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PubMed1986

Bacteriophage lambda int protein may recognize structural features of the attachment sites.

The bacteriophage lambda int protein binds to and promotes polynucleotide strand exchange within specific DNA segments called attachment sites. Previous work strongly suggests that the specificity of int protein action is based, at least in part, on its ability to recognize nucleotide sequences in the attachment sites. We suggest that int protein also recognizes structural features of the attachment sites such as the twist and roll angles between adjacent base pairs. This proposal is based on statistical analysis of the predicted twist and roll angles of a large collection of secondary attachment sites. The analysis shows that the oscillation patterns of these parameters are conserved in regions where int proteins binds.

Attachment Sites, Microbiological↗
PubMed1997

Functional properties of replication fork assemblies established by the bacteriophage lambda O and P replication proteins.

We have used a set of bacteriophage lambda and Escherichia coli replication proteins to establish rolling circle DNA replication in vitro to permit characterization of the functional properties of lambda replication forks. We demonstrate that the lambda replication fork assembly synthesizes leading strand DNA chains at a physiological rate of 650-750 nucleotides/s at 30 degrees C. This rate is identical to the fork movement rate we obtained using a minimal protein system, composed solely of E. coli DnaB helicase and DNA polymerase III holoenzyme. Our data are consistent with the conclusion that these two key bacterial replication proteins constitute the basic functional unit of a lambda replication fork. A comparison of rolling circle DNA replication in the minimal and lambda replication systems indicated that DNA synthesis proceeded for more extensive periods in the lambda system and produced longer DNA chains, which averaged nearly 200 kilobases in length. The higher potency of the lambda replication system is believed to result from its capacity to mediate efficient reloading of DnaB helicase onto rolling circle replication products, thereby permitting reinitiation of DNA chain elongation following spontaneous termination events. E. coli single-stranded DNA-binding protein and primase individually stimulated rolling circle DNA replication, but they apparently act indirectly by blocking accumulation of inhibitory free single-stranded DNA product. Finally, in the course of this work, we discovered that E. coli DNA polymerase III holoenzyme is itself capable of carrying out significant strand displacement DNA synthesis at about 50 nucleotides/s when it is supplemented with E. coli single-stranded DNA-binding protein.

Bacterial Proteins↗
PubMed1983

Early intermediates in bacteriophage lambda prohead assembly. II. Identification of biologically active intermediates.

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3, and E, as well as the E. coli genes groEL and groES. It has been previously shown that extracts prepared from cells infected with a lambda C-E- mutant accumulate biologically active gpB and gpNu3 (Murialdo, H., and Becker, A., J. Mol. Biol. 125, 57-74 (1978) ). To characterize the nature of these intermediates in prohead assembly, extracts prepared from these cells were fractionated by DEAE-cellulose chromatography as well as velocity sedimentation. Intermediates containing gpB were identified by SDS-polyacrylamide gel electrophoresis and by their ability to be assembled into biologically active proheads in vitro. The results indicate that the most abundant, biologically active intermediate (greater than 98% of the gpB activity) is a 25 S gpB-containing polymer. A second biologically active intermediate (about 1% of the total gpB activity) was identified as a gpB-gpgroEL complex.

Bacteriophage lambda↗
PubMed2004

Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12.

The orf gene of bacteriophage lambda, fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a Delta(recC ptr recB recD)::P(tac) gam bet exo pae cI DeltarecG background, lacking recF, recO, recR, ruvAB, and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.

Alleles↗
PubMed1987

Genetic and physical location of the Escherichia coli rap locus, which is essential for growth of bacteriophage lambda.

The Escherichia coli rap mutant does not support the growth of bacteriophage lambda (D. Henderson and J. Weil, Virology 71:546-559, 1976). We located the rap site at 26 min in the E. coli genetic map and determined the gene order fadR-rap-supF-trp from our transduction experiments. Plasmid pHO1 harbors a 5.6-kilobase-pair segment of the E. coli chromosome which contains the pth gene (B. Hove-Jensen, Mol. Gen. Genet. 201:269-276, 1985). This plasmid complemented rap bacteria, suggesting that it carries the dominant allele rap+. Subcloning experiments reduced the rap-complementing segment to 1.5 kilobase pairs. This segment still contained pth; thus, both loci are tightly linked. The lit mutations that inhibit phage T4 growth in E. coli are located nearby at 25 min (W. Cooley, K. Sirotkin, R. Green, and L. Snyder, J. Bacteriol. 140:83-91, 1979). We showed that rap and lit mutations are phenotypically and genetically different.

Bacteriophage lambda↗
PubMed1993

Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites. A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes'. Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex. These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein. The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites. These complexes should enable a detailed analysis of synapsis for this pathway.

Bacteriophage lambda↗
PubMed1991

RNA polymerase bound to the PR promoter of bacteriophage lambda inhibits open complex formation at the divergently transcribed PRM promoter. Implications for an indirect mechanism of transcriptional activation by lambda repressor.

We demonstrate that RNA polymerase bound at the PR promoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed PRM promoter in vitro. Using abortive initiation and run-off transcription experiments we show that inactivating mutations introduced into either the -10 or -35 regions of PR result in a significant increase in the rate of formation of transcriptionally competent complexes at the PRM promoter. This is due primarily to an increase in the rate constant for the isomerization of closed to open complexes. Gel shift and DNase I footprinting experiments were employed to further define the mechanism by which PR sequences mediate PRM repression. From these assays we were able to conclude that the formation of an open complex at the PR promoter did not exclude RNA polymerase from binding at PRM. Rather, initiation at PRM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the PR promoter. Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the PRM promoter. Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates PRM transcription by excluding RNA polymerase from the PR promoter.

Bacteriophage lambda↗
PubMed2004

Bacteriophage lambda is a highly stable DNA vaccine delivery vehicle.

The stability of whole bacteriophage lambda particles, used as a DNA vaccine delivery system has been examined. Phage were found to be highly stable under normal storage conditions. In liquid suspension, no decrease in titre was observed over a 6-month period at 4 and -70 degrees C, and phage stability was unaffected by freeze/thawing. The measured half life of phage in suspension was 36 days at 20 degrees C, 3.4 days at 37 degrees C and 2.3 days at 42 degrees C. Freeze drying of a phage suspension (with or without the stabilizers dry skim milk or trehalose) resulted in 5-20% residual viability. Following desiccation (with or without stabilizers), measured half lives ranged from 20 to 100 days at 20 degrees C, 2.6 to 38 days at 37 degrees C, 2.1 to 26 days at 42 degrees C, 7 to 33 h at 70 degrees C, and 1.3 to 6m at 100 degrees C. In all cases the addition of trehalose significantly increased the stability of the desiccated phage. When stored at -70 degrees C, desiccated phage appeared to be stable in the absence of stabilizers. When phage lambda was diluted into water, a marginal loss in titre was observed over a 2-week period. Over a 24 h period, liquid phage suspensions were stable within the pH range pH 3-11, therefore oral administration of bacteriophage DNA vaccines via drinking water may be possible.

Bacteriophage lambda↗
PubMed1998

Polyadenylation of oop RNA in the regulation of bacteriophage lambda development.

We have shown that Escherichia coli pcnB mutants are lysogenized by bacteriophage lambda with lower efficiency as compared to the pcnB+ strains. Our genetic analysis revealed that expression of the lambda cII gene is decreased in the pcnB mutants. However, using various lacZ fusions we demonstrated that neither activities of pL and pR promoters nor transcription termination at tR1 were significantly impaired in the pcnB- host. On the other hand, we found that oop RNA, an antisense RNA for cII expression, is involved in this regulation. Primer protection experiments revealed that oop RNA was polyadenylated and that this polyadenylation was impaired in the pcnB mutant. We found that the oop RNA was more abundant in the pcnB mutant than in the pcnB+ strain. Furthermore, we showed that activity of the pO promoter was not stimulated in the pcnB mutant. Such findings indicated that degradation of oop RNA in the pcnB strain was slower because of inefficient polyadenylation, which could lead to more effective inhibition of cII expression by the antisense oop RNA, resulting in less efficient lysogenization of the host. The oop RNA was found previously to play a role in phage lambda development only under conditions of overproduction of this transcript. Here we demonstrate for the first time, the physiological function of oop RNA in lambda development, confirming that this short transcript plays an important role in the negative regulation of cII gene expression during lambda infection. Moreover, polyadenylation of oop RNA is one of very few known examples of specific RNA polyadenylation by PAP I in prokaryotic cells and its role in gene expression regulation.

Bacterial Proteins↗
PubMed1983

DNA sequences necessary for packaging of bacteriophage lambda DNA.

The extent of DNA flanking the "cohered cohesive end" site of bacteriophage lambda DNA, which is required for packaging, was determined by using defined DNA fragments and a cosmid in vivo packaging assay. From the right end of lambda DNA a 20- to 36-base-pair stretch extending from the center of the cohered cohesive ends is shown to be required, whereas the packaging efficiency of cosmids extending to 70 base pairs into the left lambda arm is reduced to 10% (compared to a fragment extending until about 80 base pairs). A 60-base-pair stretch of the left arm leaves an efficiency of only 1%. The segment thus delineated, by the nature of the assay, is both necessary and sufficient for the binding of packaging proteins to the DNA, the packaging of DNA itself, the DNA cleavage, and successful injection of the DNA into a bacterial host. By contrast, in vitro packaging of restriction fragments of mature lambda DNA directly demonstrated the selectivity of the packaging proteins for the fragment originating from the left end of the DNA. The results of the two complementary experiments are discussed in terms of the various steps before, during, and after packaging for which different sequences flanking and including the cohered cohesive ends might be required.

Bacteriophage lambda↗
PubMed1982

The rex gene of bacteriophage lambda is really two genes.

Complementation tests among previously isolated rex- mutants of bacteriophage lambda reveal that the mutants comprise two complementation groups, designated rexA and rexB. Because rexB- mutants complement prm- mutants, but rexA- mutants do not, it appears that the rexA gene is coordinately controlled with the cI (repressor) gene under the direction of PRM promoter, but that some other promoter is capable of directing the expression of rexB.

Bacteriophage lambda↗
PubMed1989

Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication.

Replication of the chromosome of bacteriophage lambda depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host. As previously described, the first stage in this process is the binding of multiple copies of the lambda O initiator to the lambda replication origin (ori lambda) to form the nucleosomelike O-some. The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of lambda DNA replication to ori lambda. To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified. Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at ori lambda during the initiation of lambda DNA replication. A second-stage ori lambda.O (lambda O protein).P (lambda P protein).DnaB nucleoprotein structure is formed when O, P, and E. coli DnaB helicase are incubated with ori lambda DNA. In a third-stage reaction the E. coli DnaJ heat shock protein specifically binds to the second-stage structure to form an ori lambda.O.P.DnaB.DnaJ complex. Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of lambda DNA replication. The E. coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete ori lambda.O.P.DnaB.DnaJ.DnaK initiation complex is assembled. Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex. These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the ori lambda template. The protein disassembly reactions are described in the accompanying articles.

Bacteriophage lambda↗
PubMed1985

A cII-dependent promoter is located within the Q gene of bacteriophage lambda.

We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda. Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI. The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition. We have constructed a promoter down mutation, paq-1, by changing a single base pair in the putative cII binding site of the promoter by oligonucleotide site-directed mutagenesis. The paq-1 mutant promoter required about 4-fold higher cII concentrations for maximal activation compared to the wild-type PaQ. We tested the hypothesis that PaQ is responsible in part for the delay of lambda late-gene expression by recombining the paq-1 mutation into a phage showing severe cII-dependent inhibition. We found that the paq-1 mutation relieved the cII-dependent growth defect of this phage. The paq-1 mutation (in combination with lambda cI857) resulted in a clear-plaque phenotype at the permissive temperature of 32 degrees C. The role of the PaQ-initiated antisense transcript in the control of lambda development is discussed.

Bacteriophage lambda↗
PubMed1994

Adsorption of bacteriophage lambda on the LamB protein of Escherichia coli K-12: point mutations in gene J of lambda responsible for extended host range.

LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda group in two classes. Class I mutants block the growth of lambda with the wild-type host range (lambda h+) but support the growth of one-step host range mutants (lambda h). Class II mutants block lambda h but support the growth of two-step host range mutant (lambda hh*) phages. To identify amino acid residues in the J protein (the tail fiber of phage lambda) responsible for the extended host range phenotype of mutants of phage (lambda h+), we selected a series of one-step (lambda h) and two-step (lambda hh*) host range mutants and analyzed their corresponding J genes. Three different class I LamB missense mutants (mutations at sites 247, 245, and 148) were used to select 11 independent, new, one-step host range mutants (lambda h phages). DNA sequence analysis revealed a single-amino-acid change in each case. The 11 alterations affected only three residues in the distal part of J, corresponding to a Val-->Ala change at site 1077 in five cases, a Thr-->Met change at site 1040 in three cases, and a Leu-->Pro change at site 1127 in three cases. Recombination experiments confirmed that in the cases tested, the mutations identified were indeed responsible for the extended host range phenotype. The class II LamB mutant (Gly-->Asp at site 151) was used to select two-step extended host range mutants (lambda hh* phages) from three new lambda h phages, corresponding to different amino acid modifications in the J protein (at sites 1040, 1077, and 1127). The new lambda hh* phages analyzed corresponded to either double or triple point mutations located at the distal end of the J protein. In all, seven residues involved in the extended host range properties of lambda mutants were identified in the distal part of the J protein, suggesting that the last C-terminal portion of the J protein participates directly in the adsorption of the phage onto LamB. In agreement with the fact that the lambda h mutants (and the lambda hh* mutants) could grow on all of the lamB class I mutations tested, we found tha the nature of the J mutations did not depend on the LamB class I mutant used to select them. This is interpreted as meaning that the mutated residues in the J protein and in the LamB mutants are not involved in allele-specific protein-protein interactions. Rather, the LamB mutations would block a step in phage adsorption, and this block would be overcome by the mutations in the J protein.

Adsorption↗
PubMed1987

New mutations in the pRM promoter of bacteriophage lambda.

A pRM-cI-lacZ fusion inserted into the b2 region of bacteriophage lambda imm21 was used to isolate mutations in the lambda pRM promoter. Among the mutations causing defects in synthesis of both repressor (cI gene product) and beta-galactosidase, new promoter mutations were identified at positions -11 and -32 relative to the cI transcription start point. Both mutations are changes in conserved (consensus) nucleotides in pRM, but the mutation at -11, which alters a more highly conserved nucleotide, has a somewhat greater effect on promoter function in vitro than does the mutation at -32. We also isolated a mutation at -69 in the repressor-binding site OR1, which presumably prevents activation of pRM by repressor.

Bacteriophage lambda↗
PubMed1982

Bacteriophage lambda DNA packaging in vitro. The involvement of the lambda FI gene product, single-strand DNA, and a novel lambda-directed protein in the packaging reaction.

The FI gene product (gp) of bacteriophage lambda is required during phage head assembly in vivo. Mutations in this gene lead to an accumulation of immature concatemeric lambda DNA and of proheads that appear normal and are competent for DNA packaging in vitro. This phenotype can be taken as evidence of a failure to couple DNA and proheads for packaging/maturation. In contrast to the requirement for gpFI in vivo, the packaging of lambda DNA in vitro occurs efficiently in the complete absence of gpFI. However, if ssDNA is included at the outset of the in vitro packaging reaction, DNA packaging is blocked. This block to packaging is relieved by addition of gpFI. Thus packaging of lambda DNA in vitro can be made dependent of gpFI by the inclusion of ssDNA at the outset of the reaction. Inhibition of DNA packaging by ssDNA appears to be mediated by a lambda b region-directed protein (packaging inhibitor, ben protein) that is present in the crude extracts of cells used to support the early steps of the packaging reaction. Neither ssDNA nor the packaging inhibitor alone has significant inhibitory effect on packaging; both components are required together to effect the inhibition that is relieved by gpFI. The packaging inhibitor was extensively purified and shown to have endonucleolytic activity. Several lines of evidence are presented to support the idea that both the inhibitory and endonucleolytic activities are functions of the same protein. Although gpFI relieves the inhibition imposed by the ben protein in packaging, gpFI fails to block the DNA cleavage activity of the ben protein in the standard endonuclease assay.

Bacteriophage lambda↗
PubMed1974

Isolation of a specialized transducing bacteriophage lambda carrying the polC locus of Escherichia coli.

We have isolated a specialized transducing phage carrying the polC locus of E. coli K-12. A strain of E. coli lacking the lambda attachment site was infected with bacteriophage lambda. Lysogens carrying lambda at the tonA locus were isolated by selecting lambda-immune, T5-resistant strains. Transducing phages of dapC, dapD and polC, which map within 0.2 min of tonA, were obtained in lysates prepared from two of the lysogens. The isolated phage, lambdadpolC, is defective but can transduce four different polC temperature-sensitive mutants. After lysogenation with the transducing phage, DNA polymerase III activity is restored to normal levels in extracts of a polC strain lacking polymerase III activity. However, attempts to obtain increased levels of DNA polymerase III in extracts of induced lysogens carrying lambdadpolC have been unsuccessful.

Chromatography, Ion Exchange↗