We have determined the complete nucleotide sequences of two strains of a p1 thermoinducible mutant, p1 c 1100. Here, we present a system capable of overcoming this limitation through automated p1 phage transductions of escherichia coli. Pdf lysogenic transformation and plaque quantification. The first reports on the effectiveness of phage therapy were met with great, albeit shortlived, enthusiasm, which was followed by a collapse that was driven primarily by the introduction of antibiotics. For example, for every 100 000 particles of the wildtype lambda used at baseline, only one particle remained in circulation at 18 h. Common preconditions for safe phage therapy of pseudomonas. The wetlab procedures traditionally used rely on the identification and cloning of restriction fragment which can be delicate and cumbersome. The vectors used in this system contain a p1 packaging site pac to package vector and cloned dna into phage particles, two p1 loxp recombination sites to cyclize the packaged dna once it has been injected into a strain of escherichia coli containing the p1 cre recombinase, a kanr gene to select bacterial clones containing the cyclized dna, a. Development of a p1 phagemid system for the delivery of dna.
Download fulltext pdf bacteriophage p1 gene 10 is expressed from a promoteroperator sequence controlled by c1 and bof proteins article pdf available in journal of bacteriology 17419. Diversity of p1 phagelike elements in multidrug resistant. One method of prevention of phage infection is the use of bacterial strains harbouring particular prophages. Input dna was run alongside as a control for total amount of dna present. May 04, 2015 p1 phage as vector the development of a bacteriophage p1 cloning system capable of accepting dna fragments as large as 100 kilobase pairs kbp. For many phage types, the capsid is attached to a tail structure that is also made from phage encoded proteins. Quantification of plaque formation was attempted during the lytic phase of bacteriophage p1 when incubated with e. For optimal viewing, display the pages with the twopage view that includes the cover.
Before the actual p1 transduction of a selectable marker can be conducted, a fresh p1 stock lysate should be prepared from a wt e. P1 is larger, with a capacity of over 2% of the hosts dna. Pdf introduction to phage biology and phage display. P1 phage particles are not the great hope of universal therapy and prophylaxis, as felix dherelle believed bacteriophages that target pathogens might be quoted in reference 14. When undergoing a lysogenic cycle the phage genome exists as a plasmid in the bacterium unlike other phages e. Transfer 1 ml of overnight culture to 4 ml of rich media in a 50ml flask m9supp is optimum media, but lb will also work. This phase is devoted to synthesis of phage components and their assembly into mature phage particles.
Phage display technology applications and innovations. To increase transduction rate, we have inserted pac into the chromosome at up to five equally spaced positions. We now find that these mutants, for lack of poly p, are lysogenic for p1 and when infected with phage p1 produce only. The bacterial strains, plasmids and phage used in this study are listed in table 1. Three primer sets table 2 were used for each ip sample. The p1 genome 93,601 bp contains at least 117 genes, of which almost twothirds had not been sequenced previously and 49 have no homologs. P1 has an icosahedral head containing the phage dna attached to a 220nm long tube, contractile. An increasing number of infections are becoming harder or almost impossible to treat, carrying high morbidity, mortality, and financial cost.
Phage therapy is the application of phages that can kill bacteria to reduce in number or eliminate those bacteria. The p1 genome 93,601 bp contains at least 117 genes, of which almost twothirds had not been sequenced previously and 49 have no homologs in other. The tail also serves as a channel for delivery of the phage dna from the head into the host cell cytoplasm. The p22 phage coat can hold about 44 kb, a bit less than 1% of the hosts chromosomal dna. Development of an automated platform for highthroughput p1. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext.
In some cases the early phage proteins actually degrade the host chromosome. For each transduction, add 100 l of cells to an eppendorf tube 5. It is wellknown that bacteria bacteriophage contamination of bacterial cultures 115 figure 2. Dna carries the genetic code of the virus, so that rapid multiplication of the.
The phage particle injects its singlestranded dna into the bacterium using its tail structure like a syringe, which. Information on lysogenic bacteria is now growing rapidly. P1 has an icosahedral head containing the phage dna attached to a 220nm long tube. Packaging begins with a cut made at the pac packaging site. Pdf phage p1derived artificial chromosomes facilitate.
All lower taxonomy nodes 1 common name i synonym i. The scope of the book is general health issues and not cfsibsfm specific. Enterobacteria phage p1 an overview sciencedirect topics. Bacteriophages or phages are viruses that infect bacteria. Virulent bacteriophages lyse host cells as a result of expression of the phage genome a. Bacteriophages are composed of proteins that encapsulate a dna or rna genome, and may have structures that are. Transduction appeared to occur primarily in the first few days after addition of p1 to soil. Genome of bacteriophage p1 journal of bacteriology. Acquisition of phage sensitivity by bacteria through exchange. By combining the keio collection of singlegene deletion e. In addition to selectable markers, the vector contains the pac site and two loxp sites of phage p1. It lysogenizes its hosts as a circular, lowcopynumber plasmid.
P1 has an icosahedral head containing the dna attached to a contractile tail with six tail fibers. Bacteriophage p1 pac sites inserted into the chromosome. Inorganic polyphosphate essential for lytic growth of. Phageantibiotic synergy via delayed lysis applied and.
Pdf transduction of escherichia coli by bacteriophage pi. Phage display on the base of filamentous bacteriophages. P1 phage as vector the development of a bacteriophage p1 cloning system capable of accepting dna fragments as large as 100 kilobase pairs kbp. Infection of li cells by the ff phage starts with a specific interaction between piii and the top of the fpilus, which is a protein tube made up of pilin subunits.
Antibiotic resistance is arguably the biggest current threat to global health. Genes of the lytic pathway have been divided into early and late. Acquisition of phage sensitivity by bacteria through. The p1 genome 93,601 bp contains at least 117 genes, of which almost twothirds had not been sequenced previously and 49 have no homologs in. All phage have a chromosome encased in a capsid that is composed of phage encoded proteins.
Mar 11, 2004 p1 genes expressed in the lytic pathway are those involved in the timing of phage development, in replication from a lytic origin different from orir, in the formation of phage particles, including headful packaging, and finally in cell lysis to release the phage progeny. Bacteriophage p1 in retrospect and in prospect journal of. When undergoing a lysogenic cycle the phage genome exists as a autonomous plasmid, that is maintained at low copy number, in the bacterium. P1 is a bacteriophage of escherichia coli and other enteric bacteria. Methods bacterial strains, plasmids, phage and growth conditions.
Phage therapy has at least four advantages over conventional antibiotic therapy. Determining bacteriophage termini and packaging using ngs data. Basic microbiology techniques 06112011 phage enrichments 07122011 subculturing phage isolates 07122011 plating out phage. Recombinant molecules are packaged in vitro using various p1 packaging extracts. The phage is first grown on a strain containing the elements to be moved, and the resulting phage lysate is used to infect a second recipient strain. Jul 08, 2014 p1 is a temperate bacteriophage phage that infects escherichia coli and a some other bacteria. The transfer of hg or chloramphenicol resistance from lysogenic to nonlysogenic e.
Jul 08, 2017 a reader posted about this new to the market probiotic on cfs remissions facebook. Add 50 l of p1 lysate and incubate for 30 min at 30 c without shaking on heat block 6. Bacteriophage p1 is a temperate dna coliphage with a doublestranded, linear, cyclically permuted, terminally redundant chromosome lehnherr, 2005, yarmolinsky and sternberg, 1988. Pdf structure and morphogenesis of bacteriophage t4. T4 and p1 contain a linear doublestranded dna genome enclosed in a capsid and attached to a tail fig. This phenomenon is known as phage antibiotic synergy pas. But, unfortunately, it still does not guarantee the safety of. Phage genomes are packaged from concatemers beginning at a specific site, pac. A signalarrestrelease sequence mediates export and. This redirect is within the scope of wikiproject viruses, a collaborative effort to improve the coverage of viruses on wikipedia.
We have determined the complete nucleotide sequences of two strains of a p1 thermoinducible mutant, p1 c1100. Before the actual p1 transduction of a selectable marker can be conducted, a fresh p1 stock lysate should be prepared from a. From the beginning, dherelle was interested in the possibility of using phages. Abstract the expression of exogenous peptides on the surface of filamentous bacteriophage was initially described by smith. Certain types attach themselves to the cell membrane of the bacterium and instill a charge of dna into the cytoplasm. Here, we investigate phage dynamics in communities harboring phage resistant r and sensitive s bacteria, a common scenario in nature. Using bacillus subtilis and its lytic phage spp1, we demonstrate that r cells, lacking spp1 receptor, can be lysed by spp1 when cocultured with s cells. Dna is packaged by a headful mechanism, with 100115 kb of dna fitting into a p1 phage head. Phage particles has two p1 loxp recombination sites to cyclize the packaged dna once it has been injected into a strain of escherichia coli containing the p1 cre recombinase, a kanamycin resistant gene. Transduction frequency with phage p1 had been observed to be very low in escherichia coli k12 mutants lacking the operon ppk1ppx responsible for the synthesis of inorganic polyphosphate poly p. In this study, the observation of pas was extended to a wide variety of bacterium phage pairs using different classes of antibiotics. This genus of viruses includes many p2like phages as well as the satellite phage p4. Signals for ref were adjusted for input dna at the 30 min timepoints to account for increases in the amount of p1 phage dna compared to e. P1 is a temperate bacteriophage phage that infects escherichia coli and a some other bacteria.
P1 has an icosahedral head containing the dna attached to a contractile tail with six tail. The phage nucleic acid takes over the host biosynthetic machinery and phage specified mrnas and proteins are made. Bacteriophage p2, scientific name escherichia virus p2, is a temperate phage that infects e. It is a tailed virus with a contractile sheath and is thus classified in the genus p2virus formerly p2likevirus, subfamily peduovirinae, family myoviridae within order caudovirales.
The therapeutic use of bacteriophages, viruses that infect and kill bacteria, is well suited to be part of the multidimensional strategies to combat antibiotic resistance. Phage therapy is defined as the administration of virulent phages directly to a patient with the purpose of lysing the bacterial pathogen that is causing a clinically relevant infection. Bertani university of illinois, urbana, illinois of phage notions concerning bacterial viruses have in the past stemmed mostly from exact work done with phages unable to establish the lysogenic condition. The full p1 genome sequence has been published lobocka et al. We investigated how the information gathered by ngs technologies can be used to determine the dna termini and packaging mechanisms of dsdna phages. Stick a toothpick into an isolated plaque use sterilized forceps to handle toothpick.
Phage should be diluted such that plaques are not touching. In the more than 50 years since the discovery of p1, the phage has not been drafted for a combat role in therapy 11. Pas was shown in both grampositive and gramnegative. Phage art 2015 year of the phage conference san diego, ca. Retraction of the pilus, caused by depolymerization of pilin subunits, pulls the bacteriophage towards the cell. Phage genomes studies would exclude the use of the phages whose genomes encode such undesirable products as toxins, transposases, repressor proteins, etc. First, relatively rare encoding of exotoxins aside, phage virions are inherently safe, consisting of just benign proteins and dna, and therefore. The term was derived from bacteria and the greek phagein, meaning to devour. The phage is first grown on a strain containing the elements to be moved, and the resulting phage. This unit describes the procedure used to move portions of the e. It comprises approximately 700 phages 20% of the total drawn in figure 4.
Transduction of escherichia coli w3110r702 and j53rp4 104 to 105 cfug of soil by lysates of temperaturesensitive specialized transducing derivatives of bacteriophage p1 104 to 105 pfug of soil p1 cm cts, containing the resistance gene for chloramphenicol, or p1 cm ctstn501, containing the resistance genes for chloramphenicol and mercury hg occurred in soil. For phage plating, it is important to use solid media that is optically clear in order to be able to visualize plaques against the bacterial lawn. Lytic cycle life cycle of viruses that result in the lysis of the host bacterium. A signalarrestrelease sequence mediates export and control of the phage p1 endolysin min xu, douglas k. Enterobacteria phage p1 is known to infect and lysogenize e. After the phage dna penetrates into the cytoplasm, it is turned into a replicative plasmidlike molecule rf. P1 is a temperate bacteriophage that infects escherichia coli and some other bacteria. Researchers who have devoted their lives to phage also recount their experiences in pursuit of their quarry. The escherichia coli bacteriophage p1 packages host chromosome separately from phage dna, and transfers it to recipient cells at low frequency in a process called generalized transduction. When phages infect bacteria cultured in the presence of sublethal doses of antibiotics, the sizes of the phage plaques are significantly increased.