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PubMed1983

Morphology of complexes formed between bacteriophage lambda and structures containing the lambda receptor.

Two types of complexes can be formed between bacteriophage lambda and structures bearing the lambda receptor, either liposomes or rod-shaped particles. Type 1 complexes involve binding between the tip of the lambda tail fiber and the receptor, so that the hollow tail is positioned an average of 17 nm from the surface of the receptor-bearing structures. In type 2 complexes, the hollow tail is in direct contact with the membrane of the liposome or surface of the rod-shaped particle. Type 1 complexes are the precursors for type 2 complexes whose formation is necessary for normal DNA ejection.

Bacterial Outer Membrane Proteins↗

PubMed1995

Specific interaction of terminase, the DNA packaging enzyme of bacteriophage lambda, with the portal protein of the prohead.

Terminase, the bacteriophage lambda DNA packaging protein, is a heteromultimer of two subunits, gpNu1 and gpA, the products of genes Nu1 and A, resp. Phage 21 is a lambdoid phage that produces a terminase similar to that of lambda terminase, the subunits of 21 terminase, gp1 and gp2, have the same domain structures of their lambda analog, gpNu1 and gpA, respectively. The lambda and 21 terminases have different DNA binding and prohead binding specificities. When the C-terminal 32 amino residues of gpA replace the C-terminal 32 residues of gp2, the resulting chimeric terminase specifically uses lambda proheads, indicating that the C-terminal 32 residues of gpA are a specificity domain for prohead binding. A second chimeric terminase, in which the C-terminal six residues of gpA are replaced by the C-terminal six residues of gp2, is unable to utilize lambda proheads, and a lambda phage producing this terminase, lambda Are636, is unable to form plaques. In the present work, a pseudorevertant of lambda Are636 was isolated that contained a mutation Bms8, affecting the prohead. The B gene encodes the portal protein of lambda proheads, which forms the special vertex that is thought to serve as (1) the site of DNA entry into the prohead during packaging, (2) the site for DNA exit during DNA injection, and (3) the site of tail attachment during virion assembly. Bms8 is predicted to change residue 331 of gpB from proline to serine. Burst size measurements and in vitro DNA packaging experiments demonstrated allele-specific interactions between the Are636 terminase and Bms8 proheads. That is, wild-type terminase interacted more efficiently with wild-type proheads than with Bms8 proheads, and Are636 terminase interacted with Bms8 proheads more efficiently than with wild-type proheads. Prohead binding by lambda terminase is stimulated by an assembly catalyst, gpFI. In vitro packaging extracts lacking gpFI were used under conditions in which packaging was gpFI-independent. In the absence of gpFI, Are636 terminase interacted most efficiently with Bms8 proheads, and wild-type terminase interacted most efficiently with wild-type proheads. The allele-specific interactions in the absence of gpFI indicate that the Are636 and Bms8 mutations affect direct interactions between terminase and the portal protein, rather than acting indirectly by altering the interactions of terminase and gpB and gpFI.

Amino Acid Sequence↗

PubMed1990

Analysis of mutations in the ninR region of bacteriophage lambda that bypass a requirement for lambda N antitermination.

Two mutations in the ninR region of bacteriophage lambda that bypass a requirement for antitermination have been studied. One mutation, byp, has been cloned and mapped by marker rescue to a 417-base-pair segment in the ninR region of the genome. Analysis of the byp mutation by using promoter detection vectors, DNA sequencing, and S1 nuclease analysis showed that the byp mutation created a new promoter that transcribed gene Q. The second mutation analyzed was the deletion nin3. Sequence analysis revealed that 2,485 base pairs of the ninR region were removed, beginning within the ren gene and ending in an open reading frame termed ninG. The tR2 and tR3 terminators, and probably others, were removed by the nin3 deletion, thereby allowing the phage to be N independent and to grow in hosts defective for Nus antitermination factors.

Bacteriophage lambda↗

PubMed1996

Total modification of the bacteriophage lambda tail tube major subunit protein with foreign peptides.

The bacteriophage lambda has been shown previously to tolerate a high multiplicity of peptide additions to the C-terminus of the major tail tube subunit protein (gpV, the product of the V gene). However, it was not clear whether all gpV copies within a functional virion could tolerate such modification. Complementation tests with phage bearing either V gene amber mutations or a precisely deleted V gene were used to test the extent of possible tail tube peptide display. Expression of plasmid-encoded gpV fused C-terminally with certain foreign peptides allowed rescue of such V gene-defective phage to essentially wild-type levels. After extensive purification such phage were shown by sensitive Western blotting to contain only the modified form of gpV. Peptide-modified gpV could also form indefinite tail tube polymeric structures (polytubes).

Amino Acid Sequence↗

PubMed1992

Symmetry in the mechanism of bacteriophage lambda integrative recombination.

During the strand-exchange events of bacteriophage lambda integration, pairs of phosphodiester bonds are broken and then rejoined to form novel DNA linkages. The reaction proceeds in vitro in the absence of an external energy source; the bond energy needed to rejoin broken strands of DNA must therefore be conserved during cleavage. Although some of this conservation involves a covalent intermediate between DNA and the recombinase Int, it is possible that such an intermediate is formed with only one of the two phosphodiesters. In such an asymmetric mechanism, the second phosphodiester would be attacked by a nucleophile that is exposed by cleavage of the first DNA strand. In contrast, a symmetric mechanism hypothesizes nucleophilic attack by Int on both phosphodiesters. We have distinguished these two mechanisms by removing potential nucleophiles from the integrative recombination reaction. Our data are inconsistent with an asymmetric mechanism. We conclude that during strand exchange both phosphodiesters proceed through a covalent protein-DNA intermediate.

Bacteriophage lambda↗

PubMed1992

Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage lambda pL promoter.

The high level of efficiency of the bacteriophage lambda pL promoter is dependent upon the topological state of the promoter DNA and the binding of a DNA-bending protein, IHF, to a site centered -86 base-pairs upstream from the pL transcription start site. Abortive initiation assays indicate that DNA supercoiling stimulates open complex formation, whereas IHF enhances promoter recognition. IHF stimulates promoter recognition to the same extent on linear and supercoiled templates. We found that the pL region contains a second promoter, pL2, that initiates transcription 42 base-pairs upstream from pL. Although competitive with pL and inhibited by IHF, mutations in pL2 do not affect the regulation of pL. Stimulation by IHF is helix-face-dependent. IHF inhibits pL when the IHF binding site is displaced a helical half-turn upstream. The pL sequences protected against DNase I digestion by bound IHF and RNA polymerase do not overlap. However, DNase I-hypersensitive sites appear in the region between the two bound proteins. In addition, IHF enhances RNA polymerase binding to pL. These data suggest that stimulation of pL by IHF involves the interaction of IHF and RNA polymerase to form a loop or otherwise distort the DNA between their binding sites.

Bacterial Proteins↗

PubMed1989

Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation.

The bacteriophage lambda cIII gene product regulates the lysogenic pathway. The cIII gene is located in the leftward operon, which is transcribed from the pL promoter. We have previously shown (S. Altuvia and A. B. Oppenheim, J. Bacteriol. 167:415-419, 1986) that mutations that show elevated expression lie within the cIII coding sequence. We isolated mutants that show decreased CIII activity. All the mutations were found to cause a drastic reduction in the rate of initiation of cIII translation. Several mutations were found to be scattered within the first 40 nucleotides of the cIII coding region. Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site. Computer folding of the cIII mRNA suggested the presence of two alternative RNA structures. All the mutations within the coding region that reduce expression reduce the stability of one specific mRNA structure (structure B). Mutations that increase expression lie in the loops of this structure and may in fact stabilize it by interfering with the formation of the alternative structure (structure A). Thus, it appears that a specific mRNA secondary structure at the beginning of the cIII coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cIII expression.

Amino Acid Sequence↗

PubMed1997

Mutations in the terminase genes of bacteriophage lambda that bypass the necessity for FI.

DNA maturation in bacteriophage lambda is the process by which the concatemeric precursor DNA is cleaved at sites called cos to generate mature lambda DNA molecules. These DNA molecules are then packaged into procapsids, the empty capsid precursors. The enzyme that catalyses these events is lambda DNA terminase. It is composed of two subunits, made of 181 and 641 amino acids, the products of genes Nu1 and A, respectively. The product of the FI gene (gpFI) stimulates the formation of an intermediate in capsid assembly called complex II, which contains a procapsid, terminase and DNA. The mechanism of stimulation remains unknown. It has been suggested that gpFI may also stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. Mutants in FI fail to mature and package DNA but, in comparison with other capsid gene mutants, FI mutants are leaky. Second site mutants of FI phages, called 'fin' (for FI independence), bypass the necessity for gpFI. These mutants were originally localized to the region of Nu1 and A and are of two classes: finA includes those that induce the synthesis of fourfold more gene A product (gpA) than wild-type phages, and finB includes those that produce normal amounts of gpA. Whereas all finA mutants analysed map to Nu1, finB mutants have been found both in E and in Nu1. The existence of E mutants able to bypass the necessity for gpFI in vivo shows that gpE and gpFI interact, directly or indirectly. Here we have analysed and sequenced two finA mutants and one finB mutant. All of these map in Nu1. Of the two finA mutants, one corresponds to an Ala163Ser change and the other is a silent mutation. It is likely that the finA mutations alter mRNA conformation in a manner that results in an increase in the efficiency of A mRNA translation. The fourfold increase in gpA synthesis translates into a 10-fold increase in terminase activity. These results show that terminase overproduction is sufficient to bypass the necessity for gpFI and that such an overproduction can be achieved by changes in the efficiency of translation of A due to subtle changes in the sequence upstream of the gene. The finBcs103 mutation is a His-87-->Tyr change in Nu1. Therefore, an alternative way in which to bypass the requirement for gpFI involves an alteration in the structure of gpNu1. It is likely that the altered gpNu1 would increase cleavage and packaging efficiency directly or indirectly. We have determined that DNA cleavage in vivo does not occur in the absence of gpFI. Therefore it seems that gpFI somehow facilitates an otherwise latent capacity of terminase to autoactivate its nucleolytic activity.

Amino Acid Sequence↗

PubMed1974

The roles of the lambda c3 gene and the Escherichia coli catabolite gene activation system in the establishment of lysogeny by bacteriophage lambda.

Maximum lysogenization of E. coli by bacteriophage lambda requires both the lambdacIII gene function and the host catabolite gene activation system mediated by adenosine 3':5'-cyclic monophosphate. Whereas considerable lysogenization occurs in the presence of either system alone, lysogenization is absolutely prevented in the absence of both systems. Neither system is, however, required for efficient lysogenization when the host bears an hfl(-) mutation. It is argued that the normal function of these two systems is to negate the antagonistic effect of the Hfl(+) protein upon lysogenization. It is further argued that both the lambdacIII gene function and the Hfl(+) protein do not directly affect the host catabolite gene activation system.

Adenylyl Cyclases↗

PubMed1983

Purification of the bacteriophage lambda late gene regulator encoded by gene Q.

The product of bacteriophage lambda gene Q is a transcription antiterminator that activates phage late gene expression. We report a method to purify the lambda Q protein to near homogeneity. We have followed during purification both activity of Q protein to provoke in vitro synthesis of the lambda late protein endolysin in the DNA-dependent protein-synthesizing system, and radioactivity in a polypeptide that we show by genetic criteria to be lambda Q. These co-purify throughout the procedure.

Bacteriophage lambda↗

PubMedDate not supplied

Sequencing of cloned DNA using bacteriophage lambda gt11 templates.

A procedure for isolating and directly sequencing recombinant bacteriophage lambda gt11 DNA templates is described. Approximately 250-300 bases of sequence can be obtained directly from the lambda gt11 template, eliminating the need for subcloning prior to dideoxynucleotide sequencing of clones.

Bacteriophage lambda↗

PubMed1973

Purification and properties of the gamma-protein specified by bacteriophage lambda: an inhibitor of the host RecBC recombination enzyme.

Previous experiments have indicated that the gam gene of bacteriophage lambda is responsible for an inhibition of the RecBC DNase-an enzyme that is essential for the major host pathway of genetic recombination. We report here experiments that define the inhibitor as the protein product of the gam gene ("gamma-protein") and that characterize the inhibition reaction with highly purified preparations of gamma-protein and RecBC DNase. Genetic characterization was performed with partially purified fractions prepared from cells infected with various lambda mutants. An activity that inhibits RecBC DNase was absent in extracts prepared after infection by phage that carry nonsense or deletion mutations in the gam gene; this activity was highly thermolabile in an extract prepared after infection by phage that carry a temperature-sensitive mutation in the gam gene. For biochemical characterization, the gamma-protein has been purified more than 800-fold. This highly purified preparation inhibited all of the known catalytic activities associated with the RecBC enzyme, but exhibited no detectable DNase or ATPase activities by itself. These findings are discussed in terms of their implications for regulation of genetic recombination and bacteriophage lambda development.

Adenosine Triphosphatases↗

PubMed1993

The primary self-assembly reaction of bacteriophage lambda cI repressor dimers is to octamer.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites. As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution. Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan [Ross et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12023-12027] and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied. We report here that the primary assembly mode of all four proteins is dimer to octamer. It is not dimer to tetramer as previously assumed. While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution. Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape. This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL. Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions. Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer [Laue et al. (1993) Biochemistry 32, 2469-2472]. Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.

5-Hydroxytryptophan↗

PubMed1976

Lysogenization of Escherichia coli by bacteriophage Lambda: complementary activity of the host's DNA polymerase I and ligase and bacteriophage replication proteins Q and P.

When bacteriophage lambda DNA replication is blocked by mutation in phage genes O or P, the efficiency of lysogenization drops to a very low value unless high multiplicities of infecting phage are used. Our results show that even at high multiplicity, lambda O or P mutants cannot efficiently lysogenize some hosts that are defective in either DNA polymerase I or DNA ligase. Covalent closure of infecting DNA molecules, a preliminary step for insertion according to Campbell's model and an obvious candidate for this lysogenization defect, appears to occur normally under our conditions. In addition, prophage excision as measured by the frequency of curing O- and P- lysogens seemed normal when tested in the poll- strain. These results suggest that the Escherichia coli enzymes DNA polymerase I and ligase, and phage proteins O and P, are able to provide some complementary activity whose function is required specifically for prophage integration.

Coliphages↗

PubMed1984

Evidence for inclusion of regions of nonhomology in heteroduplex products of bacteriophage lambda recombination.

Total intracellular DNA was isolated from replication-restricted bacteriophage lambda crosses in which the infecting parents were heteroallelic for wild-type and deletion mutant alleles. This DNA was examined for the presence of heteroduplex DNA molecules that contained wild-type sequences in one strand and deletion-mutant sequences in the other. Molecules hybrid for a 689-nucleotide deletion in the immunity region of lambda were detected at significant levels only in crosses in which both the red recombination system of lambda and the rec recombination system of Escherichia coli were active. Molecules hybrid for a 1300-nucleotide deletion in the central portion of the lambda genome were detected at significant levels in DNA isolated from both red+ and red- crosses in which recA function was present.

Bacteriophage lambda↗

PubMed1988

The Nul subunit of bacteriophage lambda terminase binds to specific sites in cos DNA.

The maturation and packaging of bacteriophage lambda DNA are under the control of the multifunctional viral terminase enzyme, which is composed of the protein products of Nu1 and A, the two most leftward genes of the phage chromosome. Terminase binds selectively to the cohesive end site (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12-base single-stranded cohesive ends of the mature phage genome. The purified gpNu1 subunit of terminase forms specific complexes with cos lambda DNA. DNase I footprinting experiments showed that gpNu1 bound to three distinct regions near the extreme left end of the lambda chromosome. These regions coincided with two 16-base-pair sequences (CTGTCGTTTCCTTTCT) that were in inverted orientation, as well as a truncated version of this sequence. Bear et al. (J. Virol. 52:966-972,1984) isolated a mutant phage which contained a CG to TA transition at the 10th position of the rightmost 16-base-pair sequence, and this phage (termed lambda cos 154) exhibits a defect in DNA maturation when it replicates in Escherichia coli which is deficient in integration host factor. Footprinting experiments with cos 154 DNA showed that gpNu1 could not bind to the site which contained the mutation but could protect the other two sites. Since the DNA-packaging specificity of terminase resides in the gpNu1 subunit, these studies suggest that terminase uses these three sites as recognition sequences for specific binding to cos lambda.

Bacteriophage lambda↗

PubMed2004

Initial cos cleavage of bacteriophage lambda concatemers requires proheads and gpFI in vivo.

The development of bacteriophage lambda and double-stranded DNA viruses in general involves the convergence of two separate pathways: DNA replication and head assembly. Clearly, packaging will proceed only if an empty capsid shell, the prohead, is present to receive the DNA, but genetic evidence suggests that proheads play another role in the packaging process. For example, lambda phages with an amber mutation in any head gene or in FI, the gene encoding the accessory packaging protein gpFI, are able to produce normal amounts of DNA concatemers but they are not cut, or matured, into unit length chromosomes for packaging. Similar observations have been made for herpes simplex 1 virus. In the case of lambda, a negative model proposes that in the amber phages, unassembled capsid components are inhibitory to maturation, and a positive model suggests that assembled proheads are required for cutting. We tested the negative model by using a deletion mutant devoid of all prohead genes and FI in an in vivo cos cleavage assay; in this deleted phage, the cohesive ends were not cut. When lambda proheads and gpFI were provided in vivo via a second prophage, cutting was restored, and gpFI was required, results that support the positive model. Phage 21 is a sister phage of lambda, and although its capsid proteins share approximately 60% residue identity with lambda's, phage 21 proheads did not restore cutting, even when provided with the accessory protein gpFI. Models for the role of proheads and gpFI in cos cutting are discussed.

Bacteriophage lambda↗

PubMed1994

Degradation of bacteriophage lambda deoxyribonucleic acid in vitro by sulfur mustard.

The degradation of bacteriophage lambda (lambda) deoxyribonucleic acid (DNA) by interaction with 0.1, 0.5 and 1 mM concentrations of sulfur mustard (SM) was investigated using agarose gel electrophoresis. Alkaline agarose gel electrophoresis also revealed single strand breaks at 0.5 and 0.1 mM concentrations of SM. The presence of magnesium ions in the reaction mixture prevented DNA degradation. It is proposed that the degradation of lambda DNA by its interaction with an excess of SM may be caused by the breakage of phosphodiester backbone of DNA via the formation of an intermediate phosphotriester bond.

Bacteriophage lambda↗