the structure of the excisionase (xis) protein from conjugative transposon tn916 provides insights into the regulation of heterobivalent tyrosine recombinases.heterobivalent tyrosine recombinases play a prominent role in numerous bacteriophage and transposon recombination systems. their enzymatic activities are frequently regulated at a structural level by excisionase factors, which alter the ability of the recombinase to assemble into higher-order recombinogenic nucleoprotein structures. the tn916 conjugative transposon spreads antibiotic resistance in pathogenic bacteria and is mobilized by a heterobivalent recombinase (tn916int), whose activity is ...200515733914
the e. coli nusa carboxy-terminal domains are structurally similar and show specific rnap- and lambdan interaction.the carboxy-terminal domain of the transcription factor escherichia coli nusa, nusactd, interacts with the protein n of bacteriophage lambda, lambdan, and the carboxyl terminus of the e. coli rna polymerase alpha subunit, alphactd. we solved the solution structure of the unbound nusactd with high-resolution nuclear magnetic resonance (nmr). additionally, we investigated the binding sites of lambdan and alphactd on nusactd using nmr titrations. the solution structure of nusactd shows two structur ...200515987884
functional analysis of the lysis genes of staphylococcus aureus phage p68 in escherichia coli.double-stranded dna phages of both gram-positive and gram-negative bacteria typically use a holin-endolysin system to achieve lysis of their host. in this study, the lysis genes of staphylococcus aureus phage p68 were characterized. p68 gene lys16 was shown to encode a cell-wall-degrading enzyme, which causes cell lysis when externally added to clinical isolates of s. aureus. another gene, hol15, was identified embedded in the -1 reading frame at the 3' end of lys16. the deduced hol15 protein ha ...200516000723
two-stage continuous operation of recombinant escherichia coli using the bacteriophage lambda q- vector.a two-stage continuous culture of escherichia coli in combination with a bacteriophage lambda system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. a phage lambda vector with a q(-) mutation was used to enhance the expression of the lambda system. the optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned ...200516096763
[red/et recombination and its biomedical applications].red/et recombination, a powerful homologous recombination system based on the red operon of lambda phage or rece/ rect from rac phage, provides an innovative approach for dna engineering. deletion, insertion and mutation can be quickly and precisely performed on the target gene mediated by red/et recombination with pcr derived dna fragments or oligonucleotides. this technical platform has extensive applications in biomedical field including bacterial artificial chromosome modification, gene knoc ...200516108384
the mode of replication is a major factor in segregational plasmid instability in lactococcus lactis.the effects of the rolling-circle and theta modes of replication on the maintenance of recombinant plasmids in lactococcus lactis were studied. heterologous escherichia coli or bacteriophage lambda dna fragments of various sizes were inserted into vectors based on either the rolling-circle-type plasmid pwv01 or the theta-type plasmid pambeta1. all pambeta1 derivatives were stably maintained. pwv01 derivatives, however, showed size-dependent segregational instability, in particular when large dna ...199316348863
gene-specific random mutagenesis of escherichia coli in vivo: isolation of temperature-sensitive mutations in the acyl carrier protein of fatty acid synthesis.acyl carrier proteins (acps) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. moreover, recent data indicate that the acyl carrier protein of escherichia coli has a large protein interaction network that extends beyond lipid synthesis. despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (acpp) that encodes acp have been isolated. we report the isolation of three such mutants by a new appro ...200616352845
display of aggregation-prone ligand binding domain of human ppar gamma on surface of bacteriophage display the aggregation-prone ligand binding domain (lbd) of the human peroxisome proliferator-activated receptor gamma (ppargamma) on the surface of bacteriophages to establish an easy screening assay for the identification of ppargamma ligands.200616364215
comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure.the antibacterial working range of six lysozymes was tested under ambient and high pressure, on a panel of five gram-positive (enterococcus faecalis, bacillus subtilis, listeria innocua, staphylococcus aureus and micrococcus lysodeikticus) and five gram-negative bacteria (yersinia enterocolitica, shigella flexneri, escherichia coli o157:h7, pseudomonas aeruginosa and salmonella typhimurium). the lysozymes included two that are commercially available (hen egg white lysozyme or hewl, and mutanolys ...200616487612
a novel method for evaluating free radical scavenging abilities of antioxidants using ultraviolet induction of bacteriophage lambda.a novel biological method used to evaluate free radical scavenging abilities of antioxidants using ultraviolet (uv) induction of bacteriophage lambda in lysogenic escherichia coli kappa12 (lambda+) has been developed. this method is based on the induction of bacteriophage lambda from lysogenic cycle to lytic cycle by ultraviolet irradiation, and formation of free radicals during the course of induction. in the experiments, 10(8)cells/ml and 30s (39j/m2) were determined as the cell density of the ...200616574238
cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts.we have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and micrococcus lysodeikticus with chloroform/tris-hcl buffer (chloroform/buffer). the lysozymes included two that are commercially available (hen egg white lysozyme or hewl, and mutanolysin from streptomyces globisporus or m1l), and four that were chromatographically purified (bacteriophage lambda lysozyme or lal, bacteriophage t4 lysozyme or ...200616684100
overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.during the proteomics period, the growth in the use of recombinant proteins has increased greatly in the recent years. bacterial systems remain most attractive due to low cost, high productivity, and rapid use. however, the rational choice of the adequate promoter system and host for a specific protein of interest remains difficult. this review gives an overview of the most commonly used systems: as hosts, bacillus brevis, bacillus megaterium, bacillus subtilis, caulobacter crescentus, other str ...200616791589
[bacteriophage lambda dna replication--new discoveries made using an old experimental model].bacteriophage lamda is a model in molecular biology studies since over fifty years. nevertheless, studies of recent years (similarly to previous time periods) resulted in many new experimental results which not only expanded our knowledge on molecular mechanisms of functions of this virus, but also shed new light on general rules of the transduction and transfer of genetic information. in this review, we present recent achievements of studies on mechanisms of regulation of bacteriophage lamda dn ...200616869300
stimulation of the lambda pr promoter by escherichia coli seqa protein requires downstream gatc sequences and involves late stages of transcription initiation.escherichia coli seqa protein is a major negative regulator of chromosomal dna replication acting by sequestration, and thus inactivation, of newly formed oric regions. however, other activities of this protein have been discovered recently, one of which is regulation of transcription. seqa has been demonstrated to be a specific transcription factor acting at bacteriophage lambda promoters p(i), p(aq) and p(r). while seqa-mediated stimulation of p(i) and p(aq) occurs by facilitating functions of ...200617005979
rna-mediated destabilization of the sigma(70) region 4/beta flap interaction facilitates engagement of rna polymerase by the q antiterminator.the bacterial rna polymerase (rnap) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. during early elongation, the stability of interactions between sigma(70) (the primary sigma factor in escherichia coli) and core decreases due to an ordered displacement of segments of sigma(70) from core triggered by growth of the nascent rna. here we demonstrate that the nascent rna-mediated de ...200617081994
fis targets assembly of the xis nucleoprotein filament to promote excisive recombination by phage lambda.the phage-encoded xis protein is the major determinant controlling the direction of recombination in phage lambda. xis is a winged-helix dna binding protein that cooperatively binds to the attr recombination site to generate a curved microfilament, which promotes assembly of the excisive intasome but inhibits formation of an integrative intasome. we find that lambda synthesizes surprisingly high levels of xis immediately upon prophage induction when excision rates are maximal. however, because o ...200717275024
structure of the cooperative xis-dna complex reveals a micronucleoprotein filament that regulates phage lambda intasome assembly.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 10(6)-fold, while simultaneously inhibiting phage reintegration. control is exerted by cooperatively assembling onto a approximately 35-bp dna regulatory element, which it distorts to preferentially stabilize an ex ...200717287355
switch from theta to sigma replication of bacteriophage lambda 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 ...200717377819
multicopy plasmid modification with phage lambda red recombineering.recombineering, in vivo genetic engineering using the bacteriophage lambda red generalized recombination system, was used to create various modifications of a multicopy plasmid derived from pbr322. all genetic modifications possible on the escherichia coli chromosome and on bacterial artificial chromosomes (bacs) are also possible on multicopy plasmids and are obtained with similar frequencies to their chromosomal counterparts, including creation of point mutations (5-10% unselected frequency), ...200717434584
on kinetics of phage adsorption.adsorption of lambda-phage on sensitive bacteria escherichia coli is a classical problem but not all issues have been resolved. one of the outstanding problems is the rate of adsorption, which in some cases appears to exceed the theoretical limit imposed by the law of random diffusion. we revisit this problem by conducting experiments along with new theoretical analyses. our measurements show that upon incubating lambda-phage with bacteria ymel, the population of unbound phage in a salt buffer d ...200717434950
modelling the stability of stx lysogens.shiga-toxin-converting bacteriophages (stx phages) are temperate phages of escherichia coli, and can cause severe human disease. the spread of shiga toxins by stx phages is directly linked to lysogen stability because toxins are only synthesized and released once the lytic cycle is initiated. lysogens of stx phages are known to be less stable than those of the related lambda phage; this is often described in terms of a 'hair-trigger' molecular switch from lysogeny to lysis. we have developed a m ...200717604057
probing the antiprotease activity of lambdaciii, an inhibitor of the escherichia coli metalloprotease hflb (ftsh).the ciii protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitous escherichia coli metalloprotease hflb (ftsh). this inhibition results in the stabilization of transcription factor lambdacii, thereby helping the phage to lysogenize the host bacterium. lambdaciii, a small (54-residue) protein of unknown structure, also protects sigma(32), another specific substrate of hflb. in order to understand the details of the inhibitory mechanism of ciii, we cloned and expres ...200717890311
chromosomal model for analysis of a long ctg/cag tract stability in wild-type escherichia coli and its nucleotide excision repair mutants.many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy, and friedreich's ataxia, are associated with expansions of the triplet repeat sequences (trs) (cgg/ccg, ctg/cag, and gaa/ttc) within or near specific genes. mechanisms that mediate mutations of trs include dna replication, repair, and gene conversion and (or) recombination. the involvement of the repair systems in trs instability was investigated in escherichia coli on plasmid models, and the results s ...200717898841
development of a bacterial cloning vector for expression of scorpion toxins for biotechnological studies.scorpion venoms contain toxic peptides that recognize k(+) channels of excitable and non-excitable cells. these toxins comprise three structurally distinct groups designated alpha-ktx, beta-ktx, and gamma-ktx. it is highly desirable to develop systems for the expression of these toxins for further physiological and structural studies. in this work, an expression vector (ptev3) was constructed by inserting protein d (major capsid of phage lambda) and tev protease recognition site into plasmid pet ...200817904381
removal of deoxyinosine from the escherichia coli chromosome as studied by oligonucleotide transformation.deoxyinosine (di) is produced in dna by the hydrolytic or nitrosative deamination of deoxyadenosine. it is excised in a repair pathway that is initiated by endonuclease v, the product of the nfi gene. the repair was studied in vivo using high-efficiency oligonucleotide transformation mediated by the beta protein of bacteriophage lambda in a mismatch repair-deficient host. escherichia coli was transformed with oligonucleotides containing a selectable a-g base substitution mutation. when the mutag ...200817981100
expression using vectors with phage lambda regulatory the expression system described here, plasmids (pskf) utilize regulatory signals--such as the powerful promoter pl--from the bacteriophage lambda. transcription from pl can be fully repressed and plasmids containing it are thus stabilized by the lambda repressor, ci. the repressor is supplied by an e. coli host which contains a integrated copy of a portion of the lambda genome. this so-called defective lysogen supplies the lambda regulatory proteins ci and n but does not provide the lytic com ...200118265129
a biotin interference assay highlights two different asymmetric interaction profiles for lambda integrase arm-type binding sites in integrative versus excisive recombination.the site-specific recombinase integrase encoded by bacteriophage lambda promotes integration and excision of the viral chromosome into and out of its escherichia coli host chromosome through a holliday junction recombination intermediate. this intermediate contains an integrase tetramer bound via its catalytic carboxyl-terminal domains to the four "core-type" sites of the holliday junction dna and via its amino-terminal domains to distal "arm-type" sites. the two classes of integrase binding sit ...200818319248
identification and functional analysis of the rz/rz1-like accessory lysis genes in the membrane-containing bacteriophage prd1.bacteriophage prd1 is a tailless membrane-containing double-stranded (ds) dna virus infecting a variety of gram-negative bacteria. in order to affect cell lysis, like most dsdna phages, prd1 uses the holin-endolysin system. in this study, we identified two accessory lysis genes, xxxvi and xxxvii, coding for proteins p36 and p37, respectively. using genetic complementation assays, we show that protein pair p36/p37 is a functional and interchangeable analogue of the rz/rz1 of bacteriophage lambda. ...200818366440
analysis of some phenotypic traits of feces-borne temperate lambdoid bacteriophages from different immunity groups: a high incidence of cor+, fhua-dependent phages.a group of previously isolated heterogeneous mep lambdoid phages (43) from 19 different immunity groups for phage infection was further characterized to gain insight into some phenotypic traits and to assess their relationship with phage lambda. interestingly, the fhua host receptor was required by the majority of mep phages (37 out of 43; approximately 85%). the cor gene, which has been reported to be involved in fhua-dependent exclusion of lambdoid phages, was also found in most of the fhua-de ...200818516490
characterization and validation of a bioluminescent bioreporter for the direct detection of escherichia coli.a two-component bacteriophage-based bioluminescent reporter system was developed for the detection of escherichia coli in environmental samples. the bioreporter system consists of a luxi integrated lambda bacteriophage and a lux-based bioluminescent reporter cell that responds to the infection event through acyl-homoserine lactone (ahl) mediated quorum sensing and bioluminescent signal stimulation. this work addresses the ability of the bioreporter system to detect and quantify the target pathog ...200818593593
controlled synthesis of the coat protein of satellite tobacco necrosis virus in escherichia coli.chimeric plasmids were constructed such that the cloned complete satellite tobacco necrosis virus (stnv) rna information came under transcriptional control of the leftward promoter (pl) of bacteriophage lambda. the promoter is fully repressed at low temperatures (28 degrees) by the thermolabile repressor product of the lambdaci857 gene, present in the bacterium on a deficient prophage or as part of another plasmid. synthesis of the stnv coat protein in escherichia coli could be initiated by heat ...198418639819
importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class a penicillin-binding protein 1b of escherichia coli.the peptidoglycan glycosyltransferase (gt) module of class a penicillin-binding proteins (pbps) and monofunctional gts catalyze glycan chain elongation of the bacterial cell wall. these enzymes belong to the gt51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lambda lysozyme. in this work, we have systematically modified all the conserved amino acid residues of the gt module of escherichia coli class a pbp1b by site-directed mutagenesis and deter ...200818701463
rapid allelic exchange in enterohemorrhagic escherichia coli (ehec) and other e. coli using lambda red recombination.this unit describes an allelic exchange system for enterohemorrhagic e. coli (ehec), and similar pathogenic species of bacteria. the phage lambda red recombination system is expressed from a plasmid, inducing a hyper-recombinogenic state where electroporated pcr-generated substrates recombine with the bacterial chromosome at high efficiency. the technique can be used to substitute a drug marker for the gene of interest, or used to generate a clean in-frame deletion of the target gene. single gen ...200618770591
cleavage of bacteriophage lambda ci repressor involves the reca c-terminal domain.the sos response to dna damage in escherichia coli involves at least 43 genes, all under the control of the lexa repressor. activation of these genes occurs when the lexa repressor cleaves itself, a reaction catalyzed by an active, extended reca filament formed on dna. it has been shown that the lexa repressor binds within the deep groove of this nucleoprotein filament, and presumably, cleavage occurs in this groove. bacteriophages, such as lambda, have repressors (ci) that are structural homolo ...200919013467
production of double-stranded rna for interference with tmv infection utilizing a bacterial prokaryotic expression many species, the introduction of double-stranded rna (dsrna) induces potent and specific gene silencing, a phenomenon called rna interference (rnai). rnai is the process of sequence-specific, posttranscriptional gene silencing (ptgs) in animals and plants, mediated by dsrna homologous to the silenced genes. in plants, ptgs is part of a defense mechanism against virus infection, and dsrna is the pivotal factor that induces gene silencing. here, we report an efficient method that can produce d ...200919330324
sequence-specific recognition of dna by the c-terminal domain of nucleoid-associated protein h-ns.the molecular determinants necessary and sufficient for recognition of its specific dna target are contained in the c-terminal domain (h-nsctd) of nucleoid-associated protein h-ns. h-nsctd protects from dnasei cleavage a few short dna segments of the h-ns-sensitive hns promoter whose sequences closely match the recently identified h-ns consensus motif (tcg(t/a)t(a/t)aatt) and, alone or fused to the protein oligomerization domain of phage lambda ci repressor, inhibits transcription from the hns p ...200919740756
high adsorption rate is detrimental to bacteriophage fitness in a biofilm-like environment.bacterial 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 lambda 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 p ...200919804637
fine tuning of the e. coli nusb:nuse complex affinity to boxa rna is required for processive antitermination.phage lambda propagation in escherichia coli host cells requires transcription antitermination on the lambda chromosome mediated by lambdan protein and four host nus factors, nusa, b, e (ribosomal s10) and g. interaction of e. coli nusb:nuse heterodimer with the single stranded boxa motif of lambdanutl or lambdanutr rna is crucial for this reaction. similarly, binding of nusb:nuse to a boxa motif is essential to suppress transcription termination in the ribosomal rna (rrn) operons. we used fluor ...201019854945
tuning a genetic switch: experimental evolution and natural variation of prophage induction.genetic switches allow organisms to modulate their phenotype in response to environmental changes. understanding the evolutionary processes by which switches are tuned is central to understanding how phenotypic variation is realized. prophage induction by phage lambda is the classic example of a genetic switch and allows lambda to move between two different modes of transmission: as a lysogen it reproduces vertically as a component of the host genome; as a free phage it reproduces horizontally b ...201019891623
dna heats up: energetics of genome ejection from phage revealed by isothermal titration calorimetry.most bacteriophages are known to inject their double-stranded dna into bacteria upon receptor binding in an essentially spontaneous way. this downhill thermodynamic process from the intact virion to the empty viral capsid plus released dna is made possible by the energy stored during active packaging of the genome into the capsid. only indirect measurements of this energy have been available until now, using either single-molecule or osmotic suppression techniques. in this work, we describe for ...201019969001
escherichia coli mw005: lambda red-mediated recombineering and copy-number induction of oriv-equipped constructs in a single host.escherichia coli strain el350 contains chromosomally integrated phage lambda red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. bac and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the dna is difficult to isolate in high yield and purity. to overcome this limitation vectors, e.g. pcc1fos, have been constructed that contain the additional replica ...201020350301
a forward-genetic screen and dynamic analysis of lambda phage host-dependencies reveals an extensive interaction network and a new anti-viral strategy.latently infecting viruses are an important class of virus that plays a key role in viral evolution and human health. here we report a genome-scale forward-genetics screen for host-dependencies of the latently-infecting bacteriophage lambda. this screen identified 57 escherichia coli (e. coli) genes--over half of which have not been previously associated with infection--that when knocked out inhibited lambda phage's ability to replicate. our results demonstrate a highly integrated network betwee ...201020628568
reca-independent single-stranded dna oligonucleotide-mediated mutagenesis.the expression of beta, the single-stranded annealing protein (ssap) of bacteriophage lambda in escherichia coli promotes high levels of oligonucleotide (oligo)-mediated mutagenesis and offers a quick way to create single or multiple base pair insertions, deletions, or substitutions in the bacterial chromosome. high rates of mutagenesis can be obtained by the use of mismatch repair (mmr)-resistant mismatches or mmr-deficient hosts, which allow for the isolation of unselected mutations. it has re ...201020711416
role of dlp12 lysis genes in escherichia coli biofilm formation.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/rz(1)). the role that these dlp12 lysis genes play in biofilm formation was examined in deletion mutants of e. coli phl628, a curli-o ...201121415116
compromised factor-dependent transcription termination in a nusa mutant of escherichia coli : spectrum of termination efficiencies generated by perturbations of rho, nusg, nusa, and the h-ns family of proteins.the proteins nusa and nusg, which are essential for viability of wild-type escherichia coli, participate in various post-initiation steps of transcription including elongation, antitermination, and termination. nusg is required along with the essential rho protein for factor-dependent transcription termination (also referred to as polarity), but the role of nusa is less clear with conflicting reports that it both promotes and inhibits the process. in this study, we have found that a recessive mi ...201121602355
developing an extended genomic engineering approach based on recombineering to knock-in heterologous genes to escherichia coli genome.most existing genomic engineering protocols for manipulation of escherichia coli are primarily focused on chromosomal gene knockout. in this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage ++ (++ red) and flippase-flippase recognition targets (flp-frt) recombinations. for demonstration purposes, dna operons containing heterologous genes (i.e., pac encoding e. coli penicillin acylase and palb2 encoding pseud ...201121826554
energy-independent helicase activity of a viral genome packaging motor.the assembly of complex double-stranded dna viruses includes a genome packaging step where viral dna is translocated into the confines of a preformed procapsid shell. in most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (dna maturation) and translocation of the duplex into the capsid (dna packaging). bacteriophage λ terminase site-specific ...201122191393
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