Integration and Induction of Phage P22 in a Recombination-deficient Mutant of Salmonella typhimurium

Phage P22 can integrate as prophage into a recombination-deficient (Rec(-)) strain of Salmonella typhimurium. At 37 C, the integration efficiency is only 10% that in Rec(+) infection, but at 25 C the efficiencies in Rec(-) and Rec(+) hosts are similar. Rec(-) lysogens cannot be induced by ultraviolet irradiation or by treatments with the chemical inducing agents streptonigrin or mitomycin C. Heat induction of Rec(-) cells lysogenic for a temperature-sensitive c(2) mutant (ts c(2)) is normal, showing that the Rec(-) cell has the machinery necessary for prophage excision. Ultraviolet irradiation of Rec(-) (ts c(2)) lysogens prior to heat induction does not prevent the formation of infective centers after temperature shift. Thus, the noninducibility of Rec(-) lysogens is not due to destruction of the prophage as a result of ultraviolet irradiation. Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization experiments demonstrate that no increase in phage-specific RNA synthesis occurs after ultraviolet irradiation of a Rec(-) (c(+)) lysogen. The Rec(-) mutant appears to lack part of the mechanism required to destroy the phage repressor and allow the initiation of early phage functions such as messenger RNA synthesis. A similar conclusion was reached previously for an Escherichia coli Rec(-) strain.     

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

Summary In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage Red recombination systems on the rescue of the O ⁺ gene from the prophage by a superinfecting O ^- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants that does the Rec system, and its action requires DNA replication. The presence of UV lesions in the DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.     

Isolation and characterization of lambda transducing bacteriophages for the su1+ (supD minus) amber suppressor of Escherichia coli.

Specialized lambda transducing phages for the sul+ (supD-) amber suppressor in Escherichia coli K-12 have been isolated, using a secondary site lambda-cI857 lysogen in which we have shown the prophage to be closely linked to sul+.sul+ transducing particles were detected frequently, at 10-5 per plaque-forming unit, in lysates prepared from the secondary-site lysogens. High-frequency transducing lysates were obtained from several independently isolated sul+ transductants and were analyzed by CsCl equilibrium density gradient centrifugation. The transducing phages are defective; marker rescue analysis indicates that the lambda-N gene is not present. In lambda-cI857DELTANdSul+, a bio-type transducing phage, the genes specifying recombination and excision functions have been replaced by bacterial deoxyribonucleic acid.     

Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host''s chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed.     

Behavior of λ Bacteriophage in a Recombination Deficient Strain of Escherichia coli

The behavior of lambda phage in the Rec(-) strain JC-1569 is compared with that in the Rec(+) strain JC-1557. No difference deemed significant was noted in the adsorption rate, latent period, burst size, frequency of lysogenization, and frequency of vegetative phage recombination. The location of the prophage and its mode of insertion in the Rec(-) lysogen of wild-type lambda (lambda(+)) were inferred to be normal from the results of conjugational crosses. Spontaneous and ultraviolet (UV) irradiation induction of lambda(+) were markedly reduced in the Rec(-) lysogen. On the other hand, thermal induction of a mutant lambda (lambdacI857) lysogen of the Rec(-) strain was not reduced and was only slightly affected by UV irradiation. Phage subject to inhibition by lambda immunity failed to multiply in UV-irradiated cells of the Rec(-) lambda(+) lysogen, whereas those not inhibited by this immunity did multiply. It was concluded that the failure of UV to induce lambda(+) in the Rec(-) lysogen was not due to damage to the prophage, but rather to the inability of the irradiated cells to respond by lifting immunity. Preliminary evidence indicates that a single mutation confers recombination deficiency and the inability to lift immunity after UV irradiation. Possible relationships between recombination and the lifting of immunity are enumerated.     

Bacteriophage lambda cloning vehicles for studies of genetic recombination

A pair of bacteriophage lambda cloning vehicles has been constructed for use in studies of genetic recombination. These phages, lambda rva and lambda rvb, have the following properties: (1) Each vector has a single HindIII site in the immunity region, at which segments of DNA can be inserted. (2) These HindIII sites are flanked by selectable markers with the following phenotypes: Spi+/- (Fec+/-) to the left, and imm lambda or imm434 to the right. (3) There is essentially no sequence homology between the two phages in this region, so recombination of the markers at reasonable frequency depends on the presence of homologous inserts at the HindIII sites. As a consequence, recovered recombinants must have resulted from a crossover event within the insert DNA. Restriction enzyme maps of the vectors have been determined. Variants of the original vectors have been isolated which permit separate examination of the viral (Red) and bacterial (Rec) generalized recombination mechanisms, and which provide a standard interval to which frequencies of recombination in cloned DNAs can be compared.     

Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor

Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.     

Chaperone-assisted excisive recombination, a solitary role for DnaJ (Hsp40) chaperone in lysogeny escape

Temperate bacteriophage lytic development is intrinsically related to the stress response in particular at the DNA replication and virion maturation steps. Alternatively, temperate phages become lysogenic and integrate their genome into the host chromosome. Under stressful conditions, the prophage resumes a lytic development program, and the phage DNA is excised before being replicated. The KplE1 defective prophage of Escherichia coli K12 constitutes a model system because it is fully competent for integrative as well as excisive recombination and presents an atypical recombination module, which is conserved in various phage genomes. In this work, we identified the host-encoded stress-responsive molecular chaperone DnaJ (Hsp40) as an active participant in KplE1 prophage excision. We first show that the recombination directionality factor TorI of KplE1 specifically interacts with DnaJ. In addition, we found that DnaJ dramatically enhances both TorI binding to its DNA target and excisive recombination in vitro. Remarkably, such stimulatory effect by DnaJ was performed independently of its DnaK chaperone partner and did not require a functional DnaJ J-domain. Taken together, our results underline a novel and unsuspected functional interaction between the generic host stress-regulated chaperone and temperate bacteriophage lysogenic development.     

Isolation and Characterization of a Recombination-Deficient Hfr Strain

The isolation of a rec(-) Hfr strain of Escherichia coli K-12 is described. The method used consisted of mating AB2463 F(-) Rec(-) His(-) Lac(-) with P4X6 Hfr Rec(+) His(+) Lac(+), selecting Rec(-) His(-) Lac(+) recombinants, and searching for Hfr strains. One Hfr rec(-) strain, no. 12, was used as donor in crosses with Rec(+) and Rec(-) recipients. Crosses with Rec(+) recipients are fertile, and those with Rec(-) recipients are almost infertile, the frequency of recombinants being 10(-2) to 10(-3) that found with Rec(+) recipients. The Rec(-) mutant marker is transfered to and integrated into Rec(+) recipients. Zygotic induction of prophage lambda is observed in crosses between two Rec(-) strains. In crosses of F(-) Rec(-) with Hfr Rec(-), the gradient of integration frequencies for markers progressively more distant from the origin is steeper than in the Rec(+) x Rec(-) or the Rec(-) x Rec(+) crosses.     

Identification of Site-Specific Recombination Genes int and xis of the Rhizobium Temperate Phage 16-3

Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision). We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.     

Manipulating or Superseding Host Recombination Functions: A Dilemma That Shapes Phage Evolvability

Phages, like many parasites, tend to have small genomes and may encode autonomous functions or manipulate those of their hosts''. Recombination functions are essential for phage replication and diversification. They are also nearly ubiquitous in bacteria. The E. coli genome encodes many copies of an octamer (Chi) motif that upon recognition by RecBCD favors repair of double strand breaks by homologous recombination. This might allow self from non-self discrimination because RecBCD degrades DNA lacking Chi. Bacteriophage Lambda, an E. coli parasite, lacks Chi motifs, but escapes degradation by inhibiting RecBCD and encoding its own autonomous recombination machinery. We found that only half of 275 lambdoid genomes encode recombinases, the remaining relying on the host''s machinery. Unexpectedly, we found that some lambdoid phages contain extremely high numbers of Chi motifs concentrated between the phage origin of replication and the packaging site. This suggests a tight association between replication, packaging and RecBCD-mediated recombination in these phages. Indeed, phages lacking recombinases strongly over-represent Chi motifs. Conversely, phages encoding recombinases and inhibiting host recombination machinery select for the absence of Chi motifs. Host and phage recombinases use different mechanisms and the latter are more tolerant to sequence divergence. Accordingly, we show that phages encoding their own recombination machinery have more mosaic genomes resulting from recent recombination events and have more diverse gene repertoires, i.e. larger pan genomes. We discuss the costs and benefits of superseding or manipulating host recombination functions and how this decision shapes phage genome structure and evolvability.     

Prophage as a genetic reservoir: Promoting diversity and driving innovation in the host community

Sequencing of bacterial genomes has revealed an abundance of prophage sequences in many bacterial species. Since these sequences are accessible, through recombination, to infecting phages, bacteria carry an arsenal of genetic material that can be used by these viruses. We develop a mathematical model to isolate the effects of this phenomenon on the coevolution of temperate phage and bacteria. The model predicts that prophage sequences may play a key role in maintaining the phage population in situations that would otherwise favor host cell resistance. In addition, prophage recombination facilitates the existence of multiple phage types, thus promoting diverse co‐existence in the phage‐host ecosystem. Finally, because the host carries an archive of previous phage strategies, prophage recombination can drive waves of innovation in the host cell population.     

Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase.

The prophage lambdac1857 was inserted into the bfe gene located near rif (the structural gene for the beta subunit of deoxyribonucleic acid [DNA]-dependent ribonucleic acid polymerase) on the Escherichia coli chromosome. Induced lysates (low-frequency transducing lysates) of such a lysogen contained defective lambda phage particles (lambdadrif+) that can specifically transduce the wild-type rif+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harboring both the wild-type and the mutant rif genes were isolated. Rec+ derivatives of these heterogenotes produce high-frequency transducing lysates that contain lambdadrif+ and normal active phages at a ratio of 1 to 2. The results of marker rescue experiments and of density determination with several transducing phages indicate that most of the late genes are deleted and replaced by a segment of the chromosomal DNA carrying the bfe-rif region. The length of the chromosomal segment seems to vary between approximately 0.5 and 0.6% of the total bacterial DNA among the three independently isolated lambdadrif+ phages. Electron microscopy of heteroduplex DNA consisting of one strand from lambdadrif+-6 and the other from lambdaimm-21 phages directly confirmed that most of the phage DNA of the "left arm" was replaced by the bacterial DNA. The heteroduplex study also demonstrated that the integration of prophage lambda into the bfe region occurred at the normal cross-over point within the phage attachment site.     

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.     

A third defective lambdoid prophage of Escherichia coli K12 defined by the lambda derivative, lambdaqin111

We describe the isolation and characterization of a new Q-independent substitution mutant of lambda, lambdaqin111, which differs from other characterized Q-independent lambda phages. This mutant defines a new lambda-like prophage in the bacterial chromosome, as seen by homologous recombination between lambdaqin111 and the host DNA and by DNA/DNA hybridization methods. Genetic and electron microscopy data show that this new prophage carries, at least, genes analogous to Q-S-R of lambda and also a cos site functionally identical to lambda cos. It is located near 34 min on the Escherichia coli K12 map, i.e. in the same region but at a different site from the defective Rac prophage.     

Molecular Analysis of λbio Transducing Phage Produced by Oxolinic Acid-Induced Illegitimate Recombination in Vivo

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we examined the formation of λ Spi(-) phage during prophage induction. The frequency of Spi(-) phage was two to three orders of magnitude higher in the presence of oxolinic acid, an inhibitor of DNA gyrase A subunit, than in the absence of the drug, while it was very low in nalA(r) bacteria with the drug. RecA function is not required for the formation of these phages, indicating that this enhancement is not caused by the expression of SOS-controlled genes. Analyses of att region and recombination junctions of Spi(-) phages revealed that they have essentially the same structures as λbio transducing phages but are classified into two groups with respect to recombination sites. In the majority class of the transducing phages, there were not more than 3-bp homologies bewteen the parental E. coli bio and λ recombination sites. In the minority class of the transducing phages, on the other hand, 9-10-bp homologies were found between the parental recombination sites. These results suggested that oxolinic acid-induced illegitimate recombination takes place by two variants of a DNA gyrase-dependent mechanism.     

Improvement of recombination efficiency by mutation of red proteins

The Red recombination system of the bacteriophage lambda is a useful tool for engineering bacterial artificial chromosomes (BACs) because desired mutations can be made in any part of the DNA, even in large DNAs, independent of the presence of appropriate restriction enzyme sites. Many have tried changing the inducible promoter or decreasing the copy number of Red genes in Escherichia coli to improve the recombination efficiency. Here we describe a novel method for increasing the recombination efficiency of the Red system by mutating the Red proteins. Mutant Red alpha and Red beta independently increased recombination efficiency. We found that the 18-bp 5' untranslated region (UTR) upstream from the ATG initiation codon of Gam protein is important for recombination efficiency. Although low rates of recombination have been observed in systems using high copy number plasmids, when we performed Red recombination using a high copy number plasmid containing 5'-UTR, mutant Red alpha, and mutant Red beta, recombination increased approximately 145-fold. The novel possibility that mutant Red alpha and Red beta can increase recombination efficiency indicates a new direction for improving recombination efficiency of expression systems that use low or high copy number plasmids or a defective prophage integrated into the E. coli chromosome.     

A central coupler for recombination initiation linking chromosome architecture to s phase checkpoint

Higher-order chromosome structure is assumed to control various DNA-templated reactions in eukaryotes. Meiotic chromosomes implement developed structures called "axes" and "loops"; both are suggested to tether each other, activating Spo11 to catalyze meiotic DNA double-strand breaks (DSBs) at recombination hotspots. We found that the Schizosaccharomyces pombe Spo11 homolog Rec12 and its partners form two distinct subcomplexes, DSBC (Rec6-Rec12-Rec14) and SFT (Rec7-Rec15-Rec24). Mde2, whose expression is strictly regulated by the replication checkpoint, interacts with Rec15 to stabilize the SFT subcomplex and further binds Rec14 in DSBC. Rec10 provides a docking platform for SFT binding to axes and can partially interact with DSB sites located in loops depending upon Mde2, which is indicative of the formation of multiprotein-based tethered axis-loop complex. These data lead us to propose a mechanism by which Mde2 functions as a recombination initiation mediator to tether axes and loops, in liaison with the meiotic replication checkpoint.