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PubMed1988

Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties.

A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coli a phagemid, pBluescript SK(-), contained within the vector, can be excised by f1 or M13 helper phage. The excision process eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation. This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the f1 bacteriophage origin of replication (1). Six of 21 restriction sites in the excised pBluescript SK polylinker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated and analyzed. The lambda ZAP vector was tested by the preparation of a chicken liver cDNA library and the isolation of actin clones by screening with oligonucleotide probes. Putative actin clones were excised from the lambda vector and identified by DNA sequencing. The ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgG anti-glucocerbrosidase antibodies.

Actins↗
PubMed1974

In vitro genetic recombination of bacteriophage lambda.

DNA of bacteriophage lambda recombines in a cell-free extract prepared from an induced Escherichia coli lysogen of bacteriophage lambda. The assay for recombination in vitro takes advantage of the ability of such an extract to package lambda DNA and to assemble complete phage particles. For example, when lambda DNA that has been extracted from phage with the immunity of 434 is added to an extract, infectious lambda imm 434 particles are produced. The precursor DNA molecule in this packaging reaction is a multichromosomal polymer; circular monomers, for example, are not packaged.Nevertheless, when 434 circular DNA monomers are added to an extract, some phage that contain the imm 434 marker are produced. In this case, the circular DNA had recombined with lambda DNA in the extract and thereby had become part of a polymeric structure, which by the normal packaging process could give rise to infectious particles with the imm 434 marker. Genetic recombination is demonstrated when imm 434 circular monomer DNA carries amber mutations in genes A and B; then most of the 434 plaque formers produced in vitro are A(+)B(+), the genotype of the endogenous lambda DNA. Genetic crossing-over occurs through a region that contains the prophage attachment site, suggesting that recombination is carried out by the lambda Int functions. The 434 recombinant plaque formers are particles physically identical to wild-type 434 particles, as judged by their buoyant density in a CsCl equilibrium gradient.

Cell-Free System↗
PubMed1972

Large-scale production of lambda bacteriophage and purified lambda deoxyribonucleic acid.

Large amounts of a heat-inducible phage lambda mutant (lambdaCI857) may be obtained under standardized conditions. The phage is harvested by simple polyethylene glycol (C 6000) precipitation and purified by CsCl density gradient banding. Deoxyribonucleic acid (DNA) is extracted by cold phenol and purified by sucrose density gradient sedimentation to yield a homogenous population of unbroken lambda DNA molecules.

Cell Count↗
PubMed1974

Preparation of plasmid lambda dv from bacteriophage lambda: role of promoter-operator in the plasmid replicon.

A technique has been described for selection of bacteria carrying plasmid lambdadv. With this technique, the effects of mutations in the promoter-operators were compared on the production and perpetuation of the plasmid. It was found that "left" promoter-operator that controls leftward gene expressions can be deleted from the plasmid genome. Some mutations of "right" promoter-operator (pRoR) that controls expression of genes tof, O, and P affect the stability of the plasmid. However, the plasmid genome accomodates a variety of pRoR mutations within a reasonable but different degree of constitutivity. Some new promoter mutations that allow bypass of the pRoR cannot be carried in the plasmid genome. From these findings it was proposed that the plasmid replicon has one indispensable promoter-operator that controls expression of all the genes related to its own replication, although a variety of constitutive mutations can be accommodated in the pRorR.

Coliphages↗
PubMed1978

Correlation between UV dose requirement for lambda bacteriophage induction and lambda repressor concentration.

Escherichia coli K-12 wild type and a uvrA mutant derivative were used to construct isogenic strains bearing one, two, three, or more phage lambda cI genomes and containing increasing concentration of lambda repressor as measured by in vitro operator DNA-binding assays. The survival and phage induction in response to UV irradiation were determined. In both strains, dose-response relationships were obtained as a function of the cellular repressor concentration. The uvrA lysogens required one-tenth the UV fluence of the wild-type counterparts for induction. Lysogenic strains containing plasmids that overproduce the lambdaind+ repressor and the same lysogens with plasmids overproducing the lambdaind- repressor displayed the same survival curves as the nonlysogenic parental strain; however, only the former produced infectious centers (at a frequency of 2 x 10(-3) to 5 x 10(-4) in response to radiation.

Coliphages↗
PubMed1988

Fluorescence measurement of the kinetics of DNA injection by bacteriophage lambda into liposomes.

Bacteriophage lambda attaches to Gram-negative bacteria using the outer membrane protein LamB as its receptor. Subsequently, DNA is injected by the bacteriophage into the host cell for replication and expression. The mechanism of DNA injection, however, is poorly understood. In order to begin to characterize DNA injection, a quantitative kinetic assay to detect injection into reconstituted LamB liposomes is described. The technique involves monitoring the increase in fluorescence of liposome-encapsulated ethidium bromide, which occurs as DNA enters the aqueous compartment of the vesicles. The data indicate that injection is several times faster than indicated by earlier studies and is complete within 1 min. Such assays which allow direct observation of this process are necessary first steps toward a mechanistic understanding.

Bacterial Outer Membrane Proteins↗
PubMed2004

Morphing molecular specificities between Arm-peptide and NUT-RNA in the antitermination complexes of bacteriophages lambda and P22.

Bacteriophage lambda's N-protein includes a 17-amino-acid segment, Arm, rich in arginine and having specific affinity for a 15-nucleotide RNA stem-loop called BOX-B. Parallel but different Arm/BOX-B sequences in lambda's cousin, phage P22, account for some of the type specificity that distinguishes lambda from P22: the N of each works only with its cognate BOX-B in vivo. We find that the specificity of N(lambda) can be shifted gradually to that of N(22) by substituting sets of particular amino acids from Arm(22) into Arm of N(lambda). The determinative amino acids are generally those shown by nuclear magnetic resonance to contact BOX-B RNA; gain or loss of these contact amino acids is reasonably expected to contribute to the affinity of each amino acid sequence. Intermediate sequences may show no function with either BOX-B, or weak function with both BOX-B(lambda) and BOX-B(22), the latter suggesting possible evolutionary paths for specificity shifts.

Amino Acid Sequence↗
PubMed1991

Bacteriophage lambda P gene shows host killing which is not dependent on lambda DNA replication.

Bacteriophage lambda, having a mutation replacing glycine by glutamic acid at the 48th codon of cro, kills the host under N- conditions; we call this the hk mutation. In lambda N-N-cl-hk phage-infected bacteria, the late gene R is expressed to a significant level, phage DNA synthesis occurs with better efficiency, and the Cro activity is around 20% less, all compared to those in lambda N-N-cl-hk(+)-infected bacteria. Segments of lambda DNA from the left of pR to the right of tR2, carrying cro, cII, O, P, and the genes of the nin5 region from the above hk and hk+ phages, were cloned in pBR322. Studies with these plasmids and their derivatives having one or more of the lambda genes deleted indicate that the hk mutation is lethal only when a functional P gene is also present. When expression of P from pR is elevated, due to the deletion of tR1, host killing also occurs without the hk mutation. We conclude that the higher levels of P protein, produced either (1) when cro has the hk mutation or (2) when tR1 is deleted, are lethal to the host. We also show that due to the hk mutation, the Cro protein becomes partially defective in its negative regulation at pR, resulting in the expression of P to a lethal level even in the absence of N protein-mediated antitermination. This P protein-induced host killing depends neither on lambda DNA replication nor on any other gene functions of the phage.

Amino Acid Sequence↗
PubMed1997

Mutational analysis of protein binding sites involved in formation of the bacteriophage lambda attL complex.

Bacteriophage lambda site-specific recombination requires the formation of higher-order protein-DNA complexes to accomplish synapsis of the partner attachment (att) sites as well as for the regulation of the integration and excision reactions. The att sites are composed of a core region, the actual site of strand exchange, and flanking arm regions. The attL site consists of two core sites (C and C'), an integration host factor (IHF) binding site (H'), and three contiguous Int binding arm sites (P'1, P'2, and P'3). In this study, we employed bacteriophage P22 challenge phages to determine which protein binding sites participate in attL complex formation in vivo. The C', H', and P'1 sites were critical, because mutations in these sites severely disrupted formation of the attL complex. Mutations in the C and P'2 sites were less severe, and alteration of the P'3 site had no effect on complex formation. These results support a model in which IHF, bound to the H' site, bends the attL DNA so that the Int molecule bound to P'1 also interacts with the C' core site. This bridged complex, along with a second Int molecule bound to P'2, helps to stabilize the interaction of a third Int with the C core site. The results also indicate that nonspecific DNA binding is a significant component of the Int-core interactions and that the cooperativity of Int binding can overcome the effects of mutations in the individual arm sites and core sites.

Attachment Sites, Microbiological↗
PubMed1994

Mapping the functional domains of bacteriophage lambda integrase protein.

Bacteriophage lambda encodes a site-specific recombination system that promotes the movement of the phage genome into and out of the host bacterial chromosome. The phage-encoded integrase (Int) is composed of 356 amino acid residues and carries out the required strand exchanges by means of a type I topoisomerase activity. Int also contains two distinct DNA-binding domains that interact with two different, specific sequences (arm-type and core-type sites) on DNA. In order to help understand the mechanism of site-specific recombination, we have used a genetic approach to isolate mutants defective in different steps in the recombination reaction. We developed a genetic screen for Int mutants that are defective in catalyzing excisive recombination in vivo. These mutants were screened for proficiency in binding to the P'123 arm-type sites using the bacteriophage P22 challenge-phage assays. In all, 78 such mutants were isolated and the mutational changes mapped and sequenced. These mutants have been further characterized (1) for their ability to bind the P'1 and P'123 arm-type sites and for their ability to form the attL complex in vivo, (2) for negative dominance in vitro, (3) for the presence of type I topoisomerase activity, and (4) for the ability to resolve artificially constructed recombination intermediates. We found that (1) residues in a stretch of 88 amino acids in the middle of the protein may be involved in Int-Int interactions, (2) a region around Arg212 is involved in the catalytic site, (3) residues near the carboxyl terminus play a role in enhancing Int binding to its arm-type sites, possibly by interacting with the small amino-terminal region that has been shown to be responsible for specific recognition of the arm-type sites, and (4) residues at the very carboxyl end of the protein may be involved in modulating the cleavage or religation activities of the Int protein.

Bacteriophage lambda↗
PubMed2001

Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging.

Bacteriophage lambda is a double-stranded DNA virus that processes concatemeric DNA into virion chromosomes by cutting at specific recognition sites termed cos. A cos is composed of three subsites: cosN, the nicking site; cosB, required for packaging initiation; and cosQ, required for termination of chromosome packaging. During packaging termination, nicking of the bottom strand of cosN depends on cosQ, suggesting that cosQ is needed to deliver terminase to the bottom strand of cosN to carry out nicking. In the present work, saturation mutagenesis showed that a 7-bp segment comprises cosQ. A proposal that cosQ function requires an optimal sequence match between cosQ and cosNR, the right cosN half-site, was tested by constructing double cosQ mutants; the behavior of the double mutants was inconsistent with the proposal. Substitutions in the 17-bp region between cosQ and cosN resulted in no major defects in chromosome packaging. Insertional mutagenesis indicated that proper spacing between cosQ and cosN is required. The lethality of integral helical insertions eliminated a model in which DNA looping enables cosQ to deliver a gpA protomer for nicking at cosN. The 7 bp of cosQ coincide exactly with the recognition sequence for the Escherichia coli restriction endonuclease, EcoO109I.

Bacteriophage lambda↗
PubMed1998

The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein.

Bacteriophage lambda is not able to lysogenise the Escherichia coli rpoA341 mutant. This mutation causes a single amino acid substitution Lys271Glu in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Our previous studies indicated that the impaired lysogenisation of the rpoA341 host is due to a defect in transcriptional activation by the phage CII protein and suggested a role for alphaCTD in this process. Here we used a series of truncation and point mutants in the rpoA gene placed on a plasmid to investigate the process of transcriptional activation by the cII gene product. Our results indicate that amino-acid residues 265, 268 and 271 in the a subunit may play an important role in the CII-mediated activation of the pE promoter (most probably residue 271) or may be involved in putative interactions between alphaCTD and an UP-like element near pE (most probably residues 265 and 268). Measurement of the activity of pE-lacZ, pI-lacZ and p(aQ)-lacZ fusions in the rpoA+ and rpoA341 hosts demonstrated that the mechanism of activation of these CII-dependent promoters may be in each case different.

Artificial Gene Fusion↗
PubMed2002

Mn2+ is a native metal ion activator for bacteriophage lambda protein phosphatase.

Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large family of metal-containing phosphoesterases, including purple acid phosphatase, protein serine/threonine phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. lambdaPP can be activated several-fold by various divalent metal ions, with Mn(2+) and Ni(2+) providing the most significant activation. Despite the extensive characterization of purified lambdaPP in vitro, little is known about the identity and stoichiometry of metal ions used by lambdaPP in vivo. In this report, we describe the use of metal analysis, activity measurements, and whole cell EPR spectroscopy to investigate in vivo metal binding and activation of lambdaPP. Escherichia coli cells overexpressing lambdaPP show a 22.5-fold increase in intracellular Mn concentration and less dramatic changes in the intracellular concentration of other biologically relevant metal ions compared to control cells that do not express lambdaPP. Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing lambdaPP, indicating the presence of metal-activated enzyme in cell lysate. EPR spectra of intact cells overexpressing lambdaPP exhibit resonances previously attributed to mononuclear Mn(2+) and dinuclear [(Mn(2+))(2)] species bound to lambdaPP. Spin quantitation of EPR spectra of intact E. coli cells overexpressing lambdaPP indicates the presence of approximately 40 microM mononuclear Mn(2+)-lambdaPP and 60 microM [(Mn(2+))(2)]-lambdaPP. The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn(2+), and dinuclear-[(Mn(2+))(2)] metalloisoforms and that Mn(2+) is a physiologically relevant activating metal ion in E. coli.

Bacteriophage lambda↗
PubMed1997

Genetic analysis of second-site revertants of bacteriophage lambda integrase mutants.

Bacteriophage lambda site-specific recombination is catalyzed by the phage-encoded integrase (Int) protein. Using a collection of 21 recombination-defective Int mutants, we performed a second-site reversion analysis. One of the primary mutants contained a valine-to-glutamic acid change at position 175 (V175E), and a pseudorevertant with a lysine change at this site (V175K) was also isolated. Relative to the wild-type protein, the V175E protein was defective in its ability to form the attL complex and to catalyze excision in vivo and in vitro. A mutant containing an alanine substitution (V175A) was made by site-directed mutagenesis, and it was more efficient than the V175K protein in forming the attL complex and promoting excision. These results indicate that a nonpolar side chain at residue 175 is required for function. The second primary mutant contained a proline-to-leucine change at position 243 (P243L). A true second-site revertant was isolated that contained a glutamic acid-to-lysine change (E218K). The P243L-E218K protein promoted recombination and bound arm-type sites more efficiently than the original P243L protein but not as efficiently as the protein containing the E218K substitution alone. The E218K substitution also restored activity to a mutant with a threonine-to-isoleucine substitution at position 270 (T270I). This result showed that suppression by the E218K change is not allele specific and suggests that the substitution improves an inherent activity of Int rather than directly compensating for the defect caused by the primary substitutions. Results with challenge phages carrying attL sites with altered core sites indicate that the E218K change may improve binding to the core site.

Bacteriophage lambda↗
PubMed2004

Electrochemical studies of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of bacteriophage lambda protein phosphatase.

Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large superfamily of metallophosphoesterases, including serine/threonine protein phosphatases, purple acid phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. Members of this family share several common characteristics, including a common phosphoesterase motif, secondary structural fold (betaalphabetaalphabeta), and metal ligand environment, and often accommodate a dinuclear metal center. The identity of the active site metals often differs between family members. Despite the extensive spectroscopic studies of several family members, only the standard redox potential of porcine purple acid phosphate (PAP) has been measured. In this report, we investigate the redox properties of another member of this protein family. The standard redox potentials of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of lambdaPP were determined from anaerobic redox titration experiments. Two different S = 5/2, mono-Fe3+ lambdaPP species were identified: the first with an E/D approximately 0.17, g = 8.9 and 4.8, and an Eo' approximately +130 mV; the second with E/D approximately 0.05, g = 6.7, 5.9, and 4.4, and an Eo' approximately +120 mV. The first and second mono-Fe3+ species are thought to represent Fe present in the M2 and M1 sites, respectively. The addition of Zn2+ to mono-Fe3+ lambdaPP results in a decrease in both mono-Fe3+ species and the appearance of a new S = 5/2, Fe(3+)-Zn2+ species (E/D approximately 0.02, g = 5.9, and an Eo' > +175 mV). The Fe-Fe lambdaPP titration revealed an S = 1/2, Fe(3+)-Fe2+ (g < 2) species with an Eo' > +128 mV. These results suggest that the active site of lambdaPP supports a high oxidation potential for both metal sites and may indicate an equally oxidizing active site for other member metallophosphoesterases.

Acid Phosphatase↗
PubMed2003

High-density functional display of proteins on bacteriophage lambda.

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display. This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.

Bacteriophage M13↗
PubMedDate not supplied

[Screening substances derived from cultures of medicinal plants for antimutagenic activity in the Escherichia coli-bacteriophage lambda system].

In Escherichia coli--bacteriophage lambda system protective properties of the extracts derived from the biomass of cultured Panax ginseng, Polyscias filicifolia, Rhodiola rosea, Ungernia victoris cells, and those from intact Rhodiola roots have been studied. Escherichia coli--bacteriophage lambda system responsiveness was found to vary with the test-object state, namely: the deleted bacteriophage form (lambda-4) as well as previously mutagenized bacteriophage were more sensitive to the mutagenic and antimutagenic influence versus the native bacteriophage lambda +. The contribution of extracts in the induction and realization of the lethal injuries in phages caused by nitrite acid in extracellular phage (conditions in vitro) was estimated thus enabling to discriminate between the protective and antimutagenic extract activities. Protective extract effect in the given test-system appeared to be higher their antimutagenic action. With the most responsive bacteriophage variant the extracts from the biomass of cultured Rh. rosea and P. filicifolia cells showed high protective and somewhat lower antimutagenic activities. With other phages significant antimutagenic potential of extracts was demonstrated, which by their protective effect could be arranged in a raw as follows U. victoris > P. ginseng > P. filicifolia. The primary screening for the antimutagenic effect of preparations in the prokaryotic systems could be reduced to the investigation of their effects on the object inactivation exposed to the mutagen in vitro.

Antimutagenic Agents↗