A map of the cleavage sites for endonuclease AvaI in the chromosome of bacteriophage lambda.
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ABSTRACT:
SUBMITTER: Hughes SG
PROVIDER: S-EPMC1164730 | biostudies-other | 1977 Jun
REPOSITORIES: biostudies-other
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Project description:Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage.In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome") into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%). We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda.Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins.
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Project description:The kinetics of the reactions of the EcoRI restriction endonuclease at individual recognition sites on the DNA from bacteriophage lambda were found to differ markedly from site to site. Under certain conditions of pH and ionic strength, the rates for the cleavage of the DNA were the same at each recognition site. But under altered experimental conditions, different reaction rates were observed at each recognition site. These results are consistent with a mechanism in which the kinetic stability of the complex between the enzyme and the recognition site on the DNA differs among the sites, due to the effect of interactions between the enzyme and DNA sequences surrounding each recognition site upon the transition state of the reaction. Reactions at individual sites on a DNA molecule containing more than one recognition site were found to be independent of each other, thus excluding the possibility of a processive mechanism for the EcoRI enzyme. The consequences of these observations are discussed with regard to both DNA-protein interactions and to the application of restriction enzymes in the study of the structure of DNA molecules.
| S-EPMC1162250 | biostudies-other
Project description:The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.
| S-EPMC1162251 | biostudies-other
Project description:Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy "footprinting" assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops.
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Project description:The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.
| S-EPMC4034188 | biostudies-literature
Project description:ObjectivesIn the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs.MethodsPGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park-Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model.ResultsDespite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by evolutionarily divergent catalytic domains), the enzymes displayed varying degrees of in vitro lytic activity against numerous staphylococcal strains, including cell surface mutants and drug-resistant strains, and proved effective against static biofilms. In a mouse model of systemic MRSA infection, six PGHs provided 100% protection from death, with animals being free of clinical signs at the end of the experiment.ConclusionsOur results corroborate the high potential of PGHs for treatment of S. aureus infections and reveal unique antimicrobial and biochemical properties of the different enzymes, suggesting a high diversity of potential applications despite highly conserved peptidoglycan target sites.
| S-EPMC4398471 | biostudies-literature
Project description:Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in Escherichia coli. Biochemical characterization of gpA-DeltaN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme, DNA maturation and DNA packaging, are discussed.
| S-EPMC2082050 | biostudies-literature
Project description:BackgroundDespite identical genotypes and seemingly uniform environments, stochastic gene expression and other dynamic intracellular processes can produce considerable phenotypic diversity within clonal microbes. One trait that provides a good model to explore the molecular basis of stochastic variation is the timing of host lysis by bacteriophage (phage).ResultsIndividual lysis events of thermally-inducible λ lysogens were observed using a temperature-controlled perfusion chamber mounted on an inverted microscope. Both mean lysis time (MLT) and its associated standard deviation (SD) were estimated. Using the SD as a measure of lysis time stochasticity, we showed that lysogenic cells in controlled environments varied widely in lysis times, and that the level of lysis time stochasticity depended on allelic variation in the holin sequence, late promoter (pR') activity, and host growth rate. In general, the MLT was positively correlated with the SD. Both lower pR' activities and lower host growth rates resulted in larger SDs. Results from premature lysis, induced by adding KCN at different time points after lysogen induction, showed a negative correlation between the timing of KCN addition and lysis time stochasticity.ConclusionsTaken together with results published by others, we conclude that a large fraction of λ lysis time stochasticity is the result of random events following the expression and diffusion of the holin protein. Consequently, factors influencing the timing of reaching critical holin concentrations in the cell membrane, such as holin production rate, strongly influence the mean lysis time and the lysis time stochasticity.
| S-EPMC3166277 | biostudies-literature
Project description:Bacteriophage lambda is one of the most extensively studied organisms, and has been a primary model for understanding basic modes of genetic regulation. Here we examine the progress of lambda gene expression during phage development by ribosome profiling, and thereby provide a very high resolution view of lambda gene expression. The known genes are expressed in a predictable fashion, authenticating the analysis. But many previously unappreciated potential open reading frames become apparent in the expression analysis, revealing an unexpected complexity in the pattern of lambda gene function.
2013-07-01 | GSE47509 | GEO
Project description:Phage T4 gene 49, encoding recombination endonuclease VII, specifies, by initiation from an AUG and an internal GUG codon, two in-frame overlapping peptides (of 18 and 12 kD). The gene is transcribed early and late, albeit from different promoters. The sequence predicts that in long early transcripts, initiated far upstream of the coding sequence, the Shine-Dalgarno sequence of the first ribosome binding site can be sequestered in a hairpin and/or cleaved. These processes might reduce initiation from the first AUG and facilitate initiation of the 12-kD peptide from the internal GUG. The potential of this hairpin to participate in Y structures or cruciforms suggests possible autoregulation. Shorter, more stable late transcripts initiated from a late promoter immediately upstream of the first ribosome binding site cannot form this hairpin. More efficient translation of the longer 18-kD gene 49 peptide from these late transcripts accounts for the strong dependence of endonuclease VII activity on late gene expression. An ORF downstream from gene 49 can be translated from a motA-dependent transcript that starts inside gene 49 as well as from the gene 49 transcripts. Its initiation codon overlaps the stop codon of gene 49, suggesting some coupling of translation. The deduced protein resembles, among others, the RexA protein of phage lambda. Possible implications for T4 recombination and for the interference of lambda lysogens with T4 gene 49 and rII mutants are discussed.
| S-EPMC1203513 | biostudies-other