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PubMed1985

The terminase of bacteriophage lambda. Functional domains for cosB binding and multimer assembly.

Terminase is a protein complex involved in lambda DNA packaging. The subunits of terminase, gpNul and gpA, are the products of genes Nul and A. The actions of terminase include DNA binding, prohead binding and DNA nicking. Phage 21 is a lambdoid phage that also makes a terminase, encoded by genes 1 and 2. The terminases of 21 and lambda are not interchangeable. This specificity involves two actions of terminase; DNA binding and prohead binding. In addition, the subunits of lambda terminase will not form functional multimers with the subunits of 21 terminase. lambda-21 hybrid phages can be produced as a result of recombination. We describe here lambda-21 hybrid phages that have hybrid terminase genes. The packaging specificities of the hybrids and the structure of their genes were compared in order to identify functional domains of terminase. The packaging specificities were determined in vivo by complementation tests and helper packaging experiments. Restriction enzyme site mapping and sequencing located the sites at which recombination occurred to produce the hybrid phages. lambda-21 hybrid 51 carries the lambda A gene, and a hybrid 1/Nul gene. The crossover that produced this phage occurred near the middle of the 1 and Nul genes. The amino-terminal portion of the hybrid protein is homologous to gp1 and the carboxy-terminal portion is homologous to gpNul. It binds to 21 DNA and forms functional multimers with gpA, providing evidence that the amino-terminal portion of gpNul is involved in DNA binding and the carboxy-terminal portion of gpNul is involved in the interaction with gpA. lambda-21 hybrid 54 has a hybrid 2/A gene. The amino terminus of the hybrid protein of lambda-21 hybrid 54 is homologous with gp2. This protein forms functional multimers only with gp1, providing evidence that the amino terminus of gpA is involved in the interaction with gpNul. These studies identify three functional domains of terminase.

Bacteriophage lambda↗
PubMedDate not supplied

[Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+. I. General characteristics and genetic localization].

The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E. coli K12 strain, carring plasmid pKM101 of N-incompatibility group. By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101. We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA). It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda. Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E. coli and is practically absent in B-strains and also in E. coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII.

Bacteriophage lambda↗
PubMed1980

Transposition of IS1-lambdaBIO-IS1 from a bacteriophage lambda derivative carrying the IS1-cat-IS1 transposon (Tn9).

Tn9 is a transposable element in which a gene (cat) determining chloramphenicol resistance is flanked by directly repeated sequences that are homologous to the insertion sequence IS1. We show here that infection of Escherichia coli K12 (under Rec-Red-Int- conditions) with a lambda bio transducing phage carrying Tn9 results in the formation of lambda bio transductants as frequently as cat transductants as frequently as cat transductants (about 1 per 10(6) to 10(7) infected cells). Most of the lambda bio transductants do not carry cat, just as most of the cat transductants do not carry lambda bio. In spite of the absence of cat, the lambda bio prophage can transpose a second time, from the E. coli chromosome to different sites on an F'gal plasmid. Analysis of the structure of the transposed lambda bio element, by restriction nuclease digestion and by electron microscopy, demonstrates that the integrated lambda bio prophage is flanked by directly repeated IS1 elements. We conclude that there is no genetic information for the ability to transpose encoded in the non-repeated portion of Tn9, i.e. that the directly repeated IS1 elements alone are responsible for Tn9 transposition.

Bacteriophage lambda↗
PubMed1988

The bacteriophage lambda cohesive end site: isolation of spacing/substitution mutations that result in dependence on Escherichia coli integration host factor.

Substitution, insertion and deletion mutations have been constructed at the XmnI restriction site in cos lambda. The XmnI site is located between cosB, the site where terminase binds lambda DNA; and cosN, the site where terminase introduces staggered nicks to generate cohesive ends. Substitution mutations and deletion of a base pair (a -1 change) do not obviously affect lambda growth and DNA packaging. Changes of -2, +2 and -3 render lambda unable to grow on host cells lacking integration host factor (IHF). The -3 mutant has a reduced burst size in IHF+ cells, due to a defect in the initiation of packaging. A -7 deletion mutation is lethal. Models for the basis of these mutational effects are discussed.

Bacterial Proteins↗
PubMed1991

Complementation of the lytD1 mutation of Escherichia coli by either the cI or cro gene of bacteriophage lambda.

The lytD1 mutant of Escherichia coli exhibits temperature-sensitive growth which is attributed to cellular autolysis at the restrictive temperature. Either of two cloned phage lambda genes, identified as cI and cro, suppressed the lytD1(Ts) lysis phenotype, suggesting that lytD encodes a DNA-binding protein with a DNA-binding specificity similar to that of CI and Cro. LytD may be a repressor of a gene(s) involved in cellular autolysis.

Bacteriophage lambda↗
PubMed1988

Role of the Escherichia coli DnaK and DnaJ heat shock proteins in the initiation of bacteriophage lambda DNA replication.

We examined the role of two Escherichia coli heat shock proteins, the dnaK and dnaJ gene products, during the initiation of lambda dv DNA replication in vitro. Using 14C-labeled lambda P protein we showed that the DnaK and DnaJ heat shock proteins function together to release lambda P protein from the preprimosomal complex consisting of lambda origin of replication-lambda O-lambda P-DnaB protein. Hydrolysis of ATP, catalyzed presumably by DnaK, is required during this reaction. Substitution of DnaK protein with that of the mutant DnaK756 protein blocks lambda P release. After DnaK and DnaJ action, the preprimosomal complex, isolated on Sepharose 4B, can support lambda dv DNA replication without any additional prepriming proteins. Using DnaK-affinity chromatography we showed that both lambda O and lambda P proteins bind to DnaK protein. The lambda P protein interacts with DnaK protein in a salt-resistant, hydrophobic manner, and ATP hydrolysis is necessary to elute at least part of lambda P protein from the DnaK-affinity column. The proposed mechanism of action of the prokaryotic DnaK and DnaJ heat shock proteins agrees with the hypothesis that Hsp70, the DnaK analogue of eukaryotes, uses ATP to disrupt hydrophobic aggregates [Pelham, H. R. B. (1986) Cell 46, 959-961].

Bacteriophage lambda↗
PubMed1983

Bacteriophage lambda receptor protein in Escherichia coli K-12: lowered affinity of some mutant proteins for maltose-binding protein in vitro.

Mutant and wild-type LamB proteins (phage lambda receptor proteins) were purified by affinity chromatography with immobilized maltose-binding protein, and their transport functions were tested in reconstituted liposomes. Two mutant proteins exhibited a marked decrease in affinity for immobilized maltose-binding protein, as well as altered transport rates.

ATP-Binding Cassette Transporters↗
PubMed1988

A point mutation in the Nul gene of bacteriophage lambda facilitates phage growth in Escherichia coli with himA and gyrB mutations.

A mutant of lambda was isolated that grows in the Escherichia coli himA delta/gyrB-him320(Ts) double mutant at 42 degrees C; conditions which are non-permissive for wild-type lambda growth. The responsible mutation, ohm1, alters the 40th codon of the Nul reading frame. The Nul and A gene products comprise the terminase protein which cleaves concatameric DNA into unit-length phage genomes during DNA packaging. The Nul-ohm1 gene product acts in trans to support lambda growth in the double himA/gyrB mutant, and lambda cos154 growth in the single himA mutant. The observation that an alteration in Nul suppresses the inhibition of growth in the double himA/gyrB mutant implicates DNA gyrase, as well as integration host factor, in the DNA:protein interactions that occur at the initiation of packaging.

Bacteriophage lambda↗
PubMed1981

Purification of bacteriophage lambda O protein that specifically binds to the origin of replication.

By means of a nitrocellulose filter binding assay, DNA binding activities among proteins fractionated from extracts of Escherichia coli carrying lambda dv have been surveyed. An activity was found that binds specifically to a fragment of 164 base pairs that specifies the lambda replication origin (lambda ori). This activity was not detected in an extract of cells not carrying the lambda dv plasmid. The activity was detected in extracts of cells carrying a hybrid plasmid in which the entire lambda O gene had been cloned and placed under the control of the lac promoter. Deletion of a 60 base pair segment in the 'amino-terminal region' of the O gene abolished this activity, indicating that the lambda ori binding protein is coded for by the lambda O gene. The ori-specific binding protein was purified by five fractionation steps. The most purified preparation consists of a major polypepide that migrates with a molecular weight of 32,000 in SDS-polyacrylamide gel electrophoresis. Binding of O protein to ori occurs in the absence of other protein aceous components.

Bacteriophage lambda↗
PubMed1989

Recombination of bacteriophage lambda in recD mutants of Escherichia coli.

RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host. When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi). The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication. The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication. The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions. Recombination in rec+ cells is chi influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence.

Bacterial Proteins↗
PubMedDate not supplied

[Complexity analysis of a genome. II. Extensive homology zones in bacteriophage lambda].

The suggested earlier complexity approach for detecting structural regularities in primary structures of nucleic acids is illustrated by using lambda phage as an example. Among the most interesting regularities detected in the lambda phage genome are the following: (a) the presence of "extended homology zones" i.e. fragments in which block transpositions of duplicative type predominate explicitly; (b) the abundance of palindrome-hairpin structures and duplications in the origins and termini of many genes; (c) the hierarchy in the repeats and inversions organization.

Bacteriophage lambda↗
PubMed1983

The integrase promoter and T'I terminator in bacteriophages lambda and 434.

The lambda integrase promoter PI lies immediately downstream from a terminator T'I. The PI promoter is activated by cII protein during lysogenization. The function of T'I is unknown. A deletion (trp-lambda 29), whose fusion point lies within one stem of T'1 retains cII-activable promoter function but shows little if any transcription termination in vivo. The nucleotide sequence of PI is identical in lambda and the related phage 434. However, the T'I sequences of the two phages, though located in the same position, are not detectably homologous. When restriction fragments carrying the promoter-terminator segments from each of these phages were inserted into the trp operon, both responded identically to cII activation, and both caused termination.

Bacteriophage lambda↗
PubMed1996

Stable high-copy-number bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli.

The construction of new high-copy-number (hcn) lambda-promoter expression vectors is described. All these vectors (1) contain tandem lambda pR and pL promoters upstream of an extensive multiple cloning site (MCS) for insertion of genes, (2) direct expression of the lambda cIts857 gene, enabling their use in any Escherichia coli host strain for thermal induction of gene overexpression, and (3) bear the par locus of plasmid pSC101, ensuring their stable maintenance at hcn in the absence of continuous antibiotic selection. Six of the vectors also contain efficient ribosome-binding sites upstream of unique HpaI or NdeI sites in their MCS regions, and two contain sequences that encode N-terminal poly-His. The performance of these vectors was assessed by using them to overproduce the E. coli HMP flavohaemoprotein and the bacteriophage M13 gene II replicator protein.

Bacterial Proteins↗
PubMed1982

[Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location].

It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA. The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability. When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb. In the course of transposition of Tn9 from chromosomal attTn9N site the transposon inserted into the lambda att80 site with coordinates 26,5 +/- 5 kb. In the latter case, the increase of Tn9 single-stranded loop and the appearance of two new HindIII cleavage sites were observed in heteroduplex experiments. The data were interpreted as indicating structural rearrangements of Tn9 or linked sequences in the course of transposition.

Bacteriophage lambda↗