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PubMed1988

The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of lambda DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in lambda DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori lambda DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori lambda, however, later steps in lambda DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of lambda DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands.

Bacteriophage lambda↗
PubMed1978

Mutational analysis of the operators of bacteriophage lambda.

Oc mutations in the operators of bacteriophage lambda have been used to analyze the functional organization of the operators. In each operator, repressor binding sites 1 and 2, as identified biochemically, were found to be primarily responsible for the repressor affinity of the operators in vitro and for the repression of lytic functions in vivo. In addition, both sites were shown to be involved in the action of cro product at the operators. The data obtained have been used to estimate the repressor affinities of the individual binding sites. These affinities suggest that repressor bound at OR1 and OR2 interacts cooperatively. The results obtained support a model for repression of the early lambda operons where repressor bound at binding sites 1 and 2 interferes with RNA polymerase binding to the promotor sites.

Base Sequence↗
PubMed1978

Identification of the N gene protein of bacteriophage lambda.

The N gene protein, pN, of bacteriophage lambda stimulates early gene transcription by allowing mRNA chain elongation to proceed into genes distal to transcription termination sites normally recognized by the Escherichia coli transcription termination protein rho. pN has previously eluded detection on sodium dodecyl sulfate/polyacrylamide gels because of its small size, its instability, and the difficulty of distinguishing pN itself both from host proteins and from other early lambda proteins whose synthesis depends on pN action. These problems have now been overcome and we find that the major form of pN present in crude cell extracts of infected cells has an apparent molecular weight of 13,500. lambdabio256, a deletion-substitution mutant terminating in N, codes for a shorter pN of molecular weight 12,500. A nonsense fragment of 10,500 molecular weight coded by lambdaN(am7) has also been identified. These conclusions are based on examination of the electrophoretic profiles of the proteins synthesized after infection of UV-irradiated E. coli by various lambdaN(-) temperature-sensitive, nonsense, and deletion-substitution mutants. It has also been possible to distinguish pN itself from other early lambda polypeptides by infecting ron(-) cells with either lambdaN(mar) phage allowing pN synthesis but not pN action or lambdaN(am) phage defective in pN synthesis and pN action. Our results together with previous data are discussed with respect to the possible existence of multiple molecular weight forms of pN and the location of the coding sequences in the N gene region.

Coliphages↗
PubMed1978

Properties of the translocatable tetracycline-resistance element Tn10 in Escherichia coli and bacteriophage lambda.

A number of independent insertions into bacteriophage lambda of the translocatable tetracycline-resistance element Tn10 have been isolated and characterized. The physical positions and relative orientations of several such insertions were determined. Two independent insertions appear to lie in the same orientation at or very near the same site in the cI gene, and two more lie in opposite orientations at or near the same position in or near the rex gene. Insertions in or near genes cI, rex, and cIII have been characterized genetically for their effects on expression of nearby genes. Tn10 appears to exert a polar effect on expression of distal genes when it is inserted within an operon, even when expression of that operon is under the influence of lambda N-function. In addition, Tn10 insertions in rex appear to influence in some way expression of an "upstream" gene, cI. Lambda derivatives carrying Tn10 give rise to spontaneously occurring, tetracycline-sensitive deletions at high frequencies. It is likely that formation of these deletions is promoted in some way by the Tn10 element. Lambda::Tn10 phages carrying a Tn10 element that has undergone several successive cycles of translocation since its first isolation and characterization have been analyzed. The results confirm that Tn10 often retains its physical and functional integrity during many cycles of translocation. Lambda derivatives carrying Tn10 have been used to generate insertions of Tn10 in the chromosome of Escherichia coli. This process is independent of recA function, and seems to be quite analogous to the translocation of Tn10 in Salmonella typhimurium as studied previously.

Coliphages↗
PubMed1988

Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions.

Plasmids that express the bacteriophage lambda gam gene or the P22 abc2 gene (with and without abc1) at controllable levels were placed in Escherichia coli and tested for effects on the activity of RecBCD. Like Gam, Abc2 inhibited the ATP-dependent exonuclease activity of RecBCD, apparently not by binding to DNA. However, Abc2-mediated inhibition was partial, while Gam-mediated inhibition was complete. Both Abc2 and Gam inhibited host system-mediated homologous recombination in a Chi-containing interval in the chromosome of a hybrid lambda phage; Abc2 inhibited it more strongly than Gam. Gam but not Abc2 spared a phage T4 gene 2 mutant from restriction by RecBCD; Abc2 exhibited weak sparing activity in combination with Abc1 and substantial activity in combination with both Abc1 and P22 homologous recombination function Erf. Either Gam or the combination of the lambda recombination functions Exo and Bet was sufficient to induce a mode of plasmid replication that produced linear multimers. The combination of Abc2, Abc1, and Erf also exhibited this activity. However, Erf was inactive, both by itself and in combination with Abc1; Abc2 had weak activity. These results indicate that Gam and Abc2 modulate the activity of RecBCD in significantly different ways. In comparison with lambda Gam, P22 Abc2 has a weak effect on RecBCD nuclease activity but a strong effect on its recombination-promoting activity.

Bacteriophage lambda↗
PubMed1982

The cis-specificity of the Q-gene product of bacteriophage lambda.

A trp/lacW205 substitution, fused to the late region of bacteriophage lambda, provided a convenient assay for phage late gene expression in the presence or absence of lambda pQ. Comparison of lacZ expression from Q+ and Q- phages showed that late gene expression was markedly Q-dependent (263-fold difference). A cis/trans comparison of lambda pQ action showed a 180-fold difference in lacZ expression. The results suggest that pQ in only significantly active when supplied in cis to its site of action.

Bacteriophage lambda↗
PubMed2001

Rapid degradation of bacteriophage lambda O protein by ClpP/ClpX protease influences the lysis-versus-lysogenization decision of the phage under certain growth conditions of the host cells.

The initiator of bacteriophage lambda DNA replication, the O protein, is rapidly degraded in Escherichia coli by the ClpP/ClpX protease encoded by the host. Although the biochemical mechanism of this degradation has been investigated intensively, a physiological role for this process remained unknown since little effect of dysfunction of clpP and clpX genes on the lytic development of the phage was observed. Here we demonstrate that activities of clpP and clpX genes influence the lysis-versus-lysogenization decision of bacteriophage lambda under certain growth conditions of the host cells. This decision is influenced specifically by ClpP/ClpX-mediated O degradation and resultant inhibition of early lambda DNA replication because mutations in clpP and clpX genes have little effect on stability of other lambda proteins involved in the regulation of the phage developmental switch.

Adenosine Triphosphatases↗
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↗