Bacteriophage Lambda Genome Research Articles

Integration host factor assembly at the cohesive end site of the bacteriophage lambda genome: implications for viral DNA packaging and bacterial gene regulation.

Integration host factor (IHF) is an Escherichia coli protein involved in (i) condensation of the bacterial nucleoid and (ii) regulation of a variety of cellular functions. In its regulatory role, IHF binds to a specific sequence to introduce a strong bend into the DNA; this provides a duplex architecture conducive to the assembly of site-specific nucleoprotein complexes. Alternatively, the protein can bind in a sequence-independent manner that weakly bends and wraps the duplex to promote nucleoid formation. IHF is also required for the development of several viruses, including bacteriophage lambda, where it promotes site-specific assembly of a genome packaging motor required for lytic development. Multiple IHF consensus sequences have been identified within the packaging initiation site (cos), and we here interrogate IHF–cos binding interactions using complementary electrophoretic mobility shift (EMS) and analytical ultracentrifugation (AUC) approaches. IHF recognizes a single consensus sequence within cos (I1) to afford a strongly bent nucleoprotein complex. In contrast, IHF binds weakly but with positive cooperativity to nonspecific DNA to afford an ensemble of complexes with increasing masses and levels of condensation. Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates. At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition. The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.

ALE: a generic assembly likelihood evaluation framework for assessing the accuracy of genome and metagenome assemblies

Researchers need general purpose methods for objectively evaluating the accuracy of single and metagenome assemblies and for automatically detecting any errors they may contain. Current methods do not fully meet this need because they require a reference, only consider one of the many aspects of assembly quality or lack statistical justification, and none are designed to evaluate metagenome assemblies. In this article, we present an Assembly Likelihood Evaluation (ALE) framework that overcomes these limitations, systematically evaluating the accuracy of an assembly in a reference-independent manner using rigorous statistical methods. This framework is comprehensive, and integrates read quality, mate pair orientation and insert length (for paired-end reads), sequencing coverage, read alignment and k-mer frequency. ALE pinpoints synthetic errors in both single and metagenomic assemblies, including single-base errors, insertions/deletions, genome rearrangements and chimeric assemblies presented in metagenomes. At the genome level with real-world data, ALE identifies three large misassemblies from the Spirochaeta smaragdinae finished genome, which were all independently validated by Pacific Biosciences sequencing. At the single-base level with Illumina data, ALE recovers 215 of 222 (97%) single nucleotide variants in a training set from a GC-rich Rhodobacter sphaeroides genome. Using real Pacific Biosciences data, ALE identifies 12 of 12 synthetic errors in a Lambda Phage genome, surpassing even Pacific Biosciences' own variant caller, EviCons. In summary, the ALE framework provides a comprehensive, reference-independent and statistically rigorous measure of single genome and metagenome assembly accuracy, which can be used to identify misassemblies or to optimize the assembly process. ALE is released as open source software under the UoI/NCSA license at http://www.alescore.org. It is implemented in C and Python.

Single Virus Genomics: A New Tool for Virus Discovery

Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called ‘Single Virus Genomics’, which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.

DNA unzipping phase diagram calculated via replica theory

We show how single-molecule unzipping experiments can provide strong evidence that the zero-force melting transition of long molecules of natural dsDNA should be classified as a phase transition of the higher-order type (continuous). Toward this end, we study a statistical-mechanics model for the fluctuating structure of a long molecule of dsDNA, and compute the equilibrium phase diagram for the experiment in which the molecule is unzipped under applied force. We consider a perfect-matching dsDNA model, in which the loops are volume-excluding chains with arbitrary loop exponent c . We include stacking interactions, hydrogen bonds, and main-chain entropy. We include sequence heterogeneity at the level of random sequences; in particular, there is no correlation in the base-pairing (bp) energy from one sequence position to the next. We present heuristic arguments to demonstrate that the low-temperature macrostate does not exhibit degenerate ergodicity breaking. We use this claim to understand the results of our replica-theoretic calculation of the equilibrium properties of the system. As a function of temperature, we obtain the minimal force at which the molecule separates completely. This critical-force curve is a line in the temperature-force phase diagram that marks the regions where the molecule exists primarily as a double helix versus the region where the molecule exists as two separate strands. We compare our random-sequence model to magnetic tweezer experiments performed on the 48 502 bp genome of bacteriophage lambda . We find good agreement with the experimental data, which is restricted to temperatures between 24 and 50 degrees C . At higher temperatures, the critical-force curve of our random-sequence model is very different for that of the homogeneous-sequence version of our model. For both sequence models, the critical force falls to zero at the melting temperature T_{c} like |T-T_{c}|;{alpha} . For the homogeneous-sequence model, alpha=1/2 almost exactly, while for the random-sequence model, alpha approximately 0.9 . Importantly, the shape of the critical-force curve is connected, via our theory, to the manner in which the helix fraction falls to zero at T_{c} . The helix fraction is the property that is used to classify the melting transition as a type of phase transition. In our calculation, the shape of the critical-force curve holds strong evidence that the zero-force melting transition of long natural dsDNA should be classified as a higher-order (continuous) phase transition. Specifically, the order is 3rd or greater.

High adsorption rate is detrimental to bacteriophage fitness in a biofilm-like environment

BackgroundBacterial biofilm is ubiquitous in nature. However, it is not clear how this crowded habitat would impact the evolution of bacteriophage (phage) life history traits. In this study, we constructed isogenic λ phage strains that only differed in their adsorption rates, because of the presence/absence of extra side tail fibers or improved tail fiber J, and maker states. The high cell density and viscosity of the biofilm environment was approximated by the standard double-layer agar plate. The phage infection cycle in the biofilm environment was decomposed into three stages: settlement on to the biofilm surface, production of phage progeny inside the biofilm, and emigration of phage progeny out of the current focus of infection.ResultsWe found that in all cases high adsorption rate is beneficial for phage settlement, but detrimental to phage production (in terms of plaque size and productivity) and emigration out of the current plaque. Overall, the advantage of high adsorption accrued during settlement is more than offset by the disadvantages experienced during the production and emigration stages. The advantage of low adsorption rate was further demonstrated by the rapid emergence of low-adsorption mutant from a high-adsorption phage strain with the side tail fibers. DNA sequencing showed that 19 out of the 21 independent mutant clones have mutations in the stf gene, with the majority of them being single-nucleotide insertion/deletion mutations occurring in regions with homonucleotide runs.ConclusionWe conclude that high mutation rate of the stf gene would ensure the existence of side tail fiber polymorphism, thus contributing to rapid adaptation of the phage population between diametrically different habitats of benthic biofilm and planktonic liquid culture. Such adaptability would also help to explain the maintenance of the stf gene in phage λ's genome.

Quantitative predictions for DNA two‐dimensional display according to size and nucleotide sequence composition

2-D DNA display is a simple separation method that provides a fast and economical way of visualizing polymorphism and comparing genomes. The DNA fragments are separated first according to their size by standard gel electrophoresis and then according to their sequence composition using denaturing gradient gel electrophoresis. First developed by Fischer and Lerman (Cell 1979, 16, 191-200), this method has recently been used to distinguish strains within a bacterial species. The genomic restriction fragments are displayed as spots on a 2-D surface. Although most of the relevant physical mechanisms are understood, this technique is mostly empirical and remains essentially qualitative. In view of optimizing this procedure, we combine our understanding of the different physical mechanisms at play to develop a complete numerical model to predict the relative coordinates of the spots as a function of the corresponding DNA sequence and of the experimental conditions. We experimentally validate our model by predicting the outcome of a 2-D display of the lambda phage genome. It thus becomes possible to optimize in silico the experimental parameters, to predict whether specific mutations as well as yet undescribed genetic polymorphisms can be resolved, and to assist in interpreting the experimental data.

A study of comparability in amplified fragment length polymorphism profiling using a simple model system

A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated. The reproducibility of the AFLP model was tested by submitting both "unknown" DNA template that had been restricted and ligated with AFLP linkers (R/L mixture) and corresponding primer pairs to nine laboratories participating in the study. Participants completed the final PCR step and then used either slab gel electrophoresis or CE to detect the AFLP fragments. The predicted fragments were identified by the majority of participants with size estimates consistently up to 3 base pair (bp) larger for slab gel electrophoresis than for CE. Shadow fragments, 3 bp larger than the predicted fragments, were often observed by study participants and organizers. The nine AFLP fragments exhibited relative intensities ranging from less than 3% to 22% and, apart from the two weakest fragments, with a % CV of 16 to 25. Fragments containing the highest guanine-cytosine (GC) content of 50-56% showed the greatest stability in the AFLP profiles.

Switch from θ to σ replication of bacteriophage λ DNA: factors involved in the process and a model for its regulation

Bacteriophage lambda genome is one of the classical model replicons in studies on the regulation of DNA replication. Moreover, since genes coding for Shiga toxins are located in genomes of lambdoid phages, understanding of mechanisms controlling lambda DNA replication may be of bio-medical importance. During lytic development of bacteriophage lambda, its genome is replicated according to the theta (circle-to-circle) mode early after infection, and then it is switched to the sigma (rolling circle) mode. Two mechanisms of regulation of this switch were proposed recently and both suggested a crucial role for directionality of lambda DNA replication. Whereas one hypothesis assumed transient impairment of ClpP/ClpX-mediated proteolysis of the lambdaO initiator protein, another suggested a crucial role for transcriptional activation of the orilambda region and factors involved in the control of the p (R) promoter activity. Here we demonstrate that mutations in clpP and clpX genes had little influence on both directionality of lambda DNA replication and appearance of sigma replication intermediates. On the other hand, regulators affecting activity of the p (R) promoter (responsible for initiation of transcription, which activates orilambda) directly or indirectly influenced directionality of lambda DNA replication to various extents. Therefore, we conclude that regulation of the efficiency of transcriptional activation of orilambda, rather than transient impairment of the lambdaO proteolysis, is responsible for the control of the switch from theta to sigma replication, and propose a model for this control.

Likelihood-Based Inference for Multi-Color Optical Mapping

Multi-color optical mapping is a new technique being developed to obtain detailed physical maps (indicating relative positions of various recognition sites) of DNA molecules. We consider a study design in which the data consist of noisy observations of multiple copies of a DNA molecule marked with colors at recognition sites. The primary goal is to estimate a physical map. A secondary goal is to estimate error rates associated with the experiment, which are potentially useful for analysis and refinement of the biochemical steps in the mapping procedure. We propose statistical models for various sources of error and use maximum likelihood estimation (MLE) to construct a physical map and estimate error rates. To overcome difficulties arising in the maximization process, a latent-variable Markov chain version of the model is proposed, and the EM algorithm is used for maximization. In addition, a simulated annealing procedure is applied to maximize the profile likelihood over the discrete space of sequences of colors. We apply the methods to simulated data on the bacteriophage lambda genome.

Identification of DNA-binding protein target sequences by physical effective energy functions, free energy analysis of lambda repressor-DNA complexes

BackgroundSpecific binding of proteins to DNA is one of the most common ways gene expression is controlled. Although general rules for the DNA-protein recognition can be derived, the ambiguous and complex nature of this mechanism precludes a simple recognition code, therefore the prediction of DNA target sequences is not straightforward. DNA-protein interactions can be studied using computational methods which can complement the current experimental methods and offer some advantages. In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the λ repressor-DNA complex for which structural and thermodynamic experimental data are available.ResultsThe binding free energy of two molecules can be expressed as the sum of an intermolecular energy (evaluated using a molecular mechanics forcefield), a solvation free energy term and an entropic term. Different solvation models are used including distance dependent dielectric constants, solvent accessible surface tension models and the Generalized Born model. The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed; results show that this effect is in general negative and the reproducibility of the experimental values decreases with the increase of simulation time considered. The free energy of binding for non-specific complexes, estimated using the best energetic model, agrees with earlier theoretical suggestions. As a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences. The possibility of searching regulatory elements within the bacteriophage λ genome using this protocol is explored. Our analysis shows good prediction capabilities, even in absence of any thermodynamic data and information on the naturally recognized sequence.ConclusionThis study supports the conclusion that physics-based methods can offer a completely complementary methodology to sequence-based methods for the identification of DNA-binding protein target sequences.

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

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. 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 x 10(8) 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 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. 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. With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.

A trial of somatic gene targeting in vivo with an adenovirus vector

BackgroundGene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ+ gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro.MethodsAn 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside.ResultsThe mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (~1/10000). Our further restriction analysis did not detect any designed recombinant.ConclusionThe frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means.

Partly melted DNA conformations obtained with a probability peak finding method

Peaks in the probabilities of loops or bubbles, helical segments, and unzipping ends in melting DNA are found in this article using a peak finding method that maps the hierarchical structure of certain energy landscapes. The peaks indicate the alternative conformations that coexist in equilibrium and the range of their fluctuations. This yields a representation of the conformational ensemble at a given temperature, which is illustrated in a single diagram called a stitch profile. This article describes the methodology and discusses stitch profiles vs the ordinary probability profiles using the phage lambda genome as an example.

Somatic gene targeting with RNA/DNA chimeric oligonucleotides: an analysis with a sensitive reporter mouse system.

Targeted gene correction provides a potentially powerful method for gene therapy. RNA/DNA chimeric oligonucleotides were reported to be able to correct a point mutation with a high efficiency in cultured rodent cells, in the body of mice and rats, and in plants. The efficiency of correction in the liver of rats was claimed to be as high as 20% after tail-vein injection. However, several laboratories have failed to reproduce the high efficiency. In order to sensitively detect and measure sequence changes by the chimeric oligonucleotides, we used Muta Mouse, a transgenic mouse system for mutation detection in vivo. It carries, on its chromosome, multiple copies of the lambda phage genome with the lacZ(+) gene. Two chimeric oligonucleotides were designed to make a point mutation at the active site of the LacZ gene product. They were injected into the liver with HVJ liposomes, which were demonstrated to allow reliable gene delivery. One week later, DNA was extracted from the liver, and lambda::lacZ particles were recovered by in vitro packaging. The lacZ-negative phage was detected by selection with phenyl-beta-D-galactoside. The mutant frequency of the injected mice was at the same level as the control mouse (approximately 1/10000). Our further restriction analysis and sequencing did not detect the designed mutations. Gene correction frequency in mouse liver by these oligonucleotides was shown to be less than 1/20000 in our assay with the Muta Mouse system.

A Bayesian approach to DNA sequence segmentation.

Many deoxyribonucleic acid (DNA) sequences display compositional heterogeneity in the form of segments of similar structure. This article describes a Bayesian method that identifies such segments by using a Markov chain governed by a hidden Markov model. Markov chain Monte Carlo (MCMC) techniques are employed to compute all posterior quantities of interest and, in particular, allow inferences to be made regarding the number of segment types and the order of Markov dependence in the DNA sequence. The method is applied to the segmentation of the bacteriophage lambda genome, a common benchmark sequence used for the comparison of statistical segmentation algorithms.

Evidence of bar minigene expression and tRNA2Ile sequestration as peptidyl-tRNA2Ile during lambda bacteriophage development.

Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth). Single-base-pair mutations (bar(-)) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells). When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering tRNA(2)(Ile) as pep-tRNA(2)(Ile). Either tRNA(2)(Ile) or Pth may reverse these effects. In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester tRNA(2)(Ile) as pep-tRNA(2)(Ile). In addition, overexpression of the barI minigene impairs the development even of bar(-) phages in rap cells. Interestingly, tRNA or Pth may reestablish lambda phage development. These results suggest that lambda bar minigenes are expressed and tRNA(2)(Ile) is sequestered as pep-tRNA(2)(Ile) during lambda phage development.

Biochemical Characterization of Bacteriophage Lambda Genome Packaging in Vitro

Bacteriophage λ has been extensively studied, and the abundance of genetic and biochemical information available makes this an ideal model system to study virus DNA packaging at the molecular level. Limited in vitro packaging efficiency has hampered progress toward this end, however. It has been suggested that limited packaging efficiency is related to poor activity of purified procapsids. We describe the construction of a vector that expresses λ procapsids with a yield that is 40-fold greater than existing systems. Consistent with previous studies, packaging of a mature λ genome is very inefficient in vitro, with only 4% of the input procapsids utilized. Concatemeric DNA is the preferred packaging substrate in vivo, and procapsids interact with a nucleoprotein complex known as complex I to initiate genome packaging. When complex I is used as a packaging substrate in vitro, capsid utilization is extremely efficient, and 40% of the input DNA is packaged. Finally, we provide evidence for a packaging-stimulated ATPase activity, and kinetically characterize this reaction quantifying the energetic cost of DNA packaging in bacteriophage λ.

Revisiting the Lysogenization Control of Bacteriophage λ: IDENTIFICATION AND CHARACTERIZATION OF A NEW HOST COMPONENT, HflD

Upon infection to the Escherichia coli cell, the genome of bacteriophage lambda either replicates to form new progenies (lytic growth) or integrates into the host chromosome (lysogenization). The lambda CII protein is a key determinant in the lysis-lysogeny decision. It is a short-lived transcription activator for the lambda genes essential for lysogeny establishment. In this study, we isolated a new class of hfl (high frequency lysogenization) mutants of E. coli, using a new selection for enhancement of CII-stimulated transcription. The gene affected was termed hflD, which encodes a protein of 213 amino acids. An hflD-disrupted mutant indeed showed an Hfl phenotype, indicating that HflD acts to down-regulate lysogenization. HflD is associated peripherally with the cytoplasmic membrane. Its interaction with CII was demonstrated in vitro using purified proteins as well as in vivo using the bacterial two-hybrid system. Pulse-chase examinations demonstrated that the HflD function is required for the rapid in vivo degradation of CII, although it interfered with FtsH-mediated CII proteolysis in an in vitro reaction system using detergent-solubilized components. We suggest that HflD is a factor that sequesters CII from the target promoters and recruits it to the membrane where the FtsH protease is localized.

Whole genome sequence-enabled prediction of sequences performed for random PCR products of Escherichia coli.

The sequence of an unknown PCR product generated by random (and conventional) PCR could be determined without sequencing when it is provided with the template DNA sequence. Theoretically, this was based on formerly established ideas which assert that the amount of random PCR product mainly depends on the stability of the primer-binding structures and that the dynamic solution structure of DNA is essentially governed by the Watson-Crick base pairing. However, it has not been clear whether this holds true for larger genomes of mega- to gigabase size, beside the lambda phage genome (of 50 kb) used previously, nor has it been ascertained to uniquely specify the sequence of a random PCR product. Here, we jointly use two computer programs together with experimental data from Genome Profiling (i.e. TGGE analysis of random PCR products). The first procedure carried out by a newly remodeled computer program (PCRAna-A1) was shown to be competent to calculate a set of random PCR products from Escherichia coli genome DNA (4.7 Mb). The other procedure performed with another program (Poland-H) played a critical role in determining the final candidate sequence by theoretically offering the initial melting temperature and the melting pattern of unspecified candidate sequences. The success attained here not only proved our method to be useful for sequence prediction but also confirmed the above-mentioned ideas as rational. We believe that this is the first case to computer-utilize a genome sequence as a whole.

The terminase enzyme from bacteriophage lambda: a DNA-packaging machine.

This review focuses on the biochemical, biophysical, and catalytic properties of terminase, an enzyme involved in bacteriophage lambda genome packaging. The holoenzyme possesses ATPase, DNA strand-separation, and site-specific nuclease activities that work in concert to insert a viral genome into the confines of a performed capsid. Moreover, the terminase subunits are part of a series of nucleoprotein complexes involved in genome packaging, including remarkably stable intermediates that transition to a highly mobile DNA packaging 'machine.' Models for the assembly and interconversion of these complexes are presented. Interactions between the catalytic sites in the enzyme complex, and modulation of these catalytic activities as it relates to the assembly and relative stability of the packaging intermediates are discussed. This ordered progression of nucleoprotein intermediates is a common theme in biology as demonstrated by mechanistic similarities between viral DNA packaging, the initiation of chromosomal replication, and the initiation of transcription. Terminase is thus part of a growing number of examples of biological 'machines' or molecular 'motors.'