pac sites are indispensable for in vivo packaging of DNA by phage P22

Summary F pro ⁺ plasmids were selected and used as donors to prepare P22 transducing phages. Two types of result were observed. pro ⁺ from type I donors cannot be packaged by wild-type P22 to yield transducing particles unless a prophage pac site is introduced into the plasmid. Transposon Tn10 also allows initiation of packaging. pro ⁺ from type II plasmids can be transduced with the same efficiency as pro ⁺ DNA on the chromosome, indicating that a chromosomal pac site was included when the F pro ⁺ was excised from the Hfr strain. The usefulness of type I plasmids as a test substrate for pac signals is discussed.     

EcoRI analysis of bacteriophage P22 DNA packaging.

Bacteriophage P22 linear DNA molecules are a set of circularly permuted sequences with ends located in a limited region of the physical map. This mature form of the viral chromosome is cut in headful lengths from a concatemeric precursor during DNA encapsulation. Packaging of P22 DNA begins at a specific site, which we have termed pac, and then proceeds sequentially to cut lengths of DNA slightly longer than one complete set of P22 genes (Tye et al., 1974b). The sites of DNA maturation events have been located on the physical map of EcoRI cleavage sites in P22 DNA. EcoRI digestion products of mature P22 wild-type DNA were compared with EcoRI fragments of two deletion and two insertion mutant DNAs. These mutations decrease or increase the length of the genome, but do not alter the DNA encapsulation mechanism. Thus the position of mature molecular ends relative to EcoRI restriction sites is different in each mutant, and comparison of the digests shows which fragments come from the ends of linear molecules. From the positions of the ends of molecules processed in sequential headfuls, the location of pac and the direction of encapsulation relative to the P22 map were deduced. The pac site lies in EcoRI fragment A, 4.1 × 10³ base-pairs from EcoRI cleavage site 1. Sequential packaging of the concatemer is initiated at pac and proceeds in the counterclockwise direction relative to the circular map of P22. One-third of the linears in a population are cut from the concatemer at pac, and most packaging sequences do not extend beyond four headfuls.Fragment D is produced by EcoRI cleavage at a site near the end of a linear chromosome which has been encapsulated starting at pac. The position of the pac site is therefore defined by one end of fragment D. The pac site is not located near genes 12 and 18, the only known site for initiation of P22 DNA replication, but lies among late genes at a position on the physical gene map approximately analogous to the cohesive end site (cos) of bacteriophage λ at which λ DNA is cleaved during encapsulation. Our results suggest that P22 and λ DNA maturation mechanisms have many common properties.     

Packaging of transducing DNA by bacteriophage P1

Summary P1 transduces bacterial chromosomal markers with widely differing frequencies. We use quantitative Southern hybridisations here to show that, despite this, most markers are packaged at similar levels. Exceptions are a group of markers near 2 min and another at 90 min which seem to be packaged at levels two-to threefold higher. We thus conclude that certain marker frequency variations in transduction can be explained by differences in packaging level, but that most cannot. The limited range in packaging levels suggests that P1 can initiate the packaging of chromosomal DNA from many sites. This idea is supported by our failure to find any chromosomal sequences with homology to the phage pac site and by the occurrence of hybridising bands which seem to suggest sequential packaging from a large number of specific sites. We eliminate the possibility that chromosomal DNA packaging is the result of endonucleolytic cutting by the P1 res enzyme.     

Selective transduction of recombinant plasmids with cloned pac sites by Salmonella phage P22

Summary Recombinant plasmids having PstI fragments of P22 DNA inserted in the vector pBR322 can be transduced efficiently by Salmonella phage P22, irrespective of the cloned phage sequences. When the rec function of the donor cells and the corresponding recombination system erf of the infecting phage are simultaneously inactivated, only plasmids containing the P22 pac site can be transduced. By this selective, generalized transduction an EcoRV DNA fragment of the P22 related phage L has been identified that carries a base sequence recognized by phage P22 as a packaging signal. Experiments in which only one of the two recombination systems was inactivated, showed that the bacterial rec system obviously promotes cointegrate formation between plasmid and phage DNA much more efficiently than the phage-coded erf system, allowing the specialized plasmid transduction observed by Orbach and Jackson (1982).     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

Packaging signals for phage P22 on the chromosome of Salmonella typhimurium

Summary Numbers of transducing particles for a set of 28 different chromosomal markers were determined in a lysate of Salmonella phage P22. The results were plotted with regard to the map positions of the transduced loci. Maxima of the resulting curve are interpreted as positions of start signals (pac-sites) for packaging series on the Salmonella chromosome. 5–6 pac-sites could be found as a minimum estimate.     

Sequences within the Herpesvirus-Conserved pac1 and pac2 Motifs Are Required for Cleavage and Packaging of the Murine Cytomegalovirus Genome

The DNA sequence motifs pac1 [an A-rich region flanked by poly(C) runs] and pac2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of pac1 and pac2 were essential for cleavage whereas others were not. Within pac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.     

Regional Specificity of Illegitimate Recombination by the Translocatable Amplicillin-Resistance Element Tn1 in the Genome of Phage P22

Insertions of the translocatable ampicillin-resistance element Tn1 were selected in the genome of the temperate Salmonella phage P22 by growing the phage on hosts carrying the resistance plasmid RP4. Insertions of Tn1 into phage P22 are rare (10(-10) per phage) and nonrandomly distributed in the P22 genome. They are found mainly in the vicinity of the P22 ant gene. Insertions within the ant gene are found at many (at least 15) genetically separable sites, are found equally frequently in both orientations and cause irreversible loss of gene function. Some insertions in ant appear to be associated with an adjecent deletion. Prophage deletions were derived from P22::Tn1 phages by two methods. Low multiplicity transductants have nonrandomly distributed endpoints. One end is at or very near the site of the Tn1 insertion, and the other is in the vicinity of gene 12; however, there are many genetically distinguishable endpoints within gene 12. Prophage deletions selected as survivors of induction of a P22Ap mnt-ts lysogen have similarly nonrandom endpoints, with the Tn1-distal end frequently near the ant gene, as well as gene 12. Physical analysis of several prophage deletions suggests that the Tn1 is intact to the resolution of DNA electron microscopy and that the deletions begin at the end of the Tn1 insertion. These results suggest that illegitimate recombination associated with Tn1 shows regional specificity (i.e., preference for some large areas of the P22 genome over other areas), but that within these regions is quite nonspecific.     

Molecular analysis of the packaging signal in bacteriophage CP-T1 of Vibrio cholerae

Summary We have previously identified a unique site, pac, from which packaging of precursor concatameric viral DNA into proheads starts during the maturation process of bacteriophage CP-T1. The direction of this packaging was determined from restriction enzyme cleavage patterns of CP-T1 DNA. A restriction enzyme generated fragment containing pac was cloned and the surrounding DNA region sequenced. Analysis of the nucleotide sequence revealed numerous repeat regions related to the consensus sequence PuagttGAT.AAT.aa.t. Within the sequenced region an open reading frame encoding a 12260 M_r protein was also identified. This protein appears to share homology with the binding domains of known DNA binding proteins and may represent a putative Pac terminase possessing the specific endonuclease activity required for cleavage at the pac site. Minicell analysis of deletion derivatives of the pac-containing clone revealed a protein of approximately 12900 M_r encoded within this same region, confirming that this Pac protein is phage encoded.     

Packaging Specific Segments of the Salmonella Chromosome with Locked-in Mud-P22 Prophages

Hybrid genetic elements, Mud-P and Mud-Q (collectively, Mud-P22s), have been constructed that carry two-thirds of the temperate Salmonella phage P22 genome sandwiched between the ends of transposon Mu. Insertions of these elements in the Salmonella chromosome generate locked-in P22 prophages that cannot excise. Upon induction (as a consequence of the inactivation of P22 c2 repressor), a locked-in prophage replicates its DNA in situ, resulting in the amplification of neighboring regions of the chromosome and the processive packaging of three contiguous headsful of adjacent DNA in one direction from the P22 packaging site, pac. Phage particles in an induced lysate of a Mud-P22 lysogen contain DNA molecules corresponding to several minutes of chromosomal DNA adjacent to the site of prophage insertion and transduce nearby genetic markers with high efficiencies. Mud-P22 prophages have been introduced into an F' episome by transposition; resident Mud insertions on the Salmonella chromosome may be converted to Mud-P22 insertions by homologous recombination in P22-mediated transductional crosses.     

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages.     

Specialized Transduction by Bacteriophage P22 in SALMONELLA TYPHIMURIUM: Genetic and Physical Structure of the Transducing Genomes and the Prophage Attachment Site

P22pro-1 and P22pro-3 are specialized transducing derivatives of phage P22 that carry the proA and proB genes of Salmonella typhimurium. These genes lie immediately adjacent to the prophage attachment site on the bacterial chromosome. By examining DNA heteroduplexes in the electron microscope, we found that DNA molecules from P22pro-1 and P22pro-3 each contain a substitution which adds length to the composite genome making the intracellular replicated genome too long to fit into a single phage particle. In this respect, and in many of their biological properties, the proline-transducing phages resemble P22Tc-10, another specialized transducing phage with an oversize, intracellular replicated genome which carries a tetracycline-resistance determinant from an R-factor.—Unlike P22Tc-10, however, P22pro-1 and P22pro-3 fail to integrate normally during lysogenizing infections, even when provided with all known integration functions. These results suggest that the proline substitutions have created a defect in the phage attachment site and suggest that the Campbell model for the formation of specialized transducing phages is applicable to phage P22 with the additional feature that oversize genomes can be produced and propagated.—A physical and genetic map of the P22 genome near the prophage attachment site was constructed which shows that the insertion from the R-factor in P22Tc-10 is not at the attachment site: it is therefore unlikely that P22Tc-10 was formed in an abnormal prophage excision event as envisioned in the Campbell model, but was instead the result of a direct translocation from the R-plasmid to P22.     

Genome of Bacillus subtilis Bacteriophage SPP1: Structure and Nucleotide Sequence of pac, the Origin of DNA Packaging

The DNA of Bacillus subtilis bacteriophage SPP1 is terminally redundant and partially circularly permuted. To explain these parameters, we followed the Streisinger-Botstein models of phage maturation and assumed that packaging of SPP1 DNA begins at a unique genomic site (“pac”) and proceeds sequentially from there. We describe the sequence of about 1,000 nucleotides surrounding pac. This together with size determinations of small, pac-terminated restriction fragments has revealed heterogeneity of the natural pac ends of SPP1 DNA. Such ends fell in each DNA strand into a region of five to seven nucleotides. However, within this range more than 50% of all molecules terminated with defined cytosines on both strands, generating a 3′ protruding terminus. The nucleotide sequence of the DNA segment surrounding pac did not reveal any features which would distinguish this region.     

Ant-mediated inactivation of Salmonella phage L-specified repression at OR of prophage L.

Summary Ant product of phage P22 inactivates repression of prophage L at the right-hand operator o_R and allows for transactivation of prophage gene 12. The transactivation efficiency observed with a series of phage and prophage recombinants, using single superinfection of a lysogenic bacterium, is about the same as that recently observed at o_L of prophage L. This finding is in contrast to the failure to demonstrate derepression at o_R of prophage L in an experimental system employing double superinfection (Prell, 1978a). The reasons for the differing results are discussed and it is shown that derepression by the ant product in trans at o_R of the prophage is not modified to any significant degree by the immunity specificity (L or P22) of the prophage or of the superinfecting phage.     

The activity of ant product of the Salmonella phage P22 against the closely related but heteroimmune phage L.

Summary P22 mutants defective in the early gene 24 are complemented by phage L in mixed infection. P22 12 ^- and P22 23 ^- mutants are not complemented by phage L. Gene function 24 of an L prophage is turned on by a superinfecting P22 24 ^- mutant and complements the missing function of the defective P22 phage. Since this transactivation of prophage gene 24 depends on a functional gene ant in the superinfecting P22 mutant, it indicates derepression for leftward directed gene expression in prophage L. On the contrary neither the rightward directed expression of gene 12 nor of gene 23 in prophage L can be turned on by superinfecting P22 24 ^- 12 ^- or P22 24 ^- 23 ^- mutants (and also not by P22 12 ^- and P22 23 ^-) to a degree sufficient for complementation of simultaneously superinfecting L virB 12 ^- or L virB 23 ^- mutants. The failure to detect release of repression for rightward directed gene expression of prophage L corresponds to the earlier observation (Prell, 1975) that P22 superinfecting L lysogens cannot release replication inhibition for simultaneously infecting phage L. The results are discussed with respect to the mechanism underlying the different action of P22 antirepressor in L and in P22 lysogens.     

Ant-mediated transactivation of early genes in Salmonella prophage P22 by superinfecting virulent P22 mutants

Summary The virulent mutants P22 vir B vy and P22 vy mutants, both insensitive to mnt-repressor, transactivate the early genes of a P22 prophage. The transactivation of early P22 prophage genes depends strictly on the expression of gene ant (antirepressor-protein) by the superinfecting P22 mutant and therefore occurs by derepression.     

Mapping of mglB, the structural gene of the galactose-binding protein of Escherichia coli

Summary The tetracycline resistance transposon Tn10 was inserted into the E. coli chromosome near mglB550, a structural gene for the galactose-binding protein. P1 transductions established the position of these Tn10 insertions (zee-700, 701, 702::Tn10) close to the genes ptsF, fpk, cdd, mglB550, his, and gatA with 85%–95%, 85%, 36%, 20%–40%, 12%–15%, and 0.5% contransduction frequency. Three factor crosses revealed the relative sequence of the genes as: mglB550, zee-700::Tn10, ptsF, fpk, cdd, his. gatA was found to be 1.3% cotransducible with mglB550. Two Tn10 insertions near gatA were isolated and characterized. One, zef-704::Tn10, was 3% cotransducible with fpk, 8% with mglB550, and 42% with gatA. The other, zef-703::Tn10, was 98% cotransducible with gatA but not with mglB550 or fpk. Neither of these two Tn10 insertions was cotransducible with cdd. Four factor crosses revealed the sequence gatA, zef-704::Tn10, mglB550, fpk.Neither zee-700::Tn10 nor zef-703::Tn10 showed any (0/300) contransduction with either glpT or gyrA. The clockwise order of genes is then: his, cdd, fpk, ptsF, zee-700::Tn10, mglB550, zef-704::Tn10, gatA. With a fix-point for his at 44 min, fpk would be placed at 45 min and mglB550 at 45.5 min. During the course of this work we noticed that the cotransduction frequency between Tn10 insertions and nearby markers tended to increase when new P1 lysates were prepared from freshly reisolated strains. This may indicate loss of nonessential genes adjacent to Tn10 insertions. Using insertion zee-702::Tn10, we isolated deletions extending into an mgl gene other than mglB. Crosses between such a deletion mutant and an mglB550 mutant were done. The analysis of the periplasmic proteins of these as well as other transductants or recombinants involving the mglB550 or the mglB551 gene revealed the existence of strains synthesizing both the wild-type as well as the corresponding mutant protein. Strains containing both proteins exhibit either wild-type or mutant phenotype. These strains appeared unstable. Upon reisolation from purified stock cultures kept in glycerol at-20°C, colonies could be isolated that carried only mutant or wild-type protein.     

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     

In vivo studies of genomic packaging in the dsRNA bacteriophage Φ8

Background  

Φ8 is a bacteriophage containing a genome of three segments of double-stranded RNA inside a polyhedral capsid enveloped in a lipid-containing membrane. Plus strand RNA binds and is packaged by empty procapsids. Whereas Φ6, another member of the Cystoviridae, shows high stringency, serial dependence and precision in its genomic packaging in vitro and in vivo, Φ8 packaging is more flexible. Unique sequences (pac) near the 5' ends of plus strands are necessary and sufficient for Φ6 genomic packaging and the RNA binding sites are located on P1, the major structural protein of the procapsid.     

Interactions of Tn7 and temperate phage F116L of Pseudomonas aeruginosa

Summary Tn7 insertions into the genome of F116L, a Pseudomonas aeruginosa generalized transducing phage, were isolated by repeated cycles of transducing phage, were of strains lysogenic for F116cts mutants with selection for trimethoprim resistance (Tp¹). Two non-defective F116Lcts:Tn7 phage were characterized. They have reduced plaquing ability, produced non-lysogenic Tp^r transductants, and have yielded a deletion mutant of the phage genome upon selection for plaque formation in single infection. F116L DNA is circularly permuted and terminally redundant. A circular restriction map of 61.7 kb has been defined, and a cleavage site common to many enzymes has been identified at coordinate 23.3 kb on the map. It is presumed that this site represents the sequence for the initiation of DNA encapsidation by a headful packaging mode. The Tn7 insertion targets and a 13.4 kb deletion define regions of the F116L genome non-essential for either vegetative growth or lysogenization. The restriction map of Tn7 has been determined for five enzymes. Non-lysogenic Tp^r transuctants reveal a Tn7 insertion hot-spot in the P. aeruginosa genome.