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PubMed1982

Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene.

The bacteriophage lambda int gene product, integrase, recombines the phage DNA with the host DNA at specific sites on each to accomplish lysogeny. The int gene is transcribed from two promoters, PL and PI, each regulated positively by lambda proteins. The expression of integrase is also controlled from a site, sib, in the b region of the phage genome. This is a unique regulatory site because it is located distal to the structural gene in relation to the promoters. The expression of int from the PL promoter is inhibited when sib is present. This effect appears to be specific for PL because sib does not cause inhibition of PI-dependent int synthesis. lambda mutants that contain alterations in the site have been isolated. Sequence analyses of the mutations reveal single base changes, spanning 37 base pairs (bp) in the b region, some 240 bp beyond the int gene. Another mutant, hef13, which has a phenotype similar to that of sib, introduces a nucleotide change within the same 37-bp region. The sib and hef mutations cluster within a region of dyad symmetry. Regulation of int synthesis by sib occurs after transcription of the int gene. There is no difference in the rate of PL-promoted int mRNA synthesis in either sib+ or sib- phage infections, yet int mRNA is less stable in the sib+ infection. Because RNase III host mutants are defective in sib regulation, processing of the PL mRNA at sib by this endoribonuclease may cause int mRNA decay and decrease int synthesis.

Bacteriophage lambda↗

PubMed1999

Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins.

The beta protein of bacteriophage lambda acts in homologous genetic recombination by catalyzing the annealing of complementary single-stranded DNA produced by the lambda exonuclease. It has been shown that the beta protein binds to the products of the annealing reaction more tightly than to the initial substrates. We find that beta protein exists in three structural states. In the absence of DNA, beta protein forms inactive rings with approximately 12 subunits. The active form of the beta protein in the presence of oligonucleotides or single-stranded DNA is a ring, composed of approximately 15-18 subunits. The double-stranded products of the annealing reaction catalyzed by the rings are bound by beta protein in a left-handed helical structure, which protects the products from nucleolytic degradation. These observations suggest structural homology for a family of proteins, including the phage P22 erf, the bacterial RecT, and the eukaryotic Rad52 proteins, all of which are involved in homologous recombination.

Bacteriophage lambda↗

PubMed1993

Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster.

Genomic DNAs prepared from 168 isolates of Escherichia coli O157:H7 were analyzed for restriction fragment length polymorphisms on Southern blots probed with bacteriophage lambda DNA. The isolates analyzed included strains from a recent large multistate outbreak of E. coli O157:H7 infection associated with consumption of poorly cooked beef in restaurants, a day-care center cluster, and temporally and geographically unrelated isolates. E. coli O157:H7 isolates recovered from the incriminated meat and from 61 (96.8%) of 63 patients from Washington and Nevada possessed identical lambda restriction fragment length patterns. The lambda restriction fragment length polymorphisms observed in 11 (91.7%) of 12 day-care center patients were identical, but they differed from that of the strain associated with the multistate outbreak. E. coli O157:H7 from 42 patients temporally or geographically unrelated to either cluster of infection possessed unique and different lambda restriction fragment length patterns, except for paired isolates from three separate clusters of infection. These data demonstrate that the hybridization of DNA digests of E. coli O157:H7 with radiolabelled bacteriophage lambda DNA can be a useful, stable, and discriminatory epidemiologic tool for analyzing the linkage between strains of E. coli O157:H7.

Animals↗

PubMed1995

Mutational analysis of the prohead binding domain of the large subunit of terminase, the bacteriophage lambda DNA packaging enzyme.

Terminase, the DNA packaging enzyme of bacteriophage lambda, is made up of two subunits, gpNul and gpA, the products of the Nu1 and A genes. The activities of terminase include DNA binding, cos cleavage and prohead binding. Specificity domains within the structure of terminase have previously been defined by genetic studies of lambda-21 hybrids. The prohead binding domain of terminase is localized to the last 32 amino acid residues of gpA. Mutations in the prohead binding domain of gpA were constructed by introducing the corresponding amino acids from gp2, the gpA analog of bacteriophage 21. The last five residues of gpA can be replaced with little effect on the burst size of lambda. A phage with a replacement of the last six residues of gpA with the corresponding residues of gp2 was unable to form plaques, indicating that the sixth-to-last residues of gpA is crucial for prohead binding. Site-specific mutagenesis of the sixth-to-last position of gpA indicated that the sixth-to-last residue of gpA must be hydrophobic, of the seven amino acids tested, only isoleucine and valine can substitute for leucine at this position. Although the last five residues of gp2 were functional when they replaced the last five residues of gpA, two results indicated that the last five residues of gpA functioned better than the corresponding residues of gp2. First, the presence of a valine residue at the sixth-to-last position of gpA allowed plaque formation, whereas replacement of the last six residues of gpA with those of gp2, which substitutes a valine residue at the sixth-to-last position, was lethal. The second set of results indicating that the last five residues of gpA function better than the gp2 residues were obtained by study of revertants of lethal substitution mutations. In constructing the replacement mutations, a short linker was inserted into the C terminus of the A gene; this insertion created a short duplication of the end of the A gene, so that the normal C-terminal codons were located downstream of the stop codon of the A gene in the substitution mutants. Revertants of the lethal substitution mutations were obtained in which a mutation in the stop codon resulted in addition of the last five residues of gpA to the end of the substitution terminase.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed2000

The amino terminus of bacteriophage lambda integrase is involved in protein-protein interactions during recombination.

Bacteriophage lambda integrase (Int) catalyzes at least four site-specific recombination pathways between pairs of attachment (att) sites. Protein-protein contacts between monomers of Int are presumed to be important for these site-specific recombination events for several reasons: Int binds to the att sites cooperatively, catalytic Int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the Int family (Cre from phage P1 and Int from Haemophilus influenzae phage HP1) show extensive protein-protein contacts between monomers. We have begun to investigate interactions between Int monomers by three approaches. First, using a genetic assay, we show that regions of protein-protein interactions occur throughout Int, including in the amino-terminal domain. This domain was previously thought to be important only for high-affinity protein-DNA interactions. Second, we have found that an amino-terminal His tag reduces cooperative binding to DNA. This disruption in cooperativity decreases the stable interaction of Int with core sites, where catalysis occurs. Third, using protein-protein cross-linking to investigate the multimerization of Int during recombination, we show that Int predominantly forms dimers, trimers, and tetramers. Moreover, we show that the cysteine at position 25 is present at or near the interface between monomers that is involved in the formation of dimers and tetramers. Our evidence indicates that the amino-terminal domain of Int is involved in protein-protein interactions that are likely to be important for recombination.

Attachment Sites, Microbiological↗

PubMedDate not supplied

[Photosensitized inactivation of plasmid and bacteriophage lambda DNA: relative contribution to the lethal effect of monoadducts and diadducts of 8-methoxypsoralen and their repair in SOS-induced Escherichia coli cells].

The curves of UV (254 nm)-inactivation and inactivation by furocoumarin derivatives + UVA radiation (PUVA) of bacteriophage lambda and biologically active plasmid pBR322 were measured using Escherichia coli K12 bacteria with different defects of DNA repair system as a ghost. The ratio of mono- and diadducts (interstrand cross-links) of 8-methoxypsoralen was determined that are formed after treating the DNA of pBR322 and bacteriophage lambda with PUVA. It is shown that, on the average, about five monoadducts per one diadduct are formed in DNA of pBR322, and about 0.9 monoadducts per one diadduct are formed in lambda phage DNA. An increased (up to 50%) efficiency of SOS-repair of monoadducts of 8-methoxypsoralen in DNA of pBR322 and lambda in the presence of plasmid pKM101 muc+ (incN) was found.

Bacteriophage lambda↗

PubMed2002

Recombination-promoting activity of the bacteriophage lambda Rap protein in Escherichia coli K-12.

The rap gene of bacteriophage lambda was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the lambda red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recGDelta red(+) rap(+) strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny. The results of these experiments indicated that the lambda rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.

Bacterial Proteins↗

PubMed1986

Mutations of bacteriophage lambda that define independent but overlapping RNA processing and transcription termination sites.

Bacteriophage lambda int gene expression is regulated differentially from transcripts originated at the pL and pI promoters. Transcripts initiated at pI terminate at the site tI and express int gene product efficiently. Polymerases starting at pL do not terminate at tI, due to the antiterminating activity of lambda N protein. The pL transcripts are unable to express Int protein efficiently because sib, a control site overlapping tI in the unterminated RNA, is processed by host RNase III. We have isolated lambda sib- mutants by their inability to inhibit int expression from pL transcripts. sib mutations were genetically mapped to the left of the lambda attachment site, and do not structurally alter this site for recombination. Several sib mutations do alter the nucleotide sequence of the overlapping sib and tI sites. The lambda sib- mutants tested prevent RNA processing but do not affect transcription termination in vivo.

Attachment Sites, Microbiological↗

PubMed1980

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli.

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.

Bacteriophage lambda↗

PubMed1975

Determination of genes, restriction sites, and DNA sequences surrounding the 6S RNA template of bacteriophage lambda.

A major product of the transcription of bacteriophage lambda DNA in vitro is the 6S RNA. This article presents a detailed mapping of restriction endonuclease cleavage sites about the region of the 6S RNA template within the lambda genome. Restriction fragments defined by these sites have been used to localize the 6S RNA template within the physical and genetic maps of the lambda genome. Nucleotide sequence analysis of one of these fragments has largely confimed the nucleotide sequence of the 6S RNA reported previously and has indicated the sequence of DNA that immediately follows the 6S RNA template. This article reports the nucleotide sequence following a known site of transcription termination by RNA polymerase of Escherichia coli.

Base Sequence↗

PubMed1979

Location of the regulatory site for establishment of repression by bacteriophage lambda.

During the lysogenic response to infection by bacteriophage lambda, the phage-specified cII and cIII proteins provide for the coordinate establishment of repression and integration of the viral DNA. One critical regulatory function of cII/cIII is an activation of synthesis of the cI protein, the repressor that maintains lysogeny. The mechanism and site for regulation of the cI gene by cII/cIII have been a subject of controversy. The two principal hypotheses for cII/cIII action are: initiation of new RNA chains in the y region of lambda DNA just to the left of the cII gene; or antitermination of a short leader RNA (4S or oop RNA) initiated to the right of the cII gene. In an effort to distinguish between these hypotheses, we have studied the cII-mediated turn-on of cI protein synthesis in three classes of prophage deletion strains: deletions of the 4S RNA promoter but not the y region, deletions that remove both regions, and deletions that leave both intact. We find that an intact y region is required for normal regulation of the cI gene by cII, but the 4S RNA promoter is not. From experiments with other mutants, we conclude that rightward transcription from the early lytic promoter is also not necessary for positive regulation. Our results suggest that positive regulation by cII/cIII involves initiation of new RNA chains through activation of promoter sites.

Binding Sites↗

PubMed1977

Construction of chimeric phages and plasmids containing the origin of replication of bacteriophage lambda.

Segments of the replication control region of bacteriophage lambda (lambda) and lambda mutants defective in replication were attached in vitro to the phi80 phage vector Charon 3 and to the plasmid vector mini Col El (pVH51). The chimeric phages and plasmids have been used to localize the origin of lambda DNA replication and to facilitate a structural analysis of the lambda replicator.

Chromosome Mapping↗

PubMed1998

Involvement of boxA nucleotides in the formation of a stable ribonucleoprotein complex containing the bacteriophage lambda N protein.

The association of the transcriptional antitermination protein N of bacteriophage lambda with Escherichia coli RNA polymerase depends on nut site RNA (boxA + boxB) in the nascent transcript and the host protein, NusA. This ribonucleoprotein complex can transcribe through Rho-dependent and intrinsic termination sites located up to several hundred base pairs downstream of nut. For antitermination to occur farther downstream, this core antitermination complex must be stabilized by the host proteins NusB, NusG, and ribosomal protein S10. Here, we show that the assembly of NusB, NusG, and S10 onto the core complex involves nucleotides 2-7 of lambda boxA (CGCUCUUACACA) and is a fully cooperative process that depends on the presence of all three proteins. This assembly of NusB, NusG, and S10 also requires the carboxyl-terminal region (amino acids 73-107) of N, which interacts directly with RNA polymerase. NusB and S10 assemble in the absence of NusG when lambda boxA is altered at nucleotides 8 and 9 to create a consensus version of boxA (CGCUCUUUAACA). These experiments suggest that multiple protein-protein and protein-RNA interactions are required to convert a core antitermination complex into a complete complex.

Bacterial Proteins↗

PubMed1983

The bacteriophage lambda terminase. Partial purification and preliminary characterization of properties.

The maturation of bacteriophage lambda DNA and its packaging into preformed heads to produce infectious phage is under the control of the two leftmost genes on the lambda chromosome, i.e., Nu1 and A. Based on its ability to complement lambda A- phage-infected cell extracts for packaging of lambda DNA in vitro, a single protein, designated terminase (ter) has been extensively purified using adsorption, ion exchange, and affinity column chromatography. The final preparation represents an approximately 60,000-fold purification over the activity found in crude extracts and is about 30 to 80% homogeneous as judged by visualizing the protein after electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. In addition to packaging, terminase can also catalyze the endonucleolytic cleavage of lambda cohesive-end site DNA; both of these reactions require ATP. In some preparations, certain terminase fractions of extreme purity require protein factors present in extracts of uninfected Escherichia coli in order to catalyze the cohesive-end site cleavage reaction. On ion exchange columns purified terminase co-chromatographs with a DNA-dependent ATPase activity, hydrolyzing ATP to ADP and Pi in the presence of any of several types of DNA tested including those of non-lambda origin. The molecular weight of the native enzyme is 117,000 and appears to be a hetero-oligomer composed of 2 nonidentical subunits. The most likely composition of terminase is one gpA (gene product of A), Mr = 74,000 and two gpNu1, Mr = 21,000.

Adenosine Triphosphatases↗

PubMed1982

Bacteriophage lambda DNA packaging: scanning for the terminal cohesive end site during packaging.

Bacteriophage lambda packages the DNA of the related phage 21 poorly [Hohn, B. (1975) J. Mol. Biol. 98, 93--106]. To understand the nature of the packaging defect, the interaction of the cohesive end site (cos) specific for phage 21 (cos phi 21) with phage lambda terminase has been investigated. The ability of lambda terminase to cleave cos phi 21 was studied in vitro; lambda terminase cleaved cos phi 21 only 1% as well as it cleaved the phage lambda cohesive end site (cos lambda). In vitro packaging experiments showed that the lambda and 21 packaging specificities observed in vivo are also found in vitro. The cos cleavage reaction was modified so that competition experiments could be performed; these experiments showed that cos phi 21 was unable to bind lambda terminase, thus identifying the nature of the defect. Previous work [Feiss, M., Fisher, R. A., Siegele, D. A., Nichols, B. P. & Donelson, J. E. (1979) Virology 92, 56--67] has shown that the base pairs giving lambda or 21 packaging specificity are at the left end of the chromosome, outside the 22-base-pair symmetry region that includes the annealed cohesive ends. Therefore, terminase binding to cos requires interactions with base pairs to the Nu1 side of the cohesive end symmetry segment. The evidence supports the proposition that cos consists of adjacent sites for binding of terminase and for nicking by terminase. Because cos phi 21 can be cut by lambda terminase to terminate DNA packaging, it is proposed that the terminase that binds and nicks at the initial cos site is brought into contact with the terminal cos site by the packaging process. Terminase recognizes and nicks the cohesive end sequence of the terminal cos without requiring the binding site.

Bacteriophage lambda↗

PubMed1985

Inhibitory effect of high-level transcription of the bacteriophage lambda nutL region on transcription of rRNA in Escherichia coli.

Transcription of the bacteriophage lambda nutL region from the PL promoter on a multicopy plasmid in Escherichia coli causes a reduction in growth rate and in transcription of rRNA relative both to total transcription and to transcription of tRNAs that are not encoded in rRNA operons. These observations support the hypothesis, previously based on nut site DNA sequence homology, that the phage lambda and rRNA antitermination systems are related.

Bacteriophage lambda↗

PubMedDate not supplied

Antitermination of transcription by the N-gene protein of bacteriophage lambda: recent progress and remaining problems.

The N-gene protein of bacteriophage lambda recognizes sequences called nut in the lambda early operons and acts to prevent termination of the transcription of these operons. Part of the mechanism of action of N protein involves binding to the nusA protein of Escherichia coli, a transcription termination factor which associates directly with RNA polymerase. It is likely that N protein forms a ternary complex with the nusA protein and RNA polymerase at the nut site. Evidence is presented that translating ribosomes are not involved in N-protein action. It is still not known how RNA polymerase is modified to become termination-resistent as a result of N protein action.

Bacteriophage lambda↗

PubMed1992

Genetic analysis of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda.

cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, consists of three binding sites (called R3, R2 and R1) for gpNu1, the small subunit of terminase; and I1, a binding site for integration host factor (IHF), the DNA bending protein of Escherichia coli. cosB is located between cosN, the site where terminase introduces staggered nicks to generate cohesive ends, and the Nu1 gene; the order of sites is: cosN-R3-I1-R2-R1-Nu1. A series of lambda mutants have been constructed that have single base-pair C-to-T transition mutations in R3, R2 and R1. A single base-pair transition mutation within any one of the gpNul binding sites renders lambda dependent upon IHF for plaque formation. lambda phage with mutations in both R2 and R3 are incapable of plaque formation even in the presence of IHF. Phages that carry DNA insertions between R1 and R2, from 7 to 20 base-pairs long, are also IHF-dependent, demonstrating the requirement for a precise spacing of gpNu1 binding sites within cosB. The IHF-dependent phenotype of a lambda mutant carrying a deletion of the R1 sequence indicates that IHF obviates the need for terminase binding to the R1 site. In contrast, a lambda mutant deleted for R2 and R1 fails to form plaques on either IHF+ or IHF- cells, indicating terminase binding of R2 is involved in suppression of R mutants by IHF. A fourth R sequence, R4, is situated on the left side of cosN; a phage with a mutant R4 sequence shows a reduced burst size on both an IHF+ and an IHF- host. The inability of the R4- mutant to be suppressed by IHF, plus the fact that R4 does not bind gpNu1, suggests R4 is not part of cosB and may play a role in DNA packaging that is distinct from that of cosB.

Bacteriophage lambda↗