Lysogenization by bacteriophage lambda: II. - Identification of genes involved in the multiplicity dependent processes

We previously showed that, under conditions of rapid exponential growth, lysogenization of E. coli cells by phage λ requires that the cell is infected by at least 2 phages able to replicate their DNA, or 3 or 4 phages unable to replicate their DNA [ref. 4]. Since genes dealing with prophage integration appear not to be involved in these multiplicity dependent processes, a determination was made as to whether more than one copy of the genes involved in repressor synthesis or its activation are needed for lysogenization. The complementation patterns which we obtained indicate multiplicity effects involving gene cII (and, perhaps, cIII) in lysogenization by both phage able or unable to replicate. In the former case, we propose that cII protein (and, perhaps, cIII) both induces repressor synthesis and inhibits phage DNA replication. In lysogenization by phage unable to replicate, the data suggest that the expression of early phage genes and repressor synthesis in the course of lysogenization are mutually exclusive processes which do not take place on the same phage chromosome.     

Lysogenization by bacteriophage lambda IV inhibition of phage DNA synthesis by the products of genes cII and cIII

In direct measurements of phage λ DNA synthesis, we have detected an inhibition caused by the cII and cIII gene products. This inhibition was more clearly observed when P amber phages were grown in a permissive host, presumably because of the limitation in DNA synthesis due to uncomplete suppression. The inhibition takes place in cells infected at high multiplicity, but not in cells infected at low multiplicity. To explain these findings, we propose a model in which the bacterial population is heterogeneous with respect to its ability to support phage DNA synthesis. An initial limitation caused by host factors would be amplified by the action of the cII and cIII products, at high multiplicity only, and the resulting inhibition would be essential in the « choicetowards lysogeny.     

Lysogenization by bacteriophage lambda

Summary An easy and sensitive way of measuring the proportion of E. coli cells which are lysogenized by lambda phage or lambda mutants has been devised. With this assay it was possible to analyse the lysogenic response as a function of the average phage input per cell. The results indicate that lysogenization of exponentially growing cells requires that the cells are infected by at least two phages able to replicate, or three or four phages unable to replicate.     

Host interference with expression of the lambda N gene product

We have searched among E. coli M72 (D, bio11cI857H1) temperature resistant survivors and have found two bacterial mutants, gro100 and gro101 which block λiλ and λi⁴³⁴ phage development but allow growth of their N-independent derivatives λiλ nin and λi⁴³⁴nin. It is not known yet whether these two mutants interfere with the production of the N gene product or with its function. At least part of the gro genotype maps at 12′ of the E. coli genetic map and is co-transductible by Pl with the lac locus.     

Promiscuous mismatch extension by human DNA polymerase lambda

DNA polymerase lambda (Pol λ) is one of several DNA polymerases suggested to participate in base excision repair (BER), in repair of broken DNA ends and in translesion synthesis. It has been proposed that the nature of the DNA intermediates partly determines which polymerase is used for a particular repair reaction. To test this hypothesis, here we examine the ability of human Pol λ to extend mismatched primer-termini, either on ‘open’ template-primer substrates, or on its preferred substrate, a 1 nt gapped-DNA molecule having a 5′-phosphate. Interestingly, Pol λ extended mismatches with an average efficiency of ≈10⁻² relative to matched base pairs. The match and mismatch extension catalytic efficiencies obtained on gapped molecules were ≈260-fold higher than on template-primer molecules. A crystal structure of Pol λ in complex with a single-nucleotide gap containing a dG·dGMP mismatch at the primer-terminus (2.40 Å) suggests that, at least for certain mispairs, Pol λ is unable to differentiate between matched and mismatched termini during the DNA binding step, thus accounting for the relatively high efficiency of mismatch extension. This property of Pol λ suggests a potential role as a ‘mismatch extender’ during non-homologous end joining (NHEJ), and possibly during translesion synthesis.     

A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda

Bacteriophage lambda (λ) permits the display of many foreign peptides and proteins on the gpD major coat protein. However, some recombinant derivatives of gpD are incompatible with the assembly of stable phage particles. This presents a limitation to current λ display systems. Here we describe a novel, plasmid-based expression system in which gpD deficient λ lysogens can be co-complemented with both wild-type and recombinant forms of gpD. This dual expression system permits the generation of mosaic phage particles that contain otherwise recalcitrant recombinant gpD fusion proteins. Overall, this improved gpD display system is expected to permit the expression of a wide variety of peptides and proteins on the surface of bacteriophage λ and to facilitate the use of modified λ phage vectors in mammalian gene transfer applications.     

The production of generalized transducing phage by bacteriophage lambda

Generalized tranduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage λ to assess some of the factors that affect that process. The results of those experiments indicate: (1) the production of generalized transducing particles by bacteriophage λ is inhibited by the phage λ exonuclease (Exo). Also inhibited by λ Exo is the production of λdocR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of λdocL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by λ Exo; (2) λ-generalized transducing particles are not detected in induced lysis-defective (S⁻) λ lysogens until about 60–90 min after prophage induction. Since wild-type λ would normally lyse cells by 60 min, the production of λ-generalized transducing particles depends on the phage being lysis-defective; (3) if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10–20-fold range. In contrast, if transducing lysates are prepared by the induction of a λ lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers ; (4) if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; (5) measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the amount of prophage-proximal bacterial DNA in the cell. Measurements of DNA in transducing particles indicate that the rest of the increase is probably due to the preferential packaging of the prophage-proximal bacterial DNA.

These results are most easily interpreted in terms of a model for the initiation of bacterial DNA packaging by λ, in which the proteins involved (Ter) do not recognize any particular sequence in bacterial DNA but rather     

The enzymatic basis of processivity in lambda exonuclease

λ Exonuclease is a highly processive 5′→3′ exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by λ exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of λ biology, λ exonuclease enzymology and the availability of the X-ray crystallographic structure of λ exonuclease make it a suitable model to dissect the mechanisms of processivity. λ Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in λ exonuclease involved in recognizing the 5′-phosphate at the ends of broken dsDNA. The preference of λ exonuclease for a phosphate moiety at 5′ dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5′-phosphate is due to the formation of inert enzyme–substrate complexes. By examining a λ exonuclease mutant impaired in 5′-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of λ exonuclease has been elucidated. We propose a model in which processivity of λ exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.     

Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCλ in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKCλ (λKD or λΔNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKCλ, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by λKD or λΔNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKCλ was ~50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKCλ mutant that lacks the pseudosubstrate domain (λΔPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of λΔPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Rapid restriction mapping of DNA cloned in lambda phage vectors

A protocol for the rapid restriction mapping of phage λ clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive λ DNA termini by hybridisation with [³²P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the “ladder” of partial digestion products.     

Spermidine biases the resolution of Holliday junctions by phage lambda integrase

Holliday junctions are a central intermediate in diverse pathways of DNA repair and recombination. The isomerization of a junction determines the directionality of the recombination event. Previous studies have shown that the identity of the central sequence of the junction may favor one of the two isomers, in turn controlling the direction of the pathway. Here we demonstrate that, in the absence of DNA sequence-mediated isomer preference, polycations are the major contributor to biasing strand cleavage during junction resolution. In the case of wild-type phage λ excision junctions, spermidine plays the dominant role in controlling the isomerization state of the junction and increases the rate of junction resolution. Spermidine also counteracts the sequence-imposed bias on resolution. The spermidine-induced bias is seen equally on supercoiled and linear excisive recombination junction intermediates, and thus is not just an artefact of in vitro recombination conditions. The contribution of spermidine requires the presence of accessory factors, and results in the repositioning of Int's core-binding domains on junctions, perhaps due to DNA-spermidine–protein interactions, or by influencing DNA conformation in the core region. Our results lead us to propose that spermidine together with accessory factors promotes the formation of the second junction isomer. We propose that this rearrangement triggers the activation of the second pair of Int active sites necessary to resolve Holliday junctions during phage λ Int-mediated recombination.     

Tolerance for 8-oxoguanine but not thymine glycol in alignment-based gap filling of partially complementary double-strand break ends by DNA polymerase lambda in human nuclear extracts

Ionizing radiation induces various clustered DNA lesions, including double-strand breaks (DSBs) accompanied by nearby oxidative base damage. Previous work showed that, in HeLa nuclear extracts, DSBs with partially complementary 3′ overhangs and a one-base gap in each strand are accurately rejoined, with the gaps being filled by DNA polymerase λ. To determine the possible effect of oxidative base damage on this process, plasmid substrates were constructed containing overhangs with 8-oxoguanine or thymine glycol in base-pairing positions of 3-base (-ACG or -GTA) 3′ overhangs. In this context, 8-oxoguanine was well tolerated by the end-joining machinery when present at one end of the break, but not when present at both ends. Thymine glycol was less well tolerated than 8-oxoguanine, reducing gap filling and accurate rejoining by at least 10-fold. The results suggest that complex DSBs can be accurately rejoined despite the presence of accompanying base damage, but that nonplanar bases constitute a major barrier to this process and promote error-prone joining. A chimeric DNA polymerase, in which the catalytic domain of polymerase λ was replaced with that of polymerase β, could not substitute for polymerase λ in these assays, suggesting that this domain is specifically adapted for gap filling on aligned DSB ends.     

Analysis of cosmids using linearization by phage lambda terminase

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage λ terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested' with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage λ (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.     

Protein Components of Bacteriophages λ and λ Virulent

Parallel studies have been made of the protein coats of the temperate bacteriophage λ and of a deletion mutant, λ virulent. A new method for preparing ghosts of both phages by the action of Cu⁺⁺ is described. Protein ghosts of both phages can be dissolved in citrate at pH values below 3, more rapidly in the presence of 8 m urea. Both phages yielded three apparently identical protein components which can be separated by thin-layer gel filtration and thin-layer gel electrophoresis. The protein of molecular weight 47,000 ± 1,500 represents about 55% of the protein of the ghosts and is therefore likely to be the subunit of the head. The other proteins of molecular weight 30,000 ± 1,500 and 16,000 ± 1,500 represent approximately 25% and 20% of the protein, respectively. Amino acid analyses of the ghosts from the two phages have been carried out and show no significant differences. The buoyant density of phage λ virulent is 0.016 g/ml less than that of λ. Since no differences have been found in the protein components of the two phages, this indicates that the virulent mutant contains approximately 16% less deoxyribonucleic acid than the temperate phage.     

The maturation of coliphage lambda DNA in the absence of its packaging

In vivo, λ DNA cannot be cleaved at cos (matured) if proheads are not present; in vitro, however, cos cleavage readily takes place in the absence of proheads. In order to investigate this paradox, we have constructed plasmids that synthesize λ terminase in vivo upon induction. The plasmids also contain cos at the normal position, about 190 bp upstream of λ gene Nul. One of the plasmids, pFM3, produces levels of terminase comparable to those found after phage induction. If cells carrying pFM3 are thermoinduced, almost 100% of the intracellular plasmid DNA has a double-strand interruption at or near cos.

Since the only λ genes that pFM3 carries are Nul, A, W and B, this in vivo cleavage is occurring in the absence of proheads. Previous failure to observe 2 maturation with phages carrying prohead mutations may be due to exonucleolytic degradation of the unprotected DNA ends, a different DNA topology or compartmentalization, or terminase inhibition in the absence of prohead by the product of another λ gene that maps to the right of gene B.     

Deoxyribonucleic Acid Replication in λ Bacteriophage Mutants

After ultraviolet light induction of Escherichia coli K-12 strain W3350(λ), several structural intermediate forms of phage deoxyribonucleic acid (DNA) are synthesized. The early defective lysogens of λ, sus O₈, sus P₃, and T₁₁, were found to synthesize none of the DNA structural intermediates. A lysogen believed to be defective in all known phage activities, λsus N₇, was found to be able to synthesize an early phage DNA intermediate. The lysogen λsus Q₂₁, defective in late phage functions, is able to synthesize the early phage DNA intermediate and a concatenated molecule of greater molecular weight than the mature λ DNA.     

Some Properties of DNA from Phage-Infected Bacteria

Replicating T5 or λ phage DNA has been labeled by adding tritiated thymidine for short periods to cultures of phage-infected Escherichia coli before isolation of intracellular DNA. Two procedures are described for separating T5 replicating DNA from DNA of intracellular phage particles. Both T5 and λ replicating DNA had the same bouyant density in cesium chloride as DNA from phage particles but sedimented faster when centrifuged in sucrose density gradients. The fast sedimentation did not appear to be caused by DNA protein or DNA-RNA complexes or by aggregation of DNA, but is probably due to DNA molecules of unusual structure. Experiments involving hydrodynamic shear and sucrose density gradient centrifugation at alkaline pH have suggested that with λ the replicating form of DNA is a linear molecule considerably longer than the DNA molecules of λ-phage particles. The constituent polynucleotide chains of λ but not T5 replicating DNA also appear to be longer than those of phage DNA.