Anita C. Fenton

λ red-dependent growth and recombination of phage P22

Plasmids that express lambda recombination functions singly and in combinations, at controllable levels, have been constructed. These plasmids were placed in Escherichia coli and Salmonella strains, and their ability to complement lambda and P22 strains lacking recombination functions was examined. The combination of lambda bet and exo constitutes a minimal system that can substitute for the recombination system of P22 in allowing efficient growth and recombination in a recA- host.

Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12

Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing the red genes of bacteriophage lambda in place of recBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac(-) chloramphenicol-resistant bacterial progeny was measured. Recombinant formation was found to be reduced in ruvAB and recQ strains. In this genetic background, mutations in recF, recO, and recR had large effects on both cell viability and on recombination. In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the nin region of phage lambda partially complemented both the viability and recombination defects of the recF, recO, and recR mutants and the recombination defect of ruvC but not of ruvAB or recQ mutants.

Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12

BackgroundAn Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage λ Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants.ResultsA number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3) The Lac+ recombinants were unstable, segregating Lac- progeny. (4) A tetracycline-resistance marker in a site of the phage chromosome distant from cat was not frequently co-inherited with cat. (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7) The linked tetracycline-resistance marker was frequently co-inherited in this case.ConclusionsThe Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome. When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant.

DNA-binding properties of the Erf protein of bacteriophage P22☆

The phage P22 erf (essential recombination function) gene was placed in a small plasmid under the control of a strong, inducible promoter by manipulations in vitro. Erf protein was purified from induced cells, and characterized. Erf protein (monomer molecular weight 23,000) forms oligomers in solution. The carboxyl terminus is protease-sensitive: its removal generates a discrete amino-terminal fragment (molecular weight approximately 18,000) that also oligomerizes. At temperatures below 45 degrees C, Erf forms stable, discrete complexes with single-stranded DNA and supercoiled DNA, but not with relaxed double-stranded DNA. Binding to single-stranded DNA is stoichiometric; one Erf monomer binds approximately 15 bases of DNA, over the range of protein concentrations tested (2 to 100 micrograms/ml). At high temperatures (50 to 60 degrees C). Erf binds single- and double-stranded DNA, forming aggregates instead of discrete complexes. Heating and cooling in the absence of DNA produces a form of Erf that has single-stranded binding specificity, but forms aggregates on binding.

Sequence of the bacteriophage P22 Anti-RecBCD (abc) genes and properties of P22 abc region deletion mutants

The nucleotide sequence of a segment of the bacteriophage P22 chromosome to the left (downstream in the PL operon) of the erf gene was determined. Previous studies (A. C. Fenton and A. R. Poteete, 1984, Virology 134, 148-160) have shown that this region encodes a function that is required for efficient growth of P22 in wild-type, but not in recB- Salmonella. The gene or genes encoding this function were designated abc (anti-recBCD). The DNA sequence reveals three open reading frames that potentially encode polypeptides with molecular weights of 10,900, 11,600, and 6600 (in order of transcription). P22 deletion mutants lacking each of the open reading frames were constructed. In addition, plasmids were constructed placing each of the open reading frames under control of the lac UV5 promoter. The phenotypes of the deletion mutants, and the results of plasmid-phage complementation tests, indicate that Abc activity depends primarily on sequences that encode the 11.6-kDa polypeptide; the 10.9-kDa polypeptide-encoding sequence makes a minor contribution to Abc activity as well. These sequences have been designated abc2 and abc1, respectively. The 6.6-kDa polypeptide is apparently uninvolved.

Genetic analysis of the erf region of the bacteriophage P22 chromosome.

Derivatives of P22 with deletions of DNA sequences around and including the erf gene were obtained by crossing phages with plasmids containing fragments of the P22 chromosome. In some cases, the parent phages carried a large insertion in sequences not borne by the plasmids. In these cases, deletion of DNA from the phage chromosome to restore terminal repetition (a selectable trait) could be accomplished by recombination between phage and plasmid DNA in chosen sequences flanking the insertion on both sides and borne by the plasmid. In other cases, the parent phages had deletions of a selectable gene, which could be acquired from the plasmid parents only by acquisition of an overlapping deletion. Deletion-bearing P22 strains were tested for growth and homologous genetic recombination in wild-type, recA-, and rec(B or C)- hosts. This analysis indicated the existence of a gene, mapping to the left of erf, that is helpful (but not completely essential) for growth of P22 in a wild-type host. Because P22 lacking this gene grows as well as wild-type P22 on a recBC- host, it has been designated abc (anti-recBC). The abc gene does not appear to be essential for homologous genetic recombination in any host. A plasmid bearing a 1900 base pair fragment of P22 DNA, that expresses erf and abc under the control of the E. coli lac promoter, was constructed. It supports growth and recombination in a recA- host by a phage that lacks all of the genes known to lie between 24 and 9.

Recombination-Promoting Activity of the Bacteriophage λ Rap Protein in Escherichia coli K-12

The rap gene of bacteriophage lambda was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the lambda red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recGDelta red(+) rap(+) strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny. The results of these experiments indicated that the lambda rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.

Bacteriophage P22 accessory recombination function

The accessory recombination function (arf) gene of bacteriophage P22 is located immediately upstream of the essential recombination function (erf) gene. Three mutant alleles of arf were constructed and installed in P22 in place of the wild-type allele: an out-of-frame internal deletion, an in-frame internal deletion, and an amber mutation. The deletion mutant phages are partially defective in homologous recombination and plaque formation in wild-type and recA hosts; their defects are more severe in recB and recA recB hosts. The amber mutant phage exhibits the same growth phenotypes in nonsuppressing hosts, but not in an amber-suppressor host. Plasmids that express arf complement the growth defect of arf- phages. These plasmids stimulate erf-mediated recombination; they were also found to cause a small stimulation of recA-recBCD-mediated homologous recombination of phage lambda.