Lambda phage

About: Lambda phage is a research topic. Over the lifetime, 1609 publications have been published within this topic receiving 84675 citations. The topic is also known as: Enterobacteria phage lambda.

Structure of the cooperative Xis–DNA complex reveals a micronucleoprotein filament that regulates phage lambda intasome assembly

Abstract: Next Section Abstract The DNA architectural protein Xis regulates the construction of higher-order nucleoprotein intasomes that integrate and excise the genome of phage lambda from the Escherichia coli chromosome. Xis modulates the directionality of site-specific recombination by stimulating phage excision 106-fold, while simultaneously inhibiting phage reintegration. Control is exerted by cooperatively assembling onto a ≈35-bp DNA regulatory element, which it distorts to preferentially stabilize an excisive intasome. Here, we report the 2.6-A crystal structure of the complex between three cooperatively bound Xis proteins and a 33-bp DNA containing the regulatory element. Xis binds DNA in a head-to-tail orientation to generate a micronucleoprotein filament. Although each protomer is anchored to the duplex by a similar set of nonbase specific contacts, malleable protein–DNA interactions enable binding to sites that differ in nucleotide sequence. Proteins at the ends of the duplex sequence specifically recognize similar binding sites and participate in cooperative binding via protein–protein interactions with a bridging Xis protomer that is bound in a less specific manner. Formation of this polymer introduces ≈72° of curvature into the DNA with slight positive writhe, which functions to connect disparate segments of DNA bridged by integrase within the excisive intasome. DNA bending recombination directionality factors site-specific DNA recombination x-ray structure

Characterization of the relationship between integrase, excisionase and antirepressor activities associated with a superinfecting Shiga toxin encoding bacteriophage

Abstract: Shigatoxigenic Escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of Shiga toxin-encoding lambdoid bacteriophages. At least some of these Stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host’s pathogenic profile. Here, increasing levels of Stx phage (24B integrase expression in multiple lysogen cultures are demonstrated along with apparently negligible repression of integrase expression by the cognate CI repressor. The (24B int transcription start site and promoter region were identified and found to differ from in silico predictions. The unidirectional activity of this integrase was determined in an in situ, inducible tri-partite reaction. This indicated that (24B must encode a novel directionality factor that is controlling excision events during prophage induction. This excisionase was subsequently identified and characterized through complementation experiments. In addition, the previous proposal that a putative antirepressor was responsible for the lack of immunity to superinfection through inactivation of CI has been revisited and a new hypothesis involving the role of this protein in promoting efficient induction of the (24B prophage is proposed.

Lambda phage vectors--EMBL series.

Abstract: Publisher Summary This chapter describes the method to establish genomic libraries from large genomes and to identify specific genomic clones. Cosmids and λ replacement vectors rely on the packaging of recombinant genomes into phage heads in vitro to give infective particles as a means of introducing DNA into the bacterial cell with high efficiency. Cosmids require only a few kilobases of DNA to encode the functions essential for their replication and selection. EMBL3 and EMBL42 are replacement vectors derived from 1059, which allow the cloning of DNA fragments with sizes between 8 and 23 kb. The chapter discusses the amber derivatives of EMBL vectors, EMBL vector derivatives with further cloning sites, end fragment cloning, and others. The chapter describes the procedures for vector preparation, including the preparation of plate stock, the preparation of phage DNA, the testing of vector DNA and others.

Isolation and characterization of two sequence-specific endonucleases from Anabaena variabilis.

Abstract: Two endonucleases, AvaI and AvaII, were isolated from Anabaena variabilis on the basis of their ability to make a limited number of breaks at specific points in bacteriophage lambda DNA. Neither enzyme has cofactor requirements beyond Mg2+. Endonuclease AvaI makes eight breaks in the phage lambda chromosome at which the 5'-terminal sequence is pPy-C-G-N. AvaII endonuclease cuts phage lambda DNA more extensively, yielding fragments with the 5'-terminal sequence G-T-C-N or G-A-C-N. Neither enzyme generates cohesive ends.

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

Abstract: Aim: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. Methods and Results: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 × 108 to 1 CFU ml−1 within 1·5–10·3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 × 108 to 130 CFU ml−1 was achieved within 2·6–22·4 h, respectively. Conclusions: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. Significance and Impact of Study: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.