Showing papers on "Lambda phage published in 2002"

The murein hydrolase of the bacteriophage Φ3626 dual lysis system is active against all tested Clostridium perfringens strains

Abstract: Clostridium perfringens commonly occurs in food and feed, can produce an enterotoxin frequently implicated in food-borne disease, and has a substantial negative impact on the poultry industry As a step towards new approaches for control of this organism, we investigated the cell wall lysis system of C perfringens bacteriophage phi3626, whose dual lysis gene cassette consists of a holin gene and an endolysin gene Hol3626 has two membrane-spanning domains (MSDs) and is a group II holin A positively charged beta turn between the two MSDs suggests that both the amino terminus and the carboxy terminus of Hol3626 might be located outside the cell membrane, a very unusual holin topology Holin function was experimentally demonstrated by using the ability of the holin to complement a deletion of the heterologous phage lambda S holin in lambdadeltaSthf The endolysin gene ply3626 was cloned in Escherichia coli However, protein synthesis occurred only when bacteria were supplemented with rare tRNA(Arg) and tRNA(Ile) genes Formation of inclusion bodies could be avoided by drastically lowering the expression level Amino-terminal modification by a six-histidine tag did not affect enzyme activity and enabled purification by metal chelate affinity chromatography Ply3626 has an N-terminal amidase domain and a unique C-terminal portion, which might be responsible for the specific lytic range of the enzyme All 48 tested strains of C perfringens were sensitive to the murein hydrolase, whereas other clostridia and bacteria belonging to other genera were generally not affected This highly specific activity towards C perfringens might be useful for novel biocontrol measures in food, feed, and complex microbial communities

Stability puzzles in phage lambda.

Abstract: In the absence of RecA-mediated cleavage of the repressor, the [lambda] prophage is exceptionally stable. We develop a stochastic model that predicts the stability of such epigenetic states from affinities of the molecular components. We find that the stability, in particular, depends on the maximum possible cI protein production, and on the number of cro proteins per transcribed mRNA. We apply the model to the behavior of recently published mutants of OR and find, in particular, that a mutant that overexpress cro behaves in a different way than what was predicted, thus suggesting that the current view of the OR switch is incomplete. The approach described here should be generally applicable to the stability of expressed states.

Action at a distance in CI repressor regulation of the bacteriophage 186 genetic switch

Abstract: The non-lambdoid coliphage 186 provides an alternative model to the lytic-lysogenic switch of phage lambda. Like lambda, the key switch regulator, the CI repressor, associates to octamers. Unlike lambda, the lytic promoter (pR) and the lysogenic promoter (pL) are face-to-face, 62 bp apart and are flanked by distal CI binding sites (FL and FR) located approximately 300 bp away. Using reporter and footprinting studies, we show that the outcome, but not the mechanism, of regulation by 186 CI is very similar to lambda. 186 CI stimulates pL transcription indirectly by repressing convergent interfering transcription from pR. However, in the absence of the flanking FL and FR sites, CI bound at pR interacts co-operatively with a weak CI binding site at pL and represses both promoters. FL and FR play a critical role; they assist repression of pR and simultaneously alleviate repression of pL, thus allowing high pL activity. We propose that the 186 switch is regulated by a novel mechanism in which a CI octamer bound at pR forms alternative DNA loops to pL or to a flanking site, depending on CI concentration.

Efficient method to generate homologous recombinant baculovirus genomes in E. coli.

Abstract: Here we describe a convenient method to generate homologous recombinant baculoviral genomes in E. coli. The recombination takes place with the aid of recombination enzymes provided by the phage λ R...

Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands

Abstract: cDNA expression libraries displayed on lambda phage have been successfully employed to identify partners involved in antibody‐antigen, protein‐ protein and DNA‐protein interactions and represent a novel approach to functional genomics. However, as in all other cDNA expression libraries based on fusion to a carrier polypeptide, a major issue of this system is the absence of control over the translation frame of the cDNA. As a consequence, a large number of clones will contain lambda D/cDNA fusions, resulting in the foreign sequence being translated on alternative reading frames. Thus, many phage will not display natural proteins, but could be selected, as they mimic the binding properties of the real ligand, and will hence interfere with the selection outcome. Here we describe a novel lambda vector for display of exogenous peptides at the C-terminus of the capsid D protein. In this vector, translation of fusion peptides in the correct reading frame allows efficient in vivo biotinylation of the chimeric phage during amplification. Using this vector system we constructed three libraries from human hepatoma cells, mouse hepatocytic MMH cells and from human brain. Clones containing open reading frames (ORFs) were rapidly selected by streptavidin affinity chromatography, leading to biological repertoires highly enriched in natural polypeptides. We compared the selection outcome of two independent experiments performed using an anti-GAP-43 monoclonal antibody on the human brain cDNA library before and after ORF enrichment. A significant increase in the efficiency of identification of natural target peptides with very little background of false-positive clones was observed in the latter case.

Gene products encoded in the ninR region of phage lambda participate in Red-mediated recombination.

Abstract: Background: The ninR region of phage λ contains two recombination genes, orf (ninB) and rap (ninG), that were previously shown to have roles when the RecF and RecBCD recombination pathways of E. coli, respectively, operate on phage λ. Results: When λ DNA replication is blocked, recombination is focused at the termini of the virion chromosome. Deletion of the ninR region of λ decreases the sharpness of the focusing without diminishing the overall rate of recombination. The phenotype is accounted for in large part by the deletion of rap and of orf. Mutation of the recJ gene of the host partially suppresses the Rap– phenotype. Conclusion: ninR functions Orf and Rap participate in Red recombination, the primary pathway operating when wild-type λ grows lytically in rec+ cells. The ability of recJ mutation to suppress the Rap– phenotype indicates that RecJ exonuclease can participate in Red-mediated recombination, at least in the absence of Rap function. A model is presented for Red-mediated RecA-dependent recombination that includes these newly identified participants.

Rapid Construction of Adenoviral Vectors by Lambda Phage Genetics

Abstract: Continued improvements of adenoviral vectors require the investigation of novel genome configurations. Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production. In this way failure to generate a virus is not associated with an inability to generate the desired genome. We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli. The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability. Furthermore, the process is independent of any restriction endonucleases. The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification[s] and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E. coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid. Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule. This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions. The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations.

Isolation and characterization of Escherichia coli O157:H7 and shiga toxin-converting bacteriophages from strains of human, bovine and porcine origin

Abstract: Toxin-converting bacteriophages encoding the Stx2 gene were induced from strains of Escherichia coli O157:H7 isolated from sewage, bovine and porcine faeces. Toxin synthesis can be stimulated by the induction of integrated toxin-converting phages from the host E. colt O157:H7 organism by ultra-violet (UV) exposure. The UV-mediated DNA damage of E. coli O157:H7 triggers a bacterial SOS response resulting in phage release. Free ranging phages outside their E. coli O157:H7 hosts were detected but could not be isolated directly from environmental samples such as sewage and river water. E. coli O157:H7 colonies carrying the genes coding for Stx2 were isolated from 1 sewage sample (0.76% of positive samples), 8 cattle faecal samples (16.67% of positive samples) and 2 pig faecal samples (14. 28% of positive samples). Characterization of E. coli O157:H7 was done by repetitive sequence analysis using ERIC-PCR to determine the relationships between the individual E. colt O157:H7 strains. The ERIC-PCR analysis revealed distinct patterns for all E. coli O157:H7 strains with some small differences between the strains. DNA sequencing of some of the E. coli O157:H7 positive isolates carrying the Stx2 genes were performed confirming the amplified DNA nucleotide sequences of Stx2. Electron microscopic analysis revealed, for the first time in South Africa, that Stx2-converting phages induced from E. coli O157:H7 have different morphologies to that of phage lambda which was previously described. The role of the induced integrated Stx2 phages in natural environments such as river and dam water remains unclear. With the induction of Stx2-converting phages from environmental E. coli O157:H7 isolates, it is now possible to determine the potential of these phages to convert non-pathogenic E. colt strains and other enterobacteriaciae into pathogenic strains.

Affinity Selection of DNA-Binding Proteins from Yeast Genomic DNA Libraries by Improved λ Phage Display Vector

Abstract: Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work. After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.

Phage λ takes the first exit

Abstract: Once upon a time, biologists were accused of physics envy, of wanting universal laws and neat equations. The opposite could now be true, with physicists and mathematicians looking to biology for challenging problems and complex systems. Their contribution is often to recognize that a biological system resembles a mathematical framework for which a set of solutions already exists: the same equations have the same solutions, irrespective of their origin. This approach is illustrated by a recent model developed by Aurell and Sneppen [1xEpigenetics as a first exit problem. Aurell, E. and Sneppen, K. Phys. Rev. Lett. 2002; 88: 048101CrossrefSee all References][1] that frames the transition between lytic and lysogenic phases of bacteriophage replication as a first exit problem, which is a way of characterizing the expected time at which a random process will end.For example, the end point of a game of poker occurs when all but one player runs out of money (assuming no one has the sense to stop before his or her purse is empty). The time it takes to reach this endpoint will depend, to some extent, on chance events (the deal of the cards). When the temperate phage λ infects Escherichia coli, it enters one of two pathways: lytic (replicates and kills the host) or lysogenic (enters the genome of the host and is inherited passively by the progeny of the host). The quiescent state of a lysogenic phage is maintained by specific proteins that bind to sites on the phage genome.This protein–DNA interaction acts as a switch: if the concentration of the regulatory proteins drops below a crucial level, repression of transcription ceases, and the phage enters the lytic phase (and it's curtains for the E. coli). This system has been modelled previously by deterministic equations, for which the system has a stable equilibrium – the lysogenic state. This deterministic description might be valid if the regulating proteins were in very high concentrations in the cell. But, in reality, these molecules exist in small enough numbers that there is an element of randomness to the rate at which they bind and leave the receptor. The Brownian motion of the regulatory proteins results in the expected number of hits to the target sites growing with time only as its square root. This randomness, analogous to the effect of genetic drift on allele frequencies in small populations, introduces noise into the regulation of lysogeny. The beauty of this model is that it predicts that λ can exit the lysogenic phase and go into active replication simply through noise in the signalling system. This random model is somewhat at odds with the apparent robustness of the lysogenic state to experimental manipulation, suggesting that there could be additional factors at work. But in any case, this paper demonstrates that, rather than biologists with physics envy, we now have physicists modelling the messiness of biological systems.

An efficient method for preparing high quality DNA from plant DNA libraries in lambda phage

Abstract: An efficient method has been developed to improve preparation of phage particles by ammonium sulfate precipitation and to yield high quality DNA. The method, that has been used to screen plant DNA libraries constructed in λvectors, is inexpensive, does not require purification of phage particles, and can be used from either plate stocks or liquid lysates. Up to 1100 μg DNA was produced from 5 ml lysate obtained from agar plates.