Packaging of coliphage lambda DNA. I. The role of the cohesive end site and the gene A protein

The cohesive ends of the DNA of bacteriophage λ particles are normally formed by the action of a nuclease on the cohesive end sites (cos) of concatemeric λ DNA (reviewed by Hohn et al., 1977). The nuclease also cuts the cos site of an integrated prophage, and DNA located to the right is preferentially packaged into phage particles. This process occurs with approximately the same efficiency and rate in a single lysogen as in a tandem polylysogen. Thus, the rate of cos cutting does not increase when the number of cos sites per molecule increases, an hypothesis that has been proposed to explain why cohesive ends are not formed in circular monomers of λ DNA. We propose instead that the interaction of Ter with cos is influenced by the configuration of the DNA outside of cos during packaging, and that this configuration is different for circular monomers than for other forms of λ DNA. A model that gives rise to such a difference is described.We also found that missense mutations in the λ A gene changed the efficiency of packaging of phage relative to host DNA. This was not the case for missense mutations in several phage genes required for capsid formation. Thus, the product of gene A plays a role in determining packaging specificity, as expected if it is or is part of the nuclease that cuts λ DNA at cos.     

Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). II. The propagation of phage lambda

In the accompanying paper (Sternberg, 1973) the properties of three independently isolated strains of Escherichia coli with groE mutations (NS-1, NS-2 and NS-3) have been characterized. In this report the ability of these strains to propagate phage λ is examined in greater detail. In the temperature -sensitive groE strain NS-1, all early phage functions tested (curing, infective center formation, DNA synthesis and early messenger RNA synthesis) are expressed normally. In addition, two late phage functions (late mRNA synthesis and tail formation) are also expressed normally, and a third, phage-induced cell lysis, is expressed with only a slight delay. Based upon head-tail in vitro complementation assays, however, λ fails to make any functional heads at elevated temperatures (41 °C) in this host. Electron microscopic studies of strain NS-1 defective lysates indicate that aberrant head-like forms, including tubular forms and “monsters,” are made.Mutants of λ, designated λEP, which are able to grow in the three groE strains, have been isolated. An analysis of these mutants indicates that at least some carry a mutation in λ head gene E and these make reduced levels of active gene E protein in groE hosts.A further study of all known λ head genes indicates that it is the interaction between the gene E protein and the proteins specified by head genes B and C that is adversely affected by the groE mutation. Presumably, the relative level of gene E protein is too high in groE strains for proper head formation. The λEP mutation compensates for this effect by reducing the level of this protein, and so restoring a balance.     

Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

Structural features of lambda site-specific recombination at a secondary att site in galT.

Integration of bacteriophage λ DNA into the chromosome of its E. coli host proceeds via a site-specific recombination between specific loci (att sites) on the phage and bacterial chromosomes. Infection of an E. coli host deleted for the primary bacterial att site results in λ integration with reduced efficiency at a number of different “secondary att sites” scattered around the E. coli chromosome. The first DNA sequence analysis of such a secondary att site, that occurring in the galT gene, is reported here, and several features pertinent to the mechanism of int-dependent site-specific recombination are discussed.Previous studies have shown that the crossover in int-dependent recombination must be somewhere within a 15 bp sequence (core region) common to the phage and primary bacterial att sites, as well as to the left and right prophage att sites which are at the junctures between prophage and host DNA. Comparison of the galT secondary prophage att sites with the primary prophage att sites allows determination of the analogous “core” region in the galT secondary att site. The 15 bp sequence thus identified shows an interrupted homology (8 out of 15) with the wild-type core. The extent and arrangement of nonhomologous bases allow precise placement of the crossover point for this recombination to the +4–+5 internucleotide bond of the core region.Sequences flanking the core region show no obvious homology with analogous sequences of the phage or primary bacterial att sites. Comparison of the galT left prophage att site with the analogous wild-type site is of particular interest and is discussed in relation to binding studies with purified int protein.     

Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

Prophage lambda at unusual chromosomal locations: III. The components of the secondary attachment sites

The integration of phage λ occurs by a reciprocal genetic exchange, promoted by the product of phage int gene, at specific sites on the phage and bacterial genomes (att's). Lysogenic bacteria thus contain two att's which bracket the inserted prophage. Genetically, the phage, bacterial and prophage att's differ from each other, indicating that each site has specific elements which segregate during recombination.In hosts that lack the bacterial att, phage integration occurs at about 0.5% the normal frequency. It results from Int-promoted recombination between the phage att and any one of many secondary sites in the bacterial genome. To analyze these sites, we measured Int-promoted recombination at the secondary prophage att's. We found that they differed from the normal prophage att's and from the phage att. The secondary sites, therefore, do not appear to carry any of the specific elements of the phage or bacterial att's.The transducing phage isolated from secondary site lysogens integrate at two loci. In the absence of helper, they insert via homology with the bacterial DNA. Co-infection with helper results in their integration at the normal bacterial att.     

Restriction of lambda trp bacteriophages by Escherichia coli K

trp-transducing derivatives of phage λ have been used to study Escherichia coli K specific restriction in vivo. The expression of the trp genes from unmodified phages during infection of a rec⁺, restricting host is eliminated by restriction. In a K-restricting recB,C host, where degradation of restricted phage DNA is prevented, expression of the trp genes is little affected by the presence of a single unmodified, K-restriction recognition site, even when that site is within the trpE gene. RI restriction, in contrast to K restriction, prevents trp gene expression in a recB,C host when the restriction target is between the trp genes and the relevant promoter. The presence of two K-restriction recognition sites in a λtrp phage can have a marked effect on trp gene expression. This effect can be interpreted as the result of preferential breakage between the two restriction recognition sites. We conclude that K restriction does not break susceptible DNA at, or even preferentially near, a restriction recognition sequence.     

int-h: An int mutation of phage lambda that enhances site-specific recombination

The mutation int-h3 maps in the int gene of coliphage λ and results in the synthesis of an integrase with enhanced activity, which is manifested by an ability to support λ site-specific recombination relatively efficiently under conditions where the wild-type integrase functions inefficiently. The level of site-specific recombination seen in the presence of the int⁺ integrase in himA^? hosts is greatly reduced, as measured by lysogen formation, intramolecular site-specific integration and excision, and excision of a cryptic λ prophage. In contrast, the int-h3 integrase shows relatively high levels of activities under these conditions. Int-h3 is also more active in other host mutants (himB and hip) that reduce λ site-specific recombination. In the absence of the normal attB site, the frequency of lysogen formation (at secondary sites) by λ int⁺ is reduced 200 fold. Although λ int-h3 will integrate preferentially at the attB site if it is present, the mutant phage forms lysogens at a high frequency in attB-deleted hosts. λ int-h3 requires himA function for integration at secondary sites. The fact that the int-h3 integrase uses the same att sites as well as the same host functions as the int⁺ integrase suggests that the mutation results in a quantitative rather than a qualitative change in integrase activity; that is, the int-h3 integrase is more active. The mutant integrase supports site-specific recombination with att sites that carry the att24 mutation. We propose that the int-h3 integrase is endowed with an enhanced ability to recognize att sequences, including some that are not effectively recognized by wild-type integrase.     

Bacteriophage P2-lambda interference. III. Essential role of an early step in the initiation of lambda replication.

Induction of bacteriophage λ in the presence of a P2 prophage results in inactivation of cellular transfer RNA, inhibition of amino acid and uridine incorporation in the host, as well as inhibition of phage replication. A red gam double mutation allows λ to escape from interference, and a mutation in gene O or P abolishes the effects on the host.It is shown here that phage and plasmid DNA extracted from cells undergoing P2-λ interference are still active in a transfection assay. Mutations in bacterial gene dna B or in phage site ori suppress the inhibition of amino acid incorporation, whereas genes dnaE and dna G have no such effect. Derepression of bacterial exonuclease VIII totally suppresses the interference, and mutations in genes recA and lexA, which control the SOS functions, suppress it partially if the λ phage is red⁺. Our results suggest that P2-λ interference is due to the action of old at an early step of the initiation of λ replication.     

Physical study of prophage excision and curing of lambda prophage from lysogenic Escherichia coli

A sex factor, F′450(λ), which can be isolated as a covalent circle of DNA, has been examined by alkaline sucrose gradient centrifugation of lysates of induced cells in order to study λ prophage excision. Thermal derepression of the prophage results in loss of F′450(λ) covalent circles, which is mediated by systems involved in excision and initiation of replication. When protocols known to result in prophage curing are used, the F′450(λ) is converted to an F′450 and a λ covalent circle; in normal excision leading to phage development, F′450 covalent circles are not found. We have shown that: (1) excision usually occurs later than initiation of DNA replication of the prophage so that the excised prophage is usually already replicated or in the act of replication; (2) the DNA growing points of the prophage leave the prophage and enter the bacterial DNA; (3) the int and xis genes are involved in the earliest detectable stage of the excision process, i.e. breakage of the DNA at the attachment region; (4) the xis gene product is involved in a weak non-specific nuclease activity in addition to its highly specific activity in excision; and (5) the excision system fails to attack a single attachment site.     

Cellular levels of the prophage lambda and 434 repressors.

As a prerequisite to a quantitative study of the inactivation of phage repressors in vivo (Bailone et al., 1979), the cellular concentrations of the bacteriophage λ and 434 repressors have been measured in bacteria with varying repressor levels.Using the DNA-binding assay we have determined the conditions for optimal repressor titration. The sensitivity of the λ repressor assay was increased by adding magnesium ions to the binding mixture; this procedure was without effect on the titration of the 434 repressor. The measures of the cellular repressor concentrations varied with the method of cell disruption.The cellular concentration of λ repressor, about 140 active repressor molecules per monolysogen, was relatively constant under specific cultural conditions. The repressor concentration increased with the number of cI gene copies but not in direct proportion.The 434 repressor concentration, hardly detectable in extracts of lysogens carrying an imm434 prophage, was greatly enhanced in bacteria carrying the newly constructed plasmid pGY101, that encodes the 434 cI gene.The cellular repressor level produced by 434 is lower than that produced by λ: this indicates that the maintenance of the prophage state is ensured by a relatively small number of repressor molecules binding tightly to the operator sites.     

Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). I. Initial characterization

Three mutant strains of Escherichia coli were independently isolated based upon their inability to propagate bacteriophage λ. The strain most extensively studied, NS-1, has a pleiotropic temperature sensitive alteration that affects cell growth, stable RNA synthesis and λ propagation. Labeling experiments and colorimetric determinations of total RNA carried out in this strain demonstrate that within the first five minutes after raising the temperature to 44.5 °C the rate of total RNA accumulation is reduced to a level that is about 15% that of the control, while protein and DNA synthesis continue at nearly normal rates for at least 30 min. This effect is either due to a very rapid degradation of stable RNA species or a reduced synthesis of RNA. Although the accumulation of all stable RNA species (23, 16 and 4 S RNAs) is reduced co-ordinately to levels ranging from 12 to 16% that of the control, the synthesis of messenger RNA is affected to a lesser degree, if at all. The defect in RNA accumulation can be partially reversed by the addition of chloramphenicol at the moment of temperature shift.In addition to phage λ these strains are unable to propagate RNA phage R17 and lambdoid phages φ80, 21 and 434 at elevated temperatures. The growth of phages T4, T7, P1 and P2 is normal.A genetic analysis of strain NS-1 indicates that all of its temperature sensitive properties depend on a mutation, designated groE-1, which co-transduces with a mel (melibiose) marker. However, the expression of the RNA synthesis defect requires, in addition, a second mutation which does not co-transduce with mel.     

Deletion mutants of bacteriophage lambda : III. Physical structure of att

Deletion mutants and substitution mutants of bacteriophage λ have been used to examine the physical structure of the att^φ site in the λ chromosome which is essential for prophage integration. Integration-defective att mutants were analyzed by constructing heteroduplex DNA molecules containing one wild-type and one mutant strand and examining these heteroduplexes by electron microscopy. The results indicate that att^φ is less than 2500 base pairs in length and may be as small as 20 to 50 base pairs. Integration cross-overs between att^φ and its bacterial analog, att^B, occur within a region which is less than 12 base pairs in length. Moreover, there is no detectable base sequence homology between att^φ and att^B. These results suggest that λ integration is a truly unique system of recombination.     

Replication of bacteriophage lambda DNA. Examination of variants containing double origins and observation of a bias in directionality

Bacteriophage λ variants have been constructed that possess two λ ori sites. Replicative intermediates resulting from infection with these phages have been investigated. We find that initiation of replication from the ori site on an EcoRI fragment (containing all the DNA sequences from within the red gene to the middle of gene O) cloned in the inverted orientation is predominantly bidirectional but occurs at a decreased frequency. Double initiations were observed at low frequency. However, a second cloned ori fragment (carrying two large deletions and a small insertion) cloned in the normal orientation demonstrated insignificant levels of replication from the cloned site unless the normal ori had already initiated.A bias in directionality of λ replication has been observed. Molecules that replicate unidirectionally propagate to the right more often than to the left. If the cloned ori-containing EcoRI fragment is inserted with reversed polarity, then the bias is towards the left. Bidirectional λ replicative intermediates also appear to show a similar bias but this is superimposed on a large, apparently random, effect that results in asymmetric growing-point propagation.     

Gross map location of Escherichia coli transfer RNA genes.

Chromosomal locations of Escherichia coli genes specifying more than 20 different transfer RNA species were determined by utilizing two different methods. One was based upon gene dosage effects caused by F′ factors. In 15 different F′ strains and their corresponding F^? strains, relative contents of individual tRNAs were measured after separating the tRNAs by two-dimensional polyacrylamide gel electrophoresis. Approximate doubling of the content of particular tRNA was found in individual F′ strains, as showing gross map location of the tRNA gene. The other method was based on the amplified synthesis of tRNAs occurring after prophage induction of λ lysogens. Synthesis of individual tRNAs was measured after the induction of λ phages integrated at five different bacterial sites. Characteristic overproduction of different tRNAs was observed in individual prophage strains. This finding also gave approximate map locations of tRNA genes close to the prophage sites. The mapping data obtained by the two methods were consistent with each other and also with the reported positions in the cases where previously mapped. On the basis of map location of the tRNA_f1^Met gene newly determined, the λ-transducing phage carrying the tRNA_f1^Met gene was found.     

Physical studies of RNA involvement in bacteriophage lambda DNA replication and prophage excision

Non-diffusible genetic elements in bacteriophage λ DNA replication and λ prophage excision have been analyzed by the DNA-cutting assay of Freifelder and Kirschner (1971) and Freifelder et al. (1972). The mutant ti12, which affects a unique site for replication in or near the origin of replication (Dove et al., 1971), makes λ DNA partially refractory to replicative DNA-cutting. RNA synthesis in the vicinity of the origin, of replication seems to control the susceptibility of λ DNA to replicative DNA-cutting (Dove et al., 1969). Analogously, RNA synthesis in the vicinity of the left-hand prophage terminus seems to control excisional DNA-cutting of derepressed λ DNA, as predicted by the studies of Davies et al. (1972). These physical studies confirm previous genetic analyses and imply that the elements involved act at a very early stage in replication and in excision.     

Secondary-site attachment of coliphage lambda near the thr operon

Phage λ has been integrated at a secondary attachment site near the thr oporon of Escherichia coli. The integration caused a pleiotropic effect since both thrA and thrB enzyme activities were lost. Lysogenic, thr⁺ revertants regained thrA and thrB activities although the enzymes were synthetized constitutively at low levels. Four classes of prophage deletion strains were isolated with deletions extending into trpR and the thr operon. Genetic and biochemical analyses indicated a gene order: trpR .. λ .. thrA .. B .. C. Defective transducing phage carrying thr A, B,C were also isolated.