Showing papers on "Lambda phage published in 2011"

Bacteriophage Crosstalk: Coordination of Prophage Induction by Trans-Acting Antirepressors

Abstract: Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized.

The protein interaction map of bacteriophage lambda

Abstract: 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.

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.

Role of DLP12 lysis genes in Escherichia coli biofilm formation.

Abstract: Phages have recently been implicated as important in biofilm development, although the mechanisms whereby phages impact biofilms remain unclear. One defective lambdoid phage carried by Escherichia coli K-12 is DLP12. Among the genes found in DLP12 are essD, ybcS and rzpD/rzoD, which are homologues of the Lambda phage genes encoding cell-lysis proteins (S, R and Rz/Rz1). The role that these DLP12 lysis genes play in biofilm formation was examined in deletion mutants of E. coli PHL628, a curli-overproducing, biofilm-forming K-12 derivative. Strains lacking essD, ybcS and rzpD/rzoD were unable to form wild-type biofilms. While all mutants were compromised in attachment to abiotic surfaces and aggregated less well than the wild-type, the effect of the essD knockout on biofilm formation was less dramatic than that of deleting ybcS or rzpD/rzoD. These results were consistent with electron micrographs of the mutants, which showed a decreased number of curli fibres on cell surfaces. Also consistent with this finding, we observed that expression from the promoter of csgB, which encodes the curli subunits, was downregulated in the mutants. As curli production is transcriptionally downregulated in response to cell wall stress, we challenged the mutants with SDS and found them to be more sensitive to the detergent than the wild-type. We also examined the release of 14C-labelled peptidoglycan from the mutants and found that they did not lose labelled peptidoglycan to the same extent as the wild-type. Given that curli production is known to be suppressed by N-acetylglucosamine 6-phosphate (NAG-6P), a metabolite produced during peptidoglycan recycling, we deleted nagK, the N-acetylglucosamine kinase gene, from the lysis mutants and found that this restored curli production. This suggested that deletion of the lysis genes affected cell wall status, which was transduced to the curli operon by NAG-6P via an as yet unknown mechanism. These observations provide evidence that the S, R and Rz/Rz1 gene homologues encoded by DLP12 are not merely genetic junk, but rather play an important, though undefined, role in cell wall maintenance.

Following Cell-fate in E. coli After Infection by Phage Lambda

Abstract: The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Chemical coupling as a potent strategy for preparation of targeted bacteriophage-derived gene nanocarriers into eukaryotic cells.

Abstract: Background The ability to direct efficiently and specifically carriers toward target cells and express the transgene of interest is a critical step in gene therapy trails. The display of targeting molecules on the surface of phage particles might represent a potent solution. In the present study, we evaluated a chemical coupling strategy for displaying human holotransferrin as a targeting molecule on the surface of phage lambda particles for specifically delivering green fluorescent protein (GFP) encoding gene into a human cell line. Methods Human holotransferrin was coupled on the phage lambda particles bearing a GFP-expression cassette by a chemical coupling strategy to formulate transferrin-targeted lambda-GFP (Tf-targeted-λ-GFP) gene nanocarrier. The carrier was then characterized by phage-enzyme-linked immunosorbent assay experiments and used for transfection of the human 293T cell line. Particle internalization into the cells was evaluated by immunocytochemical staining and transfection efficacy was studied using fluorescence-activated cell sorting (FACS) analysis. Results Characterization of the nanocarrier showed a rather high copy number (274 molecules) of transferrin molecules coupled per phage particle. Immunocytochemical staining revealed efficient internalization of the Tf-targeted-λ-GFP compared to wild lambda-GFP (λ-GFP) particles. FACS analysis showed 6.72% GFP positive cells for transfections mediated by Tf-targeted-λ-GFP, whereas the value was 0.61% for wild lambda-GFP particles. Conclusions Our findings highlight chemical coupling as an efficient and straightforward strategy for displaying a targeting molecule at high density on the phage surface, which, in turn, may improve the efficiency of phage-mediated gene transfer and expression. Copyright © 2011 John Wiley & Sons, Ltd.

Probing the viral metallome: searching for metalloproteins in bacteriophage λ-- the hunt begins.

Abstract: Although the proteome and genome of bacteriophages are well developed, there is little knowledge about metals and their interactions with the phages, even though metals have been observed in stabilizing phage particles. With expanding studies of phage display and its promising applications, metalloprotein investigations in the bacteriophage areas are necessary to understand whether or not metalloproteins are included in the viral coat proteome. Since these virus studies are still in their infancy, lambda phage was chosen due to its high metal-binding potential as suggested by the cysteine/methionine rich proteins in the viral coat. After large-scale preparation and further purification of lambda phage according to standard protocols, state-of-the-art metallomics techniques via combinations of chromatographies and mass spectrometries were utilized for screening metal-associated species in lambda phage. The lambda phage sample was first separated using non-denaturing size exclusion chromatography with selective metal detection by ICPMS for screening associated metals and generating size distribution fractions for the various metal species, some of which include metalloproteins. Various molecular size distribution patterns were exhibited for the metals detected, Mn, Fe, Co, Ni, Cu and Zn, at different molecular weight ranges. On the other hand numerous other metals were not associated with the coat proteins, as they were not detected in the different molecular weight fractions. Further identification for putative metallopeptides and metalloproteins was accomplished by collecting various metal species’ fractions offline and subsequently analyzing tryptically-digested fractions via nanoLC-Chip-ESI-MS. By searching appropriate MS databases with both Spectrum Mill and MASCOT search engines, the main capsid protein, gpE, a capsid decoration protein, gpD, and main tail component protein, gpV, were found and are known for associations with the detected transition metals. These findings will likely provide valuable information for lambda phage engineered applications.

Biochemical Investigation of the Bacteriophage Protein HK97 gp74

Abstract: Bacteriophages are viruses that infect and propagate within bacteria by making use of the host’s biosynthetic machinery. With a global population of 1031, phages pose a significant influence on microbial populations. Studies of bacteriophage proteins can elucidate the influence that bacteriophages play on the evolution of bacteria, as well as, providing the basis for the use of phage proteins as possible therapeutics and bioengineering solutions. This study aims to investigate the structural and functional role of the HK97 phage protein gp74. Sequence alignments indicate that gp74 is related to homing HNH endonucleases. Homing endonucleases are predominantly double-stranded DNases, suggesting that gp74 mediates integration of phage genes into the host genome or may target foreign phage DNA. DNA digestion experiments with gp74 reveals that gp74 mediates non-specific double-stranded cleavage of lambda phage DNA and single strand cleavage of plasmid DNA. Our initial work demonstrates that HK97 gp74 is an HNH endonuclease.%%%%MAST

Probabilistic and Flux Landscapes of the Phage lambda Genetic Switch

Abstract: The phage $\lambda$ infection of an \textit{E. coli} cell has become a paradigm for understanding the molecular processes involved in gene expression and cell signaling. This system provides an example of a genetic switch, as cells with identical DNA choose either of two cell cycles: a lysogenic cycle, in which the phage genome is incorporated into the host and copied by the host; or a lytic cycle, resulting in the death of the cell and a burst of viruses. The robustness of this switch is remarkable; although the first stages of the lysogenic and lytic cycles are identical, a lysogen rarely spontaneously flips, and external stressors or instantaneous cell conditions are required to induce flipping. In particular, the cell fate decision can depend on the populations of two proteins, cI and Cro, as well as their oligomerization and subsequent binding affinities to three DNA sites. These processes in turn govern the rates at which RNAp transcribes the cI and Cro genes to produce more of their respective proteins. In this work, a dynamical model of the non-equilibrium statistical mechanics is revisited and generalized. The low number of proteins and other sources of noise are non-negligble and corrections to the kinetics are essential to understanding the stability. To this end, general integral forms for advection-diffusion equations have been developed and numerically solved for a variety of mutants and assumptions about the state of the cells. These solutions quantify the probabilistic and flux landscapes of the ensembles' evolution in concentration space and are used to predict the populations of the cell states, entropy production, passage times, and potential barriers of wild type and mutant bacteria to illuminate some structure of the configuration space from which Nature naturally selects.

Increase in susceptibility to EcoRII restriction of bacteriophage λ produced by propagation on host cells growing in 5‐azacytidine: a new in‐vivo method for demonstration of DNA‐methylation inhibition

Abstract: The efficiency of plating on EcoRII-restricting cells of bacteriophage lambda vir propagated on an Escherichia coli K-12 dcm+ host decreased with increase in concentration of 5-azacytidine (5-azaC) in the propagating medium. This illustrates, in-vivo, the inhibition of DNA-cytosine methylation induced by 5-azaC and provides a simple system for the detection of DNA-methylation inhibitors.

Reproducible gene targeting in recalcitrant Escherichia coli isolates

Abstract: A number of allele replacement methods can be used to mutate bacterial genes. For instance, the Red recombinase system of phage Lambda has been used very efficiently to inactivate chromosomal genes in E. coli K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical E. coli isolates. The procedure was modified by using longer homologous regions (85 bp and 500-600 bp), to inactivate genes in the uropathogenic E. coli strain UTI89. An lrhA regulator mutant, and deletions of the lac operon as well as the complete type 1 fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant E. coli, like the avian pathogenic E. coli strain APEC1. The lrhA regulator and lac operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic E. coli strains (APEC3E, APEC11A and APEC16A) it was very difficult or impossible to construct these mutants, with the original Red recombinase-based method, with a Red recombinase-based method using longer (85 bp) homologous regions or with our modified protocol, using 500 - 600 bp homologous regions. The method using 500-600 bp homologous regions can be used reliably in some clinical isolates, to delete single genes or entire operons by homologous recombination. However, it does not invariably show a greater efficiency in obtaining mutants, when compared to the original Red-mediated gene targeting method or to the gene targeting method with 85 bp homologous regions. Therefore the length of the homology regions is not the only limiting factor for the construction of mutants in these recalcitrant strains.

Solitons and Physics of the Lysogenic to Lytic Transition in Enterobacteria Lambda Phage

Abstract: The lambda phage is a paradigm temperate bacteriophage. Its lysogenic and lytic life cycles echo competition between the DNA binding CI and CRO proteins. Here we address the Physics of this transition in terms of an energy function that portrays the backbone as a multi-soliton configuration. The precision of the individual solitons far exceeds the B-factor accuracy of the experimentally determined protein conformations giving us confidence to conclude that three of the four loops are each composites of two closely located solitons. The only exception is the repressive DNA binding turn, it is the sole single soliton configuration of the backbone. When we compare the solitons with the Protein Data Bank we find that the one preceding the DNA recognition helix is unique to the CI protein, prompting us to conclude that the lysogenic to lytic transition is due to a saddle-node bifurcation involving a soliton-antisoliton annihilation that removes the first loop.