Prophage lambda at unusual chromosomal locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K12

An Escherichia coli strain deleted for the primary λ attachment site was lysogenized with λ at secondary sites. Some lysogens became mutants because of prophage insertion in the affected gene. Mutagenesis by phage λ is not random with respect to the gene affected: most mutants were pro, although certain other genes could be mutated at lower frequencies. In the case of several independent ilv and gal mutants, the sites of prophage insertion were in the same segment of the ilv region and galT gene respectively. The galT location may also be a preferred site for the insertion of DNAs other than prophage λ. Insertion of prophage λ within an operon can reduce the expression of operator-distal genes. A trpC λ insertion mutant expresses the operator-distal trpB function constitutively at a low level. This expression probably derives from a promoter located in the left arm of the prophage.     

Differential stability of trp messenger RNA synthesized originating at the trp promoter and pL promoter of lambda trp phage.

The trp operon translocated into the early region of phage λ can be transcribed under the control of two promoters, the authentic trp promoter (p_Ttrp mRNA) and the p_L promoter of the N gene (p_Ltrp mRNA) (Imamoto &; Tani, 1972; Segawa &; Imamoto, 1974). The p_Ltrp mRNA has a 5′-terminal λ N message. The functional and chemical stability of trp segments in these mRNA species have been assayed. To determine trp mRNA from λtrp, appropriate φ80trp DNAs were used as a DNA complement in DNA-RNA hybridization assays.When formation of mRNA is inhibited, the capacity to serve as template for enzyme synthesis decays at a comparable rate for p_L and p_Ttrp mRNA, and p_Ltrp mRNA seems to be translated as efficiently as is the normal p_Ttrp mRNA. In contrast to this similar functional stability, p_Ltrp mRNA shows a more than tenfold greater chemical stability than p_Ttrp mRNA. (p_Ttrp mRNA is degraded at the same rate as trp mRNA in uninfected bacteria. Bulk host mRNA also decays at its normal rate in cells infected with λtrp.)On the basis of those and more extensive experiments including the sedimentation analysis of those stabilized trp mRNA molecules, it is inferred that (1) the rate-limiting step to initiate bulk mRNA degradation is determined by a sequence located at or near the 5′ end of the messenger RNA; and (2) functional inactivation of each messenger is regulated independently of bulk chemical degradation of the message.Stabilization of the trp mRNA produced from the p_L promoter increases with time after phage infection. Thus, the stabilization requires a modification of the decay trigger, possibly by a phage-specific protein such as a nuclease or the N and/or tof gene that might bind to the mRNA.     

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.     

Morphogenesis of the tail of bacteriophage lambda. III. Morphogenetic pathway.

Precursors of the tail of bacteriophage λ have been detected by measurements of in vitro complementation activities and serum blocking activity in sucrose gradients of lysates defective in tail genes.On the basis of these measurements, a pathway for the assembly of the λ tail is proposed:The morphogenesis of the λ tail starts from the tail fiber (product of gene J) located at the distal end of the tail, and proceeds to the proximal end. Gene J by itself produces a 15 S structure with serum blocking activity but without any detectable in vitro complementation activity, which may be the least advanced precursor of the λ tail or an abortive product. Functions of genes J, I, K, L are required for the formation of a 15 S precursor that has in vitro complementation activities with J⁻, I⁻, K⁻ and L⁻ lysates and serum blocking activity. If the products of genes G and H act on the latter 15 S precursor, a 25 S precursor is made, but this precursor seems either to be in equilibrium with the 15 S precursor or to degrade easily into the 15 S precursor. Gene M has a function of stabilizing the 25 S precursor. After the action of gene M product, the 25 S precursor is ready to serve as a nucleus on which the product of gene V (the major tail protein) assembles. However, gene U product is also necessary at this step for the correct assembly of the major tail protein on the 25 S precursor. Without gene U product the assembly of the major tail protein does not stop at the correct length and a polytail is formed instead of a morphologically normal tail. Finally, gene Z product acts on the morphologically normal tail and makes it a biologically active tail. Without the action of gene Z product, the defective tail binds to a head and forms a phage-like particle which is only very weakly infectious. (The position of gene T in the pathway is not determined, because no sus mutant is available in gene T.)Two abnormal, less efficient pathways are also present in vitro. (1) If gene U product acts on a polytail in an U⁻ lysate, the polytail finally binds to a head and forms a phage particle with an extra long tail which is infectious to a small extent. (2) The function of gene K seems to be bypassed to some extent: K⁻ lysates accumulate particles which sediment as fast as normal phage and which are complemented by other tail⁻ lysates.     

Separation and analysis of promoter sites in bacteriophage lambda DNA by specific endonucleases

Specific endonucleases from Hemophilus influenzae, H. parainfluenzae and H. aegyptius were used to separate fragments bearing only one of the various promoters in phage λ DNA. Fragments containing these promoters were characterized by comparative analysis on polyacrylamide gels of the digestion products from λ and a variety of deletion or deletion-substitution derivatives. A single endonuclease from H. influenzae, Hin-II, is shown to cleave the early leftward and rightward promoters, p_L and p_R, at the sites of cleavage of the operators, O_L and O_R, because the corresponding cleavage sites are specifically protected by the DNA-dependent RNA polymerase. With altered p_L (mutations sex1 and sex3), the cleavage in the corresponding promoter is abolished. With X13, a mutation that presumably inactivates p_R, the cleavage in O_R still occurs.     

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.     

Packaging of coliphage lambda DNA. II. The role of the gene D protein

The gene D protein (pD) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site (cos). In this report we show that cos cutting and phage production occur without pD in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D-independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of pD; and (2) D-independence cannot be attributed to loss of a specific segment of the phage genome. pD-deficient phage resemble pD-containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept pD.pD appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of pD. In fact, pD is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head.The above results show that pD is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of pD. Yet, pD is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of pD leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. pD promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

The form of the DNA substrate required for excisive recombination of bacteriophage lambda.

We have studied the excision reaction of bacteriophage lambda, both in vivo and in vitro, using as a substrate a λatt²(L × R) phage carrying both the right and left-hand prophage attachment sites. Int and Xis are provided by induction of the heat-inducible defective prophage, λc1857 ΔH1. After a brief induction (5 min) of these cells, excisive recombination is blocked in the presence of the DNA gyrase inhibitor, coumermycin. However, after a longer induction (greater than 30 min) excisive recombination occurs efficiently under conditions where λ integrative recombination is inhibited by coumermycin. In such extensively induced coumermycin-treated cells, infecting λatt²(L × R) DNA is not supercoiled, and recombinants are found among the relaxed covalently closed circular DNA.In vitro, starting with a hydrogen-bonded λatt² DNA substrate, excision is insensitive to high concentrations of coumermycin and novobiocin. To study the DNA substrate requirements for excisive recombination in more detail, we have developed a restriction fragment assay for excisive recombination. With this assay, we demonstrate that supercoiled, hydrogen-bonded, and linear λatt² DNA molecules are all efficient substrates in the in vitro excision reaction. Spermidine is required but ATP and Mg²⁺ are not. We conclude that supercoiling is not an absolute requirement for site-specific recombination of λ.     

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.