanisms matches its skill to display resistance to several antibiotics, including β-lactams, underscoring the fact that new anti-S. aureus drugs are urgently required. In this scenario, the utilization of lytic bacteriophages that kill bacteria in a genus -or even species- specific way, has become an attractive field of study. In this report, we describe the isolation, characterization and sequencing of phages capable of killing S. aureus including methicillin resistant (MRSA) and multi-drug resistant S. aureus local strains from environmental, animal and human origin. Genome sequencing and bio-informatics analysis showed the absence of genes encoding virulence factors, toxins or antibiotic resistance determinants. Of note, there was a high similarity between our set of phages to others described in the literature such as phage K. Considering that reported phages were obtained in different continents, it seems plausible that there is a commonality of genetic features that are needed for optimum, broad host range anti-staphylococcal activity of these related phages. Importantly, the high activity and broad host range of one of our phages underscores its promising value to control the presence of S. aureus in fomites, industry and hospital environments and eventually on animal and human skin. The development of a cocktail of the reported lytic phages active against S. aureus-currently under way- is thus, a sensible strategy against this pathogen.

Infective and inactivated filamentous phage as carriers for immunogenic peptides.

PubMed

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

2012-07-01

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

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

PubMed Central

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

2015-01-01

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

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

DOE PAGES

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

2015-05-08

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

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

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

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

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

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

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2017-08-15

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

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

PubMed

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

2017-03-01

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

Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group

PubMed Central

2013-01-01

Background Comparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail. Results Here we present data on the large, virulent, broad-host-range B. cereus phage vB_BceM_Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1. Conclusions Bc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G. PMID:23388049

Advances in the T7 phage display system (Review).

PubMed

Deng, Xiangying; Wang, Li; You, Xiaolong; Dai, Pei; Zeng, Yanhua

2018-01-01

The present review describes the advantages and updated applications of the T7 phage display system in bioscience and medical science. Current phage display systems are based on various bacteriophage vectors, including M13, T7, T4 and f1. Of these, the M13 phage display is the most frequently used, however, the present review highlights the advantages of the T7 system. As a phage display platform, M13 contains single‑stranded DNA, while the T7 phage consists of double‑stranded DNA, which exhibits increased stability and is less prone to mutation during replication. Additional characteristics of the T7 phage include the following: The T7 phage does not depend on a protein secretion pathway in the lytic cycle; expressed peptides and proteins are usually located on the C‑terminal region of capsid protein gp10B, which avoids problems associated with steric hindrance; and T7 phage particles exhibit high stability under various extreme conditions, including high temperature and low pH, which facilitates effective high‑throughput affinity elutriation. Recent applications of the T7 phage display system have been instrumental in uncovering mechanisms of molecular interaction, particularly in the fields of antigen discovery, vaccine development, protein interaction, and cancer diagnosis and treatment.

Advanced Analysis to Distinguish between Physical Decrease and Inactivation of Viable Phages in Aerosol by Quantitating Phage-Specific Particles.

PubMed

Shimasaki, Noriko; Nojima, Yasuhiro; Sakakibara, Masaya; Kikuno, Ritsuko; Iizuka, Chiori; Okaue, Akira; Okuda, Shunji; Shinohara, Katsuaki

2018-01-01

　Recent studies have investigated the efficacy of air-cleaning products against pathogens in the air. A standard method to evaluate the reduction in airborne viruses caused by an air cleaner has been established using a safe bacteriophage instead of pathogenic viruses; the reduction in airborne viruses is determined by counting the number of viable airborne phages by culture, after operating the air cleaner. The reduction in the number of viable airborne phages could be because of "physical decrease" or "inactivation". Therefore, to understand the mechanism of reduction correctly, an analysis is required to distinguish between physical decrease and inactivation. The purpose of this study was to design an analysis to distinguish between the physical decrease and inactivation of viable phi-X174 phages in aerosols. We established a suitable polymerase chain reaction (PCR) system by selecting an appropriate primer-probe set for PCR and validating the sensitivity, linearity, and specificity of the primer-probe set to robustly quantify phi-X174-specific airborne particles. Using this quantitative PCR system and culture assay, we performed a behavior analysis of the phage aerosol in a small chamber (1 m 3 ) at different levels of humidity, as humidity is known to affect the number of viable airborne phages. The results revealed that the reduction in the number of viable airborne phages was caused not only by physical decrease but also by inactivation under particular levels of humidity. Our study could provide an advanced analysis to differentiate between the physical decrease and inactivation of viable airborne phages.

Genomic diversity of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum.

PubMed

Castillo, Daniel; Middelboe, Mathias

2016-12-01

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

A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins.

PubMed

Velappan, Nileena; Fisher, Hugh E; Pesavento, Emanuele; Chasteen, Leslie; D'Angelo, Sara; Kiss, Csaba; Longmire, Michelle; Pavlik, Peter; Bradbury, Andrew R M

2010-03-01

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments.

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

PubMed

Tanouchi, Yu; Covert, Markus W

2017-09-19

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-10-01

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

Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

PubMed

Ozaki, Tatsuro; Abe, Naoki; Kimura, Keitarou; Suzuki, Atsuto; Kaneko, Jun

2017-01-01

Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

The scope of phage display for membrane proteins.

PubMed

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

2011-12-09

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

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

PubMed

Colavecchio, Anna; Goodridge, Lawrence D

2017-06-01

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

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

PubMed

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

2015-01-01

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

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

PubMed Central

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

2017-01-01

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

T7 lytic phage-displayed peptide libraries: construction and diversity characterization.

PubMed

Krumpe, Lauren R H; Mori, Toshiyuki

2014-01-01

In this chapter, we describe the construction of T7 bacteriophage (phage)-displayed peptide libraries and the diversity analyses of random amino acid sequences obtained from the libraries. We used commercially available reagents, Novagen's T7Select system, to construct the libraries. Using a combination of biotinylated extension primer and streptavidin-coupled magnetic beads, we were able to prepare library DNA without applying gel purification, resulting in extremely high ligation efficiencies. Further, we describe the use of bioinformatics tools to characterize library diversity. Amino acid frequency and positional amino acid diversity and hydropathy are estimated using the REceptor LIgand Contacts website http://relic.bio.anl.gov. Peptide net charge analysis and peptide hydropathy analysis are conducted using the Genetics Computer Group Wisconsin Package computational tools. A comprehensive collection of the estimated number of recombinants and titers of T7 phage-displayed peptide libraries constructed in our lab is included.

Engineered phages for electronics.

PubMed

Cui, Yue

2016-11-15

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

Evidence of Geobacter-associated phage in a uranium-contaminated aquifer

PubMed Central

Holmes, Dawn E; Giloteaux, Ludovic; Chaurasia, Akhilesh K; Williams, Kenneth H; Luef, Birgit; Wilkins, Michael J; Wrighton, Kelly C; Thompson, Courtney A; Comolli, Luis R; Lovley, Derek R

2015-01-01

Geobacter species may be important agents in the bioremediation of organic and metal contaminants in the subsurface, but as yet unknown factors limit the in situ growth of subsurface Geobacter well below rates predicted by analysis of gene expression or in silico metabolic modeling. Analysis of the genomes of five different Geobacter species recovered from contaminated subsurface sites indicated that each of the isolates had been infected with phage. Geobacter-associated phage sequences were also detected by metagenomic and proteomic analysis of samples from a uranium-contaminated aquifer undergoing in situ bioremediation, and phage particles were detected by microscopic analysis in groundwater collected from sediment enrichment cultures. Transcript abundance for genes from the Geobacter-associated phage structural proteins, tail tube Gp19 and baseplate J, increased in the groundwater in response to the growth of Geobacter species when acetate was added, and then declined as the number of Geobacter decreased. Western blot analysis of a Geobacter-associated tail tube protein Gp19 in the groundwater demonstrated that its abundance tracked with the abundance of Geobacter species. These results suggest that the enhanced growth of Geobacter species in the subsurface associated with in situ uranium bioremediation increased the abundance and activity of Geobacter-associated phage and show that future studies should focus on how these phages might be influencing the ecology of this site. PMID:25083935

Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

PubMed

Urban-Chmiel, Renata; Wernicki, Andrzej; Stęgierska, Diana; Dec, Marta; Dudzic, Anna; Puchalski, Andrzej

2015-01-01

The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves. The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR). Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101. The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments. Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.

Genomic Organization and Molecular Analysis of Virulent Bacteriophage 2972 Infecting an Exopolysaccharide-Producing Streptococcus thermophilus Strain

PubMed Central

Lévesque, Céline; Duplessis, Martin; Labonté, Jessica; Labrie, Steve; Fremaux, Christophe; Tremblay, Denise; Moineau, Sylvain

2005-01-01

The Streptococcus thermophilus virulent pac-type phage 2972 was isolated from a yogurt made in France in 1999. It is a representative of several phages that have emerged with the industrial use of the exopolysaccharide-producing S. thermophilus strain RD534. The genome of phage 2972 has 34,704 bp with an overall G+C content of 40.15%, making it the shortest S. thermophilus phage genome analyzed so far. Forty-four open reading frames (ORFs) encoding putative proteins of 40 or more amino acids were identified, and bioinformatic analyses led to the assignment of putative functions to 23 ORFs. Comparative genomic analysis of phage 2972 with the six other sequenced S. thermophilus phage genomes confirmed that the replication module is conserved and that cos- and pac-type phages have distinct structural and packaging genes. Two group I introns were identified in the genome of 2972. They interrupted the genes coding for the putative endolysin and the terminase large subunit. Phage mRNA splicing was demonstrated for both introns, and the secondary structures were predicted. Eight structural proteins were also identified by N-terminal sequencing and/or matrix-assisted laser desorption ionization—time-of-flight mass spectrometry. Detailed analysis of the putative minor tail proteins ORF19 and ORF21 as well as the putative receptor-binding protein ORF20 showed the following interesting features: (i) ORF19 is a hybrid protein, because it displays significant identity with both pac- and cos-type phages; (ii) ORF20 is unique; and (iii) a protein similar to ORF21 of 2972 was also found in the structure of the cos-type phage DT1, indicating that this structural protein is present in both S. thermophilus phage groups. The implications of these findings for phage classification are discussed. PMID:16000821

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

PubMed

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

2016-08-01

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

Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity

PubMed Central

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; Abbazia, Patrick; Ababio, Amma; Adam, Naazneen

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. DOI: http://dx.doi.org/10.7554/eLife.06416.001 PMID:25919952

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.

Phage phenomics: Physiological approaches to characterize novel viral proteins

ScienceCinema

Sanchez, Savannah E. [San Diego State Univ., San Diego, CA (United States); Cuevas, Daniel A. [San Diego State Univ., San Diego, CA (United States); Rostron, Jason E. [San Diego State Univ., San Diego, CA (United States); Liang, Tiffany Y. [San Diego State Univ., San Diego, CA (United States); Pivaroff, Cullen G. [San Diego State Univ., San Diego, CA (United States); Haynes, Matthew R. [San Diego State Univ., San Diego, CA (United States); Nulton, Jim [San Diego State Univ., San Diego, CA (United States); Felts, Ben [San Diego State Univ., San Diego, CA (United States); Bailey, Barbara A. [San Diego State Univ., San Diego, CA (United States); Salamon, Peter [San Diego State Univ., San Diego, CA (United States); Edwards, Robert A. [San Diego State Univ., San Diego, CA (United States); Argonne National Lab. (ANL), Argonne, IL (United States); Burgin, Alex B. [Broad Institute, Cambridge, MA (United States); Segall, Anca M. [San Diego State Univ., San Diego, CA (United States); Rohwer, Forest [San Diego State Univ., San Diego, CA (United States)

2018-06-21

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.

wksl3, a New Biocontrol Agent for Salmonella enterica Serovars Enteritidis and Typhimurium in Foods: Characterization, Application, Sequence Analysis, and Oral Acute Toxicity Study

PubMed Central

Kang, Hyun-Wol; Kim, Jae-Won; Jung, Tae-Sung

2013-01-01

Of the Salmonella enterica serovars, S. Enteritidis and S. Typhimurium are responsible for most of the Salmonella outbreaks implicated in the consumption of contaminated foods in the Republic of Korea. Because of the widespread occurrence of antimicrobial-resistant Salmonella in foods and food processing environments, bacteriophages have recently surfaced as an alternative biocontrol tool. In this study, we isolated a virulent bacteriophage (wksl3) that could specifically infect S. Enteritidis, S. Typhimurium, and several additional serovars. Transmission electron microscopy revealed that phage wksl3 belongs to the family Siphoviridae. Complete genome sequence analysis and bioinformatic analysis revealed that the DNA of phage wksl3 is composed of 42,766 bp with 64 open reading frames. Since it does not encode any phage lysogeny factors, toxins, pathogen-related genes, or food-borne allergens, phage wksl3 may be considered a virulent phage with no side effects. Analysis of genetic similarities between phage wksl3 and four of its relatives (SS3e, vB_SenS-Ent1, SE2, and SETP3) allowed wksl3 to be categorized as a SETP3-like phage. A single-dose test of oral toxicity with BALB/c mice resulted in no abnormal clinical observations. Moreover, phage application to chicken skin at 8°C resulted in an about 2.5-log reduction in the number of Salmonella bacteria during the test period. The strong, stable lytic activity, the significant reduction of the number of S. Enteritidis bacteria after application to food, and the lack of clinical symptoms of this phage suggest that wksl3 may be a useful agent for the protection of foods against S. Enteritidis and S. Typhimurium contamination. PMID:23335772

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.

A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species.

PubMed

Dinsmore, P K; O'Sullivan, D J; Klaenhammer, T R

1998-05-28

The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species. Five heptad leucine repeats suggestive of a leucine zipper motif were identified between residues 232 and 266 in the predicted amino acid sequence of the AbiA protein. The biological role of residues in the repeats was investigated by incorporating amino acid substitutions via site-directed mutagenesis. Each mutant was tested for phage resistance against three phages, phi 31, sk1, and c2, belonging to species P335, 936, and c2, respectively. The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263. Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested. Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested. Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity. Replacement of L242 with an alanine elimination phage resistance against phi 31, but partial resistance to sk1 and c2 remained. Two single proline substitutions for leucines L242 and L263 virtually eliminated AbiA activity against all phages, indicating that the predicted alpha-helical structure of this region is important. Mutations in an adjacent region of basic amino acids had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity. This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.

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

Characterization of Two Virulent Phages of Lactobacillus plantarum

PubMed Central

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

2012-01-01

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

Characterization of Enterococcus faecium bacteriophage IME-EFm5 and its endolysin LysEFm5.

PubMed

Gong, Pengjuan; Cheng, Mengjun; Li, Xinwei; Jiang, Haiyan; Yu, Chuang; Kahaer, Nadire; Li, Juecheng; Zhang, Lei; Xia, Feifei; Hu, Liyuan; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Han, Wenyu; Gu, Jingmin

2016-05-01

Due to the worldwide prevalence of antibiotic resistant strains, phages therapy has been revitalized recently. In this study, an Enterococcus faecium phage named IME-EFm5 was isolated from hospital sewage. Whole genomic sequence analysis demonstrated that IME-EFm5 belong to the Siphoviridae family, and has a double-stranded genome of 42,265bp (with a 35.51% G+C content) which contains 70 putative coding sequences. LysEFm5, the endolysin of IME-EFm5, contains an amidase domain in its N-terminal and has a wider bactericidal spectrum than its parental phage IME-EFm5, including 7 strains of vancomycin-resistant E. faecium. The mutagenesis analysis revealed that the zinc ion binding residues (H27, H132, and C140), E90, and T138 are required for the catalysis of LysEFm5. However, the antibacterial activity of LysEFm5 is zinc ion independent, which is inconsistent with most of other amidase members. The phage lysin LysEFm5 might be an alternative treatment strategy for infections caused by multidrug-resistant E. faecium. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed Central

2013-01-01

Background Shigella flexneri is the major cause of shigellosis in the developing countries. The O-antigen component of the lipopolysaccharide is one of the key virulence determinants required for the pathogenesis of S. flexneri. The glucosyltransferase and/or acetyltransferase genes responsible for the modification of the O-antigen are encoded by temperate serotype converting bacteriophage present in the S. flexneri genome. Several serotype converting phages have previously been isolated and characterized, however, attempts to isolate a serotype converting phage which encodes the modification genes of serotypes 4a strain have not been successful. Results In this study, a novel temperate serotype converting bacteriophage SfIV was isolated. Lysogenisation of phage SfIV converted serotype Y strain to serotype 4a. Electron microscopy indicated that SfIV belongs to Myoviridae family. The 39,758 bp genome of phage SfIV encompasses 54 open reading frames (orfs). Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. The comparative analysis also revealed that SfIV phage contained five proteins which were not found in any other phages of S. flexneri. These proteins were: a tail fiber assembly protein, two hypothetical proteins with no clear function, and two other unknown proteins which were encoded by orfs present on a moron, that presumably got introduced in SfIV genome from another species via a transposon. These unique proteins of SfIV may play a role in the pathogenesis of the host. Conclusions This study reports the isolation and complete genome sequence analysis of bacteriophage SfIV. The SfIV phage has a host range significantly different from the other phages of Shigella. Comparative genome analysis identified several proteins unique to SfIV, which may potentially be involved in the survival and pathogenesis of its host. These findings will further our understanding on the evolution of these phages, and will also facilitate studies on development of new phage vectors and therapeutic agents to control infections caused by S. flexneri. PMID:24090466

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

PubMed

Samson, Julie E; Moineau, Sylvain

2013-01-01

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

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

PubMed

Bai, Qinqin; Yang, Yongchun; Lu, Chengping

2016-02-04

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

Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

PubMed

Seo, Moon-Hyeong; Nim, Satra; Jeon, Jouhyun; Kim, Philip M

2017-01-01

Protein-protein interactions are essential to cellular functions and signaling pathways. We recently combined bioinformatics and custom oligonucleotide arrays to construct custom-made peptide-phage libraries for screening peptide-protein interactions, an approach we call proteomic peptide-phage display (ProP-PD). In this chapter, we describe protocols for phage display for the identification of natural peptide binders for a given protein. We finally describe deep sequencing for the analysis of the proteomic peptide-phage display.

Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases

PubMed Central

De Paepe, Marianne; Hutinet, Geoffrey; Son, Olivier; Amarir-Bouhram, Jihane; Schbath, Sophie; Petit, Marie-Agnès

2014-01-01

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself. PMID:24603854

Sequencing, genome analysis and host range of a novel Ralstonia phage RsoP1EGY isolated from Egypt

USDA-ARS?s Scientific Manuscript database

A novel Ralstonia phage was isolated from soil in Egypt and designated RsPod1SoilEGY using our new four-part phage identifier naming system. When tested, this phage selectively infected only race 3 biovar 2 phylotype IIB sequevar 1, and not non-race 3 biovar 2 strains of Ralstonia solanacearum. The ...

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

PubMed Central

Forsman, Päivi; Alatossava, Tapani

1991-01-01

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

Comparative genome analysis of novel Podoviruses lytic for hypermucoviscous Klebsiella pneumoniae of K1, K2, and K57 capsular types.

PubMed

Solovieva, Ekaterina V; Myakinina, Vera P; Kislichkina, Angelina A; Krasilnikova, Valentina M; Verevkin, Vladimir V; Mochalov, Vladimir V; Lev, Anastasia I; Fursova, Nadezhda K; Volozhantsev, Nikolay V

2018-01-02

Hypermucoviscous (HV) strains of capsular types K1, K2 and K57 are the most virulent representatives of the Klebsiella pneumoniae species. Eight novel bacteriophages lytic for HV K. pneumoniae were isolated and characterized. Three bacteriophages, KpV41, KpV475, and KpV71 were found to have a lytic activity against mainly K. pneumoniae of capsular type K1. Two phages, KpV74, and KpV763 were lytic for K2 capsular type K. pneumoniae, and the phage KpV767 was specific to K57-type K. pneumoniae only. Two more phages, KpV766, and KpV48 had no capsular specificity. The phage genomes consist of a linear double-stranded DNA of 40,395-44,623bp including direct terminal repeats of 180-246 bp. The G + C contents are 52.3-54.2 % that is slightly lower than that of genomes of K. pneumoniae strains being used for phage propagation. According to the genome structures, sequence similarity and phylogenetic data, the phages are classified within the genus Kp32virus and Kp34virus of subfamily Autographivirinae, family Podoviridae. In the phage genomes, genes encoding proteins with putative motifs of polysaccharide depolymerase were identified. Depolymerase genes of phages KpV71 and KpV74 lytic for hypermucoviscous K. pneumoniae of K1 and K2 capsular type, respectively, were cloned and expressed in Escherichia coli, and the recombinant gene products were purified. The specificity and polysaccharide-degrading activity of the recombinant depolymerases were demonstrated. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Genomic, Proteomic and Morphological Characterization of Two Novel Broad Host Lytic Bacteriophages ΦPD10.3 and ΦPD23.1 Infecting Pectinolytic Pectobacterium spp. and Dickeya spp.

PubMed Central

Czajkowski, Robert; Ozymko, Zofia; de Jager, Victor; Siwinska, Joanna; Smolarska, Anna; Ossowicki, Adam; Narajczyk, Magdalena; Lojkowska, Ewa

2015-01-01

Pectinolytic Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops, including potato, worldwide. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc), P. wasabiae (Pwa) and Dickeya solani (Dso) with the objective to assess their potential as biological disease control agents. Two lytic bacteriophages infecting stains of Pcc, Pwa and Dso were isolated from potato samples collected from two potato fields in central Poland. The ΦPD10.3 and ΦPD23.1 phages have morphology similar to other members of the Myoviridae family and the Caudovirales order, with a head diameter of 85 and 86 nm and length of tails of 117 and 121 nm, respectively. They were characterized for optimal multiplicity of infection, the rate of adsorption to the Pcc, Pwa and Dso cells, the latent period and the burst size. The phages were genotypically characterized with RAPD-PCR and RFLP techniques. The structural proteomes of both phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identification was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Pulsed-field gel electrophoresis (PFGE), genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the ΦPD10.3 and ΦPD23.1 genomes. The potential use of ΦPD10.3 and ΦPD23.1 phages for the biocontrol of Pectobacterium spp. and Dickeya spp. infections in potato is discussed. PMID:25803051

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

PubMed Central

Gagic, Dragana; Ciric, Milica; Wen, Wesley X.; Ng, Filomena; Rakonjac, Jasna

2016-01-01

Microbial surface and secreted proteins (the secretome) contain a large number of proteins that interact with other microbes, host and/or environment. These proteins are exported by the coordinated activities of the protein secretion machinery present in the cell. A group of bacteriophage, called filamentous phage, have the ability to hijack bacterial protein secretion machinery in order to amplify and assemble via a secretion-like process. This ability has been harnessed in the use of filamentous phage of Escherichia coli in biotechnology applications, including screening large libraries of variants for binding to “bait” of interest, from tissues in vivo to pure proteins or even inorganic substrates. In this review we discuss the roles of secretome proteins in pathogenic and non-pathogenic bacteria and corresponding secretion pathways. We describe the basics of phage display technology and its variants applied to discovery of bacterial proteins that are implicated in colonization of host tissues and pathogenesis, as well as vaccine candidates through filamentous phage display library screening. Secretome selection aided by next-generation sequence analysis was successfully applied for selective display of the secretome at a microbial community scale, the latter revealing the richness of secretome functions of interest and surprising versatility in filamentous phage display of secretome proteins from large number of Gram-negative as well as Gram-positive bacteria and archaea. PMID:27092113

The genome of the Erwinia amylovora phage PhiEaH1 reveals greater diversity and broadens the applicability of phages for the treatment of fire blight.

PubMed

Meczker, Katalin; Dömötör, Dóra; Vass, János; Rákhely, Gábor; Schneider, György; Kovács, Tamás

2014-01-01

The enterobacterium Erwinia amylovora is the causal agent of fire blight. This study presents the analysis of the complete genome of phage PhiEaH1, isolated from the soil surrounding an E. amylovora-infected apple tree in Hungary. Its genome is 218 kb in size, containing 244 ORFs. PhiEaH1 is the second E. amylovora infecting phage from the Siphoviridae family whose complete genome sequence was determined. Beside PhiEaH2, PhiEaH1 is the other active component of Erwiphage, the first bacteriophage-based pesticide on the market against E. amylovora. Comparative genome analysis in this study has revealed that PhiEaH1 not only differs from the 10 formerly sequenced E. amylovora bacteriophages belonging to other phage families, but also from PhiEaH2. Sequencing of more Siphoviridae phage genomes might reveal further diversity, providing opportunities for the development of even more effective biological control agents, phage cocktails against Erwinia fire blight disease of commercial fruit crops.

[Peptide phage display in biotechnology and biomedicine].

PubMed

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

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

Pf16 and phiPMW: Expanding the realm of Pseudomonas putida bacteriophages

PubMed Central

Krylov, Victor N.; Shaburova, Olga V.; McGrath, John W.; Allen, Christopher C. R.; Quinn, John P.; Kulakov, Leonid A.

2017-01-01

We present the analysis of two novel Pseudomonas putida phages, pf16 and phiPMW. Pf16 represents a peripherally related T4-like phage, and is the first of its kind infecting a Pseudomonad, with evidence suggesting cyanophage origins. Extensive divergence has resulted in pf16 occupying a newly defined clade designated as the pf16-related phages, lying at the interface of the Schizo T-Evens and Exo T-Evens. Recombination with an ancestor of the P. putida phage AF is likely responsible for the tropism of this phage. phiPMW represents a completely novel Pseudomonas phage with a genome containing substantial genetic novelty through its many hypothetical proteins. Evidence suggests that this phage has been extensively shaped through gene transfer events and vertical evolution. Phylogenetics shows that this phage has an evolutionary history involving FelixO1-related viruses but is in itself highly distinct from this group. PMID:28877269

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

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

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

Characterization of novel bacteriophage phiC119 capable of lysing multidrug-resistant Shiga toxin-producing Escherichia coli O157:H7.

PubMed

Amarillas, Luis; Chaidez, Cristóbal; González-Robles, Arturo; Lugo-Melchor, Yadira; León-Félix, Josefina

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) is one of the most common and widely distributed foodborne pathogens that has been frequently implicated in gastrointestinal and urinary tract infections. Moreover, high rates of multiple antibiotic-resistant E. coli strains have been reported worldwide. Due to the emergence of antibiotic-resistant strains, bacteriophages are considered an attractive alternative to biocontrol pathogenic bacteria. Characterization is a preliminary step towards designing a phage for biocontrol. In this study, we describe the characterization of a bacteriophage designated phiC119, which can infect and lyse several multidrug-resistant STEC strains and some Salmonella strains. The phage genome was screened to detect the stx-genes using PCR, morphological analysis, host range was determined, and genome sequencing were carried out, as well as an analysis of the cohesive ends and identification of the type of genetic material through enzymatic digestion of the genome. Analysis of the bacteriophage particles by transmission electron microscopy showed that it had an icosahedral head and a long tail, characteristic of the family Siphoviridae. The phage exhibits broad host range against multidrug-resistant and highly virulent E. coli isolates. One-step growth experiments revealed that the phiC119 phage presented a large burst size (210 PFU/cell) and a latent period of 20 min. Based on genomic analysis, the phage contains a linear double-stranded DNA genome with a size of 47,319 bp. The phage encodes 75 putative proteins, but lysogeny and virulence genes were not found in the phiC119 genome. These results suggest that phage phiC119 may be a good biological control agent. However, further studies are required to ensure its control of STEC and to confirm the safety of phage use.

Composite conserved promoter-terminator motifs (PeSLs) that mediate modular shuffling in the diverse T4-like myoviruses.

PubMed

Comeau, André M; Arbiol, Christine; Krisch, Henry M

2014-06-19

The diverse T4-like phages (Tquatrovirinae) infect a wide array of gram-negative bacterial hosts. The genome architecture of these phages is generally well conserved, most of the phylogenetically variable genes being grouped together in a series hyperplastic regions (HPRs) that are interspersed among large blocks of conserved core genes. Recent evidence from a pair of closely related T4-like phages has suggested that small, composite terminator/promoter sequences (promoterearly stem loop [PeSLs]) were implicated in mediating the high levels of genetic plasticity by indels occurring within the HPRs. Here, we present the genome sequence analysis of two T4-like phages, PST (168 kb, 272 open reading frames [ORFs]) and nt-1 (248 kb, 405 ORFs). These two phages were chosen for comparative sequence analysis because, although they are closely related to phages that have been previously sequenced (T4 and KVP40, respectively), they have different host ranges. In each case, one member of the pair infects a bacterial strain that is a human pathogen, whereas the other phage's host is a nonpathogen. Despite belonging to phylogenetically distant branches of the T4-likes, these pairs of phage have diverged from each other in part by a mechanism apparently involving PeSL-mediated recombination. This analysis confirms a role of PeSL sequences in the generation of genomic diversity by serving as a point of genetic exchange between otherwise unrelated sequences within the HPRs. Finally, the palette of divergent genes swapped by PeSL-mediated homologous recombination is discussed in the context of the PeSLs' potentially important role in facilitating phage adaption to new hosts and environments. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Isolation, genome sequencing and functional analysis of two T7-like coliphages of avian pathogenic Escherichia coli.

PubMed

Chen, Mianmian; Xu, Juntian; Yao, Huochun; Lu, Chengping; Zhang, Wei

2016-05-10

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, which results in significant economic losses to the poultry industry worldwide. Due to the drug residues and increased antibiotic resistance caused by antibiotic use, bacteriophages and other alternative therapeutic agents are expected to control APEC infection in poultry. Two APEC phages, named P483 and P694, were isolated from the feces from the farmers market in China. We then studied their biological properties, and carried out high-throughput genome sequencing and homology analyses of these phages. Assembly results of high-throughput sequencing showed that the structures of both P483 and P694 genomes consist of linear and double-stranded DNA. Results of the electron microscopy and homology analysis revealed that both P483 and P694 belong to T7-like virus which is a member of the Podoviridae family of the Caudovirales order. Comparative genomic analysis showed that most of the predicted proteins of these two phages showed strongest sequence similarity to the Enterobacteria phages BA14 and 285P, Erwinia phage FE44, and Kluyvera phage Kvp1; however, some proteins such as gp0.6a, gp1.7 and gp17 showed lower similarity (<85%) with the homologs of other phages in the T7 subgroup. We also found some unique characteristics of P483 and P694, such as the two types of the genes of P694 and no lytic activity of P694 against its host bacteria in liquid medium. Our results serve to further our understanding of phage evolution of T7-like coliphages and provide the potential application of the phages as therapeutic agents for the treatment of diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

PubMed Central

Vasala, A; Dupont, L; Baumann, M; Ritzenthaler, P; Alatossava, T

1993-01-01

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module. Images PMID:8497043

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

PubMed

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

2005-06-20

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

Isolation and Characterization of phiLLS, a Novel Phage with Potential Biocontrol Agent against Multidrug-Resistant Escherichia coli

PubMed Central

Amarillas, Luis; Rubí-Rangel, Lucia; Chaidez, Cristobal; González-Robles, Arturo; Lightbourn-Rojas, Luis; León-Félix, Josefina

2017-01-01

Foodborne diseases are a serious and growing problem, and the incidence and prevalence of antimicrobial resistance among foodborne pathogens is reported to have increased. The emergence of antibiotic-resistant bacterial strains demands novel strategies to counteract this epidemic. In this regard, lytic bacteriophages have reemerged as an alternative for the control of pathogenic bacteria. However, the effective use of phages relies on appropriate biological and genomic characterization. In this study, we present the isolation and characterization of a novel bacteriophage named phiLLS, which has shown strong lytic activity against generic and multidrug-resistant Escherichia coli strains. Transmission electron microscopy of phiLLS morphology revealed that it belongs to the Siphoviridae family. Furthermore, this phage exhibited a relatively large burst size of 176 plaque-forming units per infected cell. Phage phiLLS significantly reduced the growth of E. coli under laboratory conditions. Analyses of restriction profiles showed the presence of submolar fragments, confirming that phiLLS is a pac-type phage. Phylogenetic analysis based on the amino acid sequence of large terminase subunits confirmed that this phage uses a headful packaging strategy to package their genome. Genomic sequencing and bioinformatic analysis showed that phiLLS is a novel bacteriophage that is most closely related to T5-like phages. In silico analysis indicated that the phiLLS genome consists of 107,263 bp (39.0 % GC content) encoding 160 putative ORFs, 16 tRNAs, several potential promoters and transcriptional terminators. Genome analysis suggests that the phage phiLLS is strictly lytic without carrying genes associated with virulence factors and/or potential immunoreactive allergen proteins. The bacteriophage isolated in this study has shown promising results in the biocontrol of bacterial growth under in vitro conditions, suggesting that it may prove useful as an alternative agent for the control of foodborne pathogens. However, further oral toxicity testing is needed to ensure the safety of phage use. PMID:28785246

Isolation and Characterization of phiLLS, a Novel Phage with Potential Biocontrol Agent against Multidrug-Resistant Escherichia coli.

PubMed

Amarillas, Luis; Rubí-Rangel, Lucia; Chaidez, Cristobal; González-Robles, Arturo; Lightbourn-Rojas, Luis; León-Félix, Josefina

2017-01-01

Foodborne diseases are a serious and growing problem, and the incidence and prevalence of antimicrobial resistance among foodborne pathogens is reported to have increased. The emergence of antibiotic-resistant bacterial strains demands novel strategies to counteract this epidemic. In this regard, lytic bacteriophages have reemerged as an alternative for the control of pathogenic bacteria. However, the effective use of phages relies on appropriate biological and genomic characterization. In this study, we present the isolation and characterization of a novel bacteriophage named phiLLS, which has shown strong lytic activity against generic and multidrug-resistant Escherichia coli strains. Transmission electron microscopy of phiLLS morphology revealed that it belongs to the Siphoviridae family. Furthermore, this phage exhibited a relatively large burst size of 176 plaque-forming units per infected cell. Phage phiLLS significantly reduced the growth of E. coli under laboratory conditions. Analyses of restriction profiles showed the presence of submolar fragments, confirming that phiLLS is a pac -type phage. Phylogenetic analysis based on the amino acid sequence of large terminase subunits confirmed that this phage uses a headful packaging strategy to package their genome. Genomic sequencing and bioinformatic analysis showed that phiLLS is a novel bacteriophage that is most closely related to T5-like phages. In silico analysis indicated that the phiLLS genome consists of 107,263 bp (39.0 % GC content) encoding 160 putative ORFs, 16 tRNAs, several potential promoters and transcriptional terminators. Genome analysis suggests that the phage phiLLS is strictly lytic without carrying genes associated with virulence factors and/or potential immunoreactive allergen proteins. The bacteriophage isolated in this study has shown promising results in the biocontrol of bacterial growth under in vitro conditions, suggesting that it may prove useful as an alternative agent for the control of foodborne pathogens. However, further oral toxicity testing is needed to ensure the safety of phage use.

Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks.

PubMed

Smoot, James C; Barbian, Kent D; Van Gompel, Jamie J; Smoot, Laura M; Chaussee, Michael S; Sylva, Gail L; Sturdevant, Daniel E; Ricklefs, Stacy M; Porcella, Stephen F; Parkins, Larye D; Beres, Stephen B; Campbell, David S; Smith, Todd M; Zhang, Qing; Kapur, Vivek; Daly, Judy A; Veasy, L George; Musser, James M

2002-04-02

Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human-GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.

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

PubMed

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

2014-05-01

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

Isolation and characterization of T7-like lytic bacteriophages infecting multidrug resistant Pseudomonas aeruginosa isolated from Egypt.

PubMed

El Didamony, Gamal; Askora, Ahmed; Shehata, Aya A

2015-06-01

In this study, two lytic phages designated as ϕPSZ1 and ϕPSZ2 infecting multidrug resistant Pseudomonas aeruginosa were isolated from sewage samples collected in Zagazig, Egypt. Morphological analysis by transmission electron microscopy revealed that both phages belong to the podoviridae family and resembles typical T7-like phages. ϕPSZ1 has a head of about 60 ± 5 nm in diameter with a short tail of 19 ± 2 nm in length, while ϕPSZ2 has a head of about 57 ± 5 nm in diameter with a short tail of 14 ± 2 nm in length. Both phages were shown to be able to infect 13 different P. aeruginosa strains and has no effect on other tested bacteria. In spite of morphological similarity, these phages showed diverged genomic sequences revealed by restriction enzyme digestion analysis. One-step growth curves of bacteriophages revealed eclipse and latent periods of 12 min for ϕPSZ1 and 15 min for ϕPSZ2, respectively, with burst sizes of about 100 per infected cell. Phage treatment prevented the growth of P. aeruginosa for up to 18 h with multiplicity of infection ratios of 1. These results suggest that both phages have a high potential for phage application to control P. aeruginosa.

Metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity

PubMed Central

Willner, Dana; Furlan, Mike; Schmieder, Robert; Grasis, Juris A.; Pride, David T.; Relman, David A.; Angly, Florent E.; McDole, Tracey; Mariella, Ray P.; Rohwer, Forest; Haynes, Matthew

2011-01-01

The human oropharynx is a reservoir for many potential pathogens, including streptococcal species that cause endocarditis. Although oropharyngeal microbes have been well described, viral communities are essentially uncharacterized. We conducted a metagenomic study to determine the composition of oropharyngeal DNA viral communities (both phage and eukaryotic viruses) in healthy individuals and to evaluate oropharyngeal swabs as a rapid method for viral detection. Viral DNA was extracted from 19 pooled oropharyngeal swabs and sequenced. Viral communities consisted almost exclusively of phage, and complete genomes of several phage were recovered, including Escherichia coli phage T3, Propionibacterium acnes phage PA6, and Streptococcus mitis phage SM1. Phage relative abundances changed dramatically depending on whether samples were chloroform treated or filtered to remove microbial contamination. pblA and pblB genes of phage SM1 were detected in the metagenomes. pblA and pblB mediate the attachment of S. mitis to platelets and play a significant role in S. mitis virulence in the endocardium, but have never previously been detected in the oral cavity. These genes were also identified in salivary metagenomes from three individuals at three time points and in individual saliva samples by PCR. Additionally, we demonstrate that phage SM1 can be induced by commonly ingested substances. Our results indicate that the oral cavity is a reservoir for pblA and pblB genes and for phage SM1 itself. Further studies will determine the association between pblA and pblB genes in the oral cavity and the risk of endocarditis. PMID:20547834

Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas.

PubMed

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

2014-01-01

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ~4 × 10(-12) ml cell(-1) min(-1) for X. fastidiosa strain Temecula 1 and ~5 × 10(-10) to 7 × 10(-10) ml cell(-1) min(-1) for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.

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

Virome analysis for identification of novel mammalian viruses in bat species from Chinese provinces.

PubMed

Wu, Zhiqiang; Ren, Xianwen; Yang, Li; Hu, Yongfeng; Yang, Jian; He, Guimei; Zhang, Junpeng; Dong, Jie; Sun, Lilian; Du, Jiang; Liu, Liguo; Xue, Ying; Wang, Jianmin; Yang, Fan; Zhang, Shuyi; Jin, Qi

2012-10-01

Bats are natural hosts for a large variety of zoonotic viruses. This study aimed to describe the range of bat viromes, including viruses from mammals, insects, fungi, plants, and phages, in 11 insectivorous bat species (216 bats in total) common in six provinces of China. To analyze viromes, we used sequence-independent PCR amplification and next-generation sequencing technology (Solexa Genome Analyzer II; Illumina). The viromes were identified by sequence similarity comparisons to known viruses. The mammalian viruses included those of the Adenoviridae, Herpesviridae, Papillomaviridae, Retroviridae, Circoviridae, Rhabdoviridae, Astroviridae, Flaviridae, Coronaviridae, Picornaviridae, and Parvovirinae; insect viruses included those of the Baculoviridae, Iflaviridae, Dicistroviridae, Tetraviridae, and Densovirinae; fungal viruses included those of the Chrysoviridae, Hypoviridae, Partitiviridae, and Totiviridae; and phages included those of the Caudovirales, Inoviridae, and Microviridae and unclassified phages. In addition to the viruses and phages associated with the insects, plants, and bacterial flora related to the diet and habitation of bats, we identified the complete or partial genome sequences of 13 novel mammalian viruses. These included herpesviruses, papillomaviruses, a circovirus, a bocavirus, picornaviruses, a pestivirus, and a foamy virus. Pairwise alignments and phylogenetic analyses indicated that these novel viruses showed little genetic similarity with previously reported viruses. This study also revealed a high prevalence and diversity of bat astroviruses and coronaviruses in some provinces. These findings have expanded our understanding of the viromes of bats in China and hinted at the presence of a large variety of unknown mammalian viruses in many common bat species of mainland China.

Virome Analysis for Identification of Novel Mammalian Viruses in Bat Species from Chinese Provinces

PubMed Central

Wu, Zhiqiang; Ren, Xianwen; Yang, Li; Hu, Yongfeng; Yang, Jian; He, Guimei; Zhang, Junpeng; Dong, Jie; Sun, Lilian; Du, Jiang; Liu, Liguo; Xue, Ying; Wang, Jianmin; Yang, Fan

2012-01-01

Bats are natural hosts for a large variety of zoonotic viruses. This study aimed to describe the range of bat viromes, including viruses from mammals, insects, fungi, plants, and phages, in 11 insectivorous bat species (216 bats in total) common in six provinces of China. To analyze viromes, we used sequence-independent PCR amplification and next-generation sequencing technology (Solexa Genome Analyzer II; Illumina). The viromes were identified by sequence similarity comparisons to known viruses. The mammalian viruses included those of the Adenoviridae, Herpesviridae, Papillomaviridae, Retroviridae, Circoviridae, Rhabdoviridae, Astroviridae, Flaviridae, Coronaviridae, Picornaviridae, and Parvovirinae; insect viruses included those of the Baculoviridae, Iflaviridae, Dicistroviridae, Tetraviridae, and Densovirinae; fungal viruses included those of the Chrysoviridae, Hypoviridae, Partitiviridae, and Totiviridae; and phages included those of the Caudovirales, Inoviridae, and Microviridae and unclassified phages. In addition to the viruses and phages associated with the insects, plants, and bacterial flora related to the diet and habitation of bats, we identified the complete or partial genome sequences of 13 novel mammalian viruses. These included herpesviruses, papillomaviruses, a circovirus, a bocavirus, picornaviruses, a pestivirus, and a foamy virus. Pairwise alignments and phylogenetic analyses indicated that these novel viruses showed little genetic similarity with previously reported viruses. This study also revealed a high prevalence and diversity of bat astroviruses and coronaviruses in some provinces. These findings have expanded our understanding of the viromes of bats in China and hinted at the presence of a large variety of unknown mammalian viruses in many common bat species of mainland China. PMID:22855479

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

PubMed Central

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

2017-01-01

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

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

PubMed Central

O’Hara, Brendan J.

2017-01-01

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

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

PubMed

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

2017-06-01

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

Characterization and Complete Genome Sequences of Three N4-Like Roseobacter Phages Isolated from the South China Sea.

PubMed

Li, Baolian; Zhang, Si; Long, Lijuan; Huang, Sijun

2016-09-01

Three bacteriophages (RD-1410W1-01, RD-1410Ws-07, and DS-1410Ws-06) were isolated from the surface water of Sanya Bay, northern South China Sea, on two marine bacteria type strains of the Roseobacter lineage. These phages have an isometric head and a short tail, morphologically belonging to the Podoviridae family. Two of these phages can infect four of seven marine roseobacter strains tested and the other one can infect three of them, showing relatively broader host ranges compared to known N4-like roseophages. One-step growth curves showed that these phages have similar short latent periods (1-2 h) but highly variable burst sizes (27-341 pfu cell(-1)). Their complete genomes show high level of similarities to known N4-like roseophages in terms of genome size, G + C content, gene content, and arrangement. The morphological and genomic features of these phages indicate that they belong to the N4likevirus genus. Moreover, comparative genomic analysis based on 43 N4-like phages (10 roseobacter phages and 33 phages infecting other lineages of bacteria) revealed a core genome of 18 genes shared by all the 43 phages and 38 genes shared by all the ten roseophages. The 38 core genes of N4-like roseophages nearly make up 70 % of each genome in length. Phylogenetic analysis based on the concatenated core gene products showed that our phage isolates represent two new phyletic branches, suggesting the broad genetic diversity of marine N4-like roseophages remains.

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

PubMed

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

2008-02-01

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

Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

PubMed

Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U; Severinov, Konstantin

2017-02-14

Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases. IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages. Copyright © 2017 Lavysh et al.

Isolation and characterization of vB_ArS-ArV2 - first Arthrobacter sp. infecting bacteriophage with completely sequenced genome.

PubMed

Šimoliūnas, Eugenijus; Kaliniene, Laura; Stasilo, Miroslav; Truncaitė, Lidija; Zajančkauskaitė, Aurelija; Staniulis, Juozas; Nainys, Juozas; Kaupinis, Algirdas; Valius, Mindaugas; Meškys, Rolandas

2014-01-01

This is the first report on a complete genome sequence and biological characterization of the phage that infects Arthrobacter. A novel virus vB_ArS-ArV2 (ArV2) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation. Based on transmission electron microscopy, ArV2 belongs to the family Siphoviridae and has an isometric head (∼63 nm in diameter) with a non-contractile flexible tail (∼194×10 nm) and six short tail fibers. ArV2 possesses a linear, double-stranded DNA genome (37,372 bp) with a G+C content of 62.73%. The genome contains 68 ORFs yet encodes no tRNA genes. A total of 28 ArV2 ORFs have no known functions and lack any reliable database matches. Proteomic analysis led to the experimental identification of 14 virion proteins, including 9 that were predicted by bioinformatics approaches. Comparative phylogenetic analysis, based on the amino acid sequence alignment of conserved proteins, set ArV2 apart from other siphoviruses. The data presented here will help to advance our understanding of Arthrobacter phage population and will extend our knowledge about the interaction between this particular host and its phages.

[Polyvalence of bacteriophages isolated from fruit trees, affected by bacterial fire blight].

PubMed

Tovkach, F I; Moroz, S N; Korol', N A; Faĭdiuk, Iu V; Kushkina, A I

2013-01-01

Phage populations appearing as a result of a pathogenic process caused by Erwinia amylovora have been discovered and described. They accompany bacterial fire blight development in the process of quince, pear and apple trees vegetation in Zakarpattya region of Ukraine. Phage isolates of the affected pear and quince include polyvalent virulent phages able to develop on bacterial strains associated with plants--E. amylovora. E. "horticola" and Pantoea agglomerans. E. amylovora isolated from the plant tissues affected by the fire blight and detected at the same time as phages proved to be resistant to the viral infection. It is hard to explain now this characteristic however it was noticed that resistance to phages can change drastically in case of dissociation, lysogenization and mutagenesis of erwinia in laboratory conditions. Phage population study shows that they are heterogeneous and can obviously include not only polyvalent but also specific viruses. Further studies of biology and molecular genetics of pure lines of isolated phages will help to get closer to understanding the place and role of bacteriophages in the complicated network of relations between bacterial pathogens and plants.

Bacteriophage Ecology in a Commercial Cucumber Fermentation

PubMed Central

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

2012-01-01

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

Genome-wide characterization of vibrio phage ϕpp2 with unique arrangements of the mob-like genes

PubMed Central

2012-01-01

Background Vibrio parahaemolyticus is associated with gastroenteritis, wound infections, and septicemia in human and animals. Phages can control the population of the pathogen. So far, the only one reported genome among giant vibriophages is KVP40: 244,835 bp with 26% coding regions that have T4 homologs. Putative homing endonucleases (HE) were found in Vibrio phage KVP40 bearing one segD and Vibrio cholerae phage ICP1 carrying one mobC/E and one segG. Results A newly isolated Vibrio phage ϕpp2, which was specific to the hosts of V. parahaemolyticus and V. alginolyticus, featured a long nonenveloped head of ~90 × 150 nm and tail of ~110 nm. The phage can survive at 50°C for more than one hour. The genome of the phage ϕpp2 was sequenced to be 246,421 bp, which is 1587 bp larger than KVP40. 383 protein-encoding genes (PEGs) and 30 tRNAs were found in the phage ϕpp2. Between the genomes of ϕpp2 and KVP40, 254 genes including 29 PEGs for viral structure were of high similarity, whereas 17 PEGs of KVP40 and 21 PEGs of ϕpp2 were unmatched. In both genomes, the capsid and tail genes have been identified, as well as the extensive representation of the DNA replication, recombination, and repair enzymes. In addition to the three giant indels of 1098, 1143 and 3330 nt, ϕpp2 possessed unique proteins involved in potassium channel, gp2 (DNA end protector), tRNA nucleotidyltransferase, and mob-type HEs, which were not reported in KVP40. The ϕpp2 PEG274, with strong promoters and translational initiation, was identified to be a mobE type, flanked by NrdA and NrdB/C homologs. Coincidently, several pairs of HE-flanking homologs with empty center were found in the phages of Vibrio phages ϕpp2 and KVP40, as well as in Aeromonas phages (Aeh1 and Ae65), and cyanophage P-SSM2. Conclusions Vibrio phage ϕpp2 was characterized by morphology, growth, and genomics with three giant indels and different types of HEs. The gene analysis on the required elements for transcription and translation suggested that the ϕpp2 PEG274 was an active mobE gene. The phage was signified to be a new species of T4-related, differing from KVP40. PMID:22676552

Complete genomic sequence of the Lactobacillus temperate phage LF1.

PubMed

Yoon, Bo Hyun; Chang, Hyo Ihl

2011-10-01

Bacteriophage LF1, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild type Lactobacillus fermentum, was found to contain a double-strand DNA of 42,606 base pairs (bp) with a G+C content of 45%. Bioinformatic analysis of the phage genome revealed 57 putative open reading frames (ORFs). The predicted protein products of ORFs were determined and described. According to morphological analysis by transmission electron microscopy (TEM), LF1 has an isometric head and a non-contractile tail, indicating that it belongs to the family Siphoviridae. The temperate phage LF1 has a good genetic mosaic relationship with ΦPYB5 in the packaging module. To our knowledge, this is first report of genomic sequencing and characterization of temperate phage LF1 from wild-type L. fermentum isolated from Kimchi in Korea.

[Isolation and identification of the temperate bacteriophage from isolated strains of Streptococcus suis serotype 2].

PubMed

Ma, Yuling; Lu, Chengping; Fan, Hongjie

2008-04-01

A PCR assay was developed to study the distributional characteristics of phage integrase gene in Streptococcus suis serotype 2 (SS2). A 323bp distinct DNA target can be amplified in 25 strains of virulent SS2, while can not be amplified in avirulent strain T15, 5 strains of other serotypes (SS1, SS7, SS9) and strains of group C Streptococcus strains from pigs, which suggested that the phage integrase gene may be related to the pathogenicity of SS2 and can be consider as a detection factor of the virulent gene of SS2. The sequencing and restriction endonuclease analysis of the PCR products were also done. Comparisons between the sequences of phage integrase gene with that of SS2 strain, showed a high homology with SS2 China strains 98HAH33, 05ZYH33 and North American strain 89-1591. Complete cell lysis was observed with SS2 virulent strains but not with avirulent strain T15 after the induction by mitomycin C. Electron microscopy analysis of the lysate from SS2 virulent strains HA9801 and ZY05719 revealed the presence of phage particles. The induced phage, named SS2-HA and SS2-ZY, both have a small isometric nucleocapsid approximately 50 nm in diameter and have no tail and is therefore a member of the Tectiviridae family. The phage integrase gene sequence of phage SS2-HA and SS2-ZY shared high homologue identities with virulent SS2 strains, which suggested that the phage integrase gene of SS2 has high specify. The temperate phage and phage integrase gene can only detected from SS2 virulent strains but not from avirulent strain, and the detection of phage integrase gene was related to the virulence-associate factors of SS2, such as the muramidase-released protein gene (mrp), which suggested that the temperate phage of SS2 may be related to the pathogenicity of SS2.

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

PubMed

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

2016-01-01

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

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

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

Identification of chondrocyte-binding peptides by phage display.

PubMed

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

2013-07-01

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

Cloning vector

DOEpatents

Guilfoyle, Richard A.; Smith, Lloyd M.

1994-01-01

A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

Cloning vector

DOEpatents

Guilfoyle, R.A.; Smith, L.M.

1994-12-27

A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

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

NASA Astrophysics Data System (ADS)

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

2016-04-01

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

Phage display as a technology delivering on the promise of peptide drug discovery.

PubMed

Hamzeh-Mivehroud, Maryam; Alizadeh, Ali Akbar; Morris, Michael B; Church, W Bret; Dastmalchi, Siavoush

2013-12-01

Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries can be displayed on the phage surface. The phage library can be incubated with a target of interest and the phage which bind can be isolated and sequenced to reveal the displayed peptides' primary structure. In this review, we focus on the 'mechanics' of the phage display process, whilst highlighting many diverse and subtle ways it has been used to further the drug-development process, including the potential for the phage particle itself to be used as a drug carrier targeted to a particular pathogen or cell type in the body. Copyright © 2013 Elsevier Ltd. All rights reserved.

Prophage-Encoded Staphylococcal Enterotoxin A: Regulation of Production in Staphylococcus aureus Strains Representing Different Sea Regions

PubMed Central

Zeaki, Nikoleta; Budi Susilo, Yusak; Pregiel, Anna; Rådström, Peter; Schelin, Jenny

2015-01-01

The present study investigates the nature of the link between the staphylococcal enterotoxin A (SEA) gene and the lifecycle of Siphoviridae bacteriophages, including the origin of strain variation regarding SEA production after prophage induction. Five strains representing three different genetic lines of the sea region were studied under optimal and prophage-induced growth conditions and the Siphoviridae lifecycle was followed through the phage replicative form copies and transcripts of the lysogenic repressor, cro. The role of SOS response on prophage induction was addressed through recA transcription in a recA-disruption mutant. Prophage induction was found to increase the abundance of the phage replicative form, the sea gene copies and transcripts and enhance SEA production. Sequence analysis of the sea regions revealed that observed strain variances were related to strain capacity for prophage induction, rather than sequence differences in the sea region. The impact of SOS response activation on the phage lifecycle was demonstrated by the absence of phage replicative form copies in the recA-disruption mutant after prophage induction. From this study it emerges that all aspects of SEA-producing strain, the Siphoviridae phage and the food environment must be considered when evaluating SEA-related hazards. PMID:26690218

Characterizing Phage Genomes for Therapeutic Applications

PubMed Central

Philipson, Casandra W.; Voegtly, Logan J.; Lueder, Matthew R.; Long, Kyle A.; Rice, Gregory K.; Frey, Kenneth G.; Biswas, Biswajit; Cer, Regina Z.; Hamilton, Theron; Bishop-Lilly, Kimberly A.

2018-01-01

Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA) for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates. PMID:29642590

Bacteriophages of Yersinia pestis.

PubMed

Zhao, Xiangna; Skurnik, Mikael

2016-01-01

Bacteriophage play many varied roles in microbial ecology and evolution. This chapter collates a vast body of knowledge and expertise on Yersinia pestis phages, including the history of their isolation and classical methods for their isolation and identification. The genomic diversity of Y. pestis phage and bacteriophage islands in the Y. pestis genome are also discussed because all phage research represents a branch of genetics. In addition, our knowledge of the receptors that are recognized by Y. pestis phage, advances in phage therapy for Y. pestis infections, the application of phage in the detection of Y. pestis, and clustered regularly interspaced short palindromic repeats (CRISPRs) sequences of Y. pestis from prophage DNA are all reviewed here.

Phage transposon mutagenesis.

PubMed

Siegrist, M Sloan; Rubin, Eric J

2009-01-01

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

Phage Conversion for β-Lactam Antibiotic Resistance of Staphylococcus aureus from Foods.

PubMed

Lee, Young-Duck; Park, Jong-Hyun

2016-02-01

Temperate phages have been suggested to carry virulence factors and other lysogenic conversion genes that play important roles in pathogenicity. In this study, phage TEM123 in wild-type Staphylococcus aureus from food sources was analyzed with respect to its morphology, genome sequence, and antibiotic resistance conversion ability. Phage TEM123 from a mitomycin C-induced lysate of S. aureus was isolated from foods. Morphological analysis under a transmission electron microscope revealed that it belonged to the family Siphoviridae. The genome of phage TEM123 consisted of a double-stranded DNA of 43,786 bp with a G+C content of 34.06%. A bioinformatics analysis of the phage genome identified 43 putative open reading frames (ORFs). ORF1 encoded a protein that was nearly identical to the metallo-β-lactamase enzymes that degrade β-lactam antibiotics. After transduction to S. aureus with phage TEM123, the metallo-β-lactamase gene was confirmed in the transductant by PCR and sequencing analyses. In a β-lactam antibiotic susceptibility test, the transductant was more highly resistant to β-lactam antibiotics than S. aureus S133. Phage TEM123 might play a role in the transfer of β-lactam antibiotic resistance determinants in S. aureus. Therefore, we suggest that the prophage of S. aureus with its exotoxin is a risk factor for food safety in the food chain through lateral gene transfer.

Application of a phage in decontaminating Vibrio parahaemolyticus in oysters.

PubMed

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

2018-06-20

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

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

PubMed Central

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

2017-01-01

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

Diversity in a Polymicrobial Community Revealed by Analysis of Viromes, Endolysins and CRISPR Spacers.

PubMed

Davison, Michelle; Treangen, Todd J; Koren, Sergey; Pop, Mihai; Bhaya, Devaki

2016-01-01

The polymicrobial biofilm communities in Mushroom and Octopus Spring in Yellowstone National Park (YNP) are well characterized, yet little is known about the phage populations. Dominant species, Synechococcus sp. JA-2-3B'a(2-13), Synechococcus sp. JA-3-3Ab, Chloroflexus sp. Y-400-fl, and Roseiflexus sp. RS-1, contain multiple CRISPR-Cas arrays, suggesting complex interactions with phage predators. To analyze phage populations from Octopus Spring biofilms, we sequenced a viral enriched fraction. To assemble and analyze phage metagenomic data, we developed a custom module, VIRITAS, implemented within the MetAMOS framework. This module bins contigs into groups based on tetranucleotide frequencies and CRISPR spacer-protospacer matching and ORF calling. Using this pipeline we were able to assemble phage sequences into contigs and bin them into three clusters that corroborated with their potential host range. The virome contained 52,348 predicted ORFs; some were clearly phage-like; 9319 ORFs had a recognizable Pfam domain while the rest were hypothetical. Of the recognized domains with CRISPR spacer matches, was the phage endolysin used by lytic phage to disrupt cells. Analysis of the endolysins present in the thermophilic cyanophage contigs revealed a subset of characterized endolysins as well as a Glyco_hydro_108 (PF05838) domain not previously associated with sequenced cyanophages. A search for CRISPR spacer matches to all identified phage endolysins demonstrated that a majority of endolysin domains were targets. This strategy provides a general way to link host and phage as endolysins are known to be widely distributed in bacteriophage. Endolysins can also provide information about host cell wall composition and have the additional potential to be used as targets for novel therapeutics.

Comparative analysis of multiple inducible phages from Mannheimia haemolytica.

PubMed

Niu, Yan D; Cook, Shaun R; Wang, Jiaying; Klima, Cassidy L; Hsu, Yu-hung; Kropinski, Andrew M; Turner, Dann; McAllister, Tim A

2015-08-30

Mannheimia haemolytica is a commensal bacterium that resides in the upper respiratory tract of cattle that can play a role in bovine respiratory disease. Prophages are common in the M. haemolytica genome and contribute significantly to host diversity. The objective of this research was to undertake comparative genomic analysis of phages induced from strains of M. haemolytica serotype A1 (535A and 2256A), A2 (587A and 1127A) and A6 (1152A and 3927A). Overall, four P2-like (535AP1, 587AP1, 1127AP1 and 2256AP1; genomes: 34.9-35.7 kb; G+C content: 41.5-42.1 %; genes: 51-53 coding sequences, CDSs), four λ-like (535AP2, 587AP2, 1152AP2 and 3927AP1; genomes: 48.6-52.1 kb; 41.1-41.4 % mol G+C; genes: 77-83 CDSs and 2 tRNAs) and one Mu-like (3927AP2; genome: 33.8 kb; 43.1 % mol G+C; encoding 50 CDSs) phages were identified. All P2-like phages are collinear with the temperate phage φMhaA1-PHL101 with 535AP1, 2256AP1 and 1152AP1 being most closely related, followed by 587AP1 and 1127AP1. Lambdoid phages are not collinear with any other known λ-type phages, with 587AP2 being distinct from 535AP2, 3927AP1 and 1152AP2. All λ-like phages contain genes encoding a toxin-antitoxin (TA) system and cell-associated haemolysin XhlA. The Mu-like phage induced from 3927A is closely related to the phage remnant φMhaMu2 from M. haemolytica PHL21, with similar Mu-like phages existing in the genomes of M. haemolytica 535A and 587A. This is among the first reports of both λ- and Mu-type phages being induced from M. haemolytica. Compared to phages induced from commensal strains of M. haemolytica serotype A2, those induced from the more virulent A1 and A6 serotypes are more closely related. Moreover, when P2-, λ- and Mu-like phages co-existed in the M. haemolytica genome, only P2- and λ-like phages were detected upon induction, suggesting that Mu-type phages may be more resistant to induction. Toxin-antitoxin gene cassettes in λ-like phages may contribute to their genomic persistence or the establishment of persister subpopulations of M. haemolytica. Further work is required to determine if the cell-associated haemolysin XhlA encoded by λ-like phages contributes to the pathogenicity and ecological fitness of M. haemolytica.

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

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

PubMed

Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

2012-01-01

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

Characterization and complete genome sequence analysis of a novel virulent Siphoviridae phage against Staphylococcus aureus isolated from bovine mastitis in Xinjiang, China.

PubMed

Zhang, Qian; Xing, Shaozhen; Sun, Qiang; Pei, Guangqian; Cheng, Shi; Liu, Yannan; An, Xiaoping; Zhang, Xianglilan; Qu, Yonggang; Tong, Yigang

2017-06-01

Bovine mastitis is one of the most costly diseases in dairy cows worldwide. It can be caused by over 150 different microorganisms, where Staphylococcus aureus is the most frequently isolated and a major pathogen responsible for heavy economic losses in dairy industry. Although antibiotic therapy is most widely used, alternative treatments are necessary due to the increasing antibiotic resistance. Using phage for pathogen control is a promising tool in the fight against antibiotic resistance. Mainly using high-throughput sequencing, bioinformatics and our proposed phage termini identification method, we have isolated and characterized a novel virulent phage, designated as vB_SauS_IMEP5, from manure collected from dairy farms in Shihezi, Xinjiang, China, for use as a biocontrol agent against Staphylococcus aureus infections. Its latent period was about 30 min and its burst size was approximately 272PFU/cell. Phage vB_SauS_IMEP5 survives in a wide pH range between 3 and 12. A treatment at 70 °C for 20 min can inactive the phage. Morphological analysis of vB_SauS_IMEP5 revealed that phage vB_SauS_IMEP5 morphologically resembles phages in the family Siphoviridae. Among our tested multiplicity of infections (MOIs), the optimal multiplicity of infection (MOI) of this phage was determined to be 0.001, suggesting that phage vB_SauS_IMEP5 has high bacteriolytic potential and good efficiency for reducing bacterial growth. The complete genome of IME-P5 is a 44,677-bp, linear, double-stranded DNA, with a G+C content of 34.26%, containing 69 putative ORFs. The termini of genome were determined with next-generation sequencing data using our previously proposed termini identification method, which suggests that this phage has non-redundant termini with 9nt 3' protruding cohesive ends. The genomic and proteomic characteristics of IMEP5 demonstrate that this phage does not belong to any of the previously recognized Siphoviridae Staphylococcus phage groups, suggesting the creation of a new lineage, thus adding to the knowledge on the diversity of Staphylococcus phages. An N-acetylmuramoyl-L-alanine amidase gene and several conserved genes were predicted, while no virulence or antibiotic resistance genes were identified. This study isolated and characterized a novel S. aureus phage vB_SauS_IMEP5, and our findings suggest that this phage may be potentially utilized as a therapeutic or prophylactic candidate against S.aureus infections.

Isolation and Host Range of Bacteriophage with Lytic Activity against Methicillin-Resistant Staphylococcus aureus and Potential Use as a Fomite Decontaminant.

PubMed

Jensen, Kyle C; Hair, Bryan B; Wienclaw, Trevor M; Murdock, Mark H; Hatch, Jacob B; Trent, Aaron T; White, Tyler D; Haskell, Kyler J; Berges, Bradford K

2015-01-01

Staphylococcus aureus (SA) is a commensal bacterium and opportunistic pathogen commonly associated with humans and is capable of causing serious disease and death including sepsis, pneumonia, and meningitis. Methicillin-resistant SA (MRSA) isolates are typically resistant to many available antibiotics with the common exception of vancomycin. The presence of vancomycin resistance in some SA isolates combined with the current heavy use of vancomycin to treat MRSA infections indicates that MRSA may achieve broad resistance to vancomycin in the near future. New MRSA treatments are clearly needed. Bacteriophages (phages) are viruses that infect bacteria, commonly resulting in death of the host bacterial cell. Phage therapy entails the use of phage to treat or prevent bacterial infections. In this study, 12 phages were isolated that can replicate in human SA and/or MRSA isolates as a potential way to control these infections. 5 phage were discovered through mitomycin C induction of prophage and 7 others as extracellular viruses. Primary SA strains were also isolated from environmental sources to be used as tools for phage discovery and isolation as well as to examine the target cell host range of the phage isolates by spot testing. Primary isolates were tested for susceptibility to oxacillin in order to determine which were MRSA. Experiments were performed to assess the host range and killing potential of newly discovered phage, and significant reductions in bacterial load were detected. We explored the utility of some phage to decontaminate fomites (glass and cloth) and found a significant reduction in colony forming units of MRSA following phage treatment, including tests of a phage cocktail against a cocktail of MRSA isolates. Our findings suggest that phage treatment can be used as an effective tool to decontaminate human MRSA from both hard surfaces and fabrics.

Identification of peptide sequences that target to the brain using in vivo phage display.

PubMed

Li, Jingwei; Zhang, Qizhi; Pang, Zhiqing; Wang, Yuchen; Liu, Qingfeng; Guo, Liangran; Jiang, Xinguo

2012-06-01

Phage display technology could provide a rapid means for the discovery of novel peptides. To find peptide ligands specific for the brain vascular receptors, we performed a modified phage display method. Phages were recovered from mice brain parenchyma after administrated with a random 7-mer peptide library intravenously. A longer circulation time was arranged according to the biodistributive brain/blood ratios of phage particles. Following sequential rounds of isolation, a number of phages were sequenced and a peptide sequence (CTSTSAPYC, denoted as PepC7) was identified. Clone 7-1, which encodes PepC7, exhibited translocation efficiency about 41-fold higher than the random library phage. Immunofluorescence analysis revealed that Clone 7-1 had a significant superiority on transport efficiency into the brain compared with native M13 phage. Clone 7-1 was inhibited from homing to the brain in a dose-dependent fashion when cyclic peptides of the same sequence were present in a competition assay. Interestingly, the linear peptide (ATSTSAPYA, Pep7) and a scrambled control peptide PepSC7 (CSPATSYTC) did not compete with the phage at the same tested concentration (0.2-200 pg). Labeled by Cy5.5, PepC7 exhibited significant brain-targeting capability in in vivo optical imaging analysis. The cyclic conformation of PepC7 formed by disulfide bond, and the correct structure itself play a critical role in maintaining the selectivity and affinity for the brain. In conclusion, PepC7 is a promising brain-target motif never been reported before and it could be applied to targeted drug delivery into the brain.

Isolation and characterization of a novel, T7-like phage against Aeromonas veronii.

PubMed

Anand, Taruna; Bera, Bidhan Ch; Virmani, Nitin; Vaid, Rajesh Kumar; Vashisth, Medhavi; Tripathi, Bhupendra Nath

2018-02-01

A virulent Aeromonas veronii biovar sobria and the corresponding novel, lytic bacteriophage (VTCCBPA5) were isolated from village pond water. The phage was found to belong to family Podoviridae. PCR analysis of major capsid protein gene confirmed its classification to T7-like genus. The protein profiling by SDS-PAGE indicated the major structural protein to be ~ 45 kDa. The phage (VTCCBPA5) is host specific and is stable over a range of pH (6-10) and temperatures (4-45 °C). On the basis of restriction endonuclease analysis combined with prediction mapping, it was observed to vary significantly from previously reported podophages of Aeromonas sp., viz. phiAS7 and Ahp1. The phylogenetic analysis on the basis of PCR-amplified segment of DNA polymerase gene of phage revealed it being an outgroup from podophages of Klebsiella sp. and Pseudomonas sp. though a small internal fragment (359 bp) showed the highest identity (77%) with Vibrio sp. phages. Thus, this is the first report of a novel Podoviridae phage against A. veronii. It expands the assemblage of podophages against Aeromonas sp. and BPA5 could be potentially useful in biocontrol of environmentally acquired Aeromonas veronii infections.

Essential Steps in Characterizing Bacteriophages: Biology, Taxonomy, and Genome Analysis.

PubMed

Aziz, Ramy Karam; Ackermann, Hans-Wolfgang; Petty, Nicola K; Kropinski, Andrew M

2018-01-01

Because of the rise in antimicrobial resistance there has been a significant increase in interest in phages for therapeutic use. Furthermore, the cost of sequencing phage genomes has decreased to the point where it is being used as a teaching tool for genomics. Unfortunately, the quality of the descriptions of the phage and its annotation frequently are substandard. The following chapter is designed to help people working on phages, particularly those new to the field, to accurately describe their newly isolated viruses.

Linkage of Recognition and Replication Functions by Assembling Combinatorial Antibody Fab Libraries Along Phage Surfaces

NASA Astrophysics Data System (ADS)

Kang, Angray S.; Barbas, Carlos F.; Janda, Kim D.; Benkovic, Stephen J.; Lerner, Richard A.

1991-05-01

We describe a method based on a phagemid vector with helper phage rescue for the construction and rapid analysis of combinatorial antibody Fab libraries. This approach should allow the generation and selection of many monoclonal antibodies. Antibody genes are expressed in concert with phage morphogenesis, thereby allowing incorporation of functional Fab molecules along the surface of filamentous phage. The power of the method depends upon the linkage of recognition and replication functions and is not limited to antibody molecules.

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.

Use of phages to control Campylobacter spp.

PubMed

Janež, Nika; Loc-Carrillo, Catherine

2013-10-01

The use of phages to control pathogenic bacteria has been investigated since they were first discovered in the beginning of the 1900s. Over the last century we have slowly gained an in-depth understanding of phage biology including which phage properties are desirable when considering phage as biocontrol agents and which phage characteristics to potentially avoid. Campylobacter infections are amongst the most frequently encountered foodborne bacterial infections around the world. Handling and consumption of raw or undercooked poultry products have been determined to be the main route of transmission. The ability to use phages to target these bacteria has been studied for more than a decade and although we have made progress towards deciphering how best to use phages to control Campylobacter associated with poultry production, there is still much work to be done. This review outlines methods to improve the isolation of these elusive phages, as well as methods to identify desirable characteristics needed for a successful outcome. It also highlights the body of research undertaken so far and what criteria to consider when doing in-vivo studies, especially because some in-vitro studies have not been found to translate into to phage efficacy in-vivo. © 2013. Published by Elsevier B.V. All rights reserved.

Characterization of novel bacteriophage phiC119 capable of lysing multidrug-resistant Shiga toxin-producing Escherichia coli O157:H7

PubMed Central

Amarillas, Luis; Chaidez, Cristóbal; González-Robles, Arturo; Lugo-Melchor, Yadira

2016-01-01

Background Shiga toxin-producing Escherichia coli (STEC) is one of the most common and widely distributed foodborne pathogens that has been frequently implicated in gastrointestinal and urinary tract infections. Moreover, high rates of multiple antibiotic-resistant E. coli strains have been reported worldwide. Due to the emergence of antibiotic-resistant strains, bacteriophages are considered an attractive alternative to biocontrol pathogenic bacteria. Characterization is a preliminary step towards designing a phage for biocontrol. Methods In this study, we describe the characterization of a bacteriophage designated phiC119, which can infect and lyse several multidrug-resistant STEC strains and some Salmonella strains. The phage genome was screened to detect the stx-genes using PCR, morphological analysis, host range was determined, and genome sequencing were carried out, as well as an analysis of the cohesive ends and identification of the type of genetic material through enzymatic digestion of the genome. Results Analysis of the bacteriophage particles by transmission electron microscopy showed that it had an icosahedral head and a long tail, characteristic of the family Siphoviridae. The phage exhibits broad host range against multidrug-resistant and highly virulent E. coli isolates. One-step growth experiments revealed that the phiC119 phage presented a large burst size (210 PFU/cell) and a latent period of 20 min. Based on genomic analysis, the phage contains a linear double-stranded DNA genome with a size of 47,319 bp. The phage encodes 75 putative proteins, but lysogeny and virulence genes were not found in the phiC119 genome. Conclusion These results suggest that phage phiC119 may be a good biological control agent. However, further studies are required to ensure its control of STEC and to confirm the safety of phage use. PMID:27672499

A comparative study of different strategies for removal of endotoxins from bacteriophage preparations.

PubMed

Van Belleghem, Jonas D; Merabishvili, Maya; Vergauwen, Bjorn; Lavigne, Rob; Vaneechoutte, Mario

2017-01-01

Bacterial endotoxins have high immunogenicity. Phage biology studies as well as therapeutic phage applications necessitate highly purified phage particles. In this study, we compared combinations of seven different endotoxin removal strategies and validated their endotoxin removal efficacy for five different phages (i.e. four Pseudomonas aeruginosa phages and one Staphylococcus aureus phage). These purification strategies included Endotrap HD column purification and/or CsCl density centrifugation in combination with Endotrap purification, followed by organic solvent (1-octanol), detergent (Triton X-100), enzymatic inactivation of the endotoxin using alkaline phosphatase and CIM monolytic anion exchange chromatography. We show that CsCl density purification of the P. aeruginosa phages, at an initial concentration of 10 12 -10 13 pfu/ml, led to the strongest reduction of endotoxins, with an endotoxin removal efficacy of up to 99%, whereas additional purification methods did not result in a complete removal of endotoxins from the phage preparations and only yielded an additional endotoxin removal efficacy of 23 to 99%, sometimes accompanied with strong losses in phage titer. Copyright © 2016 Elsevier B.V. All rights reserved.

Assessing the functionality and genetic diversity of lactococcal prophages.

PubMed

Kelleher, Philip; Mahony, Jennifer; Schweinlin, Katharina; Neve, Horst; Franz, Charles M; van Sinderen, Douwe

2018-05-02

Lactococcus lactis is a lactic acid bacterium that is intensively and globally exploited in commercial dairy food fermentations. Though the presence of prophages in lactococcal genomes is widely reported, only limited studies pertaining to the stability of prophages in lactococcal genomes have been performed. The current study reports on the complete genome exploration of thirty lactococcal strains for the presence of potentially intact prophages, so as to assess their genomic diversity and the associated risk or benefit of harbouring such prophages. Genomic predictions partnered with mitomycin C inductions and flow cytometric analysis of the induced cell lysates confirmed that only four strains consistently produced intact phage particles, thus indicating a relatively low risk associated with prophage induction in the fermentation setting. Our analysis revealed the widespread presence of putative phage-resistance systems encoded by lactococcal prophages, thus highlighting the potential benefits for host fitness. Many of the identified lactococcal prophages belong to the so-called P335 phage group, while a large group of phage remnants bear similarity to members of the 936 phage group. The P335 phage group was recently shown to encompass four distinct genetic lineages. Our study identified an additional lineage, thus expanding the diversity of this industrially significant phage group. Copyright © 2018 Elsevier B.V. All rights reserved.

Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection.

PubMed

Tikunova, N V; Morozova, V V

2009-10-01

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

Cytotoxic Tumor-Targeting Peptides From In Vivo Phage Display.

PubMed

Northup, Jessica R Newton; Deutscher, Susan L

2016-01-01

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

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Removal of bacteriophages with different surface charges by diverse ceramic membrane materials in pilot spiking tests.

PubMed

Hambsch, B; Bösl, M; Eberhagen, I; Müller, U

2012-01-01

This study examines mechanisms for removal of bacteriophages (MS2 and phiX174) by ceramic membranes without application of flocculants. The ceramic membranes considered included ultra- and microfiltration membranes of different materials. Phages were spiked into the feed water in pilot scale tests in a waterworks. The membranes with pore sizes of 10 nm provided a 2.5-4.0 log removal of the phages. For pore sizes of 50 nm, the log removal dropped to 0.96-1.8. The membrane with a pore size of 200 nm did not remove phages. So, the removal of both MS2- and phiX174-phages depended on the pore size of the membranes. But apart from pore size also other factors influence the removal of phages. Removal was 0.5-0.9 log higher for MS2-phages compared with phiX174-phages. Size exclusion seems to be the major but not the only mechanism which influences the efficiency of phage removal by ceramic membranes.

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

PubMed Central

Dziewit, Lukasz; Oscik, Karolina; Bartosik, Dariusz

2014-01-01

ABSTRACT ΦLM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that ΦLM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of ΦLM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage ΦLM21 shows very limited similarity to other known phage genome sequences and is thus considered unique. IMPORTANCE Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, ΦLM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of mimicking the host regulatory mechanisms by viruses is quite common in bacteriophages. PMID:25187538

Improvement and Optimization of Two Engineered Phage Resistance Mechanisms in Lactococcus lactis

PubMed Central

McGrath, Stephen; Fitzgerald, Gerald F.; van Sinderen, Douwe

2001-01-01

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17. One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages. DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case. PMID:11157223

Phage display and molecular imaging: expanding fields of vision in living subjects.

PubMed

Cochran, R; Cochran, Frank

2010-01-01

In vivo molecular imaging enables non-invasive visualization of biological processes within living subjects, and holds great promise for diagnosis and monitoring of disease. The ability to create new agents that bind to molecular targets and deliver imaging probes to desired locations in the body is critically important to further advance this field. To address this need, phage display, an established technology for the discovery and development of novel binding agents, is increasingly becoming a key component of many molecular imaging research programs. This review discusses the expanding role played by phage display in the field of molecular imaging with a focus on in vivo applications. Furthermore, new methodological advances in phage display that can be directly applied to the discovery and development of molecular imaging agents are described. Various phage library selection strategies are summarized and compared, including selections against purified target, intact cells, and ex vivo tissue, plus in vivo homing strategies. An outline of the process for converting polypeptides obtained from phage display library selections into successful in vivo imaging agents is provided, including strategies to optimize in vivo performance. Additionally, the use of phage particles as imaging agents is also described. In the latter part of the review, a survey of phage-derived in vivo imaging agents is presented, and important recent examples are highlighted. Other imaging applications are also discussed, such as the development of peptide tags for site-specific protein labeling and the use of phage as delivery agents for reporter genes. The review concludes with a discussion of how phage display technology will continue to impact both basic science and clinical applications in the field of molecular imaging.

Bacteriophages show promise as antimicrobial agents.

PubMed

Alisky, J; Iczkowski, K; Rapoport, A; Troitsky, N

1998-01-01

The emergence of antibiotic-resistant bacteria has prompted interest in alternatives to conventional drugs. One possible option is to use bacteriophages (phage) as antimicrobial agents. We have conducted a literature review of all Medline citations from 1966-1996 that dealt with the therapeutic use of phage. There were 27 papers from Poland, the Soviet Union, Britain and the U.S.A. The Polish and Soviets administered phage orally, topically or systemically to treat a wide variety of antibiotic-resistant pathogens in both adults and children. Infections included suppurative wound infections, gastroenteritis, sepsis, osteomyelitis, dermatitis, empyemas and pneumonia; pathogens included Staphylococcus, Streptococcus, Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella and Salmonella spp. Overall, the Polish and Soviets reported success rates of 80-95% for phage therapy, with rare, reversible gastrointestinal or allergic side effects. However, efficacy of phage was determined almost exclusively by qualitative clinical assessment of patients, and details of dosages and clinical criteria were very sketchy. There were also six British reports describing controlled trials of phage in animal models (mice, guinea pigs and livestock), measuring survival rates and other objective criteria. All of the British studies raised phage against specific pathogens then used to create experimental infections. Demonstrable efficacy against Escherichia, Acinetobacter, Pseudomonas and Staphylococcus spp. was noted in these model systems. Two U.S. papers dealt with improving the bioavailability of phage. Phage is sequestered in the spleen and removed from circulation. This can be overcome by serial passage of phage through mice to isolate mutants that resist sequestration. In conclusion, bacteriophages may show promise for treating antibiotic resistant pathogens. To facilitate further progress, directions for future research are discussed and a directory of authors from the reviewed papers is provided.

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

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.

Genetic discovery in Xylella fastidiosa through sequence analysis of selected randomly amplified polymorphic DNAs.

PubMed

Chen, Jianchi; Civerolo, Edwin L; Jarret, Robert L; Van Sluys, Marie-Anne; de Oliveira, Mariana C

2005-02-01

Xylella fastidiosa causes many important plant diseases including Pierce's disease (PD) in grape and almond leaf scorch disease (ALSD). DNA-based methodologies, such as randomly amplified polymorphic DNA (RAPD) analysis, have been playing key roles in genetic information collection of the bacterium. This study further analyzed the nucleotide sequences of selected RAPDs from X. fastidiosa strains in conjunction with the available genome sequence databases and unveiled several previously unknown novel genetic traits. These include a sequence highly similar to those in the phage family of Podoviridae. Genome comparisons among X. fastidiosa strains suggested that the "phage" is currently active. Two other RAPDs were also related to horizontal gene transfer: one was part of a broadly distributed cryptic plasmid and the other was associated with conjugal transfer. One RAPD inferred a genomic rearrangement event among X. fastidiosa PD strains and another identified a single nucleotide polymorphism of evolutionary value.

Genomic characteristics of vB_PpaP_PP74, a T7-like Autographivirinae bacteriophage infecting a potato pathogen of the newly proposed species Pectobacterium parmentieri.

PubMed

Kabanova, Anastasia; Shneider, Mikhail; Bugaeva, Eugenia; Ha, Vo Thi Ngoc; Miroshnikov, Kirill; Korzhenkov, Aleksei; Kulikov, Eugene; Toschakov, Stepan; Ignatov, Alexander; Miroshnikov, Konstantin

2018-06-01

Bacteriophage vB_PpaP_PP74 (PP74) is a novel virulent phage that infects members of the species Pectobacterium parmentieri, a newly established species of soft-rot-causing bacteria in the family Pectobacteriaceae, derived from potato-specific Pectobacterium wasabiae. vB_PpaP_PP74 was identified as a member of the family Podoviridae by transmission electron microscopy. The phage has a 39,790-bp dsDNA genome containing 50 open reading frames (ORFs). Because of the absence of genes encoding toxins or lysogeny factors, PP74 may be considered a candidate phage for pathogen biocontrol applications. The genome layout is similar to genomes of T7-like phages within the subfamily Autographivirinae, and therefore, functions can be attributed to most of ORFs. However, the closest nucleotide sequence homologs of phage PP74 are unclassified Escherichia phages. Based on phylogenetic analysis, vB_PpaP_PP74 is a sensu lato T7-like phage, but it forms a distant subgenus group together with homologous enterobacterial phages.

Novel application of bacteriophage for controlling foaming in wastewater treatment plant- an eco-friendly approach

PubMed Central

Khairnar, Krishna; Chandekar, Rajshree; Nair, Aparna; Pal, Preeti; Paunikar, Waman N.

2016-01-01

ABSTRACT This addendum to “Novel application of bacteriophage for controlling foaming in wastewater treatment plant- an eco-friendly approach “ includes characteristics of the phages NOC1, NOC2 and NOC3 not discussed in the previous paper. The phage adsorption and host interaction properties, their sensitivity to pH and temperature are inferred. NOC2 is seen to be more temperature resistant while others are not. All the phages show pH sensitivity. There is a variance observed in the behavior of these phages. Also, applicability of the phage based system to large scale reactors is studied and discussed here. PMID:26890996

Novel application of bacteriophage for controlling foaming in wastewater treatment plant- an eco-friendly approach.

PubMed

Khairnar, Krishna; Chandekar, Rajshree; Nair, Aparna; Pal, Preeti; Paunikar, Waman N

2016-01-01

This addendum to "Novel application of bacteriophage for controlling foaming in wastewater treatment plant- an eco-friendly approach " includes characteristics of the phages NOC1, NOC2 and NOC3 not discussed in the previous paper. The phage adsorption and host interaction properties, their sensitivity to pH and temperature are inferred. NOC2 is seen to be more temperature resistant while others are not. All the phages show pH sensitivity. There is a variance observed in the behavior of these phages. Also, applicability of the phage based system to large scale reactors is studied and discussed here.

Evolution of phage display technology: from discovery to application.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Ahmadzadeh, Vahideh; Akbari, Bahman

2017-03-01

Phage display technology as a selection-based system is an attractive method for evolution of new biological drugs. Unique ability of phage libraries for displaying proteins on bacteriophage surfaces enable them to make a major contribution in diverse fields of researches related to the diagnosis and therapy of diseases. One of the great challenges facing researchers is the modification of phage display technology and the development of new applications. This article reviews the molecular basis of phage display library, and summarizes the novel and specific applications of this technique in the field of biological drugs development including therapeutic antibodies, peptides, vaccines, and catalytic antibodies.

Biodiversity of Lactobacillus helveticus bacteriophages isolated from cheese whey starters.

PubMed

Zago, Miriam; Bonvini, Barbara; Rossetti, Lia; Meucci, Aurora; Giraffa, Giorgio; Carminati, Domenico

2015-05-01

Twenty-one Lactobacillus helveticus bacteriophages, 18 isolated from different cheese whey starters and three from CNRZ collection, were phenotypically and genetically characterised. A biodiversity between phages was evidenced both by host range and molecular (RAPD-PCR) typing. A more detailed characterisation of six phages showed similar structural protein profiles and a relevant genetic biodiversity, as shown by restriction enzyme analysis of total DNA. Latent period, burst time and burst size data evidenced that phages were active and virulent. Overall, data highlighted the biodiversity of Lb. helveticus phages isolated from cheese whey starters, which were confirmed to be one of the most common phage contamination source in dairy factories. More research is required to further unravel the ecological role of Lb. helveticus phages and to evaluate their impact on the dairy fermentation processes where whey starter cultures are used.

Application of Bacteroides fragilis phage as an alternative indicator of sewage pollution in Tampa Bay, Florida

USGS Publications Warehouse

McLaughlin, M.R.; Rose, J.B.

2006-01-01

Traditional fecal coliform bacterial indicators have been found to be severely limited in determining the significance and sources of fecal contamination in ambient waters of tropical and subtropical regions. The bacteriophages that infect Bacteroides fragilis have been suggested as better fecal indicators and at least one type may be human specific. In this study, the phages that infect B. fragilis host RYC2056 (RYC), including phage B56-3, and host ATCC 51477-HSP40 (HSP), including the human specific phage B40-8, were evaluated in the drainage basins of Tampa Bay, 7 samples (n = 62), or 11%, tested positive for the presence of phages infecting the host HSP, whereas 28 samples, or 45%, tested positive using the host RYC. A survival study was also done to compare the persistence of phages B56-3 and B40-8 to MS2 coliphage in seawater at various temperatures. The decay rates for MS2 were 0.239 log 10 d-1 at 10??C, but increased to 0.896 at 20??C and 2.62 log10 d-1 at 30??C. The two B. fragilis phages persisted much longer in the seawater compared to the coliphage and showed little variation between the temperatures. All sewage influents sampled from area wastewater treatment plants contained phages that infected the two B. fragilis hosts at levels from 1.2 ?? 104 to 1.11 ?? 10 5 pfu 100 ml-1 for host RYC and 67 to 350 pfu 100 ml -1 for host HSP. Of the 7 chlorinated effluent samples tested, 3 were positive for the presence of the phage using the host RYC and the phage enrichment method, with levels estimated to be <10 pfu 100 ml-1. No phages were detected using the host HSP in the treated sewage effluent. Coliphages were found in 3 of the 7 effluent samples at a range of 30 to 1.2 ?? 103 pfu 100 ml-1. ?? 2006 Estuarine Research Federation.

Prophage-mediated defense against viral attack and viral counter-defense

PubMed Central

Dedrick, Rebekah M.; Jacobs-Sera, Deborah; Guerrero Bustamante, Carlos A.; Garlena, Rebecca A.; Mavrich, Travis N.; Pope, Welkin H.; Reyes, Juan C Cervantes; Russell, Daniel A.; Adair, Tamarah; Alvey, Richard; Bonilla, J. Alfred; Bricker, Jerald S.; Brown, Bryony R.; Byrnes, Deanna; Cresawn, Steven G.; Davis, William B.; Dickson, Leon A.; Edgington, Nicholas P.; Findley, Ann M.; Golebiewska, Urszula; Grose, Julianne H.; Hayes, Cory F.; Hughes, Lee E.; Hutchison, Keith W.; Isern, Sharon; Johnson, Allison A.; Kenna, Margaret A.; Klyczek, Karen K.; Mageeney, Catherine M.; Michael, Scott F.; Molloy, Sally D.; Montgomery, Matthew T.; Neitzel, James; Page, Shallee T.; Pizzorno, Marie C.; Poxleitner, Marianne K.; Rinehart, Claire A.; Robinson, Courtney J.; Rubin, Michael R.; Teyim, Joseph N.; Vazquez, Edwin; Ware, Vassie C.; Washington, Jacqueline; Hatfull, Graham F.

2017-01-01

Temperate phages are common and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses infecting mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity, and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages reveals at least five distinct prophage-expressed viral defense systems that interfere with infection of lytic and temperate phages that are either closely-related (homotypic defense) or unrelated (heterotypic defense). Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defense systems include a single-subunit restriction system, a heterotypic exclusion system, and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival, and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, that acts as a highly effective counter-defense system. Prophage-mediated viral defense offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defense promotes phage co-evolution. PMID:28067906

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-09-01

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

Bacteriophages and dairy fermentations

PubMed Central

Marcó, Mariángeles Briggiler; Moineau, Sylvain; Quiberoni, Andrea

2012-01-01

This review highlights the main strategies available to control phage infection during large-scale milk fermentation by lactic acid bacteria. The topics that are emphasized include the factors influencing bacterial activities, the sources of phage contamination, the methods available to detect and quantify phages, as well as practical solutions to limit phage dispersion through an adapted factory design, the control of air flow, the use of adequate sanitizers, the restricted used of recycled products, and the selection and growth of bacterial cultures. PMID:23275866

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

PubMed

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

2013-03-28

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

Characterization and genome analysis of novel bacteriophages infecting the opportunistic human pathogens Klebsiella oxytoca and K. pneumoniae.

PubMed

Park, Eun-Ah; Kim, You-Tae; Cho, Jae-Hyun; Ryu, Sangryeol; Lee, Ju-Hoon

2017-04-01

Klebsiella is a genus of well-known opportunistic human pathogens that are associated with diabetes mellitus and chronic pulmonary obstruction; however, this pathogen is often resistant to multiple drugs. To control this pathogen, two Klebsiella-infecting phages, K. oxytoca phage PKO111 and K. pneumoniae phage PKP126, were isolated from a sewage sample. Analysis of their host range revealed that they infect K. pneumoniae and K. oxytoca, suggesting host specificity for members of the genus Klebsiella. Stability tests confirmed that the phages are stable under various temperature (4 to 60 °C) and pH (3 to 11) conditions. A challenge assay showed that PKO111 and PKP126 inhibit growth of their host strains by 2 log and 4 log, respectively. Complete genome sequencing of the phages revealed that their genome sizes are quite different (168,758 bp for PKO111 and 50,934 bp for PKP126). Their genome annotation results showed that they have no human virulence-related genes, an important safety consideration. In addition, no lysogen-formation gene cluster was detected in either phage genome, suggesting that they are both virulent phages in their bacterial hosts. Based on these results, PKO111 and PKP126 may be good candidates for development of biocontrol agents against members of the genus Klebsiella for therapeutic purposes. A comparative analysis of tail-associated gene clusters of PKO111 and PKP126 revealed relatively low homology, suggesting that they might differ in the way they recognize and infect their specific hosts.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence.

PubMed

Xie, Yicheng; Wahab, Laith; Gill, Jason J

2018-04-12

Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence

PubMed Central

Xie, Yicheng; Wahab, Laith

2018-01-01

Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format. PMID:29649135

The highly virulent 2006 Norwegian EHEC O103:H25 outbreak strain is related to the 2011 German O104:H4 outbreak strain.

PubMed

L'Abée-Lund, Trine M; Jørgensen, Hannah J; O'Sullivan, Kristin; Bohlin, Jon; Ligård, Goro; Granum, Per Einar; Lindbäck, Toril

2012-01-01

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx(2)-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages.

The Highly Virulent 2006 Norwegian EHEC O103:H25 Outbreak Strain Is Related to the 2011 German O104:H4 Outbreak Strain

PubMed Central

L'Abée-Lund, Trine M.; Jørgensen, Hannah J.; O'Sullivan, Kristin; Bohlin, Jon; Ligård, Goro; Granum, Per Einar; Lindbäck, Toril

2012-01-01

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx2-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages. PMID:22403614

Drug delivery vectors based on filamentous bacteriophages and phage-mimetic nanoparticles.

PubMed

Ju, Zhigang; Sun, Wei

2017-11-01

With the development of nanomedicine, a mass of nanocarriers have been exploited and utilized for targeted drug delivery, including liposomes, polymers, nanoparticles, viruses, and stem cells. Due to huge surface bearing capacity and flexible genetic engineering property, filamentous bacteriophage and phage-mimetic nanoparticles are attracting more and more attentions. As a rod-like bio-nanofiber without tropism to mammalian cells, filamentous phage can be easily loaded with drugs and directly delivered to the lesion location. In particular, chemical drugs can be conjugated on phage surface by chemical modification, and gene drugs can also be inserted into the genome of phage by recombinant DNA technology. Meanwhile, specific peptides/proteins displayed on the phage surface are able to conjugate with nanoparticles which will endow them specific-targeting and huge drug-loading capacity. Additionally, phage peptides/proteins can directly self-assemble into phage-mimetic nanoparticles which may be applied for self-navigating drug delivery nanovehicles. In this review, we summarize the production of phage particles, the identification of targeting peptides, and the recent applications of filamentous bacteriophages as well as their protein/peptide for targeting drug delivery in vitro and in vivo. The improvement of our understanding of filamentous bacteriophage and phage-mimetic nanoparticles will supply new tools for biotechnological approaches.

Immobilization of Active Bacteriophages on Polyhydroxyalkanoate Surfaces.

PubMed

Wang, Chanchan; Sauvageau, Dominic; Elias, Anastasia

2016-01-20

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

Development of phoH as a Novel Signature Gene for Assessing Marine Phage Diversity▿

PubMed Central

Goldsmith, Dawn B.; Crosti, Giuseppe; Dwivedi, Bhakti; McDaniel, Lauren D.; Varsani, Arvind; Suttle, Curtis A.; Weinbauer, Markus G.; Sandaa, Ruth-Anne; Breitbart, Mya

2011-01-01

Phages play a key role in the marine environment by regulating the transfer of energy between trophic levels and influencing global carbon and nutrient cycles. The diversity of marine phage communities remains difficult to characterize because of the lack of a signature gene common to all phages. Recent studies have demonstrated the presence of host-derived auxiliary metabolic genes in phage genomes, such as those belonging to the Pho regulon, which regulates phosphate uptake and metabolism under low-phosphate conditions. Among the completely sequenced phage genomes in GenBank, this study identified Pho regulon genes in nearly 40% of the marine phage genomes, while only 4% of nonmarine phage genomes contained these genes. While several Pho regulon genes were identified, phoH was the most prevalent, appearing in 42 out of 602 completely sequenced phage genomes. Phylogenetic analysis demonstrated that phage phoH sequences formed a cluster distinct from those of their bacterial hosts. PCR primers designed to amplify a region of the phoH gene were used to determine the diversity of phage phoH sequences throughout a depth profile in the Sargasso Sea and at six locations worldwide. phoH was present at all sites examined, and a high diversity of phoH sequences was recovered. Most phoH sequences belonged to clusters without any cultured representatives. Each depth and geographic location had a distinct phoH composition, although most phoH clusters were recovered from multiple sites. Overall, phoH is an effective signature gene for examining phage diversity in the marine environment. PMID:21926220

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

PubMed Central

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

2017-01-01

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

Isolation and characterization of a bacteriophage phiEap-2 infecting multidrug resistant Enterobacter aerogenes

PubMed Central

Li, Erna; Wei, Xiao; Ma, Yanyan; Yin, Zhe; Li, Huan; Lin, Weishi; Wang, Xuesong; Li, Chao; Shen, Zhiqiang; Zhao, Ruixiang; Yang, Huiying; Jiang, Aimin; Yang, Wenhui; Yuan, Jing; Zhao, Xiangna

2016-01-01

Enterobacter aerogenes (Enterobacteriaceae) is an important opportunistic pathogen that causes hospital-acquired pneumonia, bacteremia, and urinary tract infections. Recently, multidrug-resistant E. aerogenes have been a public health problem. To develop an effective antimicrobial agent, bacteriophage phiEap-2 was isolated from sewage and its genome was sequenced because of its ability to lyse the multidrug-resistant clinical E. aerogenes strain 3-SP. Morphological observations suggested that the phage belongs to the Siphoviridae family. Comparative genome analysis revealed that phage phiEap-2 is related to the Salmonella phage FSL SP-031 (KC139518). All of the structural gene products (except capsid protein) encoded by phiEap-2 had orthologous gene products in FSL SP-031 and Serratia phage Eta (KC460990). Here, we report the complete genome sequence of phiEap-2 and major findings from the genomic analysis. Knowledge of this phage might be helpful for developing therapeutic strategies against E. aerogenes. PMID:27320081

Isolation and characterization of a bacteriophage phiEap-2 infecting multidrug resistant Enterobacter aerogenes.

PubMed

Li, Erna; Wei, Xiao; Ma, Yanyan; Yin, Zhe; Li, Huan; Lin, Weishi; Wang, Xuesong; Li, Chao; Shen, Zhiqiang; Zhao, Ruixiang; Yang, Huiying; Jiang, Aimin; Yang, Wenhui; Yuan, Jing; Zhao, Xiangna

2016-06-20

Enterobacter aerogenes (Enterobacteriaceae) is an important opportunistic pathogen that causes hospital-acquired pneumonia, bacteremia, and urinary tract infections. Recently, multidrug-resistant E. aerogenes have been a public health problem. To develop an effective antimicrobial agent, bacteriophage phiEap-2 was isolated from sewage and its genome was sequenced because of its ability to lyse the multidrug-resistant clinical E. aerogenes strain 3-SP. Morphological observations suggested that the phage belongs to the Siphoviridae family. Comparative genome analysis revealed that phage phiEap-2 is related to the Salmonella phage FSL SP-031 (KC139518). All of the structural gene products (except capsid protein) encoded by phiEap-2 had orthologous gene products in FSL SP-031 and Serratia phage Eta (KC460990). Here, we report the complete genome sequence of phiEap-2 and major findings from the genomic analysis. Knowledge of this phage might be helpful for developing therapeutic strategies against E. aerogenes.

A bacteriophages journey through the human body.

PubMed

Barr, Jeremy J

2017-09-01

The human body is colonized by a diverse collective of microorganisms, including bacteria, fungi, protozoa and viruses. The smallest entity of this microbial conglomerate are the bacterial viruses. Bacteriophages, or phages for short, exert significant selective pressure on their bacterial hosts, undoubtedly influencing the human microbiome and its impact on our health and well-being. Phages colonize all niches of the body, including the skin, oral cavity, lungs, gut, and urinary tract. As such our bodies are frequently and continuously exposed to diverse collections of phages. Despite the prevalence of phages throughout our bodies, the extent of their interactions with human cells, organs, and immune system is still largely unknown. Phages physically interact with our mucosal surfaces, are capable of bypassing epithelial cell layers, disseminate throughout the body and may manipulate our immune system. Here, I establish the novel concept of an "intra-body phageome," which encompasses the collection of phages residing within the classically "sterile" regions of the body. This review will take a phage-centric view of the microbiota, human body, and immune system with the ultimate goal of inspiring a greater appreciation for both the indirect and direct interactions between bacteriophages and their mammalian hosts. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Marine Phages As Tracers: Effects of Size, Morphology, and Physico-Chemical Surface Properties on Transport in a Porous Medium.

PubMed

Ghanem, Nawras; Kiesel, Bärbel; Kallies, René; Harms, Hauke; Chatzinotas, Antonis; Wick, Lukas Y

2016-12-06

Although several studies examined the transport of viruses in terrestrial systems only few studies exist on the use of marine phages (i.e., nonterrestrial viruses infecting marine host bacteria) as sensitively detectable microbial tracers for subsurface colloid transport and water flow. Here, we systematically quantified and compared for the first time the effects of size, morphology and physicochemical surface properties of six marine phages and two coliphages (MS2, T4) on transport in sand-filled percolated columns. Phage-sand interactions were described by colloidal filtration theory and the extended Derjaguin-Landau-Verwey-Overbeek approach (XDLVO), respectively. The phages belonged to different families and comprised four phages never used in transport studies (i.e., PSA-HM1, PSA-HP1, PSA-HS2, and H3/49). Phage transport was influenced by size, morphology and hydrophobicity in an approximate order of size > hydrophobicity ≥ morphology. Two phages PSA-HP1, PSA-HS2 (Podoviridae and Siphoviridae) exhibited similar mass recovery as commonly used coliphage MS2 and were 7-fold better transported than known marine phage vB_PSPS-H40/1. Differing properties of the marine phages may be used to trace transport of indigenous viruses, natural colloids or anthropogenic nanomaterials and, hence, contribute to better risk analysis. Our results underpin the potential role of marine phages as microbial tracer for transport of colloidal particles and water flow.

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.

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

PubMed Central

Osório, Nádia; Pereira, Carla; Simões, Sara; Delgadillo, Ivonne

2018-01-01

The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells. PMID:29518018

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

PubMed

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Comparative genomic analysis of bacteriophages specific to the channel catfish pathogen Edwardsiella ictaluri

PubMed Central

2011-01-01

Background The bacterial pathogen Edwardsiella ictaluri is a primary cause of mortality in channel catfish raised commercially in aquaculture farms. Additional treatment and diagnostic regimes are needed for this enteric pathogen, motivating the discovery and characterization of bacteriophages specific to E. ictaluri. Results The genomes of three Edwardsiella ictaluri-specific bacteriophages isolated from geographically distant aquaculture ponds, at different times, were sequenced and analyzed. The genomes for phages eiAU, eiDWF, and eiMSLS are 42.80 kbp, 42.12 kbp, and 42.69 kbp, respectively, and are greater than 95% identical to each other at the nucleotide level. Nucleotide differences were mostly observed in non-coding regions and in structural proteins, with significant variability in the sequences of putative tail fiber proteins. The genome organization of these phages exhibit a pattern shared by other Siphoviridae. Conclusions These E. ictaluri-specific phage genomes reveal considerable conservation of genomic architecture and sequence identity, even with considerable temporal and spatial divergence in their isolation. Their genomic homogeneity is similarly observed among E. ictaluri bacterial isolates. The genomic analysis of these phages supports the conclusion that these are virulent phages, lacking the capacity for lysogeny or expression of virulence genes. This study contributes to our knowledge of phage genomic diversity and facilitates studies on the diagnostic and therapeutic applications of these phages. PMID:21214923

Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis

PubMed Central

Wittmann, Johannes; Gartemann, Karl-Heinz; Eichenlaub, Rudolf

2011-01-01

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections. PMID:21687530

Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis.

PubMed

Wittmann, Johannes; Gartemann, Karl-Heinz; Eichenlaub, Rudolf; Dreiseikelmann, Brigitte

2011-01-01

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections.

Bacteriophages and their derivatives for the treatment and control of food-producing animal infections.

PubMed

Carvalho, Carla; Costa, Ana Rita; Silva, Filipe; Oliveira, Ana

2017-09-01

Nowadays, the world is facing an increasing emergence of antibiotic resistant bacteria. Simultaneously, the banning of some existing antibiotics and the lack of development of new antimicrobials have created an urgent need to find new alternatives against animal infections. Bacteriophages (phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and harmless to animals. For these reasons, phages and their derivatives are being considered valuable antimicrobial alternatives and an opportunity to reduce the current use of antibiotics in agri-food production, increasing animal productivity and providing environmental protection. Furthermore, the possibility of combining phage genetic material with foreign genes encoding peptides of interest has enabled their use as vaccine delivery tools. In this case, besides bacterial infections, they might be used to prevent viral infections. This review explores current data regarding advances on the use of phages and phage-encoded proteins, such as endolysins, exolysins and depolymerases, either for therapeutic or prophylactic applications, in animal husbandry. The use of recombinant phage-derived particles or genetically modified phages, including phage vaccines, will also be reviewed.

Survey on the phage resistance mechanisms displayed by a dairy Lactobacillus helveticus strain.

PubMed

Zago, Miriam; Orrù, Luigi; Rossetti, Lia; Lamontanara, Antonella; Fornasari, Maria Emanuela; Bonvini, Barbara; Meucci, Aurora; Carminati, Domenico; Cattivelli, Luigi; Giraffa, Giorgio

2017-09-01

In this study the presence and functionality of phage defence mechanisms in Lactobacillus helveticus ATCC 10386, a strain of dairy origin which is sensitive to ΦLh56, were investigated. After exposure of ATCC 10386 to ΦLh56, the whole-genome sequences of ATCC 10386 and of a phage-resistant derivative (LhM3) were compared. LhM3 showed deletions in the S-layer protein and a higher expression of the genes involved in the restriction/modification (R/M) system. Genetic data were substantiated by measurements of bacteriophage adsorption rates, efficiency of plaquing, cell wall protein size and by gene expression analysis. In LhM3 two phage resistance mechanisms, the inhibition of phage adsorption and the upregulation of Type I R/M genes, take place and explain its resistance to ΦLh56. Although present in both ATCC 10386 and LhM3 genomes, the CRISPR machinery did not seem to play a role in the phage resistance of LhM3. Overall, the natural selection of phage resistant strains resulted successful in detecting variants carrying multiple phage defence mechanisms in L. helveticus. The concurrent presence of multiple phage-resistance systems should provide starter strains with increased fitness and robustness in dairy ecosystems. Copyright © 2017 Elsevier Ltd. All rights reserved.

The First Siphoviridae Family Bacteriophages Infecting Bordetella bronchiseptica Isolated from Environment.

PubMed

Petrovic, Aleksandra; Kostanjsek, Rok; Rakhely, Gabor; Knezevic, Petar

2017-02-01

Bordetella bronchiseptica is a well-known etiological agent of kennel cough in dogs and cats and one of the two causative agents of atrophic rhinitis, a serious swine disease. The aim of the study was to isolate B. bronchiseptica bacteriophages from environmental samples for the first time. A total of 29 phages from 65 water samples were isolated using the strain ATCC 10580 as a host. The lytic spectra of the phages were examined at 25 and 37 °C, using 12 strains of B. bronchiseptica. All phages were able to plaque on 25.0 % to 41.7 % of the strains. The selected phages showed similar morphology (Siphoviridae, morphotype B2), but variation of RFLP patterns and efficacy of plating on various strains. The partial genome sequence of phage vB_BbrS_CN1 showed its similarity to phages from genus Yuavirus. Using PCR, it was confirmed that the phages do not originate from the host strain, and environmental origin was additionally confirmed by the analysis of host genome sequence in silico and plating heated and unheated samples in parallel. Accordingly, this is the first isolation of B. bronchiseptica phages from environment and the first isolation and characterization of phages of B. bronchiseptica belonging to family Siphoviridae.

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

PubMed

Chan, Yi-Wah; Mohr, Remus; Millard, Andrew D; Holmes, Antony B; Larkum, Anthony W; Whitworth, Anna L; Mann, Nicholas H; Scanlan, David J; Hess, Wolfgang R; Clokie, Martha R J

2011-08-01

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

Isolation and Genome Characterization of the Virulent Staphylococcus aureus Bacteriophage SA97

PubMed Central

Chang, Yoonjee; Shin, Hakdong; Lee, Ju-Hoon; Park, Chul Jong; Paik, Soon-Young; Ryu, Sangryeol

2015-01-01

A novel bacteriophage that infects S. aureus, SA97, was isolated and characterized. The phage SA97 belongs to the Siphoviridae family, and the cell wall teichoic acid (WTA) was found to be a host receptor of the phage SA97. Genome analysis revealed that SA97 contains 40,592 bp of DNA encoding 54 predicted open reading frames (ORFs), and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus. PMID:26437428

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

PubMed

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

2015-03-01

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

Engineering of filamentous bacteriophage for protein sensing

NASA Astrophysics Data System (ADS)

Brasino, Michael

Methods of high throughput, sensitive and cost effective quantification of proteins enables personalized medicine by allowing healthcare professionals to better monitor patient condition and response to treatment. My doctoral research has attempted to advance these methods through the use of filamentous bacteriophage (phage). These bacterial viruses are particularly amenable to both genetic and chemical engineering and can be produced efficiently in large amounts. Here, I discuss several strategies for modifying phage for use in protein sensing assays. These include the expression of bio-orthogonal conjugation handles on the phage coat, the incorporation of specific recognition sequences within the phage genome, and the creation of antibody-phage conjugates via a photo-crosslinking non-canonical amino acid. The physical and chemical characterization of these engineered phage and the results of their use in modified protein sensing assays will be presented.

Incorporating Phage Therapy into WPI Dip Coatings for Applications on Fresh Whole and Cut Fruit and Vegetable Surfaces.

PubMed

Vonasek, Erica L; Choi, Angela H; Sanchez, Juan; Nitin, Nitin

2018-06-15

There is a significant unmet need to develop antimicrobial solutions to reduce the risk of contamination in fresh produce. Bacteriophages have been proposed as a potential approach for controlling foodborne pathogens. This study evaluated the combination of edible dip coatings with T7 bacteriophages on whole and cut produce. The evaluation includes an assessment of phage loading, phage storage stability, antimicrobial activity, and phage stability during simulated gastric digestion on sliced cucumbers, sliced apples, and whole cherry tomatoes. In this evaluation, phages coated on fresh produce using edible whey protein isolate (WPI) were compared with phages coated from an aqueous suspension (control coating). The results demonstrated that WPI coatings load more phages than the control and enhanced phage stability during cold storage (4 °C) for cut apples and whole cherry tomatoes. Phage stability decreased by 1 to 3 log(PFU) in a simulated gastric environment. Phage antimicrobial activity against Escherichia coli BL21 decreased 2 to 4 log(CFU) of bacteria on cut apples and whole cherry tomatoes, while no significant bacterial reduction was observed for sliced cucumbers. Overall, the results show that WPI dip coating provides phage loading, stability, and antimicrobial activity to produce surfaces compared to the control coating, and thus may be considered an effective approach for extending phage therapy on fresh produce. The practical application is to prevent bacterial cross contamination of fresh produce by using a combination of edible coating with bacteriophages. The results demonstrate enhanced loading and stability of phages on fresh produce when used in combination with an edible coating. © 2018 Institute of Food Technologists®.

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

PubMed Central

2013-01-01

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

Application of Fourier transform infrared spectroscopy and chemometrics for differentiation of Salmonella enterica serovar Enteritidis phage types.

PubMed

Preisner, Ornella; Guiomar, Raquel; Machado, Jorge; Menezes, José Cardoso; Lopes, João Almeida

2010-06-01

Fourier transform infrared (FT-IR) spectroscopy and chemometric techniques were used to discriminate five closely related Salmonella enterica serotype Enteritidis phage types, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a. Intact cells and outer membrane protein (OMP) extracts from bacterial cell membranes were subjected to FT-IR analysis in transmittance mode. Spectra were collected over a wavenumber range from 4,000 to 600 cm(-1). Partial least-squares discriminant analysis (PLS-DA) was used to develop calibration models based on preprocessed FT-IR spectra. The analysis based on OMP extracts provided greater separation between the Salmonella Enteritidis PT1-PT1b, PT4b, and PT6-PT6a groups than the intact cell analysis. When these three phage type groups were considered, the method based on OMP extract FT-IR spectra was 100% accurate. Moreover, complementary local models that considered only the PT1-PT1b and PT6-PT6a groups were developed, and the level of discrimination increased. PT1 and PT1b isolates were differentiated successfully with the local model using the entire OMP extract spectrum (98.3% correct predictions), whereas the accuracy of discrimination between PT6 and PT6a isolates was 86.0%. Isolates belonging to different phage types (PT19, PT20, and PT21) were used with the model to test its robustness. For the first time it was demonstrated that FT-IR analysis of OMP extracts can be used for construction of robust models that allow fast and accurate discrimination of different Salmonella Enteritidis phage types.

Phage typing or CRISPR typing for epidemiological surveillance of Salmonella Typhimurium?

PubMed

Mohammed, Manal

2017-11-07

Salmonella Typhimurium is the most dominant Salmonella serovar around the world. It is associated with foodborne gastroenteritis outbreaks but has recently been associated with invasive illness and deaths. Characterization of S. Typhimurium is therefore very crucial for epidemiological surveillance. Phage typing has been used for decades for subtyping of S. Typhimurium to determine the epidemiological relation among isolates. Recent studies however have suggested that high throughput clustered regular interspaced short palindromic repeats (CRISPR) typing has the potential to replace phage typing. This study aimed to determine the efficacy of high-throughput CRISPR typing over conventional phage typing in epidemiological surveillance and outbreak investigation of S. Typhimurium. In silico analysis of whole genome sequences (WGS) of well-documented phage types of S. Typhimurium reveals the presence of different CRISPR type among strains belong to the same phage type. Furthermore, different phage types of S. Typhimurium share identical CRISPR type. Interestingly, identical spacers were detected among outbreak and non-outbreak associated DT8 strains of S. Typhimurium. Therefore, CRISPR typing is not useful for the epidemiological surveillance and outbreak investigation of S. Typhimurium and phage typing, until it is replaced by WGS, is still the gold standard method for epidemiological surveillance of S. Typhimurium.

A cocktail of in vitro efficient phages is not a guarantee for in vivo therapeutic results against avian colibacillosis.

PubMed

Tsonos, Jessica; Oosterik, Leon H; Tuntufye, Huruma N; Klumpp, Jochen; Butaye, Patrick; De Greve, Henri; Hernalsteens, Jean-Pierre; Lavigne, Rob; Goddeeris, Bruno M

2014-07-16

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, leading to important economic losses worldwide. To cure APEC-infected chickens, a cocktail of four different APEC-specific bacteriophages (phages) was composed and tested. Specific phages were selected from a collection of phages isolated in Belgium. The selection was based on their obligate lytic infection cycle, a broad host range, low cross-resistance and low frequency of development of resistant APEC mutants. Genome analysis of the phages indicated they were close relatives of T4 and N4, considered to be safe in vivo. Chickens were intratracheally infected with APEC strain CH2 (serogroup O78), causing a mortality of about 50% during the seven days following the infection. The phage cocktail was administered 2h after the infection, via three different ways: intratracheally, intra-esophageally or via the drinking water. Treated groups did not show a significant decrease in mortality, lesion scores or weight loss compared to untreated groups, although the APEC-specific phages could be re-isolated from the lung and heart of chickens that were euthanized. Moreover, the re-isolated bacteria from infected chickens had remained sensitive to the phage cocktail. Our results indicate that the efficiency of the phage cocktail used in treating CH2-infected chickens in vivo is negligible, even though in vitro, the phages in the cocktail were able to efficiently lyse the APEC strain CH2. Our results emphasize that the 'traditional' pathway of isolation, followed by phenotypical and genotypical characterization of phages composing the cocktail, does not lead to success in phage therapy in all cases. Copyright © 2013 Elsevier B.V. All rights reserved.

Targeting GPR110 in HER2-Overexpressing Breast Cancers

DTIC Science & Technology

2015-10-01

lentiviral plasmids containing GPR110 cDNA using the pHAGE system, which includes the HA tag, under the control of inducible Tet-on promoter. The map of... pHAGE lentiviral plasmid is shown in Figure 3A. Using this, the BT474 and SKBR3 parental cells were stably infected with the lentiviral plasmid...in HER2+ breast cancer. Figure𔃽.’GPR110/overexpression’using’pHAGE’len:viral’mediated’infec:on’of’BT474’cells.’ A.#Map#of# pHAGE # len/viral

Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening.

PubMed

Mazor, Yariv; Van Blarcom, Thomas; Carroll, Sean; Georgiou, George

2010-05-01

Phage display of antibody libraries is a powerful tool for antibody discovery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab')(2). We recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluorescence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol 25, 563-565]. Although, as a single-cell analysis technique, FACS is very powerful, especially for the isolation of high-affinity binders, even with state of the art instrumentation the screening of libraries with diversity > 10(8) is technically challenging. We report here a system that takes advantage of display of full-length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid-encoded IgG with the fUSE5-ZZ phage as a helper phage. These phage particles display the Fc-binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demonstrate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without undersampling, and at the same time capitalizes on the advantages of FACS for real-time monitoring and optimization of the screening process.

A facile method to screen inhibitors of protein-protein interactions including MDM2-p53 displayed on T7 phage.

PubMed

Ishi, Kazutomo; Sugawara, Fumio

2008-05-01

Protein-protein interactions are essential in many biological processes including cell cycle and apoptosis. It is currently of great medical interest to inhibit specific protein-protein interactions in order to treat a variety of disease states. Here, we describe a facile multiwell plate assay method using T7 phage display to screen for candidate inhibitors of protein-protein interactions. Because T7 phage display is an effective method for detecting protein-protein interactions, we aimed to utilize this technique to screen for small-molecule inhibitors that disrupt these types of interaction. We used the well-characterized interaction between p53 and MDM2 and an inhibitor of this interaction, nutlin 3, as a model system to establish a new screening method. Phage particles displaying p53 interacted with GST-MDM2 immobilized on 96-well plates, and the interaction was inhibited by nutlin 3. Multiwell plate assay was then performed using a natural product library, which identified dehydroaltenusin as a candidate inhibitor of the p53-MDM2 interaction. We discuss the potential applications of this novel T7 phage display methodology, which we propose to call 'reverse phage display'.

Encapsulation Strategies of Bacteriophage (Felix O1) for Oral Therapeutic Application.

PubMed

Islam, Golam S; Wang, Qi; Sabour, Parviz M

2018-01-01

Due to emerging antibiotic-resistant strains among the pathogens, a variety of strategies, including therapeutic application of bacteriophages, have been suggested as a possible alternative to antibiotics in food animal production. As pathogen-specific biocontrol agents, bacteriophages are being studied intensively. Primarily their applications in the food industry and animal production have been recognized in the USA and Europe, for pathogens including Salmonella, Campylobacter, Escherichia coli, and Listeria. However, the viability of orally administered phage may rapidly reduce under the harsh acidic conditions of the stomach, presence of enzymes and bile. It is evident that bacteriophages, intended for phage therapy by oral administration, require efficient protection from the acidic environment of the stomach and should remain active in the animal's gastrointestinal tract where pathogen colonizes. Encapsulation of phages by spray drying or extrusion methods can protect phages from the simulated hostile gut conditions and help controlled release of phages to the digestive system when appropriate formulation strategy is implemented.

Phylogenetic Diversity of T4-Type Phages in Sediments from the Subtropical Pearl River Estuary

PubMed Central

He, Maoqiu; Cai, Lanlan; Zhang, Chuanlun; Jiao, Nianzhi; Zhang, Rui

2017-01-01

Viruses are an abundant and active component of marine sediments and play a significant role in microbial ecology and biogeochemical cycling at local and global scales. To obtain a better understanding of the ecological characteristics of the viriobenthos, the abundance and morphology of viruses and the diversity and community structure of T4-type phages were systematically investigated in the surface sediments of the subtropical Pearl River Estuary (PRE). Viral abundances ranged from 4.49 × 108 to 11.7 × 108 viruses/g and prokaryotic abundances ranged from 2.63 × 108 to 9.55 × 108 cells/g, and both decreased from freshwater to saltwater. Diverse viral morphotypes, including tailed, spherical, filamentous, and rod-shaped viruses, were observed using transmission electron microscopy. Analysis of the major capsid gene (g23) indicated that the sediment T4-type phages were highly diverse and, similar to the trend in viral abundances, their diversity decreased as the salinity increased. Phylogenetic analysis suggested that most of the g23 operational taxonomic units were affiliated with marine, paddy soil, and lake groups. The T4-type phage communities in freshwater and saltwater sediments showed obvious differences, which were related to changes in the Pearl River discharge. The results of this study demonstrated both allochthonous and autochthonous sources of the viral community in the PRE sediments and the movement of certain T4-type viral groups between the freshwater and saline water biomes. PMID:28572798

Stability and infectivity of cytolethal distending toxin type V gene-carrying bacteriophages in a water mesocosm and under different inactivation conditions.

PubMed

Allué-Guardia, Anna; Jofre, Juan; Muniesa, Maite

2012-08-01

Two cytolethal distending toxin (Cdt) type V-encoding bacteriophages (Φ62 and Φ125) were induced spontaneously from their wild-type Escherichia coli strains and from the lysogens generated in Shigella sonnei. The stability of Cdt phages was determined at various temperatures and pH values after 1 month of storage by means of infectivity tests using a plaque blot assay and analysis of phage genomes using real-time quantitative PCR (qPCR): both were highly stable. We assessed the inactivation of Cdt phages by thermal treatment, chlorination, UV radiation, and in a mesocosm in both summer and winter. The results for the two Cdt phages showed similar trends and were also similar to the phage SOM23 used for reference, but they showed a much higher persistence than Cdt-producing E. coli. Cdt phages showed maximal inactivation after 1 h at 70°C, 30 min of UV radiation, and 30 min of contact with a 10-ppm chlorine treatment. Inactivation in a mesocosm was higher in summer than in winter, probably because of solar radiation. The treatments reduced the number of infectious phages but did not have a significant effect on the Cdt phage particles detected by qPCR. Cdt phages were quantified by qPCR in 73% of river samples, and these results suggest that Cdt phages are a genetic vehicle and the natural reservoir for cdt in the environment.

Phage phenomics: Physiological approaches to characterize novel viral proteins

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

Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.

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

Phage phenomics: Physiological approaches to characterize novel viral proteins

DOE PAGES

Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; ...

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

Characterization and complete genome sequence of a novel N4-like bacteriophage, pSb-1 infecting Shigella boydii.

PubMed

Jun, Jin Woo; Yun, Sae Kil; Kim, Hyoun Joong; Chai, Ji Young; Park, Se Chang

2014-10-01

Shigellosis is one of major foodborne pathogens in both developed and developing countries. Although antibiotic therapy is considered an effective treatment for shigellosis, the imprudent use of antibiotics has led to the increase of multiple-antibiotic-resistant Shigella species globally. In this study, we isolated a virulent Podoviridae bacteriophage (phage), pSb-1, that infects Shigella boydii. One-step growth analysis revealed that this phage has a short latent period (15 min) and a large burst size (152.63 PFU/cell), indicating that pSb-1 has good host infectivity and effective lytic activity. The double-stranded DNA genome of pSb-1 is composed of 71,629 bp with a G + C content of 42.74%. The genome encodes 103 putative ORFs, 9 putative promoters, 21 transcriptional terminators, and one tRNA region. Genome sequence analysis of pSb-1 and comparative analysis with the homologous phage EC1-UPM, N4-like phage revealed that there is a high degree of similarity (94%, nucleotide sequence identity) between pSb-1 and EC1-UPM in 73 of the 103 ORFs of pSb-1. The results of this investigation indicate that pSb-1 is a novel virulent N4-like phage infecting S. boydii and that this phage might have potential uses against shigellosis. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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

PubMed

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

2017-10-01

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

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

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

Crown, Kevin K.; Santarpia, Joshua

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

Phage-Enabled Nanomedicine: From Probes to Therapeutics in Precision Medicine.

PubMed

Sunderland, Kegan S; Yang, Mingying; Mao, Chuanbin

2017-02-13

Both lytic and temperate bacteriophages (phages) can be applied in nanomedicine, in particular, as nanoprobes for precise disease diagnosis and nanotherapeutics for targeted disease treatment. Since phages are bacteria-specific viruses, they do not naturally infect eukaryotic cells and are not toxic to them. They can be genetically engineered to target nanoparticles, cells, tissues, and organs, and can also be modified with functional abiotic nanomaterials for disease diagnosis and treatment. This Review will summarize the current use of phage structures in many aspects of precision nanomedicine, including ultrasensitive biomarker detection, enhanced bioimaging for disease diagnosis, targeted drug and gene delivery, directed stem cell differentiation, accelerated tissue formation, effective vaccination, and nanotherapeutics for targeted disease treatment. We will also propose future directions in the area of phage-based nanomedicines, and discuss the state of phage-based clinical trials. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

Cao, Binrui; Yang, Mingying; Mao, Chuanbin

2016-06-21

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

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

PubMed Central

Cao, Binrui; Yang, Mingying; Mao, Chuanbin

2016-01-01

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

Metagenomic Analysis of Therapeutic PYO Phage Cocktails from 1997 to 2014

PubMed Central

Larsen, Mette Voldby

2017-01-01

Phage therapy has regained interest in recent years due to the alarming spread of antibiotic resistance. Whilst phage cocktails are commonly sold in pharmacies in countries such as Georgia and Russia, this is not the case in western countries due to western regulatory agencies requiring a thorough characterization of the drug. Here, DNA sequencing of constituent biological entities constitutes a first step. The pyophage (PYO) cocktail is one of the main commercial products of the Georgian Eliava Institute of Bacteriophage, Microbiology and Virology and is used to cure skin infections. Since its first production in the 1930s, the composition of the cocktail has been periodically modified to add phages effective against emerging pathogenic strains. In this paper, we compared the composition of three PYO cocktails from 1997 (PYO97), 2000 (PYO2000) and 2014 (PYO2014). Based on next generation sequencing, de novo assembly and binning of contigs into draft genomes based on tetranucleotide distance, thirty and twenty-nine phage draft genomes were predicted in PYO97 and PYO2014, respectively. Of these, thirteen and fifteen shared high similarity to known phages. Eleven draft genomes were found to be common in the two cocktails. One of these showed no similarity to publicly available phage genomes. Representatives of phages targeting E. faecalis, E. faecium, E. coli, Proteus, P. aeruginosa and S. aureus were found in both cocktails. Finally, we estimated larger overlap of the PYO2000 cocktail to PYO97 compared to PYO2014. Using next generation sequencing and metagenomics analysis, we were able to characterize and compare the content of PYO cocktails separated by 17 years in time. Even though the cocktail composition is upgraded every six months, we found it to remain relatively stable over the years. PMID:29099783

Genomic analysis of cold-active Colwelliaphage 9A and psychrophilic phage-host interactions.

PubMed

Colangelo-Lillis, Jesse R; Deming, Jody W

2013-01-01

The 104 kb genome of cold-active bacteriophage 9A, which replicates in the marine psychrophilic gamma-proteobacterium Colwellia psychrerythraea strain 34H (between -12 and 8 °C), was sequenced and analyzed to investigate elements of molecular adaptation to low temperature and phage-host interactions in the cold. Most characterized ORFs indicated closest similarity to gamma-proteobacteria and their phages, though no single module provided definitive phylogenetic grouping. A subset of primary structural features linked to psychrophily suggested that the majority of annotated phage proteins were not psychrophilic; those that were, primarily serve phage-specific functions and may also contribute to 9A's restricted temperature range for replication as compared to host. Comparative analyses suggest ribonucleotide reductase genes were acquired laterally from host. Neither restriction modification nor the CRISPR-Cas system appeared to be the predominant phage defense mechanism of Cp34H or other cold-adapted bacteria; we hypothesize that psychrophilic hosts rely more on the use of extracellular polymeric material to block cell surface receptors recognized by phages. The relative dearth of evidence for genome-specific defenses, genetic transfer events or auxiliary metabolic genes suggest that the 9A-Cp34H system may be less tightly coupled than are other genomically characterized marine phage-host systems, with possible implications for phage specificity under different environmental conditions.

Novel N4 Bacteriophages Prevail in the Cold Biosphere.

PubMed

Zhan, Yuanchao; Buchan, Alison; Chen, Feng

2015-08-01

Coliphage N4 is a lytic bacteriophage discovered nearly half a century ago, and it was considered to be a "genetic orphan" until very recently, when several additional N4-like phages were discovered to infect nonenteric bacterial hosts. Interest in this genus of phages is stimulated by their unique genetic features and propagation strategies. To better understand the ecology of N4-like phages, we investigated the diversity and geographic patterns of N4-like phages by examining 56 Chesapeake Bay viral communities, using a PCR-clone library approach targeting a diagnostic N4-like DNA polymerase gene. Many new lineages of N4-like phages were found in the bay, and their genotypes shift from the lower to the upper bay. Interestingly, signature sequences of N4-like phages were recovered only from winter month samples, when water temperatures were below 4°C. An analysis of existing metagenomic libraries from various aquatic environments supports the hypothesis that N4-like phages are most prolific in colder waters. In particular, a high number of N4-like phages were detected in Organic Lake, Antarctica, a cold and hypersaline system. The prevalence of N4-like phages in the cold biosphere suggests these viruses possess yet-to-be-determined mechanisms that facilitate lytic infections under cold conditions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization and Genomic Study of Phage vB_EcoS-B2 Infecting Multidrug-Resistant Escherichia coli

PubMed Central

Xu, Yue; Yu, Xinyan; Gu, Yu; Huang, Xu; Liu, Genyan; Liu, Xiaoqiu

2018-01-01

The potential of bacteriophage as an alternative antibacterial agent has been reconsidered for control of pathogenic bacteria due to the widespread occurrence of multi-drug resistance bacteria. More and more lytic phages have been isolated recently. In the present study, we isolated a lytic phage named vB_EcoS-B2 from waste water. VB_EcoS-B2 has an icosahedral symmetry head and a long tail without a contractile sheath, indicating that it belongs to the family Siphoviridae. The complete genome of vB_EcoS-B2 is composed of a circular double stranded DNA of 44,283 bp in length, with 54.77% GC content. vB_EcoS-B2 is homologous to 14 relative phages (such as Escherichia phage SSL-2009a, Escherichia phage JL1, and Shigella phage EP23), but most of these phages exhibit different gene arrangement. Our results serve to extend our understanding toward phage evolution of family Siphoviridae of coliphages. Sixty-five putative open reading frames were predicted in the complete genome of vB_EcoS-B2. Twenty-one of proteins encoded by vB_EcoS-B2 were determined in phage particles by Mass Spectrometry. Bacteriophage genome and proteome analysis confirmed the lytic nature of vB_EcoS-B2, namely, the absence of toxin-coding genes, islands of pathogenicity, or genes through lysogeny or transduction. Furthermore, vB_EcoS-B2 significantly reduced the growth of E. coli MG1655 and also inhibited the growth of several multi-drug resistant clinical stains of E. coli. Phage vB_EcoS-B2 can kill some of the MRD E. coli entirely, strongly indicating us that it could be one of the components of phage cocktails to treat multi-drug resistant E. coli. This phage could be used to interrupt or reduce the spread of multi-drug resistant E. coli. PMID:29780362

Integrated Multivariate Analysis with Nondetects for the Development of Human Sewage Source-Tracking Tools Using Bacteriophages of Enterococcus faecalis.

PubMed

Wangkahad, Bencharong; Mongkolsuk, Skorn; Sirikanchana, Kwanrawee

2017-02-21

We developed sewage-specific microbial source tracking (MST) tools using enterococci bacteriophages and evaluated their performance with univariate and multivariate analyses involving data below detection limits. Newly isolated Enterococci faecalis bacterial strains AIM06 (DSM100702) and SR14 (DSM100701) demonstrated 100% specificity and 90% sensitivity to human sewage without detecting 68 animal manure pooled samples of cats, chickens, cows, dogs, ducks, pigs, and pigeons. AIM06 and SR14 bacteriophages were present in human sewage at 2-4 orders of magnitude. A principal component analysis confirmed the importance of both phages as main water quality parameters. The phages presented only in the polluted water, as classified by a cluster analysis, and at median concentrations of 1.71 × 10 2 and 4.27 × 10 2 PFU/100 mL, respectively, higher than nonhost specific RYC2056 phages and sewage-specific KS148 phages (p < 0.05). Interestingly, AIM06 and SR14 phages exhibited significant correlations with each other and with total coliforms, E. coli, enterococci, and biochemical oxygen demand (Kendall's tau = 0.348 to 0.605, p < 0.05), a result supporting their roles as water quality indicators. This research demonstrates the multiregional applicability of enterococci hosts in MST application and highlights the significance of multivariate analysis with nondetects in evaluating the performance of new MST host strains.

Molecular characterization of a genomic region in a Lactococcus bacteriophage that is involved in its sensitivity to the phage defense mechanism AbiA.

PubMed

Dinsmore, P K; Klaenhammer, T R

1997-05-01

A spontaneous mutant of the lactococcal phage phi31 that is insensitive to the phage defense mechanism AbiA was characterized in an effort to identify the phage factor(s) involved in sensitivity of phi31 to AbiA. A point mutation was localized in the genome of the AbiA-insensitive phage (phi31A) by heteroduplex analysis of a 9-kb region. The mutation (G to T) was within a 738-bp open reading frame (ORF245) and resulted in an arginine-to-leucine change in the predicted amino acid sequence of the protein. The mutant phi31A-ORF245 reduced the sensitivity of phi31 to AbiA when present in trans, indicating that the mutation in ORF245 is responsible for the AbiA insensitivity of phi31A. Transcription of ORF245 occurs early in the phage infection cycles of phi31 and phi31A and is unaffected by AbiA. Expansion of the phi31 sequence revealed ORF169 (immediately upstream of ORF245) and ORF71 (which ends 84 bp upstream of ORF169). Two inverted repeats lie within the 84-bp region between ORF71 and ORF169. Sequence analysis of an independently isolated AbiA-insensitive phage, phi31B, identified a mutation (G to A) in one of the inverted repeats. A 118-bp fragment from phi31, encompassing the 84-bp region between ORF71 and ORF169, eliminates AbiA activity against phi31 when present in trans, establishing a relationship between AbiA and this fragment. The study of this region of phage phi31 has identified an open reading frame (ORF245) and a 118-bp DNA fragment that interact with AbiA and are likely to be involved in the sensitivity of this phage to AbiA.

CRISPR analysis of bacteriophage-insensitive mutants (BIMs) of industrial Streptococcus thermophilus--implications for starter design.

PubMed

Mills, S; Griffin, C; Coffey, A; Meijer, W C; Hafkamp, B; Ross, R P

2010-03-01

An efficient approach for generation of bacteriophage-insensitive mutants (BIMs) of Streptococcus thermophilus starters was described in our laboratory [Mills et al. (2007) J Microbiol Methods70, 159-164]. The aim of this study was to analyse the phage resistance mechanism responsible for BIM formation. Three clustered regularly interspaced short palindromic repeat (CRISPR) regions have been identified in Strep. thermophilus, and Strep. thermophilus can integrate novel spacers into these loci in response to phage attack. Characterization of three sets of BIMs indicated that two sets had altered CRISPR1 and/or CRISPR3 loci. A range of BIMs of yoghurt starter CSK938 were generated with the same phage in different phage challenge experiments, and each acquired unique spacer regions ranging between one and four new spacers in CRISPR1. In addition, the BIM that acquired only one new spacer in CRISPR1 also acquired an additional spacer in CRISPR3. A fourth BIM, generated with a different phage, had two spacers deleted from CRISPR1 but acquired two spacers in CRISPR3. Analysis of the Mozzarella starter CSK939 and its associated BIMs indicated that formation of second generation BIMs does not lead to increases in spacer number but to alterations in spacer regions. BIMs of an exopolysaccharide (EPS)-producing strain that lost the ability to produce EPS did not harbour an altered CRISPR, suggesting that phage sensitivity may be related to the EPS-producing phenotype. Acquisition/deletion of new spacers in CRISPR loci in response to phage attack generates distinctly individual variants. It also demonstrates that other modifications may be responsible for the phage resistance of Strep. thermophilus BIMs. Isolation of individual BIMs that have unique spacers towards the leader region of the CRISPR locus may be a very useful approach for rotation strategies with the same starter backbone. Upon phage infection, BIMs 'in reserve' can be slotted into the rotation scheme.

Immunocontraception: Filamentous Bacteriophage as a Platform for Vaccine Development.

PubMed

Samoylova, Tatiana I; Braden, Timothy D; Spencer, Jennifer A; Bartol, Frank F

2017-11-20

Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to address this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Filamentous bacteriophages can be used as platforms for development of such vaccines. In this review authors highlight structural and immunogenic properties of filamentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in animals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those administering the vaccines. Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be produced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Immunocontraception: Filamentous Bacteriophage as a Platform for Vaccine Development

PubMed Central

Samoylova, Tatiana I.; Braden, Timothy D.; Spencer, Jennifer A.; Bartol, Frank F.

2017-01-01

Background: Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to ad-dress this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Fil-amentous bacteriophages can be used as platforms for development of such vaccines. Objective: In this review authors highlight structural and immunogenic properties of fila-mentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. Results: Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in ani-mals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those ad-ministering the vaccines. Conclusion: Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be pro-duced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents. PMID:28901276

Comparative studies with tox plus and tox minus corynebacteriophages.

PubMed

Holmes, R K; Barksdale, L

1970-06-01

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

Comparative Studies with tox+ and tox− Corynebacteriophages 1

PubMed Central

Holmes, Randall K.; Barksdale, Lane

1970-01-01

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

Genomic characterization of key bacteriophages to formulate the potential biocontrol agent to combat enteric pathogenic bacteria.

PubMed

Parmar, Krupa M; Dafale, Nishant A; Tikariha, Hitesh; Purohit, Hemant J

2018-05-01

Combating bacterial pathogens has become a global concern especially when the antibiotics and chemical agents are failing to control the spread due to its resistance. Bacteriophages act as a safe biocontrol agent by selectively lysing the bacterial pathogens without affecting the natural beneficial microflora. The present study describes the screening of prominent enteric pathogens NDK1, NDK2, NDK3, and NDK4 (Escherichia, Klebsiella, Enterobacter, and Serratia) mostly observed in domestic wastewater; against which KNP1, KNP2, KNP3, and KNP4 phages were isolated. To analyze their potential role in eradicating enteric pathogens and toxicity issue, these bacteriophages were sequenced using next-generation sequencing and characterized based on its genomic content. The isolated bacteriophages were homologous to Escherichia phage (KNP1), Klebsiella phage (KNP2), Enterobacter phage (KNP3), Serratia phage (KNP4), and belonged to Myoviridae family of Caudovirales except for the unclassified KNP4 phage. Draft genome analysis revealed the presence of lytic enzymes such as holing and lysozyme in KNP1 phage, endolysin in KNP2 phage, and endopeptidase with holin in KNP3 phage. The absence of any lysogenic and virulent genes makes this bacteriophage suitable candidate for preparation of phage cocktail to combat the pathogens present in wastewater. However, KNP4 contained a virulent gene rendering it unsuitable to be used as a biocontrol agent. These findings make the phages (KNP1-KNP3) as a promising alternative for the biocontrol of pathogens in wastewater which is the main culprit to spread these dominated pathogens in different natural water bodies. This study also necessitates for genomic screening of bacteriophages for lysogenic and virulence genes prior to its use as a biocontrol agent.

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

Phage-host interactions analysis of newly characterized Oenococcus oeni bacteriophages: Implications for malolactic fermentation in wine.

PubMed

Costantini, Antonella; Doria, Francesca; Saiz, Juan-Carlos; Garcia-Moruno, Emilia

2017-04-04

Nowadays, only few phages infecting Oenococcus oeni, the principal lactic acid bacteria (LAB) species responsible for malolactic fermentation (MLF) in wine, have been characterized. In the present study, to better understanding the factors affecting the lytic activity of Oenococcus phages, fifteen O. oeni bacteriophages have been studied in detail, both with molecular and microbiological methods. No correlations were found between genome sizes, type of integrase genes, or morphology and the lytic activity of the 15 tested phages. Interestingly, though phage attack in a wine at the end of alcoholic fermentation seems not to be a problem, it can indeed represent a risk factor for MLF when the alcohol content is low, feature that may be a key point for choosing the appropriate time for malolactic starter inoculation. Additionally, it was observed that some phages genomes bear 2 or 3 types of integrase genes, which point to horizontal gene transfer between O. oeni bacteriophages. Copyright © 2017. Published by Elsevier B.V.

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

Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.

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

The True Story and Advantages of RNA Phage Capsids as Nanotools.

PubMed

Pumpens, Paul; Renhofa, Regina; Dishlers, Andris; Kozlovska, Tatjana; Ose, Velta; Pushko, Peter; Tars, Kaspars; Grens, Elmars; Bachmann, Martin F

2016-01-01

RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools. © 2016 S. Karger AG, Basel.

H-NS Mutation-Mediated CRISPR-Cas Activation Inhibits Phage Release and Toxin Production of Escherichia coli Stx2 Phage Lysogen.

PubMed

Fu, Qiang; Li, Shiyu; Wang, Zhaofei; Shan, Wenya; Ma, Jingjiao; Cheng, Yuqiang; Wang, Hengan; Yan, Yaxian; Sun, Jianhe

2017-01-01

Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an Escherichia coli ( E. coli ) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune system has on Stx phage lysogen. We investigated heat-stable nucleoid-structuring (H-NS) mutation-mediated CRISPR-Cas activation and its effect on E. coli Stx2 phage lysogen. The Δ hns mutant (MG1655Δ hns ) of the E. coli K-12 strain MG1655 was obtained. The Δ hns mutant lysogen that was generated after Stx phage lysogenic infection had a repressed growth status and showed subdued group behavior, including biofilm formation and swarming motility, in comparison to the wild-type strain. The de-repression effect of the H-NS mutation on CRISPR-Cas activity was then verified. The results showed that cas gene expression was upregulated and the transformation efficiency of the wild-type CRISPR plasmids was decreased, which may indicate activation of the CRISPR-Cas system. Furthermore, the function of CRISPR-Cas on Stx2 phage lysogen was investigated by activating the CRISPR-Cas system, which contains an insertion of the protospacer regions of the Stx2 phage Min27. The phage release and toxin production of four lysogens harboring the engineered CRISPRs were investigated. Notably, in the supernatant of the Δ hns mutant lysogen harboring the Min27 spacer, both the progeny phage release and the toxin production were inhibited after mitomycin C induction. These observations demonstrate that the H-NS mutation-activated CRISPR-Cas system plays a role in modifying the effects of the Stx2 phage lysogen. Our findings indicated that H-NS mutation-mediated CRISPR-Cas activation in E. coli protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.

Liquid based formulations of bacteriophages for the management of waterborne bacterial pathogens in water microcosms.

PubMed

Ahiwale, Sangeeta; Tagunde, Sujata; Khopkar, Sushama; Karni, Mrudula; Gajbhiye, Milind; Kapadnis, Balasaheb

2013-11-01

Water resources are contaminated by life-threatening multidrug resistant pathogenic bacteria. Unfortunately, these pathogenic bacteria do not respond to the traditional water purification methods. Therefore, there is a need of environmentally friendly strategies to overcome the problems associated with the antimicrobial resistant bacterial pathogens. In the present study, highly potent lytic phages against multidrug-resistant Salmonella enterica serovar Paratyphi B, Pseudomonas aeruginosa and Klebsiella pneumoniae were isolated from the Pavana river water. They belonged to the Podoviridae and Siphoviridae families. These phages were purified and enriched in the laboratory. Monovalent formulations of phiSPB, BVPaP-3 and KPP phages were prepared in three different liquids viz., phage broth, saline and distilled water. The phages were stable for almost 8-10 months in the phage broth at 4 degrees C. The stability of the phages in saline and distilled water was 5-6 months at 4 degrees C. All of the phages were stable only for 4-6 months in the phage broth at 30 degrees C. The monovalent phage formulation of psiSPB was applied at MOI < 1, as disinfectant against an exponential and stationary phase cells of Salmonella enterica serovar Paratyphi B in various water microcosms. The results indicated that there was almost 80 % reduction in the log phase cells of Salmonella serovar Paratyphi B in 24 h. In stationary phase cells, the reduction was comparatively less within same period. At the same time, there was concomitant increase in the phage population by 80% in all the microcosms indicating that psiSPB phage is highly potent in killing pathogen in water. Results strongly support that the formulation of psiSPB in the phage broth in monovalent form could be used as an effective biological disinfectant for preventing transmission of water-borne bacterial pathogens, including antimicrobial resistant ones.

Biological responses to M13 bacteriophage modified titanium surfaces in vitro.

PubMed

Sun, Yuhua; Li, Yiting; Wu, Baohua; Wang, Jianxin; Lu, Xiong; Qu, Shuxin; Weng, Jie; Feng, Bo

2017-08-01

Phage-based materials have showed great potential in tissue engineering application. However, it is unknown what inflammation response will happen to this kind of materials. This work is to explore the biological responses to M13 bacteriophage (phage) modified titanium surfaces in vitro from the aspects of their interaction with macrophages, osteoblasts and mineralization behavior. Pretreated Ti surface, Ti surfaces with noncrosslinked phage film (APP) and crosslinked phage film (APPG) were compared. Phage films could limit the macrophage adhesion and activity due to inducing adherent-cell apoptosis. The initial inflammatory activity (24h) caused by phage films was relatively high with more production of TNF-α, but in the later stage (7-10days) inflammatory response was reduced with lower TNF-α, IL-6 and higher IL-10. In addition, phage films improved osteoblast adhesion, differentiation, and hydroapatite (HA)-forming via a combination of topographical and biochemcial cues. The noncrosslinked phage film displayed the best immunomodulatory property, osteogenic activity and HA mineralization ability. This work provides better understanding of inflammatory and osteogenetic activity of phage-based materials and contributes to their future application in tissue engineering. In vivo, the bone and immune cells share a common microenvironment, and are being affected by similar cytokines, signaling molecules, transcription factors and membrane receptors. Ideal implants should cause positive biological response, including adequate and appropriate inflammatory reaction, well-balanced bone formation and absorption. Phage-based materials have showed great potential in tissue engineering application. However, at present it is unknown what inflammation response will happen to this kind of materials. A good understanding of the immune response possibly induced by phage-based materials is needed. This work studied the osteoimmunomodulation property of phage films on titanium surface, involving inflammatory response, osteogenic activity and biomineralization ability. It provides more understanding of the phage-based materials and contributes to their future application in tissue engineering. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Isolation and characterisation of lytic bacteriophages of Klebsiella pneumoniae and Klebsiella oxytoca.

PubMed

Karumidze, Natia; Kusradze, Ia; Rigvava, Sophio; Goderdzishvili, Marine; Rajakumar, Kumar; Alavidze, Zemphira

2013-03-01

Klebsiella bacteria have emerged as an increasingly important cause of community-acquired nosocomial infections. Extensive use of broad-spectrum antibiotics in hospitalised patients has led to both increased carriage of Klebsiella and the development of multidrug-resistant strains that frequently produce extended-spectrum β-lactamases and/or other defences against antibiotics. Many of these strains are highly virulent and exhibit a strong propensity to spread. In this study, six lytic Klebsiella bacteriophages were isolated from sewage-contaminated river water in Georgia and characterised as phage therapy candidates. Two of the phages were investigated in greater detail. Biological properties, including phage morphology, nucleic acid composition, host range, growth phenotype, and thermal and pH stability were studied for all six phages. Limited sample sequencing was performed to define the phylogeny of the K. pneumoniae- and K. oxytoca-specific bacteriophages vB_Klp_5 and vB_Klox_2, respectively. Both of the latter phages had large burst sizes, efficient rates of adsorption and were stable under different adverse conditions. Phages reported in this study are double-stranded DNA bacterial viruses belonging to the families Podoviridae and Siphoviridae. One or more of the six phages was capable of efficiently lysing ~63 % of Klebsiella strains comprising a collection of 123 clinical isolates from Georgia and the United Kingdom. These phages exhibit a number of properties indicative of potential utility in phage therapy cocktails.

Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials

PubMed Central

Merabishvili, Maya; Pirnay, Jean-Paul; Verbeken, Gilbert; Chanishvili, Nina; Tediashvili, Marina; Lashkhi, Nino; Glonti, Thea; Krylov, Victor; Mast, Jan; Van Parys, Luc; Lavigne, Rob; Volckaert, Guido; Mattheus, Wesley; Verween, Gunther; De Corte, Peter; Rose, Thomas; Jennes, Serge; Zizi, Martin; De Vos, Daniel; Vaneechoutte, Mario

2009-01-01

We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on succesive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, φKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee. PMID:19300511

Mycobacteriophages: an important tool for the diagnosis of Mycobacterium tuberculosis (review).

PubMed

Fu, Xiaoyan; Ding, Mingxing; Zhang, Ning; Li, Jicheng

2015-07-01

The prevention and control of tuberculosis (TB) on a global scale has become increasingly important with the emergence of multidrug‑resistant TB. Mycobacterium tuberculosis phages have been identified as an important investigative tool. Phage genomes exhibit a significant level of diversity and mosaic genome architecture, however, they are simple structures, which are amenable to genetic manipulation. Based on these characteristics, the phages may be used to construct a shuttle plasmid, which is an indispensable tool in the investigation of TB. Furthermore, they may be used for rapid diagnosis and assessing drug susceptibility of TB, including phage amplified assessment and reporter phage technology. With an improved understanding of mycobacteriophages, further clarification of the pathogenesis of TB, and of the implications for its diagnosis and therapy, may be elucidated.

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

Abbasifar, Reza; Griffiths, Mansel W.; Sabour, Parviz M.

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 havemore » 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.« less

Longitudinal monitoring of Listeria monocytogenes and Listeria phages in seafood processing environments in Thailand.

PubMed

Vongkamjan, Kitiya; Benjakul, Soottawat; Kim Vu, Hue Thi; Vuddhakul, Varaporn

2017-09-01

Listeria monocytogenes is a foodborne pathogen commonly found in environments of seafood processing, thus presenting a challenge for eradication from seafood processing facilities. Monitoring the prevalence and subtype diversity of L. monocytogenes together with phages that are specific to Listeria spp. ("Listeria phages") will provide knowledge on the bacteria-phage ecology in food processing plants. In this work, a total of 595 samples were collected from raw material, finished seafood products and environmental samples from different sites of a seafood processing plant during 17 sampling visits in 1.5 years of study. L. monocytogenes and Listeria spp. (non-monocytogenes) were found in 22 (3.7%) and 43 (7.2%) samples, respectively, whereas 29 Listeria phages were isolated from 9 (1.5%) phage-positive samples. DNA fingerprint analysis of L. monocytogenes isolates revealed 11 Random Amplified Polymorphic DNA (RAPD) profiles, with two subtypes were frequently observed over time. Our data reveal a presence of Listeria phages within the same seafood processing environments where a diverse set of L. monocytogenes subtypes was also found. Although serotype 4b was observed at lower frequency, data indicate that isolates from this seafood processing plant belonged to both epidemiologically important serotypes 1/2a and 4b, which may suggest a potential public health risk. Phages (all showed a unique genome size of 65 ± 2 kb) were classified into 9 host range groups, representing both broad- and narrow-host range. While most L. monocytogenes isolates from this facility were susceptible to phages, five isolates showed resistance to 12-20 phages. Variations in phage host range among Listeria phages isolated from food processing plant may affect a presence of a diverse set of L. monocytogenes isolates derived from the same processing environment in Thailand. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacteriophages of methanotrophic bacteria.

PubMed Central

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

1980-01-01

Bacteriophages of methanotrophic bacteria have been found in 16 out of 88 studied samples (underground waters, pond water, soil, gas and oil installation waters, fermentor cultural fluids, bacterial paste, and rumen of cattle) taken in different geographic zones of the Soviet Union. Altogether, 23 phage strains were isolated: 10 strains that specifically lysed only Methylosinus sporium strains, 2 strains that each lysed 1 of 5 Methylosinus trichosporium strains studied, and 11 strains that lysed Flavobacterium gasotypicum and, at the same time, 1 M. sporium strain. By fine structure, the phages were divided into two types (with very short or long noncontractile tails); by host range and serological properties, they fell into three types. One-step growth characteristics of the phages differed only slightly; the latent period varied from 6 to 8 h, the rise period varied from 4 to 6 h, and the average burst size was 100. All phages had guanine- and cytosine-rich double-stranded deoxyribonucleic acid consisting of common nitrogen bases. The molecular mass of the deoxyribonucleic acid as determined by restriction endonuclease analysis was 29.4 X 10(6) for M. sporium phages and 44 X 10(6) for F. gasotypicum phages. By all of the above-mentioned properties, all phages within each of the groups were completely identical to one another, but differed from phages of other groups. Bacteriophages lysing M. sporium and M. trichosporium GB2 were identical to phages M1 and M4, respectively, which were isolated earlier in the German Democratic Republic on the same methanotrophic species. Images PMID:6774962

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages.

PubMed

Cheng, Lucy; Marinelli, Laura J; Grosset, Noël; Fitz-Gibbon, Sorel T; Bowman, Charles A; Dang, Brian Q; Russell, Daniel A; Jacobs-Sera, Deborah; Shi, Baochen; Pellegrini, Matteo; Miller, Jeff F; Gautier, Michel; Hatfull, Graham F; Modlin, Robert L

2018-03-01

A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

PubMed Central

Schnabel, Elise L.; Jones, Alan L.

2001-01-01

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages φEa1 and φEa7 and 3 novel phages named φEa100, φEa125, and φEa116C, were identified based on differences in genome size and restriction fragment pattern. φEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages φEa100, φEa7, and φEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. φEa116C contained an approximately 75-kb genome. φEa1, φEa7, φEa100, φEa125, and φEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. φEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 105 CFU. PMID:11133428

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

PubMed Central

Kasman, Laura M.; Lukowiak, Andrew A.; Garczynski, Stephen F.; McNall, Rebecca J.; Youngman, Phil; Adang, Michael J.

1998-01-01

Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning. PMID:9687463

Application of streptavidin mass spectrometric immunoassay tips for immunoaffinity based antibody phage display panning.

PubMed

Chin, Chai Fung; Ler, Lian Wee; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Tye, Gee Jun; Lim, Theam Soon

2016-01-01

Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model.

PubMed

Lin, Abraham; Jimenez, Jose; Derr, Julien; Vera, Pedro; Manapat, Michael L; Esvelt, Kevin M; Villanueva, Laura; Liu, David R; Chen, Irene A

2011-01-01

Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

Inhibition of Bacterial Conjugation by Phage M13 and Its Protein g3p: Quantitative Analysis and Model

PubMed Central

Lin, Abraham; Jimenez, Jose; Derr, Julien; Vera, Pedro; Manapat, Michael L.; Esvelt, Kevin M.; Villanueva, Laura; Liu, David R.; Chen, Irene A.

2011-01-01

Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes. PMID:21637841

Continuous microfluidic assortment of interactive ligands (CMAIL)

NASA Astrophysics Data System (ADS)

Hsiao, Yi-Hsing; Huang, Chao-Yang; Hu, Chih-Yung; Wu, Yen-Yu; Wu, Chung-Hsiun; Hsu, Chia-Hsien; Chen, Chihchen

2016-08-01

Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields. During the weak horizontal electric field phase, phages were differentially swept laterally depending on their affinity for the antigen, and all phages were electrophoresed down to be collected during the strong vertical electric field phase. Phages of different affinity were spatially separated, allowing the continuous operation. More than 105 CFU (colony forming unit) antigen-interacting phages were isolated with ~100% specificity from a phage library containing 3 × 109 individual members within 40 minutes of sorting using CMAIL. CMAIL is rapid, sensitive, specific, and does not employ washing, elution or magnetic beads. In conclusion, we have developed an efficient and cost-effective method for isolating and sorting affinity reagents involving phage display.

Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

PubMed

Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

2014-03-04

Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

Investigating the biocontrol and anti-biofilm potential of a three phage cocktail against Cronobacter sakazakii in different brands of infant formula.

PubMed

Endersen, Lorraine; Buttimer, Colin; Nevin, Eoghan; Coffey, Aidan; Neve, Horst; Oliveira, Hugo; Lavigne, Rob; O'Mahony, Jim

2017-07-17

In recent years, the microbiological safety of powdered infant formula has gained increasing attention due to the identification of contaminating C. sakazakii and its epidemiological link with life-threatening neonatal infections. Current intervention strategies have fallen short of ensuring the production of infant formula that is free from C. sakazakii. In this study, we describe the isolation and characterisation of three bacteriophages (phages) and their application as a phage cocktail to inhibit the growth of C. sakazakii in different brands of infant formula, while also assessing the phages ability to prevent biofilm formation. All three phages, isolated from slurry, possess a relatively broad host range, verified by their ability to infect across genera and species. When all three phages were combined and used as part of a phage cocktail, 73% coverage was obtained across all Cronobacter strains tested. Optimum thermo-tolerance and pH stability were determined between 4°C-37°C, and pH6-8, respectively, well within the normal range of application of infant formula. Genome sequencing and analysis revealed all the phages to be free from lysogenic properties, a trait which renders each favourable for phage therapy applications. As such, the combined-phage preparation (3×10 8 pfu/mL) was found to possess a strong bactericidal effect on C. sakazakii/C. sakazakii LUX cells (≤10 4 cfu/mL), resulting in a significant reduction in cell numbers, to below the limit of detection (<10cfu/mL). This was observed following a 20h challenge in different brands of infant formula, where samples in the absence of the phage cocktail reached concentrations of ~10 9 cfu/mL. The phage cocktail also demonstrated promise in preventing the establishment of biofilm, as biofilm formation could not be detected for up to 48h post treatment. These results highlight the potential application of this phage preparation for biocontrol of C. sakazakii contamination in reconstituted infant formula and also as a preventative agent against biofilm formation. Copyright © 2017. Published by Elsevier B.V.

Discovery and Complete Genome Sequence of a Bacteriophage from an Obligate Intracellular Symbiont of a Cellulolytic Protist in the Termite Gut

PubMed Central

Pramono, Ajeng K.; Kuwahara, Hirokazu; Itoh, Takehiko; Toyoda, Atsushi; Yamada, Akinori; Hongoh, Yuichi

2017-01-01

Termites depend nutritionally on their gut microbes, and protistan, bacterial, and archaeal gut communities have been extensively studied. However, limited information is available on viruses in the termite gut. We herein report the complete genome sequence (99,517 bp) of a phage obtained during a genome analysis of “Candidatus Azobacteroides pseudotrichonymphae” phylotype ProJPt-1, which is an obligate intracellular symbiont of the cellulolytic protist Pseudotrichonympha sp. in the gut of the termite Prorhinotermes japonicus. The genome of the phage, designated ProJPt-Bp1, was circular or circularly permuted, and was not integrated into the two circular chromosomes or five circular plasmids composing the host ProJPt-1 genome. The phage was putatively affiliated with the order Caudovirales based on sequence similarities with several phage-related genes; however, most of the 52 protein-coding sequences had no significant homology to sequences in the databases. The phage genome contained a tRNA-Gln (CAG) gene, which showed the highest sequence similarity to the tRNA-Gln (CAA) gene of the host “Ca. A. pseudotrichonymphae” phylotype ProJPt-1. Since the host genome lacked a tRNA-Gln (CAG) gene, the phage tRNA gene may compensate for differences in codon usage bias between the phage and host genomes. The phage genome also contained a non-coding region with high nucleotide sequence similarity to a region in one of the host plasmids. No other phage-related sequences were found in the host ProJPt-1 genome. To the best of our knowledge, this is the first report of a phage from an obligate, mutualistic endosymbiont permanently associated with eukaryotic cells. PMID:28321010

Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

PubMed Central

Bonanno, Ludivine; Petit, Marie-Agnès; Loukiadis, Estelle; Michel, Valérie

2016-01-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event. PMID:26826235

Locating and Activating Molecular ‘Time Bombs’: Induction of Mycolata Prophages

PubMed Central

Dyson, Zoe A.; Brown, Teagan L.; Farrar, Ben; Doyle, Stephen R.; Tucci, Joseph; Seviour, Robert J.; Petrovski, Steve

2016-01-01

Little is known about the prevalence, functionality and ecological roles of temperate phages for members of the mycolic acid producing bacteria, the Mycolata. While many lytic phages infective for these organisms have been isolated, and assessed for their suitability for use as biological control agents of activated sludge foaming, no studies have investigated how temperate phages might be induced for this purpose. Bioinformatic analysis using the PHAge Search Tool (PHAST) on Mycolata whole genome sequence data in GenBank for members of the genera Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella revealed 83% contained putative prophage DNA sequences. Subsequent prophage inductions using mitomycin C were conducted on 17 Mycolata strains. This led to the isolation and genome characterization of three novel Caudovirales temperate phages, namely GAL1, GMA1, and TPA4, induced from Gordonia alkanivorans, Gordonia malaquae, and Tsukamurella paurometabola, respectively. All possessed highly distinctive dsDNA genome sequences. PMID:27487243

The Impact of Prophage on the Equilibria and Stability of Phage and Host

NASA Astrophysics Data System (ADS)

Yu, Pei; Nadeem, Alina; Wahl, Lindi M.

2017-06-01

In this paper, we present a bacteriophage model that includes prophage, that is, phage genomes that are incorporated into the host cell genome. The general model is described by an 18-dimensional system of ordinary differential equations. This study focuses on asymptotic behaviour of the model, and thus the system is reduced to a simple six-dimensional model, involving uninfected host cells, infected host cells and phage. We use dynamical system theory to explore the dynamic behaviour of the model, studying in particular the impact of prophage on the equilibria and stability of phage and host. We employ bifurcation and stability theory, centre manifold and normal form theory to show that the system has multiple equilibrium solutions which undergo a series of bifurcations, finally leading to oscillating motions. Numerical simulations are presented to illustrate and confirm the analytical predictions. The results of this study indicate that in some parameter regimes, the host cell population may drive the phage to extinction through diversification, that is, if multiple types of host emerge; this prediction holds even if the phage population is likewise diverse. This parameter regime is restricted, however, if infecting phage are able to recombine with prophage sequences in the host cell genome.

Phage Life Cycles Behind Bacterial Biodiversity.

PubMed

Olszak, Tomasz; Latka, Agnieszka; Roszniowski, Bartosz; Valvano, Miguel A; Drulis-Kawa, Zuzanna

2017-11-24

Bacteriophages (phages or bacterial viruses) are the most abundant biological entities in our planet; their influence reaches far beyond the microorganisms they parasitize. Phages are present in every environment and shape up every bacterial population in both active and passive ways. They participate in the circulation of organic matter and drive the evolution of microorganisms by horizontal gene transfer at unprecedented scales. The mass flow of genetic information in the microbial world influences the biosphere and poses challenges for science and medicine. The genetic flow, however, depends on the fate of the viral DNA injected into the bacterial cell. The archetypal notion of phages only engaging in predatorprey relationships is slowly fading. Because of their varied development cycles, environmental conditions, and the diversity of microorganisms they parasitize, phages form a dense and highly complex web of dependencies, which has important consequences for life on Earth. The sophisticated phage-bacteria interplay includes both aggressive action (bacterial lysis) and "diplomatic negotiations" (prophage domestication). Here, we review the most important mechanisms of interactions between phages and bacteria and their evolutionary consequences influencing their biodiversity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Phage idiotype vaccination: first phase I/II clinical trial in patients with multiple myeloma

PubMed Central

2014-01-01

Background Multiple myeloma is characterized by clonal expansion of B cells producing monoclonal immunoglobulins or fragments thereof, which can be detected in the serum and/or urine and are ideal target antigens for patient-specific immunotherapies. Methods Using phage particles as immunological carriers, we employed a novel chemically linked idiotype vaccine in a clinical phase I/II trial including 15 patients with advanced multiple myeloma. Vaccines composed of purified paraproteins linked to phage were manufactured successfully for each patient. Patients received six intradermal immunizations with phage idiotype vaccines in three different dose groups. Results Phage idiotype was well tolerated by all study participants. A subset of patients (80% in the middle dose group) displayed a clinical response indicated by decrease or stabilization of paraprotein levels. Patients exhibiting a clinical response to phage vaccines also raised idiotype-specific immunoglobulins. Induction of a cellular immune response was demonstrated by a cytotoxicity assay and delayed type hypersensitivity tests. Conclusion We present a simple, time- and cost-efficient phage idiotype vaccination strategy, which represents a safe and feasible patient-specific therapy for patients with advanced multiple myeloma and produced promising anti-tumor activity in a subset of patients. PMID:24885819

Targeted delivery of siRNA into breast cancer cells via phage fusion proteins.

PubMed

Bedi, Deepa; Gillespie, James W; Petrenko, Vasily A; Ebner, Andreas; Leitner, Michael; Hinterdorfer, Peter; Petrenko, Valery A

2013-02-04

Nucleic acids, including antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), which were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active, and physiologically acceptable cancer nanomedicine.

Linkage and homology analysis divides the eight genes for the small subunit of petunia ribulose 1,5-bisphosphate carboxylase into three gene families

PubMed Central

Dean, Caroline; van den Elzen, Peter; Tamaki, Stanley; Dunsmuir, Pamela; Bedbrook, John

1985-01-01

Twenty-six λ phage clones with homology to coding sequences of the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase have been isolated from an EMBL3 λ phage bank of Petunia (Mitchell) DNA. Restriction mapping of the phage inserts shows that the clones were obtained from five nonoverlapping regions of petunia DNA that carry seven SSU genes. Comparison of the HindIII genomic fragments of petunia DNA with the HindIII restriction fragments of the isolated phage indicates that petunia nuclear DNA encodes eight SSU genes, seven of which are present in the phage clones. Two incomplete genes, which contain only the 3′ end of an SSU gene, were also found in the phage clones. We demonstrate that the eight SSU genes of petunia can be divided into three gene families based on homology to three petunia cDNA clones. Two gene families contain single SSU genes and the third contains six genes, four of which are closely linked within petunia nuclear DNA. Images PMID:16593584

Exploring the contribution of bacteriophages to antibiotic resistance.

PubMed

Lekunberri, Itziar; Subirats, Jèssica; Borrego, Carles M; Balcázar, José Luis

2017-01-01

Bacteriophages (phages) are the most abundant and diverse biological entities in our planet. They infect susceptible bacterial hosts into which they either multiply or persist. In the latter case, phages can confer new functions to their hosts as a result of gene transfer, thus contributing to their adaptation (short-term) and evolution (long-term). In this regard, the role of phages on the dissemination of antibiotic resistance genes (ARGs) among bacterial hosts in natural environments has not yet been clearly resolved. Here, we carry out a comprehensive analysis of thirty-three viromes from different habitats to investigate whether phages harbor ARGs. Our results demonstrate that while human-associated viromes do not or rarely carry ARGs, viromes from non-human sources (e.g. pig feces, raw sewage, and freshwater and marine environments) contain a large reservoir of ARGs, thus pointing out that phages could play a part on the spread of antibiotic resistance. Given this, the role of phages should not be underestimated and it should be considered when designing strategies to tackle the global crisis of antibiotic resistance. Copyright © 2016 Elsevier Ltd. All rights reserved.

Propionibacterium acnes bacteriophages display limited genetic diversity and broad killing activity against bacterial skin isolates.

PubMed

Marinelli, Laura J; Fitz-Gibbon, Sorel; Hayes, Clarmyra; Bowman, Charles; Inkeles, Megan; Loncaric, Anya; Russell, Daniel A; Jacobs-Sera, Deborah; Cokus, Shawn; Pellegrini, Matteo; Kim, Jenny; Miller, Jeff F; Hatfull, Graham F; Modlin, Robert L

2012-01-01

Investigation of the human microbiome has revealed diverse and complex microbial communities at distinct anatomic sites. The microbiome of the human sebaceous follicle provides a tractable model in which to study its dominant bacterial inhabitant, Propionibacterium acnes, which is thought to contribute to the pathogenesis of the human disease acne. To explore the diversity of the bacteriophages that infect P. acnes, 11 P. acnes phages were isolated from the sebaceous follicles of donors with healthy skin or acne and their genomes were sequenced. Comparative genomic analysis of the P. acnes phage population, which spans a 30-year temporal period and a broad geographic range, reveals striking similarity in terms of genome length, percent GC content, nucleotide identity (>85%), and gene content. This was unexpected, given the far-ranging diversity observed in virtually all other phage populations. Although the P. acnes phages display a broad host range against clinical isolates of P. acnes, two bacterial isolates were resistant to many of these phages. Moreover, the patterns of phage resistance correlate closely with the presence of clustered regularly interspaced short palindromic repeat elements in the bacteria that target a specific subset of phages, conferring a system of prokaryotic innate immunity. The limited diversity of the P. acnes bacteriophages, which may relate to the unique evolutionary constraints imposed by the lipid-rich anaerobic environment in which their bacterial hosts reside, points to the potential utility of phage-based antimicrobial therapy for acne. Propionibacterium acnes is a dominant member of the skin microflora and has also been implicated in the pathogenesis of acne; however, little is known about the bacteriophages that coexist with and infect this bacterium. Here we present the novel genome sequences of 11 P. acnes phages, thereby substantially increasing the amount of available genomic information about this phage population. Surprisingly, we find that, unlike other well-studied bacteriophages, P. acnes phages are highly homogeneous and show a striking lack of genetic diversity, which is perhaps related to their unique and restricted habitat. They also share a broad ability to kill clinical isolates of P. acnes; phage resistance is not prevalent, but when detected, it appears to be conferred by chromosomally encoded immunity elements within the host genome. We believe that these phages display numerous features that would make them ideal candidates for the development of a phage-based therapy for acne.

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 group is proposed as the founding cohort of a new phage type, the phiCbK-like phages. This work will serve as a foundation for future studies on morphogenesis, infection and phage-host interactions in C. crescentus. PMID:23050599

Genomic and phenotypic variation in epidemic-spanning Salmonella enterica serovar Enteritidis isolates

PubMed Central

2009-01-01

Background Salmonella enterica serovar Enteritidis (S. Enteritidis) has caused major epidemics of gastrointestinal infection in many different countries. In this study we investigate genome divergence and pathogenic potential in S. Enteritidis isolated before, during and after an epidemic in Uruguay. Results 266 S. Enteritidis isolates were genotyped using RAPD-PCR and a selection were subjected to PFGE analysis. From these, 29 isolates spanning different periods, genetic profiles and sources of isolation were assayed for their ability to infect human epithelial cells and subjected to comparative genomic hybridization using a Salmonella pan-array and the sequenced strain S. Enteritidis PT4 P125109 as reference. Six other isolates from distant countries were included as external comparators. Two hundred and thirty three chromosomal genes as well as the virulence plasmid were found as variable among S. Enteritidis isolates. Ten out of the 16 chromosomal regions that varied between different isolates correspond to phage-like regions. The 2 oldest pre-epidemic isolates lack phage SE20 and harbour other phage encoded genes that are absent in the sequenced strain. Besides variation in prophage, we found variation in genes involved in metabolism and bacterial fitness. Five epidemic strains lack the complete Salmonella virulence plasmid. Significantly, strains with indistinguishable genetic patterns still showed major differences in their ability to infect epithelial cells, indicating that the approach used was insufficient to detect the genetic basis of this differential behaviour. Conclusion The recent epidemic of S. Enteritidis infection in Uruguay has been driven by the introduction of closely related strains of phage type 4 lineage. Our results confirm previous reports demonstrating a high degree of genetic homogeneity among S. Enteritidis isolates. However, 10 of the regions of variability described here are for the first time reported as being variable in S. Enteritidis. In particular, the oldest pre-epidemic isolates carry phage-associated genetic regions not previously reported in S. Enteritidis. Overall, our results support the view that phages play a crucial role in the generation of genetic diversity in S. Enteritidis and that phage SE20 may be a key marker for the emergence of particular isolates capable of causing epidemics. PMID:19922635

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

Branched Lateral Tail Fiber Organization in T5-Like Bacteriophages DT57C and DT571/2 is Revealed by Genetic and Functional Analysis.

PubMed

Golomidova, Alla K; Kulikov, Eugene E; Prokhorov, Nikolai S; Guerrero-Ferreira, Ricardo С; Knirel, Yuriy A; Kostryukova, Elena S; Tarasyan, Karina K; Letarov, Andrey V

2016-01-21

The T5-like siphoviruses DT57C and DT571/2, isolated from horse feces, are very closely related to each other, and most of their structural proteins are also nearly identical to T5 phage. Their LTFs (L-shaped tail fibers), however, are composed of two proteins, LtfA and LtfB, instead of the single Ltf of bacteriophage T5. In silico and mutant analysis suggests a possible branched structure of DT57C and DT571/2 LTFs, where the LtfB protein is connected to the phage tail via the LtfA protein and with both proteins carrying receptor recognition domains. Such adhesin arrangement has not been previously recognized in siphoviruses. The LtfA proteins of our phages are found to recognize different host O-antigen types: E. coli O22-like for DT57C phage and E. coli O87 for DT571/2. LtfB proteins are identical in both phages and recognize another host receptor, most probably lipopolysaccharide (LPS) of E. coli O81 type. In these two bacteriophages, LTF function is essential to penetrate the shield of the host's O-antigens. We also demonstrate that LTF-mediated adsorption becomes superfluous when the non-specific cell protection by O-antigen is missing, allowing the phages to bind directly to their common secondary receptor, the outer membrane protein BtuB. The LTF independent adsorption was also demonstrated on an O22-like host mutant missing O-antigen O-acetylation, thus showing the biological value of this O-antigen modification for cell protection against phages.

Branched Lateral Tail Fiber Organization in T5-Like Bacteriophages DT57C and DT571/2 is Revealed by Genetic and Functional Analysis

PubMed Central

Golomidova, Alla K.; Kulikov, Eugene E.; Prokhorov, Nikolai S.; Guerrero-Ferreira, Ricardo С.; Knirel, Yuriy A.; Kostryukova, Elena S.; Tarasyan, Karina K.; Letarov, Andrey V.

2016-01-01

The T5-like siphoviruses DT57C and DT571/2, isolated from horse feces, are very closely related to each other, and most of their structural proteins are also nearly identical to T5 phage. Their LTFs (L-shaped tail fibers), however, are composed of two proteins, LtfA and LtfB, instead of the single Ltf of bacteriophage T5. In silico and mutant analysis suggests a possible branched structure of DT57C and DT571/2 LTFs, where the LtfB protein is connected to the phage tail via the LtfA protein and with both proteins carrying receptor recognition domains. Such adhesin arrangement has not been previously recognized in siphoviruses. The LtfA proteins of our phages are found to recognize different host O-antigen types: E. coli O22-like for DT57C phage and E. coli O87 for DT571/2. LtfB proteins are identical in both phages and recognize another host receptor, most probably lipopolysaccharide (LPS) of E. coli O81 type. In these two bacteriophages, LTF function is essential to penetrate the shield of the host’s O-antigens. We also demonstrate that LTF-mediated adsorption becomes superfluous when the non-specific cell protection by O-antigen is missing, allowing the phages to bind directly to their common secondary receptor, the outer membrane protein BtuB. The LTF independent adsorption was also demonstrated on an O22-like host mutant missing O-antigen O-acetylation, thus showing the biological value of this O-antigen modification for cell protection against phages. PMID:26805872

Molecular Analysis of Arthrobacter Myovirus vB_ArtM-ArV1: We Blame It on the Tail

PubMed Central

Šimoliūnas, Eugenijus; Truncaitė, Lidija; Zajančkauskaitė, Aurelija; Nainys, Juozas; Kaupinis, Algirdas; Valius, Mindaugas; Meškys, Rolandas

2017-01-01

ABSTRACT This is the first report on a myophage that infects Arthrobacter. A novel virus, vB_ArtM-ArV1 (ArV1), was isolated from soil using Arthrobacter sp. strain 68b for phage propagation. Transmission electron microscopy showed its resemblance to members of the family Myoviridae: ArV1 has an isometric head (∼74 nm in diameter) and a contractile, nonflexible tail (∼192 nm). Phylogenetic and comparative sequence analyses, however, revealed that ArV1 has more genes in common with phages from the family Siphoviridae than it does with any myovirus characterized to date. The genome of ArV1 is a linear, circularly permuted, double-stranded DNA molecule (71,200 bp) with a GC content of 61.6%. The genome includes 101 open reading frames (ORFs) yet contains no tRNA genes. More than 50% of ArV1 genes encode unique proteins that either have no reliable identity to database entries or have homologues only in Arthrobacter phages, both sipho- and myoviruses. Using bioinformatics approaches, 13 ArV1 structural genes were identified, including those coding for head, tail, tail fiber, and baseplate proteins. A further 6 ArV1 ORFs were annotated as encoding putative structural proteins based on the results of proteomic analysis. Phylogenetic analysis based on the alignment of four conserved virion proteins revealed that Arthrobacter myophages form a discrete clade that seems to occupy a position somewhat intermediate between myo- and siphoviruses. Thus, the data presented here will help to advance our understanding of genetic diversity and evolution of phages that constitute the order Caudovirales. IMPORTANCE Bacteriophages, which likely originated in the early Precambrian Era, represent the most numerous population on the planet. Approximately 95% of known phages are tailed viruses that comprise three families: Podoviridae (with short tails), Siphoviridae (with long noncontractile tails), and Myoviridae (with contractile tails). Based on the current hypothesis, myophages, which may have evolved from siphophages, are thought to have first emerged among Gram-negative bacteria, whereas they emerged only later among Gram-positive bacteria. The results of the molecular characterization of myophage vB_ArtM-ArV1 presented here conform to the aforementioned hypothesis, since, at a glance, bacteriophage vB_ArtM-ArV1 appears to be a siphovirus that possesses a seemingly functional contractile tail. Our work demonstrates that such “chimeric” myophages are of cosmopolitan nature and are likely characteristic of the ecologically important soil bacterial genus Arthrobacter. PMID:28122988

A Yersinia pestis-specific, lytic phage preparation significantly reduces viable Y. pestis on various hard surfaces experimentally contaminated with the bacterium

PubMed Central

Rashid, Mohammed H.; Revazishvili, Tamara; Dean, Timothy; Butani, Amy; Verratti, Kathleen; Bishop-Lilly, Kimberly A.; Sozhamannan, Shanmuga; Sulakvelidze, Alexander; Rajanna, Chythanya

2012-01-01

Five Y. pestis bacteriophages obtained from various sources were characterized to determine their biological properties, including their taxonomic classification, host range and genomic diversity. Four of the phages (YpP-G, Y, R and YpsP-G) belong to the Podoviridae family, and the fifth phage (YpsP-PST) belongs to the Myoviridae family, of the order Caudovirales comprising of double-stranded DNA phages. The genomes of the four Podoviridae phages were fully sequenced and found to be almost identical to each other and to those of two previously characterized Y. pestis phages Yepe2 and φA1122. However, despite their genomic homogeneity, they varied in their ability to lyse Y. pestis and Y. pseudotuberculosis strains. The five phages were combined to yield a “phage cocktail” (tentatively designated “YPP-100”) capable of lysing the 59 Y. pestis strains in our collection. YPP-100 was examined for its ability to decontaminate three different hard surfaces (glass, gypsum board and stainless steel) experimentally contaminated with a mixture of three genetically diverse Y. pestis strains CO92, KIM and 1670G. Five minutes of exposure to YPP-100 preparations containing phage concentrations of ca. 109, 108 and 107 PFU/mL completely eliminated all viable Y. pestis cells from all three surfaces, but a few viable cells were recovered from the stainless steel coupons treated with YPP-100 diluted to contain ca. 106 PFU/mL. However, even that highly diluted preparation significantly (p = < 0.05) reduced Y. pestis levels by ≥ 99.97%. Our data support the idea that Y. pestis phages may be useful for decontaminating various hard surfaces naturally- or intentionally-contaminated with Y. pestis. PMID:23275868

Intravenous infusion of phage-displayed antibody library in human cancer patients: enrichment and cancer-specificity of tumor-homing phage-antibodies.

PubMed

Shukla, Girja S; Krag, David N; Peletskaya, Elena N; Pero, Stephanie C; Sun, Yu-Jing; Carman, Chelsea L; McCahill, Laurence E; Roland, Thomas A

2013-08-01

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 × 10(11) TUs/kg of an scFv library 1 to 4 h 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 (0-5) 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 (07-2,355) 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.

Rapid Identification of Intact Staphylococcal Bacteriophages Using Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry.

PubMed

Štveráková, Dana; Šedo, Ondrej; Benešík, Martin; Zdráhal, Zbyněk; Doškař, Jiří; Pantůček, Roman

2018-04-04

Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae , Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus , as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus -specific bacteriophages.

Rapid Identification of Intact Staphylococcal Bacteriophages Using Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

PubMed Central

Štveráková, Dana; Šedo, Ondrej; Benešík, Martin; Zdráhal, Zbyněk; Doškař, Jiří

2018-01-01

Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae, Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus, as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus-specific bacteriophages. PMID:29617332

Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display.

PubMed

Kügler, Jonas; Nieswandt, Simone; Gerlach, Gerald F; Meens, Jochen; Schirrmann, Thomas; Hust, Michael

2008-09-01

The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen-host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.

Novel Podoviridae Family Bacteriophage Infecting Weissella cibaria Isolated from Kimchi

PubMed Central

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

2012-01-01

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

T7 phage displaying latent membrane protein 1 of Epstein-Barr virus elicits humoral and cellular immune responses in rats.

PubMed

Gao, J; Liu, Z; Huang, M; Li, X; Wang, Z

2011-01-01

The latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus (EBV) has become a potential target in EBV-associated tumor prevention and treatment due to its multiple biological effects. In this study, the recombinant T7 phage displaying full-length LMP1 protein was cloned and used as an immunogen to immunize rats. Results of flow cytometry, Western blot analysis, and ELISA confirmed that both humoral and cellular immune responses were elicited in the immunized rats. Our data suggested that T7 phage was an efficient antigen carrier. The recombinant T7-LMP1 phage reconstitutes the antigenic and immunogenic properties of LMP1 and can serve as a vaccine against EBV.

An electron microscopic study of bacteriophages from marine waters

NASA Astrophysics Data System (ADS)

Frank, Hermann; Moebus, Karlheinz

1987-12-01

The morphology of 75 bacteriophage strains isolated from water samples collected in the North Sea or in the northern Atlantic was studied by electron microscopy. Only tailed phages were observed (bradley groups A, B, and C). According to structural similarities, the strains are ascribed to 12 groups, 5 of which comprise types of marine phages not reported before. Four of these 5 groups include phage types that have not been detected from any other source. Among the phages isolated from northern Atlantic water a high incidence was observed of strains the particles of which have long appendages. Certain types of the northern Atlantic phages investigated were derived only from samples collected either east or west of the Azores. This finding agrees with former observations pointing to the existence of different populations of closely related bacteria east and west, respectively, of the northern Mid-Atlantic Ridge.

Role of phages in the pathogenesis of Burkholderia or “Where are the toxin genes in Burkholderia phages?”

PubMed Central

Summer, Elizabeth J.; Gill, Jason J.; Upton, Chris; Gonzalez, Carlos F.; Young, Ry

2007-01-01

Summary Most bacteria of the genus Burkholderia are soil- and rhizosphere- associated, noted for their metabolic plasticity in the utilization of a wide range of organic compounds as carbon sources. Many Burkholderia species are also opportunistic human and plant pathogens and the distinction between environmental, plant, and human pathogens is not always clear. Burkholderia phages are not uncommon and multiple cryptic prophages are identifiable in the sequenced Burkholderia genomes. Phages have played a crucial role in the transmission of virulence factors among many important pathogens, however, the data does not yet support a significant correlation between phages and pathogenicity in the Burkholderia. This may be due to the role of Burkholderia as a “versaphile” such that selection is occurring in several niches, including roles as a pathogen and in the context of environmental survival. PMID:17719265

Chemical modulation of M13 bacteriophage and its functional opportunities for nanomedicine

PubMed Central

Chung, Woo-Jae; Lee, Doe-Young; Yoo, So Young

2014-01-01

M13 bacteriophage (phage) has emerged as an attractive bionanomaterial owing to its genetically tunable surface chemistry and its potential to self-assemble into hierarchical structures. Furthermore, because of its unique nanoscopic structure, phage has been proposed as a model system in soft condensed physics and as a biomimetic building block for structured functional materials. Genetic engineering of phage provides great opportunities to develop novel nanomaterials with functional surface peptide motifs; however, this biological approach is generally limited to peptides containing the 20 natural amino acids. To extend the scope of phage applications, strategies involving chemical modification have been employed to incorporate a wider range of functional groups, including synthetic chemical compounds. In this review, we introduce the design of chemoselective phage functionalization and discuss how such a strategy is combined with genetic engineering for a variety of medical applications, as reported in recent literature. PMID:25540583

Chemical modulation of M13 bacteriophage and its functional opportunities for nanomedicine.

PubMed

Chung, Woo-Jae; Lee, Doe-Young; Yoo, So Young

2014-01-01

M13 bacteriophage (phage) has emerged as an attractive bionanomaterial owing to its genetically tunable surface chemistry and its potential to self-assemble into hierarchical structures. Furthermore, because of its unique nanoscopic structure, phage has been proposed as a model system in soft condensed physics and as a biomimetic building block for structured functional materials. Genetic engineering of phage provides great opportunities to develop novel nanomaterials with functional surface peptide motifs; however, this biological approach is generally limited to peptides containing the 20 natural amino acids. To extend the scope of phage applications, strategies involving chemical modification have been employed to incorporate a wider range of functional groups, including synthetic chemical compounds. In this review, we introduce the design of chemoselective phage functionalization and discuss how such a strategy is combined with genetic engineering for a variety of medical applications, as reported in recent literature.

Phages in nature

PubMed Central

Millard, Andrew D; Letarov, Andrey V; Heaphy, Shaun

2011-01-01

Bacteriophages or phages are the most abundant organisms in the biosphere and they are a ubiquitous feature of prokaryotic existence. A bacteriophage is a virus which infects a bacterium. Archaea are also infected by viruses, whether these should be referred to as ‘phages’ is debatable, but they are included as such in the scope this article. Phages have been of interest to scientists as tools to understand fundamental molecular biology, as vectors of horizontal gene transfer and drivers of bacterial evolution, as sources of diagnostic and genetic tools and as novel therapeutic agents. Unraveling the biology of phages and their relationship with their hosts is key to understanding microbial systems and their exploitation. In this article we describe the roles of phages in different host systems and show how modeling, microscopy, isolation, genomic and metagenomic based approaches have come together to provide unparalleled insights into these small but vital constituents of the microbial world. PMID:21687533

Analysis of Bacteriophage Motion Through a Non-Static Medium

NASA Astrophysics Data System (ADS)

Dickey, Samuel A.

In this work, I investigated the motion of bacteriophages (phages) through their mucosal environment. Recently, biologists here at San Diego State University have proposed a model in which phages move sub-diffusively through mucosal fibers in their hunt for bacteria to prey upon. Through a Hoc protein located upon the capsid of the wild type phages, these phages are allowed to bind to mucosal fibers, and extend the amount of time spent in a single location hunting for bacteria. Contrarily, the delta hoc phages are unable to. The ability of the wild type phages to attach itself to mucosal fibers is what enables its subdiffusive behavior. This study investigates the diffusive behavior of these phages in different mucus concentrations. It expands on previous studies in which only short tracks could be observed. In the study at hand, phages are imaged in a highly doped optical fiber with varying concentrations of mucus present in solution. Through rigorous image processing techniques, trajectories of these phages are created with a minimized noise level. We developed code that created position-versus-time files for each phage present in the experimental data. These files were then further analyzed. The sub-diffusive behavior is investigated via mean squared displacement versus time. The diffusive exponent can be obtained from fits to these data. For large enough time intervals, I always obtained an exponent of one for space and time averaged data. This indicates that the diffusion is normal, or sub-diffusive of the CTRW type. CTRW sub-diffusive motion is characterized by waiting times that resemble a power law distribution and have long tails. I investigate these stuck time distributions, however am unable to determine if a power law or exponential fits the data best. Moreover, the distribution gives the same power law exponent for phages moving through water, or mucus, for wild type and delta hoc phages. These exponents would predict super-diffusive instead of sub-diffusive behavior. We conclude that many of these problems result from the small amount of data available to us and the still primitive conditions of the setup at the time the data were collected.

Identifying the cellular targets of natural products using T7 phage display.

PubMed

Piggott, Andrew M; Karuso, Peter

2016-05-04

Covering: up to the end of 2015While Nature continues to deliver a myriad of potent and structurally diverse biologically active small molecules, the cellular targets and modes of action of these natural products are rarely identified, significantly hindering their development as new chemotherapeutic agents. This article provides an introductory tutorial on the use of T7 phage display as a tool to rapidly identify the cellular targets of natural products and is aimed specifically at natural products chemists who may have only limited experience in molecular biology. A brief overview of T7 phage display is provided, including its strengths, weaknesses, and the type of problems that can and cannot be tackled with this technology. Affinity probe construction is reviewed, including linker design and natural product derivatisation strategies. A detailed description of the T7 phage biopanning procedure is provided, with valuable tips for optimising each step in the process, as well as advice for identifying and avoiding the most commonly encountered challenges and pitfalls along the way. Finally, a brief discussion is provided on techniques for validating the cellular targets identified using T7 phage display.

Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates

PubMed Central

Marinelli, Laura J.; Fitz-Gibbon, Sorel; Hayes, Clarmyra; Bowman, Charles; Inkeles, Megan; Loncaric, Anya; Russell, Daniel A.; Jacobs-Sera, Deborah; Cokus, Shawn; Pellegrini, Matteo; Kim, Jenny; Miller, Jeff F.; Hatfull, Graham F.; Modlin, Robert L.

2012-01-01

ABSTRACT Investigation of the human microbiome has revealed diverse and complex microbial communities at distinct anatomic sites. The microbiome of the human sebaceous follicle provides a tractable model in which to study its dominant bacterial inhabitant, Propionibacterium acnes, which is thought to contribute to the pathogenesis of the human disease acne. To explore the diversity of the bacteriophages that infect P. acnes, 11 P. acnes phages were isolated from the sebaceous follicles of donors with healthy skin or acne and their genomes were sequenced. Comparative genomic analysis of the P. acnes phage population, which spans a 30-year temporal period and a broad geographic range, reveals striking similarity in terms of genome length, percent GC content, nucleotide identity (>85%), and gene content. This was unexpected, given the far-ranging diversity observed in virtually all other phage populations. Although the P. acnes phages display a broad host range against clinical isolates of P. acnes, two bacterial isolates were resistant to many of these phages. Moreover, the patterns of phage resistance correlate closely with the presence of clustered regularly interspaced short palindromic repeat elements in the bacteria that target a specific subset of phages, conferring a system of prokaryotic innate immunity. The limited diversity of the P. acnes bacteriophages, which may relate to the unique evolutionary constraints imposed by the lipid-rich anaerobic environment in which their bacterial hosts reside, points to the potential utility of phage-based antimicrobial therapy for acne. PMID:23015740

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

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

Lara, Elena; Holmfeldt, Karin; Solonenko, Natalie

Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. We isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. In the host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested,more » >99.9% 16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new 'rare virosphere' phage-host model system.« less

A Newly Isolated Bacteriophage, PBES 02, Infecting Cronobacter sakazakii.

PubMed

Lee, Hyung Ju; Kim, Wan Il; Kwon, Young Chan; Cha, Kyung Eun; Kim, Minjin; Myung, Heejoon

2016-09-28

A novel bacteriophage, PBES 02, infecting Cronobacter sakazakii was isolated and characterized. It has a spherical head of 90 nm in diameter and a tail of 130 nm in length, and belongs to Myoviridae as observed under a transmission electron microscope. The major virion protein appears to be 38 kilodaltons (kDa) in size. The latent period of PBES 02 is 30 min and the burst size is 250. Infectivity of the phage remained intact after exposure to temperatures ranging from 4°C to 55°C for 1 h. It was also stable after exposure to pHs ranging from 6 to 10 for 1 h. The phage effectively removed contaminating Cronobacter sakazakii from broth infant formula. PBES 02 has a double-stranded DNA genome of 149,732 bases. Its GC ratio is 50.7%. Sequence analysis revealed that PBES 02 has 299 open reading frames (ORFs) and 14 tRNA genes. Thirty-nine ORFs were annotated, including 24 related to replication and regulation functions, 10 related to structural proteins, and 5 related to DNA packaging. The genome of PBES 02 is closely related to that of two other C. sakazakii phages, CR3 and CR8. Comparison of DNA sequences of genes encoding the major capsid protein revealed a wide geographical distribution of related phages over Asia, Europe, and America.

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

PubMed

Abramov, Gili; Morag, Omry; Goldbourt, Amir

2011-08-11

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

Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to "Megavirales".

PubMed

Mushegian, Arcady; Karin, Eli Levy; Pupko, Tal

2018-01-01

The order Herpesvirales includes animal viruses with large double-strand DNA genomes replicating in the nucleus. The main capsid protein in the best-studied family Herpesviridae contains a domain with HK97-like fold related to bacteriophage head proteins, and several virion maturation factors are also homologous between phages and herpesviruses. The origin of herpesvirus DNA replication proteins is less well understood. While analyzing the genomes of herpesviruses in the family Malacohepresviridae, we identified nearly 30 families of proteins conserved in other herpesviruses, including several phage-related domains in morphogenetic proteins. Herpesvirus DNA replication factors have complex evolutionary history: some are related to cellular proteins, but others are closer to homologs from large nucleocytoplasmic DNA viruses. Phylogenetic analyses suggest that the core replication machinery of herpesviruses may have been recruited from the same pool as in the case of other large DNA viruses of eukaryotes. Published by Elsevier Inc.

Biocontrol and Rapid Detection of Food-Borne Pathogens Using Bacteriophages and Endolysins

PubMed Central

Bai, Jaewoo; Kim, You-Tae; Ryu, Sangryeol; Lee, Ju-Hoon

2016-01-01

Bacteriophages have been suggested as natural food preservatives as well as rapid detection materials for food-borne pathogens in various foods. Since Listeria monocytogenes-targeting phage cocktail (ListShield) was approved for applications in foods, numerous phages have been screened and experimentally characterized for phage applications in foods. A single phage and phage cocktail treatments to various foods contaminated with food-borne pathogens including E. coli O157:H7, Salmonella enterica, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Cronobacter sakazakii, and Vibrio spp. revealed that they have great potential to control various food-borne pathogens and may be alternative for conventional food preservatives. In addition, phage-derived endolysins with high host specificity and host lysis activities may be preferred to food applications rather than phages. For rapid detection of food-borne pathogens, cell-wall binding domains (CBDs) from endolysins have been suggested due to their high host-specific binding. Fluorescence-tagged CBDs have been successfully evaluated and suggested to be alternative materials of expensive antibodies for various detection applications. Most recently, reporter phage systems have been developed and tested to confirm their usability and accuracy for specific detection. These systems revealed some advantages like rapid detection of only viable pathogenic cells without interference by food components in a very short reaction time, suggesting that these systems may be suitable for monitoring of pathogens in foods. Consequently, phage is the next-generation biocontrol agent as well as rapid detection tool to confirm and even identify the food-borne pathogens present in various foods. PMID:27092128

Efficient identification of tubby-binding proteins by an improved system of T7 phage display.

PubMed

Caberoy, Nora B; Zhou, Yixiong; Jiang, Xiaoyu; Alvarado, Gabriela; Li, Wei

2010-01-01

Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions. (c) 2009 John Wiley & Sons, Ltd.

Characterization of a new ViI-like Erwinia amylovora bacteriophage phiEa2809.

PubMed

Lagonenko, Alexander L; Sadovskaya, Olga; Valentovich, Leonid N; Evtushenkov, Anatoly N

2015-04-01

Erwinia amylovora is a Gram-negative plant pathogenic bacteria causing fire blight disease in many Rosaceae species. A novel E. amylovora bacteriophage, phiEa2809, was isolated from symptomless apple leaf sample collected in Belarus. This phage was also able to infect Pantoea agglomerans strains. The genome of phiEa2809 is a double-stranded linear DNA 162,160 bp in length, including 145 ORFs and one tRNA gene. The phiEa2809 genomic sequence is similar to the genomes of the Serratia plymutica phage MAM1, Shigella phage AG-3, Dickeya phage vB DsoM LIMEstone1 and Salmonella phage ViI and lacks similarity to described E. amylovora phage genomes. Based on virion morphology (an icosahedral head, long contractile tail) and genome structure, phiEa2809 was classified as a member of Myoviridae, ViI-like bacteriophages group. PhiEa2809 is the firstly characterized ViI-like bacteriophage able to lyse E. amylovora. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Modular architecture of the T4 phage superfamily: A conserved core genome and a plastic periphery

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

Comeau, Andre M.; Bertrand, Claire; Letarov, Andrei

2007-06-05

Among the most numerous objects in the biosphere, phages show enormous diversity in morphology and genetic content. We have sequenced 7 T4-like phages and compared their genome architecture. All seven phages share a core genome with T4 that is interrupted by several hyperplastic regions (HPRs) where most of their divergence occurs. The core primarily includes homologues of essential T4 genes, such as the virion structure and DNA replication genes. In contrast, the HPRs contain mostly novel genes of unknown function and origin. A few of the HPR genes that can be assigned putative functions, such as a series of novelmore » Internal Proteins, are implicated in phage adaptation to the host. Thus, the T4-like genome appears to be partitioned into discrete segments that fulfil different functions and behave differently in evolution. Such partitioning may be critical for these large and complex phages to maintain their flexibility, while simultaneously allowing them to conserve their highly successful virion design and mode of replication.« less

“French Phage Network”—Second Meeting Report

PubMed Central

Torres-Barceló, Clara; Kaltz, Oliver; Froissart, Rémy; Gandon, Sylvain; Ginet, Nicolas; Ansaldi, Mireille

2017-01-01

The study of bacteriophages (viruses of bacteria) includes a variety of approaches, such as structural biology, genetics, ecology, and evolution, with increasingly important implications for therapeutic and industrial uses. Researchers working with phages in France have recently established a network to facilitate the exchange on complementary approaches, but also to engage new collaborations. Here, we provide a summary of the topics presented during the second meeting of the French Phage Network that took place in Marseille in November 2016. PMID:28430166

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

PubMed Central

Letarov, A V; Krisch, H M

2013-01-01

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

Three novel bacteriophages isolated from the East African Rift Valley soda lakes.

PubMed

van Zyl, Leonardo Joaquim; Nemavhulani, Shonisani; Cass, James; Cowan, Donald Arthur; Trindade, Marla

2016-12-03

Soda lakes are unique environments in terms of their physical characteristics and the biology they harbour. Although well studied with respect to their microbial composition, their viral compositions have not, and consequently few bacteriophages that infect bacteria from haloalkaline environments have been described. Bacteria were isolated from sediment samples of lakes Magadi and Shala. Three phages were isolated on two different Bacillus species and one Paracoccus species using agar overlays. The growth characteristics of each phage in its host was investigated and the genome sequences determined and analysed by comparison with known phages. Phage Shbh1 belongs to the family Myoviridae while Mgbh1 and Shpa belong to the Siphoviridae family. Tetranucleotide usage frequencies and G + C content suggests that Shbh1 and Mgbh1 do not regularly infect, and have therefore not evolved with, the hosts they were isolated on here. Shbh1 was shown capable of infecting two different Bacillus species from the two different lakes demonstrating its potential broad-host range. Comparative analysis of their genome sequence with known phages revealed that, although novel, Shbh1 does share substantial amino acid similarity with previously described Bacillus infecting phages (Grass, phiNIT1 and phiAGATE) and belongs to the Bastille group, while Mgbh1 and Shpa are highly novel. The addition of these phages to current databases should help with metagenome/metavirome annotation efforts. We describe a highly novel Paracoccus infecting virus (Shpa) which together with NgoΦ6 and vB_PmaS_IMEP1 is one of only three phages known to infect Paracoccus species but does not show similarity to these phages.

Isolation and characterization of a virulent bacteriophage SPW specific for Staphylococcus aureus isolated from bovine mastitis of lactating dairy cattle.

PubMed

Li, Longping; Zhang, Zhiying

2014-09-01

Mastitis in dairy cattle continues to be an economically important disease. However, control is complicated by a high prevalence of resistance to antibiotics. Phage therapy, therefore, is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a highly virulent phage SPW from wastewater of dairy farm, which possesses a strong lytic capability against mastitis-associated Staphylococcus aureus, the most important pathogen in bovine clinical and subclinical mastitis. The phage SPW produced large, round and clear plaques on bacterial culture plates. TEM showed phage SPW has an icosahedral head 62.5 nm in diameter and long tail of 106 nm, head and tail were held together by a connector of 18 ± 1.5 nm long and can be classified as a member of the Myoviridae family. Restriction analysis indicated that phage SPW was a dsDNA virus with an approximate genome size of 65-69 kb. One-step growth kinetics showed a short latency period of about 10-15 min and a rise period of 50 min and a relatively small burst size was 44 ± 3 phages particles/infected cell. Moreover, adsorption rates were not influenced by calcium ions and phage SPW was relatively stable in a wide range of temperature and pH values, and resistant to chloroform and isopropanol. The optimal multiplicity of infection (MOI) was 0.01. When phage SPW was used to infect five other clinically isolated pathogenic isolates, it showed relatively wide spectrum host range. Phage SPW was capable of eliciting efficient lysis of S. aureus, revealing it potentially as an effective approach to prophylaxis or treatment of S. aureus-associated mastitis in dairy cows.

The X-ray Crystal Structure of the Phage Tail Terminator Protein Reveals the Biologically Relevant Hexameric Rang Structure and Demonstrates a Conserved mechanism of Tail Termination among Divrse Long Tailed Phages

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

Pell, L.; Liu, A; Edmonds, L

The tail terminator protein (TrP) plays an essential role in phage tail assembly by capping the rapidly polymerizing tail once it has reached its requisite length and serving as the interaction surface for phage heads. Here, we present the 2.7-A crystal structure of a hexameric ring of gpU, the TrP of phage ?. Using sequence alignment analysis and site-directed mutagenesis, we have shown that this multimeric structure is biologically relevant and we have delineated its functional surfaces. Comparison of the hexameric crystal structure with the solution structure of gpU that we previously solved using NMR spectroscopy shows large structural changesmore » occurring upon multimerization and suggests a mechanism that allows gpU to remain monomeric at high concentrations on its own, yet polymerize readily upon contact with an assembled tail tube. The gpU hexamer displays several flexible loops that play key roles in head and tail binding, implying a role for disorder-to-order transitions in controlling assembly as has been observed with other ? morphogenetic proteins. Finally, we have found that the hexameric structure of gpU is very similar to the structure of a putative TrP from a contractile phage tail even though it displays no detectable sequence similarity. This finding coupled with further bioinformatic investigations has led us to conclude that the TrPs of non-contractile-tailed phages, such as ?, are evolutionarily related to those of contractile-tailed phages, such as P2 and Mu, and that all long-tailed phages may utilize a conserved mechanism for tail termination.« less

A human gut phage catalog correlates the gut phageome with type 2 diabetes.

PubMed

Ma, Yingfei; You, Xiaoyan; Mai, Guoqin; Tokuyasu, Taku; Liu, Chenli

2018-02-01

Substantial efforts have been made to link the gut bacterial community to many complex human diseases. Nevertheless, the gut phages are often neglected. In this study, we used multiple bioinformatic methods to catalog gut phages from whole-community metagenomic sequencing data of fecal samples collected from both type II diabetes (T2D) patients (n = 71) and normal Chinese adults (n = 74). The definition of phage operational taxonomic units (pOTUs) and identification of large phage scaffolds (n = 2567, ≥ 10 k) revealed a comprehensive human gut phageome with a substantial number of novel sequences encoding genes that were unrelated to those in known phages. Interestingly, we observed a significant increase in the number of gut phages in the T2D group and, in particular, identified 7 pOTUs specific to T2D. This finding was further validated in an independent dataset of 116 T2D and 109 control samples. Co-occurrence/exclusion analysis of the bacterial genera and pOTUs identified a complex core interaction between bacteria and phages in the human gut ecosystem, suggesting that the significant alterations of the gut phageome cannot be explained simply by co-variation with the altered bacterial hosts. Alterations in the gut bacterial community have been linked to the chronic disease T2D, but the role of gut phages therein is not well understood. This is the first study to identify a T2D-specific gut phageome, indicating the existence of other mechanisms that might govern the gut phageome in T2D patients. These findings suggest the importance of the phageome in T2D risk, which warrants further investigation.

Bacteriophages and medical oncology: targeted gene therapy of cancer.

PubMed

Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

2014-08-01

Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology.

Targeting the Atypical Chemokine Receptor ACKR3/CXCR7: Phase 1 - Phage Display Peptide Identification and Characterization.

PubMed

Vestal, R D; LaJeunesse, D R; Taylor, E W

2016-01-01

One of the greatest challenges in fighting cancer is cell targeting and biomarker selection. The Atypical Chemokine Receptor ACKR3/CXCR7 is expressed on many cancer cell types, including breast cancer and glioblastoma, and binds the endogenous ligands SDF1/CXCL12 and ITAC/CXCL11. A 20 amino acid region of the ACKR3/CXCR7 N-terminus was synthesized and targeted with the NEB PhD-7 Phage Display Peptide Library. Twenty-nine phages were isolated and heptapeptide inserts sequenced; of these, 23 sequences were unique. A 3D molecular model was created for the ACKR3/CXCR7 N-terminus by mutating the corresponding region of the crystal structure of CXCR4 with bound SDF1/CXCL12. A ClustalW alignment was performed on each peptide sequence using the entire SDF1/CXCL12 sequence as the template. The 23-peptide sequences showed similarity to three distinct regions of the SDF1/CXCL12 molecule. A 3D molecular model was made for each of the phage peptide inserts to visually identify potential areas of steric interference of peptides that simulated CXCL12 regions not in contact with the receptor's Nterminus. An ELISA analysis of the relative binding affinity between the peptides identified 9 peptides with statistically significant results. The candidate pool of 9 peptides was further reduced to 3 peptides based on their affinity for the targeted N-terminus region peptide versus no target peptide present or a scrambled negative control peptide. The results clearly show the Phage Display protocol can be used to target a synthesized region of the ACKR3/CXCR7 N-terminus. The 3 peptides chosen, P20, P3, and P9, will be the basis for further targeting studies.

The temperate marine phage PhiHAP-1 of Halomonas aquamarina possesses a linear plasmid-like prophage genome.

PubMed

Mobberley, Jennifer M; Authement, R Nathan; Segall, Anca M; Paul, John H

2008-07-01

A myovirus-like temperate phage, PhiHAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 x 10(10) VLP ml(-1)) than control cultures (3.83 x 10(7) VLP ml(-1)) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G+C content of 59%. The PhiHAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10(-3)) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the PhiHAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and PhiHAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of PhiHAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The PhiHAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor.

Characterization of antimicrobial resistance, molecular and phage types of Salmonella enterica serovar Typhi isolations.

PubMed

Demczuk, W H B; Finley, R; Nadon, C; Spencer, A; Gilmour, M; Ng, L-K

2010-10-01

Isolation rates in Canada of Salmonella enterica serovar Typhi increased from 0.29 to 0.55 isolations/100,000 population during 2000-2006. Although no ciprofloxacin resistance was detected, nalidixic acid resistance increased from 41% to 80%. Multidrug-resistant S. Typhi represented 18% of the strains tested. Pulsed-field gel electrophoresis (PFGE) analysis of 222 isolates resulted in 91 distinct patterns clustering into four major genetic similarity groups. The five most frequently occurring PFGE patterns accounted for 46% of the isolates. Drug-resistant isolates predominantly occurred in one PFGE similarity group. There were 39 phage types identified in 826 isolates analysed with 60% described by five phage types; 134 were untypable. The phage types associated with multidrug resistance were phage types 53, B1, D1, E1, E9, G3 and M1. Improved integration of epidemiological and laboratory case data will facilitate the protection of public health in Canada during an era of increasing travel and globalization.

Interaction Analysis through Proteomic Phage Display

PubMed Central

2014-01-01

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

Bacterial detection using bacteriophages and gold nanorods by following time-dependent changes in Raman spectral signals.

PubMed

Moghtader, Farzaneh; Tomak, Aysel; Zareie, Hadi M; Piskin, Erhan

2018-03-27

This study attemps to develop bacterial detection strategies using bacteriophages and gold nanorods (GNRs) by Raman spectral analysis. Escherichia coli was selected as the target and its specific phage was used as the bioprobe. Target bacteria and phages were propagated/purified by traditional techniques. GNRs were synthesized by using hexadecyltrimethyl ammonium bromide (CTAB) as stabilizer. A two-step detection strategy was applied: Firstly, the target bacteria were interacted with GNRs in suspensions, and then they were dropped onto silica substrates for detection. It was possible to obtain clear surface-enchanced Raman spectroscopy (SERS) peaks of the target bacteria, even without using phages. In the second step, the phage nanoemulsions were droped onto the bacterial-GNRs complexes on those surfaces and time-dependent changes in the Raman spectra were monitored at different time intervals upto 40 min. These results demonstrated that how one can apply phages with plasmonic nanoparticles for detection of pathogenic bacteria very effectively in a quite simple test.

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

PubMed Central

Dasa, Siva Sai Krishna; Kelly, Kimberly A.

2016-01-01

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

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

PubMed Central

de Mik, G.; de Groot, I.

1977-01-01

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

Virucidal Influence of Ionic Liquids on Phages P100 and MS2

PubMed Central

Fister, Susanne; Mester, Patrick; Sommer, Julia; Witte, Anna K.; Kalb, Roland; Wagner, Martin; Rossmanith, Peter

2017-01-01

An increasing number of publications describe the potential of ionic liquids (ILs) as novel antimicrobials, antibacterial coatings and even as active pharmaceutical ingredients. Nevertheless, a major research area, notably their impact on viruses, has so far been neglected. Consequently the aim of this study was to examine the effects of ILs on the infectivity of viruses. A systematic analysis to investigate the effects of defined structural elements of ILs on virus activity was performed using 55 ILs. All structure activity relationships (SARs) were tested on the human norovirus surrogate phage MS2 and phage P100 representing non-enveloped DNA viruses. Results demonstrate that IL SAR conclusions, established for prokaryotes and eukaryotes, are not readily applicable to the examined phages. A virus-type-dependent IL influence was also apparent. Overall, four ILs, covering different structural elements, were found to reduce phage P100 infectivity by ≥4 log10 units, indicating a virucidal effect, whereas the highest reduction for phage MS2 was about 3 log10 units. Results indicate that future applications of ILs as virucidal agents will require development of novel SARs and the obtained results serve as a good starting point for future studies. PMID:28883814

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

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

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinitymore » to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 {+-} 0.7 x 10{sup 5} M{sup -1} which indicates a strong binding close to that of antibody.« less

T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries.

PubMed

Krumpe, Lauren R H; Atkinson, Andrew J; Smythers, Gary W; Kandel, Andrea; Schumacher, Kathryn M; McMahon, James B; Makowski, Lee; Mori, Toshiyuki

2006-08-01

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.

A novel lineage of myoviruses infecting cyanobacteria is widespread in the oceans.

PubMed

Sabehi, Gazalah; Shaulov, Lihi; Silver, David H; Yanai, Itai; Harel, Amnon; Lindell, Debbie

2012-02-07

Viruses infecting bacteria (phages) are thought to greatly impact microbial population dynamics as well as the genome diversity and evolution of their hosts. Here we report on the discovery of a novel lineage of tailed dsDNA phages belonging to the family Myoviridae and describe its first representative, S-TIM5, that infects the ubiquitous marine cyanobacterium, Synechococcus. The genome of this phage encodes an entirely unique set of structural proteins not found in any currently known phage, indicating that it uses lineage-specific genes for virion morphogenesis and represents a previously unknown lineage of myoviruses. Furthermore, among its distinctive collection of replication and DNA metabolism genes, it carries a mitochondrial-like DNA polymerase gene, providing strong evidence for the bacteriophage origin of the mitochondrial DNA polymerase. S-TIM5 also encodes an array of bacterial-like metabolism genes commonly found in phages infecting cyanobacteria including photosynthesis, carbon metabolism and phosphorus acquisition genes. This suggests a common gene pool and gene swapping of cyanophage-specific genes among different phage lineages despite distinct sets of structural and replication genes. All cytosines following purine nucleotides are methylated in the S-TIM5 genome, constituting a unique methylation pattern that likely protects the genome from nuclease degradation. This phage is abundant in the Red Sea and S-TIM5 gene homologs are widespread in the oceans. This unusual phage type is thus likely to be an important player in the oceans, impacting the population dynamics and evolution of their primary producing cyanobacterial hosts.

A Review of Phage Therapy against Bacterial Pathogens of Aquatic and Terrestrial Organisms.

PubMed

Doss, Janis; Culbertson, Kayla; Hahn, Delilah; Camacho, Joanna; Barekzi, Nazir

2017-03-18

Since the discovery of bacteriophage in the early 1900s, there have been numerous attempts to exploit their innate ability to kill bacteria. The purpose of this report is to review current findings and new developments in phage therapy with an emphasis on bacterial diseases of marine organisms, humans, and plants. The body of evidence includes data from studies investigating bacteriophage in marine and land environments as modern antimicrobial agents against harmful bacteria. The goal of this paper is to present an overview of the topic of phage therapy, the use of phage-derived protein therapy, and the hosts that bacteriophage are currently being used against, with an emphasis on the uses of bacteriophage against marine, human, animal and plant pathogens.

Multi-locus variable-number tandem repeat analysis for outbreak studies of Salmonella enterica serotype Enteritidis

PubMed Central

Malorny, Burkhard; Junker, Ernst; Helmuth, Reiner

2008-01-01

Background Salmonella enterica subsp. enterica serotype Enteritidis is known as an important and pathogenic clonal group which continues to cause worldwide sporadic cases and outbreaks in humans. Here a new multiple-locus variable-number tandem repeat analysis (MLVA) method is reported for highly-discriminative subtyping of Salmonella Enteritidis. Emphasis was given on the most predominant phage types PT4 and PT8. The method comprises multiplex PCR specifically amplifying repeated sequences from nine different loci followed by an automatic fragment size analysis using a multicolor capillary electrophoresis instrument. A total of 240 human, animal, food and environmental isolates of S. Enteritidis including 23 definite phage types were used for development and validation. Furthermore, the MLVA types were compared to the phage types of several isolates from two recent outbreaks to determine the concordance between both methods and to estimate their in vivo stability. The in vitro stability of the two MLVA types specifically for PT4 and PT8 strains were determined by multiple freeze-thaw cycles. Results Seventy-nine different MLVA types were identified in 240 S. Enteritidis strains. The Simpson's diversity index for the MLVA method was 0.919 and Nei diversity values for the nine VNTR loci ranged from 0.07 to 0.65. Twenty-four MLVA types could be assigned to 62 PT4 strains and 21 types to 81 PT8 strains. All outbreak isolates had an indistinguishable outbreak specific MLVA type. The in vitro stability experiments showed no changes of the MLVA type compared to the original isolate. Conclusion This MLVA method is useful to discriminate S. Enteritidis strains even within a single phage type. It is easy in use, fast, and cheap compared to other high-resolution molecular methods and therefore an important tool for surveillance and outbreak studies for S. Enteritidis. PMID:18513386

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

PubMed

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

2014-03-06

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging. © 2013.

Phage particles harboring antibiotic resistance genes in fresh-cut vegetables and agricultural soil.

PubMed

Larrañaga, Olatz; Brown-Jaque, Maryury; Quirós, Pablo; Gómez-Gómez, Clara; Blanch, Anicet R; Rodríguez-Rubio, Lorena; Muniesa, Maite

2018-06-01

Bacteriophages are ubiquitously distributed prokaryotic viruses that are more abundant than bacteria. As a consequence of their life cycle, phages can kidnap part of their host's genetic material, including antibiotic resistance genes (ARGs), which released phage particles transfer in a process called transduction. The spread of ARGs among pathogenic bacteria currently constitutes a serious global health problem. In this study, fresh vegetables (lettuce, spinach and cucumber), and cropland soil were screened by qPCR for ten ARGs (bla TEM , bla CTX-M-1 group, bla CTX-M-9 group, bla OXA-48 , bla VIM , mecA, sul1, qnrA, qnrS and armA) in their viral DNA fraction. The presence of ARGs in the phage DNA was analyzed before and after propagation experiments in an Escherichia coli host strain to evaluate the ability of the phage particles to infect a host. ARGs were found in the phage DNA fraction of all matrices, although with heterogeneous values. ARG prevalence was significantly higher in lettuce and soil, and the most common overall were β-lactamases. After propagation experiments, an increase in ARG densities in phage particles was observed in samples of all four matrices, confirming that part of the isolated phage particles were infectious. This study reveals the abundance of free, replicative ARG-containing phage particles in vegetable matrices and cropland soil. The particles are proposed as vehicles for resistance transfer in these environments, where they can persist for a long time, with the possibility of generating new resistant bacterial strains. Ingestion of these mobile genetic elements may also favor the emergence of new resistances, a risk not previously considered. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable evolution among core genes with therapeutic potential

PubMed Central

2011-01-01

Background Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen. Results Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. Conclusions Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products. PMID:21631945

Novel “Superspreader” Bacteriophages Promote Horizontal Gene Transfer by Transformation

PubMed Central

Bliskovsky, Valery V.; Malagon, Francisco; Baker, James D.; Prince, Jeffrey S.; Klaus, James S.; Adhya, Sankar L.

2017-01-01

ABSTRACT Bacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub “superspreaders,” releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications. PMID:28096488

Leuconostoc bacteriophages from blue cheese manufacture: long-term survival, resistance to thermal treatments, high pressure homogenization and chemical biocides of industrial application.

PubMed

Pujato, Silvina A; Guglielmotti, Daniela M; Ackermann, Hans-W; Patrignani, Francesca; Lanciotti, Rosalba; Reinheimer, Jorge A; Quiberoni, Andrea

2014-05-02

Nine Leuconostoc mesenteroides phages were isolated during blue cheese manufacture yielding faulty products with reduced eye formation. Their morphologies, restriction profiles, host ranges and long-term survival rates (25°C, 8°C, -20°C and -80°C) were analysed. Based on restriction analysis, six of them were further examined regarding resistance to physical (heat and high pressure homogenization, HPH) and chemical treatments (ethanol, sodium hypochlorite, peracetic acid, biocides A, C, E and F). According to their morphology, L. mesenteroides phages studied in the present work belonged to the Caudovirales order and Siphoviridae family. Six distinct restriction patterns were obtained with EcoRV, HindIII, ClaI and XhoI enzymes, revealing interesting phage diversity in the dairy environment. No significant reductions in phage counts were observed after ten months of storage at -20°C and -80°C, while slightly and moderate decrease in phage numbers were noticed at 8°C and 25°C, respectively. The phages subjected to heat treatments generally showed high resistance at 63°C and moderate resistance at 72°C. However, 80°C for 30 min and 90°C for 2 min led to complete inactivation of viral particles. In general, the best ethanol concentration tested was 75%, as complete inactivation for most Leuconostoc phages within 30 min of incubation was achieved. Peracetic acid, and biocides A, C, E and F were highly effective when used at the same or at a moderately lower concentration as recommended by the producer. Usually, moderate or high concentrations (600-1,600 ppm) of sodium hypochlorite were necessary to completely inactivate phage particles. Leuconostoc phages were partially inactivated by HPH treatments as remaining viral particles were found even after 8 passes at 100 MPa. This is the first report of L. mesenteroides phages isolated from an Argentinean dairy cheese plant. The results of this work could be useful for establishing the most effective physical and chemical treatments for inactivating phages in industrial plants and laboratory environments. Copyright © 2014 Elsevier B.V. All rights reserved.

Abundant and diverse clustered regularly interspaced short palindromic repeat spacers in Clostridium difficile strains and prophages target multiple phage types within this pathogen.

PubMed

Hargreaves, Katherine R; Flores, Cesar O; Lawley, Trevor D; Clokie, Martha R J

2014-08-26

Clostridium difficile is an important human-pathogenic bacterium causing antibiotic-associated nosocomial infections worldwide. Mobile genetic elements and bacteriophages have helped shape C. difficile genome evolution. In many bacteria, phage infection may be controlled by a form of bacterial immunity called the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system. This uses acquired short nucleotide sequences (spacers) to target homologous sequences (protospacers) in phage genomes. C. difficile carries multiple CRISPR arrays, and in this paper we examine the relationships between the host- and phage-carried elements of the system. We detected multiple matches between spacers and regions in 31 C. difficile phage and prophage genomes. A subset of the spacers was located in prophage-carried CRISPR arrays. The CRISPR spacer profiles generated suggest that related phages would have similar host ranges. Furthermore, we show that C. difficile strains of the same ribotype could either have similar or divergent CRISPR contents. Both synonymous and nonsynonymous mutations in the protospacer sequences were identified, as well as differences in the protospacer adjacent motif (PAM), which could explain how phages escape this system. This paper illustrates how the distribution and diversity of CRISPR spacers in C. difficile, and its prophages, could modulate phage predation for this pathogen and impact upon its evolution and pathogenicity. Clostridium difficile is a significant bacterial human pathogen which undergoes continual genome evolution, resulting in the emergence of new virulent strains. Phages are major facilitators of genome evolution in other bacterial species, and we use sequence analysis-based approaches in order to examine whether the CRISPR/Cas system could control these interactions across divergent C. difficile strains. The presence of spacer sequences in prophages that are homologous to phage genomes raises an extra level of complexity in this predator-prey microbial system. Our results demonstrate that the impact of phage infection in this system is widespread and that the CRISPR/Cas system is likely to be an important aspect of the evolutionary dynamics in C. difficile. Copyright © 2014 Hargreaves et al.

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.

Life-Style and Genome Structure of Marine Pseudoalteromonas Siphovirus B8b Isolated from the Northwestern Mediterranean Sea

DOE PAGES

Lara, Elena; Holmfeldt, Karin; Solonenko, Natalie; ...

2015-01-14

Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. We isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. In the host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested,more » >99.9% 16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new 'rare virosphere' phage-host model system.« less

Bacteriophage Can Prevent Encrustation and Blockage of Urinary Catheters by Proteus mirabilis

PubMed Central

Nzakizwanayo, Jonathan; Hanin, Aurélie; Alves, Diana R.; McCutcheon, Benjamin; Dedi, Cinzia; Salvage, Jonathan; Knox, Karen; Stewart, Bruce; Metcalfe, Anthony; Clark, Jason; Gilmore, Brendan F.; Gahan, Cormac G. M.; Jenkins, A. Toby A.

2015-01-01

Proteus mirabilis forms dense crystalline biofilms on catheter surfaces that occlude urine flow, leading to serious clinical complications in long-term catheterized patients, but there are presently no truly effective approaches to control catheter blockage by this organism. This study evaluated the potential for bacteriophage therapy to control P. mirabilis infection and prevent catheter blockage. Representative in vitro models of the catheterized urinary tract, simulating a complete closed drainage system as used in clinical practice, were employed to evaluate the performance of phage therapy in preventing blockage. Models mimicking either an established infection or early colonization of the catheterized urinary tract were treated with a single dose of a 3-phage cocktail, and the impact on time taken for catheters to block, as well as levels of crystalline biofilm formation, was measured. In models of established infection, phage treatment significantly increased time taken for catheters to block (∼3-fold) compared to untreated controls. However, in models simulating early-stage infection, phage treatment eradicated P. mirabilis and prevented blockage entirely. Analysis of catheters from models of established infection 10 h after phage application demonstrated that phage significantly reduced crystalline biofilm formation but did not significantly reduce the level of planktonic cells in the residual bladder urine. Taken together, these results show that bacteriophage constitute a promising strategy for the prevention of catheter blockage but that methods to deliver phage in sufficient numbers and within a key therapeutic window (early infection) will also be important to the successful application of phage to this problem. PMID:26711744

CRISPR-Cas-Mediated Phage Resistance Enhances Horizontal Gene Transfer by Transduction.

PubMed

Watson, Bridget N J; Staals, Raymond H J; Fineran, Peter C

2018-02-13

A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through transformation, conjugation, and transduction, which can be advantageous, neutral, or detrimental to fitness. Bacteria and archaea control HGT and phage infection through CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immunity. Although the benefits of resisting phage infection are evident, this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Despite the ability of CRISPR-Cas to limit HGT through conjugation and transformation, its role in transduction is largely overlooked. Transduction is the phage-mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT. We demonstrate that in Pectobacterium atrosepticum , CRISPR-Cas can inhibit the transduction of plasmids and chromosomal loci. In addition, we detected phage-mediated transfer of a large plant pathogenicity genomic island and show that CRISPR-Cas can inhibit its transduction. Despite these inhibitory effects of CRISPR-Cas on transduction, its more common role in phage resistance promotes rather than diminishes HGT via transduction by protecting bacteria from phage infection. This protective effect can also increase transduction of phage-sensitive members of mixed populations. CRISPR-Cas systems themselves display evidence of HGT, but little is known about their lateral dissemination between bacteria and whether transduction can contribute. We show that, through transduction, bacteria can acquire an entire chromosomal CRISPR-Cas system, including cas genes and phage-targeting spacers. We propose that the positive effect of CRISPR-Cas phage immunity on enhancing transduction surpasses the rarer cases where gene flow by transduction is restricted. IMPORTANCE The generation of genetic diversity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through transformation, conjugation, and transduction. Of these, transduction, the phage-mediated transfer of bacterial DNA, is arguably the major route for genetic exchange. CRISPR-Cas adaptive immune systems control gene transfer by conjugation and transformation, but transduction has been mostly overlooked. Our results indicate that CRISPR-Cas can impede, but typically enhances the transduction of plasmids, chromosomal genes, and pathogenicity islands. By limiting wild-type phage replication, CRISPR-Cas immunity increases transduction in both phage-resistant and -sensitive members of mixed populations. Furthermore, we demonstrate mobilization of a chromosomal CRISPR-Cas system containing phage-targeting spacers by generalized transduction, which might partly account for the uneven distribution of these systems in nature. Overall, the ability of CRISPR-Cas to promote transduction reveals an unexpected impact of adaptive immunity on horizontal gene transfer, with broader implications for microbial evolution. Copyright © 2018 Watson et al.

Using the QCM Biosensor-Based T7 Phage Display Combined with Bioinformatics Analysis for Target Identification of Bioactive Small Molecule.

PubMed

Takakusagi, Yoichi; Takakusagi, Kaori; Sugawara, Fumio; Sakaguchi, Kengo

2018-01-01

Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 phage capsid. Here, we describe the T7 phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.

Temperate bacteriophage {phi}O18P from an Aeromonas media isolate: Characterization and complete genome sequence

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

Beilstein, Frauke; Dreiseikelmann, Brigitte

2008-03-30

A group of 74 Aeromonas isolates from surface water of three ponds in Bielefeld, Germany was screened for prophage induction after UV irradiation. The phage {phi}O18P was induced from the Aeromonas media isolate O18. {phi}O18P belongs to the Myoviridae phage family. The complete nucleotide sequence of the double stranded DNA genome of bacteriophage {phi}O18P consists of 33,985 bp. The genome has 5' protruding cohesive ends of 16 bases. On the {phi}O18P genome 46 open reading frames (orfs) were identified which are organized in the modules integration and regulation, replication, head, packaging, tail and lysis. Additionally the phage DNA includes amore » methylase gene. Comparison of the genome architecture with those of other bacteriophages revealed significant similarities to the P2 phage family and especially to the prophages of Aeromonas salmonicida and the Vibrio cholerae phage K139.« less

Phage Display Technology in Biomaterials Engineering: Progress and Opportunities for Applications in Regenerative Medicine.

PubMed

Martins, Ivone M; Reis, Rui L; Azevedo, Helena S

2016-11-18

The field of regenerative medicine has been gaining momentum steadily over the past few years. The emphasis in regenerative medicine is to use various in vitro and in vivo approaches that leverage the intrinsic healing mechanisms of the body to treat patients with disabling injuries and chronic diseases such as diabetes, osteoarthritis, and degenerative disorders of the cardiovascular and central nervous system. Phage display has been successfully employed to identify peptide ligands for a wide variety of targets, ranging from relatively small molecules (enzymes, cell receptors) to inorganic, organic, and biological (tissues) materials. Over the past two decades, phage display technology has advanced tremendously and has become a powerful tool in the most varied fields of research, including biotechnology, materials science, cell biology, pharmacology, and diagnostics. The growing interest in and success of phage display libraries is largely due to its incredible versatility and practical use. This review discusses the potential of phage display technology in biomaterials engineering for applications in regenerative medicine.

The Presence of Phage Orthologous Genes in Helicobacter pylori Correlates with the Presence of the Virulence Factors CagA and VacA.

PubMed

Kyrillos, Alexandra; Arora, Gaurav; Murray, Bradley; Rosenwald, Anne G

2016-06-01

The bacterium Helicobacter pylori is associated with ulcers and the development of gastric cancer. Several genes, including cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), are associated with increased gastric cancer risk. Some strains of H. pylori also contain sequences related to bacteriophage phiHP33; however, the significance of these phage-related sequences remains unknown. We assessed the extent to which phiHP33-related sequences are present in 335 H. pylori strains using homology searches then mapped shared genes between phiHP33 and H. pylori strains onto an existing phylogeny. One hundred and twenty-one H. pylori strains contain phage orthologous sequences, and the presence of the phage-related sequences correlates with the presence of CagA and VacA. Mapping of the phage orthologs onto a phylogeny of H. pylori is consistent with the hypothesis that these genes were acquired by horizontal gene transfer. phiHP33 phage orthologous sequences might be of significance in understanding virulence of different H. pylori strains. © 2015 John Wiley & Sons Ltd.

ssrA (tmRNA) Plays a Role in Salmonella enterica Serovar Typhimurium Pathogenesis

PubMed Central

Julio, Steven M.; Heithoff, Douglas M.; Mahan, Michael J.

2000-01-01

Escherichia coli ssrA encodes a small stable RNA molecule, tmRNA, that has many diverse functions, including tagging abnormal proteins for degradation, supporting phage growth, and modulating the activity of DNA binding proteins. Here we show that ssrA plays a role in Salmonella enterica serovar Typhimurium pathogenesis and in the expression of several genes known to be induced during infection. Moreover, the phage-like attachment site, attL, encoded within ssrA, serves as the site of integration of a region of Salmonella-specific sequence; adjacent to the 5′ end of ssrA is another region of Salmonella-specific sequence with extensive homology to predicted proteins encoded within the unlinked Salmonella pathogenicity island SPI4. S. enterica serovar Typhimurium ssrA mutants fail to support the growth of phage P22 and are delayed in their ability to form viable phage particles following induction of a phage P22 lysogen. These data indicate that ssrA plays a role in the pathogenesis of Salmonella, serves as an attachment site for Salmonella-specific sequences, and is required for the growth of phage P22. PMID:10692360

Phage diabody repertoires for selection of large numbers of bispecific antibody fragments.

PubMed

McGuinness, B T; Walter, G; FitzGerald, K; Schuler, P; Mahoney, W; Duncan, A R; Hoogenboom, H R

1996-09-01

Methods for the generation of large numbers of different bispecific antibodies are presented. Cloning strategies are detailed to create repertoires of bispecific diabody molecules with variability on one or both of the antigen binding sites. This diabody format, when combined with the power of phage display technology, allows the generation and analysis of thousands of different bispecific molecules. Selection for binding presumably also selects for more stable diabodies. Phage diabody libraries enable screening or selection of the best combination bispecific molecule with regards to affinity of binding, epitope recognition and pairing before manufacture of the best candidate.

Why Be Temperate: Lessons from Bacteriophage λ.

PubMed

Gandon, Sylvain

2016-05-01

Many pathogens have evolved the ability to induce latent infections of their hosts. The bacteriophage λ is a classical model for exploring the regulation and the evolution of latency. Here, I review recent experimental studies on phage λ that identify specific conditions promoting the evolution of lysogenic life cycles. In addition, I present specific adaptations of phage λ that allow this virus to react plastically to variations in the environment and to reactivate its lytic life cycle. All of these different examples are discussed in the light of evolutionary epidemiology theory to disentangle the different evolutionary forces acting on temperate phages. Understanding phage λ adaptations yield important insights into the evolution of latency in other microbes, including several life-threatening human pathogens. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

RNA-seq analysis of prophage induction in multidrug-resistant salmonella enterica serovar typhimurium DT104 following exposure to the agricultural antibiotic carbadox

USDA-ARS?s Scientific Manuscript database

Non-typhoidal Salmonella is a leading cause of U.S. foodborne disease and food-related deaths. Multidrug-resistant (MDR) Salmonella Typhimurium DT104 contains 5 prophages in the genome that may be induced to produce phage under various environmental conditions, including antibiotic exposure. We inve...

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2009-11-18

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

Detection of Foodborne Pathogenic Bacteria using Bacteriophage Tail Spike Proteins

NASA Astrophysics Data System (ADS)

Poshtiban, Somayyeh

Foodborne infections are worldwide health problem with tremendous social and financial impacts. Efforts are focused on developing accurate and reliable technologies for detection of food contaminations in early stages preferably on-site. This thesis focuses on interfacing engineering and biology by combining phage receptor binding proteins (RBPs) with engineered platforms including microresonator-based biosensors, magnetic particles and polymerase chain reaction (PCR) to develop bacterial detection sensors. We used phage RBPs as target specific bioreceptors to develop an enhanced microresonator array for bacterial detection. These resonator beams are optimized to feature a high natural frequency while offer large surface area for capture of bacteria. Theoretical analysis indicates a high mass sensitivity with a threshold for the detection of a single bacterial cell. We used phage RBPs as target specific bioreceptors, and successfully demonstrated the application of these phage RBB-immobilized arrays for specific detection of C. jejuni cells. We also developed a RBP-derivatized magnetic pre-enrichment method as an upstream sample preparation method to improve sensitivity and specificity of PCR for detection of bacterial cells in various food samples. The combination of RBP-based magnetic separation and real-time PCR allowed the detection of small number of bacteria in artificially contaminated food samples without any need for time consuming pre-enrichment step through culturing. We also looked into integration of the RBP-based magnetic separation with PCR onto a single microfluidic lab-on-a-chip to reduce the overall turnaround time.

Basic Phage Mathematics.

PubMed

Abedon, Stephen T; Katsaounis, Tena I

2018-01-01

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

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

PubMed

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

2016-08-01

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

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

PubMed

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

2014-02-01

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

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

PubMed

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

2008-01-01

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

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

PubMed Central

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

2008-01-01

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

Recombinant human antibody fragment against tetanus toxoid produced by phage display.

PubMed

Neelakantam, B; Sridevi, N V; Shukra, A M; Sugumar, P; Samuel, S; Rajendra, L

2014-03-01

Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen.

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

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

Cardarelli, Lia; Lam, Robert; Tuite, Ashleigh

2010-08-17

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

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

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

Cardarelli, Lia; Lam, Robert; Tuite, Ashleigh

2011-11-23

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

Exposing the Secrets of Two Well-Known Lactobacillus casei Phages, J-1 and PL-1, by Genomic and Structural Analysis

PubMed Central

Dieterle, Maria Eugenia; Bowman, Charles; Batthyany, Carlos; Lanzarotti, Esteban; Turjanski, Adrián; Hatfull, Graham

2014-01-01

Bacteriophage J-1 was isolated in 1965 from an abnormal fermentation of Yakult using Lactobacillus casei strain Shirota, and a related phage, PL-1, was subsequently recovered from a strain resistant to J-1. Complete genome sequencing shows that J-1 and PL-1 are almost identical, but PL-1 has a deletion of 1.9 kbp relative to J-1, resulting in the loss of four predicted gene products involved in immunity regulation. The structural proteins were identified by mass spectrometry analysis. Similarly to phage A2, two capsid proteins are generated by a translational frameshift and undergo proteolytic processing. The structure of gene product 16 (gp16), a putative tail protein, was modeled based on the crystal structure of baseplate distal tail proteins (Dit) that form the baseplate hub in other Siphoviridae. However, two regions of the C terminus of gp16 could not be modeled using this template. The first region accounts for the differences between J-1 and PL-1 gp16 and showed sequence similarity to carbohydrate-binding modules (CBMs). J-1 and PL-1 GFP-gp16 fusions bind specifically to Lactobacillus casei/paracasei cells, and the addition of l-rhamnose inhibits binding. J-1 gp16 exhibited a higher affinity than PL-1 gp16 for cell walls of L. casei ATCC 27139 in phage adsorption inhibition assays, in agreement with differential adsorption kinetics observed for both phages in this strain. The data presented here provide insights into how Lactobacillus phages interact with their hosts at the first steps of infection. PMID:25217012

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

PubMed Central

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

2013-01-01

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

Characterization of bacteriophages Cp1 and Cp2, the strain-typing agents for Xanthomonas axonopodis pv. citri.

PubMed

Ahmad, Abdelmonim Ali; Ogawa, Megumi; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

2014-01-01

The strains of Xanthomonas axonopodis pv. citri, the causative agent of citrus canker, are historically classified based on bacteriophage (phage) sensitivity. Nearly all X. axonopodis pv. citri strains isolated from different regions in Japan are lysed by either phage Cp1 or Cp2; Cp1-sensitive (Cp1(s)) strains have been observed to be resistant to Cp2 (Cp2(r)) and vice versa. In this study, genomic and molecular characterization was performed for the typing agents Cp1 and Cp2. Morphologically, Cp1 belongs to the Siphoviridae. Genomic analysis revealed that its genome comprises 43,870-bp double-stranded DNA (dsDNA), with 10-bp 3'-extruding cohesive ends, and contains 48 open reading frames. The genomic organization was similar to that of Xanthomonas phage phiL7, but it lacked a group I intron in the DNA polymerase gene. Cp2 resembles morphologically Escherichia coli T7-like phages of Podoviridae. The 42,963-bp linear dsDNA genome of Cp2 contained terminal repeats. The Cp2 genomic sequence has 40 open reading frames, many of which did not show detectable homologs in the current databases. By proteomic analysis, a gene cluster encoding structural proteins corresponding to the class III module of T7-like phages was identified on the Cp2 genome. Therefore, Cp1 and Cp2 were found to belong to completely different virus groups. In addition, we found that Cp1 and Cp2 use different molecules on the host cell surface as phage receptors and that host selection of X. axonopodis pv. citri strains by Cp1 and Cp2 is not determined at the initial stage by binding to receptors.

Isolation and Characterization of Lytic Phage vB_EcoM_JS09 against Clinically Isolated Antibiotic-Resistant Avian Pathogenic Escherichia coli and Enterotoxigenic Escherichia coli.

PubMed

Zhou, Yan; Bao, Hongduo; Zhang, Hui; Wang, Ran

2015-01-01

To characterize the lytic coliphage vB_EcoM_JS09 (phage JS09) isolated from sewage samples of a swine farm in Jiangsu Province, China, which infects antibiotic-resistant avian pathogenic Escherichia coli (APEC) and enterotoxigenic E. coli (ETEC). Transmission electron microscopy revealed that phage JS09 has an isometric icosahedral head (76 nm in diameter) and a long contractile tail (140 nm in length) and features a T-even morphology. Its latent period was 30 min and the average burst size was 79 phage particles per infected cell. It attached to the host cells within 9 min. JS09 could infect 16 clinically isolated APEC and ETEC strains and the laboratory-engineered E. coli K and B strains. Ten of the clinical isolates of E. coli were resistant to antibiotics. At a multiplicity of infection of 10, 3, 1, or 0.3, the phage caused rapid cell lysis within 2 h, resulting in 5- to 10-fold reductions in cell concentration. Sequencing of the JS09 genome revealed a 169.148-kb linear but circularly permuted and terminally redundant dsDNA with 37.98% G+C content. Two hundred seventy-three open reading frames were predicted to be coding sequences, 135 of which were functionally defined and organized in a modular format which includes modules for DNA replication, DNA packaging, structural proteins, and host cell lysis proteins. Phage JS09 is assigned to the Caudovirales order (Myoviridae phage family), and it is considered a T4-like phage based on its morphological, genomic, and growth characteristics. JS09 gp37, a receptor-binding protein (RBP) important for host cell infection, shares little homology with other RBP in the NCBI database, which suggests that the variable regions in gp37 determine the unique host range of phage JS09. Protein sequence comparisons cluster the putative 'RBP' of JS09 much more closely with those of Yersinia phage phiD1, phage TuIa, and phage TuIb. A novel lytic coliphage named JS09 was isolated from sewage samples of a swine farm in Jiangsu Province, China. It could infect antibiotic-resistant APEC and ETEC. The morphological, genomic, and growth characteristics of JS09 were studied, and this will be helpful for phage therapy in controlling diseases caused by APEC and ETEC. © 2015 S. Karger AG, Basel.

Genetic evidence for the involvement of the S-layer protein gene sap and the sporulation genes spo0A, spo0B, and spo0F in Phage AP50c infection of Bacillus anthracis.

PubMed

Plaut, Roger D; Beaber, John W; Zemansky, Jason; Kaur, Ajinder P; George, Matroner; Biswas, Biswajit; Henry, Matthew; Bishop-Lilly, Kimberly A; Mokashi, Vishwesh; Hannah, Ryan M; Pope, Robert K; Read, Timothy D; Stibitz, Scott; Calendar, Richard; Sozhamannan, Shanmuga

2014-03-01

In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.

Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A, spo0B, and spo0F in Phage AP50c Infection of Bacillus anthracis

PubMed Central

Beaber, John W.; Zemansky, Jason; Kaur, Ajinder P.; George, Matroner; Biswas, Biswajit; Henry, Matthew; Bishop-Lilly, Kimberly A.; Mokashi, Vishwesh; Hannah, Ryan M.; Pope, Robert K.; Read, Timothy D.; Stibitz, Scott; Calendar, Richard; Sozhamannan, Shanmuga

2014-01-01

In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer. PMID:24363347

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

PubMed

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

2018-04-05

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

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

PubMed Central

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

2018-01-01

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

Difunctional bacteriophage conjugated with photosensitizers for Candida albicans-targeting photodynamic inactivation.

PubMed

Dong, Shuai; Shi, Hongxi; Zhang, Xintong; Chen, Xi; Cao, Donghui; Mao, Chuanbin; Gao, Xiang; Wang, Li

2018-01-01

Candida albicans is the most prevalent fungal pathogen of the human microbiota, causing infections ranging from superficial infections of the skin to life-threatening systemic infections. Due to the increasing occurrence of antibiotic-resistant C. albicans strains, new approaches to control this pathogen are needed. Photodynamic inactivation is an emerging alternative to treat infections based on the interactions between visible light and photosensitisers, in which pheophorbide a (PPA) is a chlorophyll-based photosensitizer that could induce cell death after light irradiation. Due to PPA's phototoxicity and low efficiency, the main challenge is to implement photosensitizer cell targeting and attacking. In this study, PPA was conjugated with JM-phage by EDC/NHS crosslinking. UV-Vis spectra was used to determine the optimum conjugation percentages of PPA and JM-phage complex for photodynamic inactivation. After photodynamic inactivation, the efficacy of PPA-JM-phage was assessed by performing in vitro experiments, such as MTS assay, scanning electron microscopy, measurement of dysfunctional mitochondria, ROS accumulation, S cell arrest and apoptotic pathway. A single-chain variable-fragment phage (JM) with high affinity to MP65 was screened from human single-fold single-chain variable-fragment libraries and designed as a binding target for C. albicans cells. Subsequently, PPa was integrated into JM phage to generate a combined nanoscale material, which was called PPA-JM-phage. After photodynamic inactivation, the growth of C. albicans was inhibited by PPA-JM-phage and apoptosis was observed. Scanning electron microscopy analysis revealed shrinking and rupturing of C. albicans . We also found that depolarization of mitochondrial membrane potential was decreased and intracellular reactive oxygen species levels were elevated significantly in C. albicans inhibited by PPA-JM-phage. Additionally, PPA-JM-phage also lead to S-phase arrest, and metacaspase activation resulting from mitochondrial dysfunction was also found to be involved in C. albicans apoptosis. PPa-JM-phage may induce C. albicans apoptosis through a caspase-dependent pathway and the results herein shed light on the potential application of phtototherapeutic nanostructures in fungal inactivation.

Difunctional bacteriophage conjugated with photosensitizers for Candida albicans-targeting photodynamic inactivation

PubMed Central

Dong, Shuai; Shi, Hongxi; Zhang, Xintong; Chen, Xi; Cao, Donghui; Mao, Chuanbin; Gao, Xiang; Wang, Li

2018-01-01

Background Candida albicans is the most prevalent fungal pathogen of the human microbiota, causing infections ranging from superficial infections of the skin to life-threatening systemic infections. Due to the increasing occurrence of antibiotic-resistant C. albicans strains, new approaches to control this pathogen are needed. Photodynamic inactivation is an emerging alternative to treat infections based on the interactions between visible light and photosensitisers, in which pheophorbide a (PPA) is a chlorophyll-based photosensitizer that could induce cell death after light irradiation. Due to PPA’s phototoxicity and low efficiency, the main challenge is to implement photosensitizer cell targeting and attacking. Methods In this study, PPA was conjugated with JM-phage by EDC/NHS crosslinking. UV-Vis spectra was used to determine the optimum conjugation percentages of PPA and JM-phage complex for photodynamic inactivation. After photodynamic inactivation, the efficacy of PPA-JM-phage was assessed by performing in vitro experiments, such as MTS assay, scanning electron microscopy, measurement of dysfunctional mitochondria, ROS accumulation, S cell arrest and apoptotic pathway. Results A single-chain variable-fragment phage (JM) with high affinity to MP65 was screened from human single-fold single-chain variable-fragment libraries and designed as a binding target for C. albicans cells. Subsequently, PPa was integrated into JM phage to generate a combined nanoscale material, which was called PPA-JM-phage. After photodynamic inactivation, the growth of C. albicans was inhibited by PPA-JM-phage and apoptosis was observed. Scanning electron microscopy analysis revealed shrinking and rupturing of C. albicans. We also found that depolarization of mitochondrial membrane potential was decreased and intracellular reactive oxygen species levels were elevated significantly in C. albicans inhibited by PPA-JM-phage. Additionally, PPA-JM-phage also lead to S-phase arrest, and metacaspase activation resulting from mitochondrial dysfunction was also found to be involved in C. albicans apoptosis. Conclusion PPa-JM-phage may induce C. albicans apoptosis through a caspase-dependent pathway and the results herein shed light on the potential application of phtototherapeutic nanostructures in fungal inactivation. PMID:29692614

Shigella Phages Isolated during a Dysentery Outbreak Reveal Uncommon Structures and Broad Species Diversity.

PubMed

Doore, Sarah M; Schrad, Jason R; Dean, William F; Dover, John A; Parent, Kristin N

2018-04-15

In 2016, Michigan experienced the largest outbreak of shigellosis, a type of bacillary dysentery caused by Shigella spp., since 1988. Following this outbreak, we isolated 16 novel Shigella -infecting bacteriophages (viruses that infect bacteria) from environmental water sources. Most well-known bacteriophages infect the common laboratory species Escherichia coli and Salmonella enterica , and these phages have built the foundation of molecular and bacteriophage biology. Until now, comparatively few bacteriophages were known to infect Shigella spp., which are close relatives of E. coli We present a comprehensive analysis of these phages' host ranges, genomes, and structures, revealing genome sizes and capsid properties that are shared by very few previously described phages. After sequencing, a majority of the Shigella phages were found to have genomes of an uncommon size, shared by only 2% of all reported phage genomes. To investigate the structural implications of this unusual genome size, we used cryo-electron microscopy to resolve their capsid structures. We determined that these bacteriophage capsids have similarly uncommon geometry. Only two other viruses with this capsid structure have been described. Since most well-known bacteriophages infect Escherichia or Salmonella , our understanding of bacteriophages has been limited to a subset of well-described systems. Continuing to isolate phages using nontraditional strains of bacteria can fill gaps that currently exist in bacteriophage biology. In addition, the prevalence of Shigella phages during a shigellosis outbreak may suggest a potential impact of human health epidemics on local microbial communities. IMPORTANCE Shigella spp. bacteria are causative agents of dysentery and affect more than 164 million people worldwide every year. Despite the need to combat antibiotic-resistant Shigella strains, relatively few Shigella -infecting bacteriophages have been described. By specifically looking for Shigella -infecting phages, this work has identified new isolates that (i) may be useful to combat Shigella infections and (ii) fill gaps in our knowledge of bacteriophage biology. The rare qualities of these new isolates emphasize the importance of isolating phages on "nontraditional" laboratory strains of bacteria to more fully understand both the basic biology and diversity of bacteriophages. Copyright © 2018 American Society for Microbiology.

Excision of thymine dimers in vitro by extracts of bacteriophage-infected Escherichia coli

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

Friedberg, E.C.; Minton, K.; Pawl, G.

1974-05-01

Extracts of DNA polymerase I defective Escherichia coli infected with phage T4 contain an exonuclease activity that removes thymine dimers from uv- irradiated DNA previously nicked with T4 uv endonuclease. This activity is not expressed if cells are infected in the presence of chloramphenicol. The enzyme has a requirement for divalent cation and is not affected by caffeine, but excision is inhibited in the presence of proflavine. The enzyme is present in all phage T4 mutants thus far examined, including 25 uv-sensitive mutants isolated during the course of the experiments, all of which are defective in the v gene. Amore » similar activity can be detected in cells infected with phages T2, T3, and T6, but not in cells infected with phage T7. (auth)« less

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.

Genomic characterization of bacteriophage vB_PcaP_PP2 infecting Pectobacterium carotovorum subsp. carotovorum, a new member of a proposed genus in the subfamily Autographivirinae.

PubMed

Lim, Jeong-A; Heu, Sunggi; Park, Jinwoo; Roh, Eunjung

2017-08-01

Bacteriophage vB_PcaP_PP2 (PP2) is a novel virulent phage that infects the plant-pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. PP2 phage has a 41,841-bp double-stranded DNA encoding 47 proteins, and it was identified as a member of the family Podoviridae by transmission electron microscopy. Nineteen of its open reading frames (ORFs) show homology to functional proteins, and 28 ORFs have been characterized as hypothetical proteins. PP2 phage is homologous to Cronobacter phage vB_CskP_GAP227 and Dev-CD-23823. Based on phylogenetic analysis, PP2 and its homologous bacteriophages form a new group within the subfamily Autographivirinae in the family Podoviridae, suggesting the need to establish a new genus. No lysogenic-cycle-related genes or bacterial toxins were identified.

Graphical analysis of flow cytometer data for characterizing controlled fluorescent protein display on λ phage.

PubMed

Sokolenko, Stanislav; Nicastro, Jessica; Slavcev, Roderick; Aucoin, Marc G

2012-12-01

As native virus particles typically cannot be resolved using a flow cytometer, the general practice is to use fluorescent dyes to label the particles. In this work, an attempt was made to use a common commercial flow cytometer to characterize a phage display strategy that allows for controlled levels of protein display, in this case, eGFP. To achieve this characterization, a number of data processing steps were needed to ensure that the observed phenomena were indeed capturing differences in the phages produced. Phage display of eGFP resulted in altered side scatter and fluorescence profile, and sub-populations could be identified within what would otherwise be considered uniform populations. Surprisingly, this study has found that side scatter may be used in the future to characterize the display of nonfluorescent proteins. Copyright © 2012 International Society for Advancement of Cytometry.

Complete genome sequence of a phage hyperparasite of Candidatus Xenohaliotis californiensis (Rickettsiales) - a pathogen of Haliotis spp (Gasteropoda).

PubMed

Cruz-Flores, Roberto; Cáceres-Martínez, Jorge; Del Río-Portilla, Miguel Ángel; Licea-Navarro, Alexei F; Gonzales-Sánchez, Ricardo; Guerrero, Abraham

2018-04-01

Bacteriophages are recognized as major mortality agents of microbes, among them intracellular marine rickettsiales-like bacteria. Recently, a phage hyperparasite of Candidatus Xenohaliotis californiensis (CXc) has been described. This bacterium is considered the causal agent of Withering Syndrome (WS) which is a chronic and potentially lethal disease of abalone species from California, USA and the peninsula of Baja California, Mexico. This hyperparasite which infects CXc could be used as a biocontrol agent for WS. Therefore, it is necessary to obtain genomic information to characterize this phage. In this study, the first complete genome sequence of a novel phage, Xenohaliotis phage (pCXc) was determined. The complete genome of pCXc from red abalone (Haliotis rufescens) is 35,728 bp, while the complete genome of pCXc from yellow abalone (Haliotis corrugata) is 35,736 bp. Both phage genomes consist of double-stranded DNA with a G + C content of 38.9%. In both genomes 33 open reading frames (ORFs) were predicted. Only 10 ORFs encode proteins that have identifiable functional homologues. These 10 ORFs were classified by function, including structural, DNA replication, DNA packaging, nucleotide transport and metabolism, life cycle regulation, recombination and repair, and additional functions. A PCR method for the specific detection of pCXc was developed. This information will help to understand a new group of phages that infect intracellular marine rickettsiales-like bacteria in mollusks.

Quantification of Shiga Toxin-Converting Bacteriophages in Wastewater and in Fecal Samples by Real-Time Quantitative PCR ▿

PubMed Central

Imamovic, Lejla; Ballesté, Elisenda; Jofre, Juan; Muniesa, Maite

2010-01-01

Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 103 gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 1010 GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 105 GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 104 GC per g of sample). The stx2 genes and stx2 variants were detected in the viral fraction of some of the samples after sequencing of stx2 fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment. PMID:20622134

Genetically Engineered Virulent Phage Banks in the Detection and Control of Emergent Pathogenic Bacteria

PubMed Central

Blois, Hélène; Iris, François

2010-01-01

Natural outbreaks of multidrug-resistant microorganisms can cause widespread devastation, and several can be used or engineered as agents of bioterrorism. From a biosecurity standpoint, the capacity to detect and then efficiently control, within hours, the spread and the potential pathological effects of an emergent outbreak, for which there may be no effective antibiotics or vaccines, become key challenges that must be met. We turned to phage engineering as a potentially highly flexible and effective means to both detect and eradicate threats originating from emergent (uncharacterized) bacterial strains. To this end, we developed technologies allowing us to (1) concurrently modify multiple regions within the coding sequence of a gene while conserving intact the remainder of the gene, (2) reversibly interrupt the lytic cycle of an obligate virulent phage (T4) within its host, (3) carry out efficient insertion, by homologous recombination, of any number of engineered genes into the deactivated genomes of a T4 wild-type phage population, and (4) reactivate the lytic cycle, leading to the production of engineered infective virulent recombinant progeny. This allows the production of very large, genetically engineered lytic phage banks containing, in an E. coli host, a very wide spectrum of variants for any chosen phage-associated function, including phage host-range. Screening of such a bank should allow the rapid isolation of recombinant T4 particles capable of detecting (ie, diagnosing), infecting, and destroying hosts belonging to gram-negative bacterial species far removed from the original E. coli host. PMID:20569057

Marine viruses discovered via metagenomics shed light on viral strategies throughout the oceans

NASA Astrophysics Data System (ADS)

Coutinho, Felipe H.; Silveira, Cynthia B.; Gregoracci, Gustavo B.; Thompson, Cristiane C.; Edwards, Robert A.; Brussaard, Corina P. D.; Dutilh, Bas E.; Thompson, Fabiano L.

2017-07-01

Marine viruses are key drivers of host diversity, population dynamics and biogeochemical cycling and contribute to the daily flux of billions of tons of organic matter. Despite recent advancements in metagenomics, much of their biodiversity remains uncharacterized. Here we report a data set of 27,346 marine virome contigs that includes 44 complete genomes. These outnumber all currently known phage genomes in marine habitats and include members of previously uncharacterized lineages. We designed a new method for host prediction based on co-occurrence associations that reveals these viruses infect dominant members of the marine microbiome such as Prochlorococcus and Pelagibacter. A negative association between host abundance and the virus-to-host ratio supports the recently proposed Piggyback-the-Winner model of reduced phage lysis at higher host densities. An analysis of the abundance patterns of viruses throughout the oceans revealed how marine viral communities adapt to various seasonal, temperature and photic regimes according to targeted hosts and the diversity of auxiliary metabolic genes.

Phage or foe: an insight into the impact of viral predation on microbial communities.

PubMed

Fernández, Lucía; Rodríguez, Ana; García, Pilar

2018-05-01

Since their discovery, bacteriophages have been traditionally regarded as the natural enemies of bacteria. However, recent advances in molecular biology techniques, especially data from "omics" analyses, have revealed that the interplay between bacterial viruses and their hosts is far more intricate than initially thought. On the one hand, we have become more aware of the impact of viral predation on the composition and genetic makeup of microbial communities thanks to genomic and metagenomic approaches. Moreover, data obtained from transcriptomic, proteomic, and metabolomic studies have shown that responses to phage predation are complex and diverse, varying greatly depending on the bacterial host, phage, and multiplicity of infection. Interestingly, phage exposure may alter different phenotypes, including virulence and biofilm formation. The complexity of the interactions between microbes and their viral predators is also evidenced by the link between quorum-sensing signaling pathways and bacteriophage resistance. Overall, new data increasingly suggests that both temperate and virulent phages have a positive effect on the evolution and adaptation of microbial populations. From this perspective, further research is still necessary to fully understand the interactions between phage and host under conditions that allow co-existence of both populations, reflecting more accurately the dynamics in natural microbial communities.

Targeting Pancreatic Islets with Phage Display Assisted by Laser Pressure Catapult Microdissection

PubMed Central

Yao, Virginia J.; Ozawa, Michael G.; Trepel, Martin; Arap, Wadih; McDonald, Donald M.; Pasqualini, Renata

2005-01-01

Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature. PMID:15681844

Immunological basis of M13 phage vaccine: Regulation under MyD88 and TLR9 signaling.

PubMed

Hashiguchi, Shuhei; Yamaguchi, Yuya; Takeuchi, Osamu; Akira, Shizuo; Sugimura, Kazuhisa

2010-11-05

Peptide-displaying bacteriophages induce mimotope-specific antibody responses, suggesting a novel application of phage-display library as bacteriophage vaccine. We examined the antibody response against M13 phage in mice induced by an i.p. administration of M13 phage in phosphate-buffered saline. We showed here that firstly, mice showed strong IgG antibody responses, particularly, in IgG2b, IgG2c, and IgG3 subclasses even in primary responses. Secondly, IgG production in primary response is totally dependent on MyD88 signaling. These responses were almost comparable, but slightly weaker, in TLR2-, TLR4- and TLR7-deficient mice relative to wild-type mice, suggesting that this enhancing effect is not due to plausible LPS contamination. Thirdly, although primary IgG1 response was not detected in wild-type mice, remarkable IgG1 response was induced in TLR9-deficient mice, suggesting that TLR9 pathway functions as regulatory, but not a simple augmenting signaling cascade, and furthermore, the enhanced IgG1 response was not due to adjuvant effect of single-stranded DNA derived from M13 phage. Thus, innate immunity including TLR regulation is crucial for M13 phage vaccine design. Copyright © 2010 Elsevier Inc. All rights reserved.

Using lytic bacteriophages to eliminate or significantly reduce contamination of food by foodborne bacterial pathogens.

PubMed

Sulakvelidze, Alexander

2013-10-01

Bacteriophages (also called 'phages') are viruses that kill bacteria. They are arguably the oldest (3 billion years old, by some estimates) and most ubiquitous (total number estimated to be 10(30) -10(32) ) known organisms on Earth. Phages play a key role in maintaining microbial balance in every ecosystem where bacteria exist, and they are part of the normal microflora of all fresh, unprocessed foods. Interest in various practical applications of bacteriophages has been gaining momentum recently, with perhaps the most attention focused on using them to improve food safety. That approach, called 'phage biocontrol', typically includes three main types of applications: (i) using phages to treat domesticated livestock in order to reduce their intestinal colonization with, and shedding of, specific bacterial pathogens; (ii) treatments for decontaminating inanimate surfaces in food-processing facilities and other food establishments, so that foods processed on those surfaces are not cross-contaminated with the targeted pathogens; and (iii) post-harvest treatments involving direct applications of phages onto the harvested foods. This mini-review primarily focuses on the last type of intervention, which has been gaining the most momentum recently. Indeed, the results of recent studies dealing with improving food safety, and several recent regulatory approvals of various commercial phage preparations developed for post-harvest food safety applications, strongly support the idea that lytic phages may provide a safe, environmentally-friendly, and effective approach for significantly reducing contamination of various foods with foodborne bacterial pathogens. However, some important technical and nontechnical problems may need to be addressed before phage biocontrol protocols can become an integral part of routine food safety intervention strategies implemented by food industries in the USA. © 2013 Society of Chemical Industry.

Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans.

PubMed

Kang, Ilnam; Oh, Hyun-Myung; Kang, Dongmin; Cho, Jang-Cheon

2013-07-23

The abundance, genetic diversity, and crucial ecological and evolutionary roles of marine phages have prompted a large number of metagenomic studies. However, obtaining a thorough understanding of marine phages has been hampered by the low number of phage isolates infecting major bacterial groups other than cyanophages and pelagiphages. Therefore, there is an urgent requirement for the isolation of phages that infect abundant marine bacterial groups. In this study, we isolated and characterized HMO-2011, a phage infecting a bacterium of the SAR116 clade, one of the most abundant marine bacterial lineages. HMO-2011, which infects "Candidatus Puniceispirillum marinum" strain IMCC1322, has an ~55-kb dsDNA genome that harbors many genes with novel features rarely found in cultured organisms, including genes encoding a DNA polymerase with a partial DnaJ central domain and an atypical methanesulfonate monooxygenase. Furthermore, homologs of nearly all HMO-2011 genes were predominantly found in marine metagenomes rather than cultured organisms, suggesting the novelty of HMO-2011 and the prevalence of this phage type in the oceans. A significant number of the viral metagenome sequences obtained from the ocean surface were best assigned to the HMO-2011 genome. The number of reads assigned to HMO-2011 accounted for 10.3%-25.3% of the total reads assigned to viruses in seven viromes from the Pacific and Indian Oceans, making the HMO-2011 genome the most or second-most frequently assigned viral genome. Given its ability to infect the abundant SAR116 clade and its widespread distribution, Puniceispirillum phage HMO-2011 could be an important resource for marine virus research.

Microencapsulation of Clostridium difficile specific bacteriophages using microfluidic glass capillary devices for colon delivery using pH triggered release

PubMed Central

Vinner, Gurinder K.; Vladisavljević, Goran T.; Clokie, Martha R. J.

2017-01-01

The prevalence of pathogenic bacteria acquiring multidrug antibiotic resistance is a global health threat to mankind. This has motivated a renewed interest in developing alternatives to conventional antibiotics including bacteriophages (viruses) as therapeutic agents. The bacterium Clostridium difficile causes colon infection and is particularly difficult to treat with existing antibiotics; phage therapy may offer a viable alternative. The punitive environment within the gastrointestinal tract can inactivate orally delivered phages. C. difficile specific bacteriophage, myovirus CDKM9 was encapsulated in a pH responsive polymer (Eudragit® S100 with and without alginate) using a flow focussing glass microcapillary device. Highly monodispersed core-shell microparticles containing phages trapped within the particle core were produced by in situ polymer curing using 4-aminobenzoic acid dissolved in the oil phase. The size of the generated microparticles could be precisely controlled in the range 80 μm to 160 μm through design of the microfluidic device geometry and by varying flow rates of the dispersed and continuous phase. In contrast to free ‘naked’ phages, those encapsulated within the microparticles could withstand a 3 h exposure to simulated gastric fluid at pH 2 and then underwent a subsequent pH triggered burst release at pH 7. The significance of our research is in demonstrating that C. difficile specific phage can be formulated and encapsulated in highly uniform pH responsive microparticles using a microfluidic system. The microparticles were shown to afford significant protection to the encapsulated phage upon prolonged exposure to an acid solution mimicking the human stomach environment. Phage encapsulation and subsequent release kinetics revealed that the microparticles prepared using Eudragit® S100 formulations possess pH responsive characteristics with phage release triggered in an intestinal pH range suitable for therapeutic purposes. The results reported here provide proof-of-concept data supporting the suitability of our approach for colon targeted delivery of phages for therapeutic purposes. PMID:29023522

Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

PubMed Central

Matochko, Wadim L.; Derda, Ratmir

2013-01-01

Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N × 1 frequency vector n = ||ni||, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N × N matrix and a stochastic sampling operator (S a). The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of S a and use them to define the sequencing operator (S e q). Sequencing without any bias and errors is S e q = S a IN, where IN is a N × N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (C E N), which describes elimination or statistically significant downsampling, of specific reads during the sequencing process. PMID:24416071

Influence of environmental variables in the efficiency of phage therapy in aquaculture.

PubMed

Silva, Yolanda J; Costa, Liliana; Pereira, Carla; Cunha, Ângela; Calado, Ricardo; Gomes, Newton C M; Almeida, Adelaide

2014-09-01

Aquaculture facilities worldwide continue to experience significant economic losses because of disease caused by pathogenic bacteria, including multidrug-resistant strains. This scenario drives the search for alternative methods to inactivate pathogenic bacteria. Phage therapy is currently considered as a viable alternative to antibiotics for inactivation of bacterial pathogens in aquaculture systems. While phage therapy appears to represent a useful and flexible tool for microbiological decontamination of aquaculture effluents, the effect of physical and chemical properties of culture waters on the efficiency of this technology has never been reported. The present study aimed to evaluate the effect of physical and chemical properties of aquaculture waters (e.g. pH, temperature, salinity and organic matter content) on the efficiency of phage therapy under controlled experimental conditions in order to provide a basis for the selection of the most suitable protocol for subsequent experiments. A bioluminescent genetically transformed Escherichia coli was selected as a model microorganism to monitor real-time phage therapy kinetics through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU). For all experiments, a bacterial concentration of ≈ 10(5) CFU ml(-1) and a phage concentration of ≈ 10(6-8) plaque forming unit ml(-1) were used. Phage survival was not significantly affected by the natural variability of pH (6.5-7.4), temperature (10-25 °C), salinity (0-30 g NaCl l(-1) ) and organic matter concentration of aquaculture waters in a temperate climate. Nonetheless, the efficiency of phage therapy was mostly affected by the variation of salinity and organic matter content. As the effectiveness of phage therapy increases with water salt content, this approach appears to be a suitable choice for marine aquaculture systems. The success of phage therapy may also be enhanced in non-marine systems through the addition of salt, whenever this option is feasible and does not affect the survival of aquatic species being cultured. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage▿

PubMed Central

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

2008-01-01

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 (ΦAb-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 ΦAb-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 ΦAb-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. PMID:18065619

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil.

PubMed

Lu, Lan; Li, Zhi Jie; Li, Long Fei; Shen, Jing; Zhang, Lin; Li, Ming Xing; Xiao, Zhan Gang; Wang, Jian Hao; Cho, Chi Hin

2017-11-01

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs. Copyright © 2017. Published by Elsevier Inc.

Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

PubMed

Bakhshinejad, Babak; Zade, Hesam Motaleb; Shekarabi, Hosna Sadat Zahed; Neman, Sara

2016-12-01

Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

Life-Style and Genome Structure of Marine Pseudoalteromonas Siphovirus B8b Isolated from the Northwestern Mediterranean Sea

PubMed Central

Lara, Elena; Holmfeldt, Karin; Solonenko, Natalie; Sà, Elisabet Laia; Ignacio-Espinoza, J. Cesar; Cornejo-Castillo, Francisco M.; Verberkmoes, Nathan C.; Vaqué, Dolors; Sullivan, Matthew B.; Acinas, Silvia G.

2015-01-01

Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. Here we isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. Host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested, >99.9% 16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new ‘rare virosphere’ phage–host model system. PMID:25587991

Use of phages to control Vibrio splendidus infection in the juvenile sea cucumber Apostichopus japonicus.

PubMed

Li, Zhen; Li, Xiaoyu; Zhang, Jiancheng; Wang, Xitao; Wang, Lili; Cao, Zhenhui; Xu, Yongping

2016-07-01

In the present study, we isolated 3 bacteriophages with the ability to control Vibrio splendidus, a bacterium known to cause disease in the juvenile sea cucumber. These bacteriophages were designated as vB_VspS_VS-ABTNL-1 (PVS-1), vB_VspS_VS-ABTNL-2 (PVS-2) and vB_VspS_VS-ABTNL-3 (PVS-3). The ability of the 3 phages to inhibit the growth of V. splendidus VS-ABTNL was tested in vitro using each of the 3 phages individually or in the form of a cocktail of all 3 phages in the proportion of 1:1:1. All treated cultures produced a significant (P < 0.05) inhibition of growth of V. splendidus VS-ABTNL compared with untreated V. splendidus VS-ABTNL with the cocktail being superior to any of the 3 phages used individually. The lytic capability of the 3 phages was subsequently determined with a Spot Assay Technique performed with 4 isolates of V. splendidus, 3 other Vibrio species and 2 environmental isolates. Both PVS-1 and PVS-2 were lytic to all 4 isolates of V. splendidus while PVS-3 only inhibited the growth of 3 of them. V. splendidus VS-ABTNL was more susceptible to phage PVS-2 than the other 2 phages. In an in vivo performance trial, 360 sea cucumbers (23 ± 2 g) were randomly assigned to 1 of 6 treatments. Each treatment was housed in 3 PVC tanks (38 cm × 54 cm × 80 cm) with 20 sea cucumbers per tank. Six diets were prepared including an unsupplemented control diet, antibiotic treatment diet, 3 diets containing 1 of the 3 phages individually and a diet containing a cocktail of all 3 phages. After 60 days of feeding, all sea cucumber were challenged with V. splendidus VS-ABTNL by immersion in sea water containing a bacterial concentration of 6 × 10(6) CFU/mL for 2 days. The survival rate of sea cucumbers during the next 10 days was 18% for the unsupplemented diet, 82% for the antibiotic treatment, 82% for the phage cocktail, 65% for phage PVS-1, 58% for phage PVS-2 and 50% for phage PVS-3. There were no significant differences in weight gain, ingestion rate or feed conversion among sea cucumber fed the 4 phage treatments compared with those fed the unsupplemented diet (P > 0.05). The levels of nitric oxide synthase and acid phosphatase of sea cucumbers fed phage-containing diets were significantly (P < 0.05) increased compared with those fed the control diet. However, no significant differences (P > 0.05) were detected among the 4 phage-fed treatments. An additional study was conducted in which 60 healthy sea cucumbers (23 ± 2 g) were randomly assigned to a control, an untreated group and a test group to investigate the effects of injecting phages by coelomic injection on the survival rate and enzyme activities in the coelomic fluid of the sea cucumbers. The control was injected with 1 ml of sterilized seawater while the untreated group and the test group were injected with the same volume of V. splendidus-ABTNL culture (3 × 10(5) CFU/mL). Then, the test group was injected with 1 ml of the 3 phage cocktail (MOI = 10). After 48 h, the activities of lysozyme, acid phosphatase and superoxide dismutase were elevated in the untreated group while the levels of these enzymes in the test group were similar to the blank control. After 10-day observation, the survival rate of the sea cucumber was 100% for the blank control, 80% for the test group and 20% for the negative control. The overall results of this experiment indicate that phage therapy increased the survival of sea cucumber infected with V. splendidus VS-ABTNL. The above results demonstrate that using phages, especially a combination of different phages, may be a feasible way to control Vibrio infection in the sea cucumber industry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe

PubMed Central

Haley, Joshua A.; Stark, W. Marshall

2016-01-01

ABSTRACT Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo. Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases, we identified and validated the integrase from a newly isolated Streptomyces phage, ϕJoe. ϕJoe integrase is active in vitro and in vivo. The specific recognition site for integration is present in a wide range of different actinobacteria, including Streptomyces venezuelae, an emerging model bacterium in Streptomyces research. PMID:28003200

Isolation and characterization of bacteriophages specific to hydrogen-sulfide-producing bacteria.

PubMed

Gong, Chao; Heringa, Spencer; Singh, Randhir; Kim, Jinkyung; Jiang, Xiuping

2013-01-01

The objectives of this study were to isolate and characterize bacteriophages specific to hydrogen-sulfide-producing bacteria (SPB) from raw animal materials, and to develop a SPB-specific bacteriophage cocktail for rendering application. Meat, chicken offal, and feather samples collected from local supermarkets and rendering processing plants were used to isolate SPB (n = 142). Bacteriophages (n = 52) specific to SPB were isolated and purified from the above samples using 18 of those isolated SPB strains as hosts. The host ranges of bacteriophages against 5 selected SPB strains (Escherichia coli, Citrobacter freundii, and Hafnia alvei) were determined. Electron microscopy observation of 9 phages selected for the phage cocktail revealed that 6 phages belonged to the family of Siphoviridae and 3 belonged to the Myoviridae family. Restriction enzyme digestion analysis with endonuclease DraI detected 6 distinguished patterns among the 9 phages. Phage treatment prevented the growth of SPB for up to 10 h with multiplicity of infection ratios of 1, 10, 100, and 1000 in tryptic soy broth at 30 °C, and extended the lag phase of SPB growth for 2 h at 22 °C with multiplicities of infection of 10, 100, and 1000. These results suggest that the selected bacteriophage cocktail has a high potential for phage application to control SPB in raw animal materials destined for the rendering process.

Molecular Basis for Lytic Bacteriophage Resistance in Enterococci.

PubMed

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

2016-08-30

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

Application of physico-chemical typing methods for the epidemiological analysis of Salmonella enteritidis strains of phage type 25/17.

PubMed Central

Seltmann, G.; Voigt, W.; Beer, W.

1994-01-01

Eighty-nine Salmonella enteritidis phage type 25/17 strains isolated from a localized outbreak in the German state Nordrhein-Westfalen (outbreak NWI) could not be further differentiated by biochemotyping and plasmid pattern analysis. They were submitted to a complex typing system consisting of modern physico-chemical analytical procedures. In lipopolysaccharide pattern analysis the strains proved to be homogeneous. In multilocus enzyme electrophoresis, outer membrane and whole cell protein pattern (WCPP) analysis, and Fourier-transform infrared (FT-IR) spectroscopy (increasing extent of differentiation in the given order) strains deviating from each basal pattern were found. The extent of correspondence in these deviations was satisfactory. Forty-six strains of the same sero- and phage type, however, obtained from different outbreaks, were additionally typed. The results obtained with them indicate that the data of the first group were not restricted to strains from outbreak NWI, but of general validity. It was found that both WCPP and FT-IR represent valuable methods for the sub-grouping of bacteria. Images Fig. 1 Fig. 2 Fig. 3 PMID:7995351

Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

PubMed Central

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

2015-01-01

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

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

PubMed Central

Gillis, Annika; Mahillon, Jacques

2014-01-01

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

An inclusive Research Education Community (iREC): Impact of the SEA-PHAGES program on research outcomes and student learning.

PubMed

Hanauer, David I; Graham, Mark J; Betancur, Laura; Bobrownicki, Aiyana; Cresawn, Steven G; Garlena, Rebecca A; Jacobs-Sera, Deborah; Kaufmann, Nancy; Pope, Welkin H; Russell, Daniel A; Jacobs, William R; Sivanathan, Viknesh; Asai, David J; Hatfull, Graham F

2017-12-19

Engaging undergraduate students in scientific research promises substantial benefits, but it is not accessible to all students and is rarely implemented early in college education, when it will have the greatest impact. An inclusive Research Education Community (iREC) provides a centralized scientific and administrative infrastructure enabling engagement of large numbers of students at different types of institutions. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) is an iREC that promotes engagement and continued involvement in science among beginning undergraduate students. The SEA-PHAGES students show strong gains correlated with persistence relative to those in traditional laboratory courses regardless of academic, ethnic, gender, and socioeconomic profiles. This persistent involvement in science is reflected in key measures, including project ownership, scientific community values, science identity, and scientific networking. Copyright © 2017 the Author(s). Published by PNAS.

An inclusive Research Education Community (iREC): Impact of the SEA-PHAGES program on research outcomes and student learning

PubMed Central

Hanauer, David I.; Graham, Mark J.; Betancur, Laura; Bobrownicki, Aiyana; Cresawn, Steven G.; Garlena, Rebecca A.; Jacobs-Sera, Deborah; Kaufmann, Nancy; Pope, Welkin H.; Russell, Daniel A.; Jacobs, William R.; Sivanathan, Viknesh; Asai, David J.

2017-01-01

Engaging undergraduate students in scientific research promises substantial benefits, but it is not accessible to all students and is rarely implemented early in college education, when it will have the greatest impact. An inclusive Research Education Community (iREC) provides a centralized scientific and administrative infrastructure enabling engagement of large numbers of students at different types of institutions. The Science Education Alliance–Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) is an iREC that promotes engagement and continued involvement in science among beginning undergraduate students. The SEA-PHAGES students show strong gains correlated with persistence relative to those in traditional laboratory courses regardless of academic, ethnic, gender, and socioeconomic profiles. This persistent involvement in science is reflected in key measures, including project ownership, scientific community values, science identity, and scientific networking. PMID:29208718

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

DTIC Science & Technology

2005-10-01

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

Landscape Phage: Evolution from Phage Display to Nanobiotechnology.

PubMed

Petrenko, Valery A

2018-06-07

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

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

PubMed

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

2017-11-01

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

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

PubMed

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

2015-02-19

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

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

PubMed Central

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

2012-01-01

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

Recombinant human antibody fragment against tetanus toxoid produced by phage display

PubMed Central

Neelakantam, B.; Sridevi, N. V.; Shukra, A. M.; Sugumar, P.; Samuel, S.

2014-01-01

Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen. PMID:24678405

Identification of Relevant Conformational Epitopes on the HER2 Oncoprotein by Using Large Fragment Phage Display (LFPD)

PubMed Central

Gabrielli, Federico; Salvi, Roberto; Garulli, Chiara; Kalogris, Cristina; Arima, Serena; Tardella, Luca; Monaci, Paolo; Pupa, Serenella M.; Tagliabue, Elda; Montani, Maura; Quaglino, Elena; Stramucci, Lorenzo; Curcio, Claudia

2013-01-01

We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines. PMID:23555577

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device.

PubMed

Nishiyama, Kazusa; Takakusagi, Yoichi; Kusayanagi, Tomoe; Matsumoto, Yuki; Habu, Shiori; Kuramochi, Kouji; Sugawara, Fumio; Sakaguchi, Kengo; Takahashi, Hideyo; Natsugari, Hideaki; Kobayashi, Susumu

2009-01-01

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.

Characterization, Genome Sequence, and Analysis of Escherichia Phage CICC 80001, a Bacteriophage Infecting an Efficient L-Aspartic Acid Producing Escherichia coli.

PubMed

Xu, Youqiang; Ma, Yuyue; Yao, Su; Jiang, Zengyan; Pei, Jiangsen; Cheng, Chi

2016-03-01

Escherichia phage CICC 80001 was isolated from the bacteriophage contaminated medium of an Escherichia coli strain HY-05C (CICC 11022S) which could produce L-aspartic acid. The phage had a head diameter of 45-50 nm and a tail of about 10 nm. The one-step growth curve showed a latent period of 10 min and a rise period of about 20 min. The average burst size was about 198 phage particles per infected cell. Tests were conducted on the plaques, multiplicity of infection, and host range. The genome of CICC 80001 was sequenced with a length of 38,810 bp, and annotated. The key proteins leading to host-cell lysis were phylogenetically analyzed. One protein belonged to class II holin, and the other two belonged to the endopeptidase family and N-acetylmuramoyl-L-alanine amidase family, respectively. The genome showed the sequence identity of 82.7% with that of Enterobacteria phage T7, and carried ten unique open reading frames. The bacteriophage resistant E. coli strain designated CICC 11021S was breeding and its L-aspartase activity was 84.4% of that of CICC 11022S.

Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine.

PubMed

Tran, Ngoc Q; Rezende, Lisa F; Qimron, Udi; Richardson, Charles C; Tabor, Stanley

2008-07-08

Bacteriophage T7 DNA polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.

Therapeutic and prophylactic applications of bacteriophage components in modern medicine.

PubMed

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

2014-01-01

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

Current insights into phage biodiversity and biogeography.

PubMed

Thurber, Rebecca Vega

2009-10-01

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

FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

PubMed Central

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

1946-01-01

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

Activity of Bacteriophages in Removing Biofilms of Pseudomonas aeruginosa Isolates from Chronic Rhinosinusitis Patients

PubMed Central

Fong, Stephanie A.; Drilling, Amanda; Morales, Sandra; Cornet, Marjolein E.; Woodworth, Bradford A.; Fokkens, Wytske J.; Psaltis, Alkis J.; Vreugde, Sarah; Wormald, Peter-John

2017-01-01

Introduction: Pseudomonas aeruginosa infections are prevalent amongst chronic rhinosinusitis (CRS) sufferers. Many P. aeruginosa strains form biofilms, leading to treatment failure. Lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death. Aim: To assess the activity of a phage cocktail in eradicating biofilms of ex vivo P.aeruginosa isolates from CRS patients. Methods: P. aeruginosa isolates from CRS patients with and without cystic fibrosis (CF) across three continents were multi-locus sequence typed and tested for antibiotic resistance. Biofilms grown in vitro were treated with a cocktail of four phages (CT-PA). Biofilm biomass was measured after 24 and 48 h, using a crystal violet assay. Phage titrations were performed to confirm replication of the phages. A linear mixed effects model was applied to assess the effects of treatment, time, CF status, and multidrug resistance on the biomass of the biofilm. Results: The isolates included 44 strain types. CT-PA treatment significantly reduced biofilm biomass at both 24 and 48 h post-treatment (p < 0.0001), regardless of CF status or antibiotic resistance. Biomass was decreased by a median of 76% at 48 h. Decrease in biofilm was accompanied by a rise in phage titres for all except one strain. Conclusion: A single dose of phages is able to significantly reduce biofilms formed in vitro by a range of P.aeruginosa isolates from CRS patients. This represents an exciting potential and novel targeted treatment for P. aeruginosa biofilm infections and multidrug resistant bacteria. PMID:29018773

Prevalence and Characterization of Salmonella enterica and Salmonella Bacteriophages Recovered from Beef Cattle Feedlots in South Texas.

PubMed

Xie, Yicheng; Savell, Jeffrey W; Arnold, Ashley N; Gehring, Kerri B; Gill, Jason J; Taylor, T Matthew

2016-08-01

Asymptomatic Salmonella carriage in beef cattle is a food safety concern, and the beef feedlot environment may function as a reservoir of this pathogen. The goal of this study was to identify and isolate Salmonella and Salmonella bacteriophages from beef cattle feedlot environments in order to better understand the microbial ecology of Salmonella and identify phages that might be useful as anti-Salmonella beef safety interventions. Three feedlots in south Texas were visited, and 27 distinct samples from each source were collected from dropped feces, feed from feed bunks, drinking water from troughs, and soil in cattle pens (n = 108 samples). Preenrichment, selective enrichment, and selective/differential isolation of Salmonella were performed on each sample. A representative subset of presumptive Salmonella isolates was prepared for biochemical identification and serotyping. Samples were pooled by feedlot and sample type to create 36 samples and enriched to recover phages. Recovered phages were tested for host range against two panels of Salmonella hosts. Salmonella bacteria were identified in 20 (18.5%) of 108 samples by biochemical and/or serological testing. The serovars recovered included Salmonella enterica serovars Anatum, Muenchen, Altona, Kralingen, Kentucky, and Montevideo; Salmonella Anatum was the most frequently recovered serotype. Phage-positive samples were distributed evenly over the three feedlots, suggesting that phage prevalence is not strongly correlated with the presence of culturable Salmonella. Phages were found more frequently in soil and feces than in feed and water samples. The recovery of bacteriophages in the Salmonella-free feedlot suggests that phages might play a role in suppressing the Salmonella population in a feedlot environment.

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

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

PubMed

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

2009-10-10

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

Phages of lactic acid bacteria: The role of genetics in understanding phage-host interactions and their co-evolutionary processes

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

Mahony, Jennifer, E-mail: j.mahony@ucc.ie; Ainsworth, Stuart; Stockdale, Stephen

Dairy fermentations are among the oldest food processing applications, aimed at preservation and shelf-life extension through the use of lactic acid bacteria (LAB) starter cultures, in particular strains of Lactococcus lactis, Streptococcus thermophilus, Lactobacillus spp. and Leuconostoc spp. Traditionally this was performed by continuous passaging of undefined cultures from a finished fermentation to initiate the next fermentation. More recently, consumer demands on consistent and desired flavours and textures of dairy products have led to a more defined approach to such processes. Dairy (starter) companies have responded to the need to define the nature and complexity of the starter culture mixes,more » and dairy fermentations are now frequently based on defined starter cultures of low complexity, where each starter component imparts specific technological properties that are desirable to the product. Both mixed and defined starter culture approaches create the perfect environment for the proliferation of (bacterio)phages capable of infecting these LAB. The repeated use of the same starter cultures in a single plant, coupled to the drive towards higher and consistent production levels, increases the risk and negative impact of phage infection. In this review we will discuss recent advances in tracking the adaptation of phages to the dairy industry, the advances in understanding LAB phage-host interactions, including evolutionary and genomic aspects.« less

Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix.

PubMed

Smith, Nicola L; Taylor, Edward J; Lindsay, Anna-Marie; Charnock, Simon J; Turkenburg, Johan P; Dodson, Eleanor J; Davies, Gideon J; Black, Gary W

2005-12-06

Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyogenes is frequently mediated by bacteriophage infection. One of the key bacteriophage-encoded virulence factors is a putative "hyaluronidase," HylP1, a phage tail-fiber protein responsible for the digestion of the S. pyogenes hyaluronan capsule during phage infection. Here we demonstrate that HylP1 is a hyaluronate lyase. The 3D structure, at 1.8-angstroms resolution, reveals an unusual triple-stranded beta-helical structure and provides insight into the structural basis for phage tail assembly and the role of phage tail proteins in virulence. Unlike the triple-stranded beta-helix assemblies of the bacteriophage T4 injection machinery and the tailspike endosialidase of the Escherichia coli K1 bacteriophage K1F, HylP1 possesses three copies of the active center on the triple-helical fiber itself without the need for an accessory catalytic domain. The triple-stranded beta-helix is not simply a structural scaffold, as previously envisaged; it is harnessed to provide a 200-angstroms-long substrate-binding groove for the optimal reduction in hyaluronan viscosity to aid phage penetration of the capsule.

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.

Prevalence and Evolution of Core Photosystem II Genes in Marine Cyanobacterial Viruses and Their Hosts

PubMed Central

Lee, Jessica A; Thompson, Luke R; Bielawski, Joseph P

2006-01-01

Cyanophages (cyanobacterial viruses) are important agents of horizontal gene transfer among marine cyanobacteria, the numerically dominant photosynthetic organisms in the oceans. Some cyanophage genomes carry and express host-like photosynthesis genes, presumably to augment the host photosynthetic machinery during infection. To study the prevalence and evolutionary dynamics of this phenomenon, 33 cultured cyanophages of known family and host range and viral DNA from field samples were screened for the presence of two core photosystem reaction center genes, psbA and psbD. Combining this expanded dataset with published data for nine other cyanophages, we found that 88% of the phage genomes contain psbA, and 50% contain both psbA and psbD. The psbA gene was found in all myoviruses and Prochlorococcus podoviruses, but could not be amplified from Prochlorococcus siphoviruses or Synechococcus podoviruses. Nearly all of the phages that encoded both psbA and psbD had broad host ranges. We speculate that the presence or absence of psbA in a phage genome may be determined by the length of the latent period of infection. Whether it also carries psbD may reflect constraints on coupling of viral- and host-encoded PsbA–PsbD in the photosynthetic reaction center across divergent hosts. Phylogenetic clustering patterns of these genes from cultured phages suggest that whole genes have been transferred from host to phage in a discrete number of events over the course of evolution (four for psbA, and two for psbD), followed by horizontal and vertical transfer between cyanophages. Clustering patterns of psbA and psbD from Synechococcus cells were inconsistent with other molecular phylogenetic markers, suggesting genetic exchanges involving Synechococcus lineages. Signatures of intragenic recombination, detected within the cyanophage gene pool as well as between hosts and phages in both directions, support this hypothesis. The analysis of cyanophage psbA and psbD genes from field populations revealed significant sequence diversity, much of which is represented in our cultured isolates. Collectively, these findings show that photosynthesis genes are common in cyanophages and that significant genetic exchanges occur from host to phage, phage to host, and within the phage gene pool. This generates genetic diversity among the phage, which serves as a reservoir for their hosts, and in turn influences photosystem evolution. PMID:16802857

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

PubMed

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

2017-01-16

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

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

PubMed

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

2012-02-01

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

Molecular Insights Into the Evolutionary Pathway of Vibrio cholerae O1 Atypical El Tor Variants

PubMed Central

Kim, Eun Jin; Lee, Dokyung; Moon, Se Hoon; Lee, Chan Hee; Kim, Sang Jun; Lee, Jae Hyun; Kim, Jae Ouk; Song, Manki; Das, Bhabatosh; Clemens, John D.; Pape, Jean William; Nair, G. Balakrish; Kim, Dong Wook

2014-01-01

Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor. PMID:25233006

Filamentous Phage: Structure and Biology.

PubMed

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

2017-01-01

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

Advances in phage display technology for drug discovery.

PubMed

Omidfar, Kobra; Daneshpour, Maryam

2015-06-01

Over the past decade, several library-based methods have been developed to discover ligands with strong binding affinities for their targets. These methods mimic the natural evolution for screening and identifying ligand-target interactions with specific functional properties. Phage display technology is a well-established method that has been applied to many technological challenges including novel drug discovery. This review describes the recent advances in the use of phage display technology for discovering novel bioactive compounds. Furthermore, it discusses the application of this technology to produce proteins and peptides as well as minimize the use of antibodies, such as antigen-binding fragment, single-chain fragment variable or single-domain antibody fragments like VHHs. Advances in screening, manufacturing and humanization technologies demonstrate that phage display derived products can play a significant role in the diagnosis and treatment of disease. The effects of this technology are inevitable in the development pipeline for bringing therapeutics into the market, and this number is expected to rise significantly in the future as new advances continue to take place in display methods. Furthermore, a widespread application of this methodology is predicted in different medical technological areas, including biosensing, monitoring, molecular imaging, gene therapy, vaccine development and nanotechnology.

Prospects for Biocontrol of Vibrio parahaemolyticus Contamination in Blue Mussels (Mytilus edulus)—A Year-Long Study

PubMed Central

Onarinde, Bukola A.; Dixon, Ronald A.

2018-01-01

Vibrio parahaemolyticus is an environmental organism normally found in subtropical estuarine environments which can cause seafood-related human infections. Clinical disease is associated with diagnostic presence of tdh and/or trh virulence genes and identification of these genes in our preliminary isolates from retail shellfish prompted a year-long surveillance of isolates from a temperate estuary in the north of England. The microbial and environmental analysis of 117 samples of mussels, seawater or sediment showed the presence of V. parahaemolyticus from mussels (100%) at all time-points throughout the year including the colder months although they were only recovered from 94.9% of seawater and 92.3% of sediment samples. Throughout the surveillance, 96 isolates were subjected to specific PCR for virulence genes and none tested positive for either. The common understanding that consuming poorly cooked mussels only represents a risk of infection during summer vacations therefore is challenged. Further investigations with V. parahaemolyticus using RAPD-PCR cluster analysis showed a genetically diverse population. There was no distinct clustering for “environmental” or “clinical” reference strains although a wide variability and heterogeneity agreed with other reports. Continued surveillance of isolates to allay public health risks are justified since geographical distribution and composition of V. parahaemolyticus varies with Future Ocean warming and the potential of environmental strains to acquire virulence genes from pathogenic isolates. The prospects for intervention by phage-mediated biocontrol to reduce or eradicate V. parahaemolyticus in mussels was also investigated. Bacteriophages isolated from enriched samples collected from the river Humber were assessed for their ability to inhibit the growth of V. parahaemolyticus strains in-vitro and in-vivo (with live mussels). V. parahaemolyticus were significantly reduced in-vitro, by an average of 1 log−2 log units and in-vivo, significant reduction of the organisms in mussels occurred in three replicate experimental tank set ups with a “phage cocktail” containing 12 different phages. Our perspective biocontrol study suggests that a cocktail of specific phages targeted against strains of V. parahaemolyticus provides good evidence in an experimental setting of the valuable potential of phage as a decontamination agent in natural or industrial mussel processing (343w). PMID:29922246

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

PubMed Central

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

1973-01-01

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

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

PubMed Central

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

2009-01-01

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

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

PubMed Central

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

2009-01-01

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

Distinct Bacteriophages Encoding Panton-Valentine Leukocidin (PVL) among International Methicillin-Resistant Staphylococcus aureus Clones Harboring PVL▿

PubMed Central

Boakes, E.; Kearns, A. M.; Ganner, M.; Perry, C.; Hill, R. L.; Ellington, M. J.

2011-01-01

Genetically diverse community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) can harbor a bacteriophage encoding Panton-Valentine leukocidin (PVL) lysogenized into its chromosome (prophage). Six PVL phages (ΦPVL, Φ108PVL, ΦSLT, ΦSa2MW, ΦSa2USA, and ΦSa2958) are known, and single-nucleotide polymorphisms (SNPs) in the PVL genes have been reported. We sought to determine the distribution of lysogenized PVL phages among MRSA strains with PVL (PVL-MRSA strains), the PVL gene sequences, and the chromosomal phage insertion sites in 114 isolates comprising nine clones of PVL-MRSA that were selected for maximal underlying genetic diversity. The six PVL phages were identified by PCR; ΦSa2USA was present in the highest number of different lineages (multilocus sequence type clonal complex 1 [CC1], CC5, CC8, and sequence type 93 [ST93]) (n = 37 isolates). Analysis of 92 isolates confirmed that PVL phages inserted into the same chromosomal insertion locus in CC22, -30, and -80 but in a different locus in isolates of CC1, -5, -8, -59, and -88 and ST93 (and CC22 in two isolates). Within the two different loci, specific attachment motifs were found in all cases, although some limited inter- and intralineage sequence variation occurred. Overall, lineage-specific relationships between the PVL phage, the genes that encode the toxin, and the position at which the phage inserts into the host chromosome were identified. These analyses provide important insights into the microepidemiology of PVL-MRSA, will prove a valuable adjunct in outbreak investigation, and may help predict the emergence of new strains. PMID:21106787

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

PubMed

Somers, Klaartje; Stinissen, Piet; Somers, Veerle

2011-06-01

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

On The Influence Of Vector Design On Antibody Phage Display

PubMed Central

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

2007-01-01

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

On the influence of vector design on antibody phage display.

PubMed

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

2007-01-20

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

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

PubMed

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

2013-11-14

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

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

PubMed Central

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

2013-01-01

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

Differential Decomposition of Bacterial and Viral Fecal ...

EPA Pesticide Factsheets

Understanding the decomposition of microorganisms associated with different human fecal pollution types is necessary for proper implementation of many water qualitymanagement practices, as well as predicting associated public health risks. Here, thedecomposition of select cultivated and molecular indicators of fecal pollution originating from fresh human feces, septage, and primary effluent sewage in a subtropical marine environment was assessed over a six day period with an emphasis on the influence of ambient sunlight and indigenous microbiota. Ambient water mixed with each fecal pollution type was placed in dialysis bags and incubated in situ in a submersible aquatic mesocosm. Genetic and cultivated fecal indicators including fecal indicator bacteria (enterococci, E. coli, and Bacteroidales), coliphage (somatic and F+), Bacteroides fragilis phage (GB-124), and human-associated geneticindicators (HF183/BacR287 and HumM2) were measured in each sample. Simple linearregression assessing treatment trends in each pollution type over time showed significant decay (p ≤ 0.05) in most treatments for feces and sewage (27/28 and 32/40, respectively), compared to septage (6/26). A two-way analysis of variance of log10 reduction values for sewage and feces experiments indicated that treatments differentially impact survival of cultivated bacteria, cultivated phage, and genetic indicators. Findings suggest that sunlight is critical for phage decay, and indigenous microbio

Host Specific Diversity in Lactobacillus johnsonii as Evidenced by a Major Chromosomal Inversion and Phage Resistance Mechanisms

PubMed Central

Guinane, Caitriona M.; Kent, Robert M.; Norberg, Sarah; Hill, Colin; Fitzgerald, Gerald F.; Stanton, Catherine; Ross, R. Paul

2011-01-01

Genetic diversity and genomic rearrangements are a driving force in bacterial evolution and niche adaptation. We sequenced and annotated the genome of Lactobacillus johnsonii DPC6026, a strain isolated from the porcine intestinal tract. Although the genome of DPC6026 is similar in size (1.97mbp) and GC content (34.8%) to the sequenced human isolate L. johnsonii NCC 533, a large symmetrical inversion of approximately 750 kb differentiated the two strains. Comparative analysis among 12 other strains of L. johnsonii including 8 porcine, 3 human and 1 poultry isolate indicated that the genome architecture found in DPC6026 is more common within the species than that of NCC 533. Furthermore a number of unique features were annotated in DPC6026, some of which are likely to have been acquired by horizontal gene transfer (HGT) and contribute to protection against phage infection. A putative type III restriction-modification system was identified, as were novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) elements. Interestingly, these particular elements are not widely distributed among L. johnsonii strains. Taken together these data suggest intra-species genomic rearrangements and significant genetic diversity within the L. johnsonii species and indicate towards a host-specific divergence of L. johnsonii strains with respect to genome inversion and phage exposure. PMID:21533100

Host specific diversity in Lactobacillus johnsonii as evidenced by a major chromosomal inversion and phage resistance mechanisms.

PubMed

Guinane, Caitriona M; Kent, Robert M; Norberg, Sarah; Hill, Colin; Fitzgerald, Gerald F; Stanton, Catherine; Ross, R Paul

2011-04-20

Genetic diversity and genomic rearrangements are a driving force in bacterial evolution and niche adaptation. We sequenced and annotated the genome of Lactobacillus johnsonii DPC6026, a strain isolated from the porcine intestinal tract. Although the genome of DPC6026 is similar in size (1.97 mbp) and GC content (34.8%) to the sequenced human isolate L. johnsonii NCC 533, a large symmetrical inversion of approximately 750 kb differentiated the two strains. Comparative analysis among 12 other strains of L. johnsonii including 8 porcine, 3 human and 1 poultry isolate indicated that the genome architecture found in DPC6026 is more common within the species than that of NCC 533. Furthermore a number of unique features were annotated in DPC6026, some of which are likely to have been acquired by horizontal gene transfer (HGT) and contribute to protection against phage infection. A putative type III restriction-modification system was identified, as were novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) elements. Interestingly, these particular elements are not widely distributed among L. johnsonii strains. Taken together these data suggest intra-species genomic rearrangements and significant genetic diversity within the L. johnsonii species and indicate towards a host-specific divergence of L. johnsonii strains with respect to genome inversion and phage exposure.

Characterization of Bacillus phage-K2 isolated from chungkookjang, a fermented soybean foodstuff.

PubMed

Kim, Eun Ju; Hong, Jeong Won; Yun, Na-Rae; Lee, Young Nam

2011-01-01

An investigation of a virulent Bacillus phage-K2 (named Bp-K2) isolated from chungkookjang (a fermented soybean foodstuff) was made. Bp-K2 differed in infectivity against a number of Bacillus subtilis strains including starter strains of chungkookjang and natto, being more infectious to Bacillus strains isolated from the chungkookjang, but much less active against a natto strain. Bp-K2 is a small DNA phage whose genome size is about 21 kb. Bp-K2 is a tailed bacteriophage with an isometric icosahedral head (50 nm long on the lateral side, 80 nm wide), a long contractile sheath (85-90 nm × 28 nm), a thin tail fiber (80-85 nm long, 10 nm wide), and a basal plate (29 nm long, 47 nm wide) with a number of spikes, but no collar. The details of the structures of Bp-K2 differ from natto phage ϕBN100 as well as other known Bacillus phages such as SPO1-like or ϕ 29-like viruses. These data suggest that Bp-K2 would be a new member of the Myoviridae family of Bacillus bacteriophages.

Phage displayed scFv: pIII scaffold may fine tune binding specificity.

PubMed

Goswami, Pooja; Saini, Deepti; Sinha, Subrata

2009-10-01

The fine specificity of antibodies is important for their discriminating powers during diagnostics and in vivo therapy. We have attempted to isolate human scFv antibodies to the oncofetal antigen, the placental isozyme of alkaline phosphatase (PLAP) in which it is important to distinguish between the closely related intestinal alkaline phosphatase (IAP) and bone alkaline phosphatase (BAP) isozymes. As the antibodies are selected in the phage displayed form and might be finally used as different entities, including the soluble scFv form, it may be important to look at the influence of scaffolds in determining specificity. There have been earlier reports of the role of the constant region and other scaffolding proteins in determining specificity. In this paper, we report isolation of one such clone, E6, which showed specificity to PLAP in phage antibody form but lost the specificity when soluble scFv was tested for same, and showed partial cross reactivity to BAP. We suggest that the altered specificity of scFv might be the result of loss of phage pIII scaffold, which is present in phage-displayed antibody and may help the displayed antibody to assume specific conformational structure, which may govern binding characteristics of the same.

Protein complex purification from Thermoplasma acidophilum using a phage display library.

PubMed

Hubert, Agnes; Mitani, Yasuo; Tamura, Tomohiro; Boicu, Marius; Nagy, István

2014-03-01

We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available. Copyright © 2013 Elsevier B.V. All rights reserved.

A novel chimeric prophage vB_LdeS-phiJB from commercial Lactobacillus delbrueckii subsp. bulgaricus.

PubMed

Guo, Tingting; Zhang, Chenchen; Xin, Yongping; Xin, Min; Kong, Jian

2016-05-01

Prophage vB_LdeS-phiJB (phiJB) was induced by mitomycin C and UV radiation from the Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 isolated from a Chinese yoghurt sample. It has an isometric head and a non-contractile tail with 36,969 bp linear double-stranded DNA genome, which is classified into the group a of Lb. delbrueckii phages. The genome of phiJB is highly modular with functionally related genes clustered together. Unexpectedly, there is no similarity of its DNA replication module to any phages that have been reported, while it consists of open-reading frames homologous to the proteins of Lactobacillus strains. Comparative genomic analysis indicated that its late gene clusters, integration/lysogeny modules and DNA replication module derived from different evolutionary ancestors and integrated into a chimera. Our results revealed a novel chimeric phage of commercial Lb. delbrueckii and will broaden the knowledge of phage diversity in the dairy industry.

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

PubMed

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

2008-04-01

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

A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2

PubMed Central

Hambly, Emma; Tétart, Francoise; Desplats, Carine; Wilson, William H.; Krisch, Henry M.; Mann, Nicholas H.

2001-01-01

Sequence analysis of a 10-kb region of the genome of the marine cyanomyovirus S-PM2 reveals a homology to coliphage T4 that extends as a contiguous block from gene (g)18 to g23. The order of the S-PM2 genes in this region is similar to that of T4, but there are insertions and deletions of small ORFs of unknown function. In T4, g18 codes for the tail sheath, g19, the tail tube, g20, the head portal protein, g21, the prohead core protein, g22, a scaffolding protein, and g23, the major capsid protein. Thus, the entire module that determines the structural components of the phage head and contractile tail is conserved between T4 and this cyanophage. The significant differences in the morphology of these phages must reflect the considerable divergence of the amino acid sequence of their homologous virion proteins, which uniformly exceeds 50%. We suggest that their enormous diversity in the sea could be a result of genetic shuffling between disparate phages mediated by such commonly shared modules. These conserved sequences could facilitate genetic exchange by providing partially homologous substrates for recombination between otherwise divergent phage genomes. Such a mechanism would thus expand the pool of phage genes accessible by recombination to all those phages that share common modules. PMID:11553768

Twelve previously unknown phage genera are ubiquitous in global oceans

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

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

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

Somatostatin displayed on filamentous phage as a receptor-specific agonist

PubMed Central

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

1998-01-01

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

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.

To Be or Not To Be T4: Evidence of a Complex Evolutionary Pathway of Head Structure and Assembly in Giant Salmonella Virus SPN3US

PubMed Central

Ali, Bazla; Desmond, Maxim I.; Mallory, Sara A.; Benítez, Andrea D.; Buckley, Larry J.; Weintraub, Susan T.; Osier, Michael V.; Black, Lindsay W.; Thomas, Julie A.

2017-01-01

Giant Salmonella phage SPN3US has a 240-kb dsDNA genome and a large complex virion composed of many proteins for which the functions of most are undefined. We recently determined that SPN3US shares a core set of genes with related giant phages and sequenced and characterized 18 amber mutants to facilitate its use as a genetic model system. Notably, SPN3US and related giant phages contain a bolus of ejection proteins within their heads, including a multi-subunit virion RNA polymerase (vRNAP), that enter the host cell with the DNA during infection. In this study, we characterized the SPN3US virion using mass spectrometry to gain insight into its head composition and the features that its head shares with those of related giant phages and with T4 phage. SPN3US has only homologs to the T4 proteins critical for prohead shell formation, the portal and major capsid proteins, as well as to the major enzymes essential for head maturation, the prohead protease and large terminase subunit. Eight of ~50 SPN3US head proteins were found to undergo proteolytic processing at a cleavage motif by the prohead protease gp245. Gp245 undergoes auto-cleavage of its C-terminus, suggesting this is a conserved activation and/or maturation feature of related phage proteases. Analyses of essential head gene mutants showed that the five subunits of the vRNAP must be assembled for any subunit to be incorporated into the prohead, although the assembled vRNAP must then undergo subsequent major conformational rearrangements in the DNA packed capsid to allow ejection through the ~30 Å diameter tail tube for transcription from the injected DNA. In addition, ejection protein candidate gp243 was found to play a critical role in head assembly. Our analyses of the vRNAP and gp243 mutants highlighted an unexpected dichotomy in giant phage head maturation: while all analyzed giant phages have a homologous protease that processes major capsid and portal proteins, processing of ejection proteins is not always a stable/defining feature. Our identification in SPN3US, and related phages, of a diverged paralog to the prohead protease further hints toward a complicated evolutionary pathway for giant phage head structure and assembly. PMID:29187846

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

PubMed

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

2013-09-01

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

Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

PubMed

Scott, Benjamin M; Matochko, Wadim L; Gierczak, Richard F; Bhakta, Varsha; Derda, Ratmir; Sheffield, William P

2014-01-01

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of "designer serpins" with novel reactivity and/or specificity.

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

PubMed

Petrov, Vasiliy M; Ratnayaka, Swarnamala; Nolan, James M; Miller, Eric S; Karam, Jim D

2010-10-28

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

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

PubMed Central

Scott, Benjamin M.; Matochko, Wadim L.; Gierczak, Richard F.; Bhakta, Varsha; Derda, Ratmir; Sheffield, William P.

2014-01-01

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2–P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352–356 (P7–P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7–P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of “designer serpins” with novel reactivity and/or specificity. PMID:24427287

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

PubMed Central

2010-01-01

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

[Susceptibilities of multidrug-resistant pathogens responsible for complicated skin and soft tissue infections to standard bacteriophage cocktails].

PubMed

Gündoğdu, Aycan; Kılıç, Hüseyin; Ulu Kılıç, Ayşegül; Kutateladze, Mzia

2016-04-01

Skin and soft tissue infections (SSTIs) may represent a wide clinical spectrum from cellulitis to high-mortality associated necrotizing fasciitis. Limitations in therapy due to the multiple drug resistance, leads to increase in the morbidity and mortality rates, especially in complicated SSTIs such as diabetic foot, decubitus, and surgical wound infections. Therefore, alternative treatment strategies other than antibiotics are needed in appropriate clinical conditions. "Bacteriophage therapy", which is an old method and has been used as part of standard treatment in some countries such as Georgia and Russia, has again become popular worldwide. The aim of this study was to investigate the in vitro susceptibilities of multidrug-resistant (MDR) pathogens isolated from patients with complicated SSTIs, against standard bacteriophage (phage) cocktails. Six different ready-made phage preparations [Pyophage, Intestiphage, ENKO, SES, Fersisi and Staphylococcal Bacteriophage (Sb)] used in this study have been provided by G. Eliava Institute, Georgia. Because of the absence of ready-made phage preparations for Acinetobacter baumannii and Klebsiella pneumoniae, Φ1-Φ7 and ΦKL1- ΦKL3 phages were used provided from the same institute's phage library, respectively. Isolation and identification of the pathogens from abscess and wound samples of patients with SSTIs were performed by conventional methods and automatized VITEK(®)-2 (bioMerieux, ABD) system. Antimicrobial susceptibility testing was conducted complying CLSI standards' and the bacteria that were resistant to at least two different antibiotic groups were considered as MDR. Accordingly, a total of 33 isolates, nine of them were E.coli (8 ESBL and 1 ESBL + carbapenemase positive); nine were MDR P.aeruginosa; nine were MDR A.baumannii; three were methicillin-resistant Staphylococcus aureus (MRSA) and three were K.pneumoniae (1 ESBL, 1 carbapenemase and 1 ESBL + carbapenemase positive) were included in the study. The phage susceptibilities of the pathogens were performed by using spot test. In the study, 29 (87.9%) out of 33 MDR pathogens were found to be susceptible to at least one of the tested phage/phage preparations. All MRSA (3/3) strains were susceptible to ENKO, SES, Fersisi and Sb phage cocktails, while all A.baumannii isolates (9/9) were susceptible to Φ5 and Φ7 phages. However, two E.coli, one K. pneumoniae and one P.aeruginosa strains were resistant to the all phage preparations tested. Although the clinical use of phages has not been approved yet, except a few Eastern European countries, this study exhibits the potential use of the topical bacteriophage therapy in the treatment of complicated SSTIs caused by MDR pathogens with limited treatment options, such as diabetic foot, decubitus, and surgical wound infections.

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

NASA Astrophysics Data System (ADS)

Moebus, K.

1983-12-01

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

EFFECT OF SODIUM CHLORIDE ON STAPHYLOCOCCUS-PHAGE RELATIONSHIPS

PubMed Central

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

1963-01-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-06-14

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

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

PubMed

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

2012-01-01

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

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

PubMed

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

2015-11-01

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

Modified Filamentous Bacteriophage as a Scaffold for Carbon Nanofiber.

PubMed

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

2016-12-21

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

Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of Pseudomonas aeruginosa Mediated by Increased Expression of Phage-Related Genes

PubMed Central

Heussler, Gary E.; Cady, Kyle C.; Koeppen, Katja; Bhuju, Sabin; Stanton, Bruce A.

2015-01-01

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting. PMID:25968642

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

PubMed

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

2012-01-01

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

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

PubMed Central

Imamovic, Lejla; Muniesa, Maite

2012-01-01

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

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2012-01-01

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

Genome Sequences of 12 Cluster AN Arthrobacter Phages

PubMed Central

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

2017-01-01

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

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

PubMed

Dang, Vinh T; Sullivan, Matthew B

2014-01-01

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

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

PubMed

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

2017-08-01

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

Efficacy of an Optimised Bacteriophage Cocktail to Clear Clostridium difficile in a Batch Fermentation Model

PubMed Central

Nale, Janet Y.; Redgwell, Tamsin A.; Millard, Andrew; Clokie, Martha R. J.

2018-01-01

Clostridium difficile infection (CDI) is a major cause of infectious diarrhea. Conventional antibiotics are not universally effective for all ribotypes, and can trigger dysbiosis, resistance and recurrent infection. Thus, novel therapeutics are needed to replace and/or supplement the current antibiotics. Here, we describe the activity of an optimised 4-phage cocktail to clear cultures of a clinical ribotype 014/020 strain in fermentation vessels spiked with combined fecal slurries from four healthy volunteers. After 5 h, we observed ~6-log reductions in C. difficile abundance in the prophylaxis regimen and complete C. difficile eradication after 24 h following prophylactic or remedial regimens. Viability assays revealed that commensal enterococci, bifidobacteria, lactobacilli, total anaerobes, and enterobacteria were not affected by either regimens, but a ~2-log increase in the enterobacteria, lactobacilli, and total anaerobe abundance was seen in the phage-only-treated vessel compared to other treatments. The impact of the phage treatments on components of the microbiota was further assayed using metagenomic analysis. Together, our data supports the therapeutic application of our optimised phage cocktail to treat CDI. Also, the increase in specific commensals observed in the phage-treated control could prevent further colonisation of C. difficile, and thus provide protection from infection being able to establish. PMID:29438355

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

PubMed

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

2007-12-01

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

Novel groups and unique distribution of phage phoH genes in paddy waters in northeast China

PubMed Central

Wang, Xinzhen; Liu, Junjie; Yu, Zhenhua; Jin, Jian; Liu, Xiaobing; Wang, Guanghua

2016-01-01

Although bacteriophages are ubiquitous in various environments, their genetic diversity is primarily investigated in pelagic marine environments. Corresponding studies in terrestrial environments are few. In this study, we conducted the first survey of phage diversity in the paddy ecosystem by targeting a new viral biomarker gene, phoH. A total of 424 phoH sequences were obtained from four paddy waters generated from a pot experiment with different soils collected from open paddy fields in northeast China. The majority of phoH sequences in paddy waters were novel, with the highest identity of ≤70% with known phoH sequences. Four unique groups (Group α, Group β, Group γ and Group δ) and seven new subgroups (Group 2b, Group 3d, Group 3e, Group 6a, Group 6b, Group 6c and Group 6d) were formed exclusively with the clones from the paddy waters, suggesting novel phage phoH groups exist in the paddy ecosystem. Additionally, the distribution proportions of phoH clones in different groups varied among paddy water samples, suggesting the phage community in paddy fields is biogeographically distributed. Furthermore, non-metric multidimensional scaling analysis indicated that phage phoH assemblages in paddy waters were distinct from those in marine waters. PMID:27910929

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

PubMed

Hobbs, Zack; Abedon, Stephen T

2016-04-01

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

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

PubMed

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

2015-09-09

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

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe.

PubMed

Fogg, Paul C M; Haley, Joshua A; Stark, W Marshall; Smith, Margaret C M

2017-03-01

Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces , most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor , by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases, we identified and validated the integrase from a newly isolated Streptomyces phage, ϕJoe. ϕJoe integrase is active in vitro and in vivo The specific recognition site for integration is present in a wide range of different actinobacteria, including Streptomyces venezuelae , an emerging model bacterium in Streptomyces research. Copyright © 2017 Fogg et al.

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.

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2015-12-20

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

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

PubMed

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

2012-01-01

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

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

PubMed Central

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

2015-01-01

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

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.

Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

PubMed

Phipps, M Lisa; Lillo, Antoinetta M; Shou, Yulin; Schmidt, Emily N; Paavola, Chad D; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I; Bradbury, Andrew R M; Martinez, Jennifer S

2016-01-01

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

PubMed Central

Sotelo, Pablo H.; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen

2012-01-01

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles. PMID:22692130

Metagenomic characterization of viral communities in Goseong Bay, Korea

NASA Astrophysics Data System (ADS)

Hwang, Jinik; Park, So Yun; Park, Mirye; Lee, Sukchan; Jo, Yeonhwa; Cho, Won Kyong; Lee, Taek-Kyun

2016-12-01

In this study, seawater samples were collected from Goseong Bay, Korea in March 2014 and viral populations were examined by metagenomics assembly. Enrichment of marine viral particles using FeCl3 followed by next-generation sequencing produced numerous sequences. De novo assembly and BLAST search showed that most of the obtained contigs were unknown sequences and only 0.74% of sequences were associated with known viruses. As a result, 138 viruses, including bacteriophages (87%), viruses infecting algae and others (13%) were identified. The identified 138 viruses were divided into 11 orders, 14 families, 34 genera, and 133 species. The dominant viruses were Pelagibacter phage HTVC010P and Roseobacter phage SIO1. The viruses infecting algae, including the Ostreococcus species, accounted for 9.4% of total identified viruses. In addition, we identified pathogenic herpes viruses infecting fishes and giant viruses infecting parasitic acanthamoeba species. This is a comprehensive study to reveal the viral populations in the Goseong Bay using metagenomics. The information associated with the marine viral community in Goseong Bay, Korea will be useful for comparative analysis in other marine viral communities.

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

PubMed Central

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

2015-01-01

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

Total coliphages removal by activated sludge process and their morphological diversity by transmission electron microscopy.

PubMed

Jebri, Sihem; Hmaied, Fatma; Yahya, Mariem; Ben Ammar, Aouatef; Hamdi, Moktar

This study was conducted to isolate phages in treated sewage collected from wastewater treatment plant, and explore their morphological diversity by transmission electron microscopy (TEM). Fates of total bacteriophages and their reduction by biological treatment were also assayed. Phages were isolated using the plaque assay then negatively stained and observed by electron microscope. Electron micrographs showed different types of phages with different shapes and sizes. The majority of viruses found in treated sewage ranged from 30 to 100 nm in capsid diameter. Many of them were tailed, belonging to Siphoviridae, Myoviridae and Podoviridae families. Non-tailed phage particles were also found at a low rate, presumably belonging to Leviviridae or Microviridae families. This study shows the diversity and the abundance of bacteriophages in wastewater after biological treatment. Their persistence in wastewater reused in agriculture should raise concerns about their potential role in controlling bacterial populations in the environment. They should be also included in water treatment quality controlling guidelines as fecal and viral indicators.

Microselection – affinity selecting antibodies against a single rare cell in a heterogeneous population

PubMed Central

Sørensen, Morten Dræby; Agerholm, Inge Errebo; Christensen, Britta; Kølvraa, Steen; Kristensen, Peter

2010-01-01

Abstract Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non-invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV-irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific. PMID:20726925

A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

PubMed

Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

2016-11-04

When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of V H and V L genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of Host-Takeover During SPO1 Infection of Bacillus subtilis.

PubMed

Stewart, Charles R

2018-01-01

When Bacillus subtilis is infected by bacteriophage SPO1, the phage directs the remodeling of the host cell, converting it into a factory for phage reproduction. Much synthesis of host DNA, RNA, and protein is shut off, and cell division is prevented. Here I describe the protocols by which we have demonstrated those processes, and identified the roles played by specific SPO1 gene products in causing those processes.

Isolation and characterization of a "phiKMV-like" bacteriophage and its therapeutic effect on mink hemorrhagic pneumonia.

PubMed

Cao, Zhenhui; Zhang, Jiancheng; Niu, Yan D; Cui, Naizhong; Ma, Yongsheng; Cao, Fang; Jin, Liji; Li, Zhen; Xu, Yongping

2015-01-01

The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink both in vitro and in vivo. Five Pseudomonas aeruginosa (P. aeruginosa) strains were isolated from lungs of mink with suspected hemorrhagic pneumonia and their identity was confirmed by morphological observation and 16S rDNA sequence analysis. Compared to P. aeruginosa strains isolated from mink with hemorrhagic pneumonia in 2002, these isolates were more resistant to antibiotics selected. A lytic phage vB_PaeP_PPA-ABTNL (PPA-ABTNL) of the Podoviridae family was isolated from hospital sewage using a P. aeruginosa isolate as host, showing broad host range against P. aeruginosa. A one-step growth curve analysis of PPA-ABTNL revealed eclipse and latent periods of 20 and 35 min, respectively, with a burst size of about 110 PFU per infected cell. Phage PPA-ABTNL significantly reduced the growth of P. aeruginosa isolates in vitro. The genome of PPA-ABTNL was 43,227 bp (62.4% G+C) containing 54 open reading frames and lacked regions encoding known virulence factors, integration-related proteins and antibiotic resistance determinants. Genome architecture analysis showed that PPA-ABTNL belonged to the "phiKMV-like Viruses" group. A repeated dose inhalational toxicity study using PPA-ABTNL crude preparation was conducted in mice and no significantly abnormal histological changes, morbidity or mortality were observed. There was no indication of any potential risk associated with using PPA-ABTNL as a therapeutic agent. The results of a curative treatment experiment demonstrated that atomization by ultrasonic treatment could efficiently deliver phage to the lungs of mink and a dose of 10 multiplicity of infection was optimal for treating mink hemorrhagic pneumonia. Our work demonstrated the potential for phage to fight P. aeruginosa involved in mink lung infections when administered by means of ultrasonic nebulization.

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

USDA-ARS?s Scientific Manuscript database

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

Phage display for generating peptide reagents.

PubMed

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

2008-02-01

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

Thioredoxin is required for filamentous phage assembly.

PubMed Central

Russel, M; Model, P

1985-01-01

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

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

PubMed

Tangney, Mark; Fitzgerald, Gerald F

2002-04-23

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

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

PubMed

Cui, Haiying; Yuan, Lu; Lin, Lin

2017-12-01

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

Polar flagella rotation in Vibrio parahaemolyticus confers resistance to bacteriophage infection

PubMed Central

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

2016-01-01

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

Phage display—A powerful technique for immunotherapy

PubMed Central

Bazan, Justyna; Całkosiński, Ireneusz; Gamian, Andrzej

2012-01-01

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

Bacteriophage Procurement for Therapeutic Purposes

PubMed Central

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

2016-01-01

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

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

PubMed

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

2017-04-28

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

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

PubMed

Grose, Julianne H; Casjens, Sherwood R

2014-11-01

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

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

PubMed Central

Grose, Julianne H.; Casjens, Sherwood R.

2014-01-01

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

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

PubMed Central

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-01-01

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

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

PubMed

Abedon, Stephen T

2016-01-01

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

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

PubMed

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-12-24

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

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

NASA Astrophysics Data System (ADS)

Chapman, Emily; Wu, Xiao-Lun

2006-03-01

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

Phage Therapy: Eco-Physiological Pharmacology

PubMed Central

Abedon, Stephen T.

2014-01-01

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

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

PubMed

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

2010-02-05

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

Molecular characterization of bacteriophages for microbial source tracking in Korea.

PubMed

Lee, Jung Eun; Lim, Mi Young; Kim, Sei Yoon; Lee, Sunghee; Lee, Heetae; Oh, Hyun-Myung; Hur, Hor-Gil; Ko, Gwangpyo

2009-11-01

We investigated coliphages from various fecal sources, including humans and animals, for microbial source tracking in South Korea. Both somatic and F+-specific coliphages were isolated from 43 fecal samples from farms, wild animal habitats, and human wastewater plants. Somatic coliphages were more prevalent and abundant than F+ coliphages in all of the tested fecal samples. We further characterized 311 F+ coliphage isolates using RNase sensitivity assays, PCR and reverse transcription-PCR, and nucleic acid sequencing. Phylogenetic analyses were performed based on the partial nucleic acid sequences of 311 F+ coliphages from various sources. F+ RNA coliphages were most prevalent among geese (95%) and were least prevalent in cows (5%). Among the genogroups of F+ RNA coliphages, most F+ coliphages isolated from animal fecal sources belonged to either group I or group IV, and most from human wastewater sources were in group II or III. Some of the group I coliphages were present in both human and animal source samples. F+ RNA coliphages isolated from various sources were divided into two main clusters. All F+ RNA coliphages isolated from human wastewater were grouped with Qbeta-like phages, while phages isolated from most animal sources were grouped with MS2-like phages. UniFrac significance statistical analyses revealed significant differences between human and animal bacteriophages. In the principal coordinate analysis (PCoA), F+ RNA coliphages isolated from human waste were distinctively separate from those isolated from other animal sources. However, F+ DNA coliphages were not significantly different or separate in the PCoA. These results demonstrate that proper analysis of F+ RNA coliphages can effectively distinguish fecal sources.

Evaluating the Use of Multilocus Variable Number Tandem Repeat Analysis of Shiga Toxin-Producing Escherichia coli O157 as a Routine Public Health Tool in England

PubMed Central

Byrne, Lisa; Elson, Richard; Dallman, Timothy J.; Perry, Neil; Ashton, Philip; Wain, John; Adak, Goutam K.; Grant, Kathie A.; Jenkins, Claire

2014-01-01

Multilocus variable number tandem repeat analysis (MLVA) provides microbiological support for investigations of clusters of cases of infection with Shiga toxin-producing E. coli (STEC) O157. All confirmed STEC O157 isolated in England and submitted to the Gastrointestinal Bacteria Reference Unit (GBRU) during a six month period were typed using MLVA, with the aim of assessing the impact of this approach on epidemiological investigations. Of 539 cases investigated, 341 (76%) had unique (>2 single locus variants) MLVA profiles, 12% of profiles occurred more than once due to known household transmission and 12% of profiles occurred as part of 41 clusters, 21 of which were previously identified through routine public health investigation of cases. The remaining 20 clusters were not previously detected and STEC enhanced surveillance data for associated cases were retrospectively reviewed for epidemiological links including shared exposures, geography and/or time. Additional evidence of a link between cases was found in twelve clusters. Compared to phage typing, the number of sporadic cases was reduced from 69% to 41% and the diversity index for MLVA was 0.996 versus 0.782 for phage typing. Using MLVA generates more data on the spatial and temporal dispersion of cases, better defining the epidemiology of STEC infection than phage typing. The increased detection of clusters through MLVA typing highlights the challenges to health protection practices, providing a forerunner to the advent of whole genome sequencing as a diagnostic tool. PMID:24465775

Evaluating the use of multilocus variable number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 as a routine public health tool in England.

PubMed

Byrne, Lisa; Elson, Richard; Dallman, Timothy J; Perry, Neil; Ashton, Philip; Wain, John; Adak, Goutam K; Grant, Kathie A; Jenkins, Claire

2014-01-01

Multilocus variable number tandem repeat analysis (MLVA) provides microbiological support for investigations of clusters of cases of infection with Shiga toxin-producing E. coli (STEC) O157. All confirmed STEC O157 isolated in England and submitted to the Gastrointestinal Bacteria Reference Unit (GBRU) during a six month period were typed using MLVA, with the aim of assessing the impact of this approach on epidemiological investigations. Of 539 cases investigated, 341 (76%) had unique (>2 single locus variants) MLVA profiles, 12% of profiles occurred more than once due to known household transmission and 12% of profiles occurred as part of 41 clusters, 21 of which were previously identified through routine public health investigation of cases. The remaining 20 clusters were not previously detected and STEC enhanced surveillance data for associated cases were retrospectively reviewed for epidemiological links including shared exposures, geography and/or time. Additional evidence of a link between cases was found in twelve clusters. Compared to phage typing, the number of sporadic cases was reduced from 69% to 41% and the diversity index for MLVA was 0.996 versus 0.782 for phage typing. Using MLVA generates more data on the spatial and temporal dispersion of cases, better defining the epidemiology of STEC infection than phage typing. The increased detection of clusters through MLVA typing highlights the challenges to health protection practices, providing a forerunner to the advent of whole genome sequencing as a diagnostic tool.

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

Genome Sequences of Mycobacteriophages Amgine, Amohnition, Bella96, Cain, DarthP, Hammy, Krueger, LastHope, Peanam, PhelpsODU, Phrank, SirPhilip, Slimphazie, and Unicorn

PubMed Central

Anders, Kirk R.; Mavrodi, Dmitri V.; Vazquez, Edwin; Amoh, Nana Yaa A.; Baliraine, Frederick N.; Buchser, William J.; Cast, Thomas P.; Chamberlain, Carmen E.; Chung, Hui-Min; D’Angelo, William A.; Farris, Christian T.; Fernandez-Martinez, Mariceli; Fischman, Haley D.; Forsyth, Mark H.; Fortier, Anna G.; Gallo, Kara F.; Held, Greta J.; Lomas, Miguel A.; Maldonado-Vazquez, Natalia Y.; Moonsammy, Claudia H.; Namboote, Peace; Paudel, Sudip; Reyes, Gabriella M.; Rubin, Michael R.; Saha, Margaret S.; Stukey, Joseph; Tobias, Tristan D.; Garlena, Rebecca A.; Stoner, Ty H.; Russell, Daniel A.

2017-01-01

ABSTRACT We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc²155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites. PMID:29217790

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

PubMed

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

2012-02-28

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

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

PubMed

Ploss, Martin; Kuhn, Andreas

2011-09-26

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

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

PubMed Central

2011-01-01

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

Stop Codon Reassignment in the Wild

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

Ivanova, Natalia; Schwientek, Patrick; Tripp, H. James

Since the discovery of the genetic code and protein translation mechanisms (1), a limited number of variations of the standard assignment between unique base triplets (codons) and their encoded amino acids and translational stop signals have been found in bacteria and phages (2-3). Given the apparent ubiquity of the canonical genetic code, the design of genomically recoded organisms with non-canonical codes has been suggested as a means to prevent horizontal gene transfer between laboratory and environmental organisms (4). It is also predicted that genomically recoded organisms are immune to infection by viruses, under the assumption that phages and their hostsmore » must share a common genetic code (5). This paradigm is supported by the observation of increased resistance of genomically recoded bacteria to phages with a canonical code (4). Despite these assumptions and accompanying lines of evidence, it remains unclear whether differential and non-canonical codon usage represents an absolute barrier to phage infection and genetic exchange between organisms. Our knowledge of the diversity of genetic codes and their use by viruses and their hosts is primarily derived from the analysis of cultivated organisms. Advances in single-cell sequencing and metagenome assembly technologies have enabled the reconstruction of genomes of uncultivated bacterial and archaeal lineages (6). These initial findings suggest that large scale systematic studies of uncultivated microorganisms and viruses may reveal the extent and modes of divergence from the canonical genetic code operating in nature. To explore alternative genetic codes, we carried out a systematic analysis of stop codon reassignments from the canonical TAG amber, TGA opal, and TAA ochre codons in assembled metagenomes from environmental and host-associated samples, single-cell genomes of uncultivated bacteria and archaea, and a collection of phage sequences« less