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PubMed2005

Quantitative kinetic analysis of the bacteriophage lambda genetic network.

The lysis-lysogeny decision of bacteriophage lambda has been a paradigm for a developmental genetic network, which is composed of interlocked positive and negative feedback loops. This genetic network is capable of responding to environmental signals and to the number of infecting phages. An interplay between CI and Cro functions suggested a bistable switch model for the lysis-lysogeny decision. Here, we present a real-time picture of the execution of lytic and lysogenic pathways with unprecedented temporal resolution. We monitor, in vivo, both the level and function of the CII and Q gene regulators. These activators are cotranscribed yet control opposite developmental pathways. Conditions that favor the lysogenic response show severe delay and down-regulation of Q activity, in both CII-dependent and CII-independent ways. Whereas CII activity correlates with its protein level, Q shows a pronounced threshold before its function is observed. Our quantitative analyses suggest that by regulating CII and CIII, Cro plays a key role in the ability of the lambda genetic network to sense the difference between one and more than one phage particles infecting a cell. Thus, our results provide an improved framework to explain the longstanding puzzle of the decision process.

Bacteriophage lambda↗
PubMed1982

The Fi-gene product of bacteriophage lambda. Purification and properties.

The FI gene product of bacteriophage lambda has been purified extensively using a biochemical assay that measures assembly of lambda phage particles in vitro. The molecular weight of the native protein was estimated to be 21700 with an S20, w of 2.1 S and a Stokes radius of 2.5 nm. The molecular weight in dodecylsulfate was estimated to be 19000. The protein is highly acidic with an isoelectric point less than 4.1.

Bacteriophage lambda↗
PubMedDate not supplied

Recruitment of host ATP-dependent proteases by bacteriophage lambda.

Upon infection of a bacterial cell, the temperate bacteriophage lambda executes a regulated temporal program with two possible outcomes: (1) Cell lysis and virion production or (2) establishment of a dormant state, lysogeny, in which the phage genome (prophage) is integrated into the host chromosome. The prophage is replicated passively as part of the host chromosome until it is induced to resume the lytic cycle. In this review, we summarize the evidence that implicates every known ATP-dependent protease in the regulation of specific steps in the phage life cycle. The proteolysis of specific regulatory proteins appears to fine-tune phage gene expression. The bacteriophage utilizes multiple proteases to irreversibly inactivate specific regulators resulting in a temporally regulated program of gene expression. Evolutionary forces may have favored the utilization of overlapping protease specificities for differential proteolysis of phage regulators according to different phage life styles.

ATP-Dependent Proteases↗
PubMed1990

Integration of bacteriophage lambda into the cryptic lambdoid prophages of Escherichia coli.

Bacteriophage lambda missing its chromosomal attachment site will integrate into recA+ Escherichia coli K-12 and C at the sites of cryptic prophages. The specific regions in which these recombination events occur were identified in both lambda and the bacterial chromosomes. A NotI restriction site on the prophage allowed its physical mapping. This allowed us to identify the locations of Rac, Qin, and Qsr' cryptic prophages on the NotI map of E. coli K-12 and, by analogy, to identify the cryptic prophage in E. coli C as Qin. No new cryptic prophages were detected in E. coli K-12.

Bacteriophage lambda↗
PubMed1988

Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor.

Escherichia coli integration host factor (IHF) is a small basic protein that is required for efficient integrative recombination of bacteriophage lambda. IHF binds specifically to sequences within attP, the site in bacteriophage lambda that undergoes recombination. It has been suggested that the binding of IHF creates bends in DNA so as to help attP condense into a compact structure that is activated for recombination. In this work we show that IHF binding to either of two sites found within attP does indeed produce bending of DNA. In contrast, the other recombination protein needed for integrative recombination, Int, does not appreciably bend the DNA to which it is bound. In agreement with the proposal that IHF bending is important for creating a condensed attP, bending by IHF persists in the presence of bound Int. Our conclusions about protein-directed bends in DNA are based on the study of the electrophoretic mobility of a set of permuted DNA fragments in the presence or absence of IHF and/or Int. To facilitate this study, we have constructed a novel vector that simplifies the generation of permuted fragments. This vector should be useful in studying the bending of other DNA sequences by specific binding proteins.

Bacteriophage lambda↗
PubMed1983

Genetic recombination of bacteriophage lambda DNAs in Xenopus oocytes.

Pairs of genetically marked bacteriophage lambda DNAs have been injected into Xenopus laevis oocyte nuclei. After suitable incubation, DNA was recovered and packaged into phage particles in vitro. When these were plated onto a selective host, phage recombinant for parental markers were observed. Recombination was dependent on both parents being present in the same oocyte nucleus and was roughly proportional to the physical separation of the markers. Thus, the oocytes appear to contain the machinery necessary for performing typical genetic recombination. This system offers a great deal of scope and flexibility for future studies of recombination mechanisms at the molecular level in vertebrates.

Animals↗
PubMed2004

Distribution of minigenes in the bacteriophage lambda chromosome.

The bar loci in the chromosome of bacteriophage lambda inhibit phage vegetative growth in bacteria defective for peptidyl-tRNA hydrolase (Pth). Expression of the bar regions results in accumulation of peptidyl-tRNA, inhibition of protein synthesis, and arrest of mutant cell growth. These effects have been ascribed to the expression of two-codon ORFs present in translatable sequences named 'minigenes' in the lambda bar regions. To investigate the nature, frequency, and distribution of minigenes in the phage genome, we conducted a survey of their location in lambda DNA. A short-fragment random genomic DNA library was constructed for the identification of clones inhibitory of Pth-defective cells (bar-like phenotype). Three new bar-like minigenes were identified in the library but only one was on the sense strand and it had a rare initiation codon. This result contrasted with the in silico identification of over a hundred putative minigenes using an ad hoc computer program on both strands of lambda DNA. Unlike bar constructs, most of the toxic constructed clones were also toxic to wild-type bacteria, thus suggesting a different inhibition mechanism. Sequence analysis of these cloned inserts showed that they harbored minigenes, mini-ORFs, gene starts, gene ends, or combinations thereof. Our data suggest that minigene-like sequences may, at least partly, account for toxicity in wild-type cells. We propose that clustering of minigenes at gene ends may play a role in gene expression. Other minigenes identified in silico were non-toxic. It is still an open question what the in vivo function of these and toxic minigenes might be.

Bacteriophage lambda↗
PubMed1989

Alternative mRNA structures of the cIII gene of bacteriophage lambda determine the rate of its translation initiation.

The bacteriophage lambda cIII gene product has a regulatory function in the lysis-lysogeny decision following infection. The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA. We demonstrate, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium. Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30 S ribosomal subunit. Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A). In this structure, the translation initiation region is occluded, thereby preventing 30 S ribosomal subunit binding. By varying the temperature or Mg2+ concentration it was possible to alter the relative proportion of the alternative structures in wild-type mRNA. We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression.

Bacteriophage lambda↗
PubMed1998

Analysis of higher order intermediates and synapsis in the bent-L pathway of bacteriophage lambda site-specific recombination.

The integrase protein of bacteriophage lambda mediates recombination via four distinct pathways. The recent in vitro reconstitution of the efficient bidirectional reaction between two attLtenP'1 target sites now allows comparisons of this pathway, known as the bent-L pathway, with the inefficient bidirectional straight-L pathway and with the efficient but unidirectional pathways of integration and excision. To this end, a series of higher order intermediates of the bent-L pathway was characterized using gel mobility shift assays, two-dimensional gel analysis, and footprinting. The analysis spans the initial binding of proteins to individual DNA target sites, synapsis of two partner DNA targets, and strand exchange. This study identifies a presynaptic "checkpoint" of recombination. It shows that synapsis is a slow step in the recombination reaction, while subsequent strand exchange is comparatively fast. Synaptic complexes contain a preponderance of recombinant products, suggesting that an energetically favorable but somewhat subtle conformational change drives strand exchange. In addition, comparison of wild-type integrase with a catalytically defective mutant of integrase, IntF, showed that, in addition to the catalysis defect, this mutant has different DNA-binding properties than the wild-type protein.

Bacteriophage lambda↗
PubMed1980

Integrative recombination of bacteriophage lambda: extent of the DNA sequence involved in attachment site function.

We have investigated the minimum extent of DNA sequence required for the attachment site of bacteriophage lambda to function in integrative recombination. A DNA fragment carrying the phage attachment site (attP) of bacteriophage lambda was trimmed, recloned, and tested for recombination proficiency. In order to recombine with the bacterial attachment site (attB), the phage attachment site must retain about 250 base pairs of its original sequence. On the left side, the essential sequence extends beyond 106 base pairs from the center of the 15-base-pair common core sequence but not beyond 152 base pairs. On the right side the required sequence extends beyond 68 base pairs but not beyond 99 base pairs from the center of the core. A trimmed site that has lost part of the sequence mentioned above cannot function as the phage attachment site. However, depending on which part of the sequence is present, such a site can still act in reactions normally requiring one of the prophage attachment sites or the bacterial attachment site. The results also suggest that the essential suquence of the bacterial attachment site consists only of the sequence common to the phage and bacterial attachment sites.

Attachment Sites, Microbiological↗
PubMed1987

Activated RecA protein may induce expression of a gene that is not controlled by the LexA repressor and whose function is required for mutagenesis and repair of UV-irradiated bacteriophage lambda.

The activated form of the RecA protein (RecA) is known to be involved in the reactivation and mutagenesis of UV-irradiated bacteriophage lambda and in the expression of the SOS response in Escherichia coli K-12. The expression of the SOS response requires cleavage of the LexA repressor by RecA and the subsequent expression of LexA-controlled genes. The evidence presented here suggests that RecA induces the expression of a gene(s) that is not under LexA control and that is also necessary for maximal repair and mutagenesis of damaged phage. This conclusion is based on the chloramphenicol sensitivity of RecA -dependent repair and mutagenesis of damaged bacteriophage lambda in lexA(Def) hosts.

Bacterial Proteins↗
PubMed1966

Intracellular forms of lambda deoxyribonucleic acid in Escherichia coli infected with clear or virulent mutants of bacteriophage lambda.

Weissbach, Arthur (National Institutes of Health, Bethesda, Md.), Allan Lipton, and Arnold Lisio. Intracellular forms of lambda deoxyribonucleic acid in Escherichia coli infected with clear or virulent mutants of bacteriophage lambda. J. Bacteriol. 91:1489-1493. 1966.-Infection of either the sensitive or lysogenic strain of Escherichia coli K-112S by lambda(+) leads to the formation of a new phage deoxyribonucleic acid (DNA) species having the properties of a twisted circular DNA duplex. This new phage DNA species is also seen in cells infected with clear or virulent mutants of lambda which cannot lysogenize, or do so at a low frequency. The sedimentation rate of circular lambda DNA duplex at various pH values and its lability were examined.

Carbon Isotopes↗
PubMed2001

Bacteriophage lambda DNA packaging: DNA site requirements for termination and processivity.

Bacteriophage lambda chromosomes are processively packaged into preformed shells, using end-to-end multimers of intracellular viral DNA as the packaging substate. A 200 bp long DNA segment, cos, contains all the sequences needed for DNA packaging. The work reported here shows that efficient DNA packaging termination requires cos's I2 segment, in addition to the required termination subsite, cosQ, and the nicking site, cosN. Efficient processivity requires cosB, in addition to cosQ and cosN. An initiation-defective mutant form of cosB sponsored efficient processivity, indicating that the terminase-cosB interactions required for termination are less stringent than those required at initiation. The finding that an initiation-defective form of cosB is functional for processivity allows a re-interpretation of a similar finding, obtained previously, that the initiation-defective cosB of phage 21 is functional for processivity by the lambda packaging machinery. The cosBphi21 result can now be interpreted as indicating that interactions between cosBphi21 and lambda terminase, while insufficient for initiation, function for processivity.

Bacteriophage lambda↗
PubMed1971

The thermal inactivation of T4 and lambda bacteriophage.

Thermal inactivation of T(4) and lambda bacteriophage shows two components of differing sensitivity are present. These cannot be interpreted as owing to nucleic acid and protein. One protein function-the inhibition of radiation-induced DNA degradation-is lost with quite different thermal kinetics. lambda heated in the presence of DNase is more rapidly inactivated; lambda is also protected by slow cooling after heat. These results suggest that the packing of the DNA in the head occurs so as to permit different degrees of thermal expansion in the outer coils. These can rupture the coat and this is one form of inactivation. Killed vaccines could be more safely made by heating in the presence of a nuclease followed by rapid cooling.

Coliphages↗
PubMed1976

Red system of bacteriophage lambda complements the growth of a bacteriophage T1 gene 4 mutant.

The ability of phage lambda to complement the growth of T1am23, a T1 gene 4 mutant with a DNA arrest phenotype, has been shown to require both lambda Red functions, redX and redB. lambdagam function, however, is not required. Therefore, the lambda Red function can substitute for T1 gene 4 function. However, T1+ does not substitute for lambda Red in allowing lambda to grow in a polA host.

Chromosome Mapping↗
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↗
PubMed2006

Interaction of bacteriophage lambda with its cell surface receptor: an in vitro study of binding of the viral tail protein gpJ to LamB (Maltoporin).

The cell surface receptor for bacteriophage Lambda is LamB (maltoporin). Responsible for phage binding to LamB is the C-terminal part, gpJ, of phage tail protein J. To study the interaction between LamB and gpJ, a chimera protein composed of maltose binding protein (MBP or MalE) connected to the C-terminal part of J (gpJ, amino acids 684-1131) of phage tail protein J of bacteriophage Lambda was expressed in Escherichia coli and purified to homogeneity. The interaction of the MBP-gpJ chimera protein with reconstituted LamB and its mutants LamB Y118G and the loop deletion mutant LamB Delta4+Delta6+Delta9v was studied using planar lipid bilayer membranes on a single-channel and multichannel level. Titration with the MBP-gpJ chimera blocked completely the ion current through reconstituted LamB when it was added to the cis side, the extracellular side of LamB with a half-saturation constant of approximately 6 nM in 1 M KCl. Control experiments with LamB Delta4+Delta6+Delta9v from which all major external loops had been removed showed similar blocking, whereas MBP alone caused no visible effect. Direct conductance measurement with His(6)-gpJ that contained a hexahistidyl tag (His(6) tag) at the N-terminal end of the protein for easy purification revealed no blocking of the ion current, requiring other measurements for the binding constant. However, when maltoporin was preincubated with His-gpJ, MBP-gpJ could not block the channel, which indicated that also His(6)-gpJ bound to the channel. High-molecular mass bands on SDS-PAGE and Western blots, confirming the planar lipid bilayer experiment results, also demonstrated stable complex formation between His(6)-gpJ and LamB or LamB mutants. The results revealed that phage Lambda binding includes not only the extracellular loops.

Bacterial Outer Membrane Proteins↗