Bacteriophage P2: Interaction with phage lambda and with recombination-deficient bacteria

Sironi, Gianpiero; Bialy, Harvey; Lozeron, Homer A.; Calendar, Richard

doi:10.1016/0042-6822(71)90040-7

Cited by 53 publications

(29 citation statements)

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“…We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.Temperate coliphage P2 has three nonessential lysogenic conversion genes, Z/fun, tin, and old, conferring resistance to infections by phage T5, T-even phages, and lambda phages, respectively (1,8,11,12,15,21). These genes have been shown to have a higher AT content than the rest of the genome and a codon usage that differs from that of the host, which suggests that they are horizontally transferred genes (3).…”

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Identification of a Gene Encoding a Functional Reverse Transcriptase within a Highly Variable Locus in the P2-Like Coliphages

2006

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The P2-like coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.Temperate coliphage P2 has three nonessential lysogenic conversion genes, Z/fun, tin, and old, conferring resistance to infections by phage T5, T-even phages, and lambda phages, respectively (1,8,11,12,15,21). These genes have been shown to have a higher AT content than the rest of the genome and a codon usage that differs from that of the host, which suggests that they are horizontally transferred genes (3). P2-like prophages are common in Escherichia coli; about 30% of the ECOR collection (20) contain P2-like prophages. At the region equivalent to the P2 Z/fun gene, i.e., the Z region, which is located between the well-conserved tail genes G and F I , these P2-like prophages have been shown to contain different gene cassettes surrounded by a highly similar inverted repeat, indicating a site-specific integration event. Similar inverted repeats, spacing other genes, can be found in genetically unstable regions in pathogenic enterobacteria (19). The tin and old genes are located at the right end of the P2 genetic map close to the cos site. The old gene encodes an exonuclease that blocks multiplication of lambdoid phages (16), and tin encodes a protein that inhibits T4 DNA synthesis by poisoning the T4 single-stranded binding protein (15). These lysogenic conversion genes have an unusual AT content and codon utilization and are believed to have been additions from foreign genomes. This is supported by the fact that genes for hypothetical proteins similar to Old are found in various bacterial genomes in the UniProt GenBank (Table 1), while the Tin protein so far is unique. Furthermore, P2-related phages found in other enterobacteria contain other genes at this locus (4-6, 14, 17, 18). To clarify the nature of this locus in the P2-like coliphages, we have sequenced and characterized the region equivalent to the P2 tin and old genes, i.e., the TO region, in seven of the P2-like prophages in the ECOR library.Sequence variation between the A genes and the cos sites of the prophages. To sequence the region located between the A genes and the cos sites of the prophages, DNA was extracted from seven strains of the ECOR collection, which are known to contain P2-like prophages (P2-ECnb). A primer located at the catalytic site of the A gene and a primer located to the left of the cos sequence were used for the PCR amplifications, and specific amplified DNA fragments of variable length were obtained. The fragments were either sequenced directly or first cloned and then sequenced. To obtain the complete region, plasmid primers and internal primers were used. All strains contained different D...

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Identification of a Gene Encoding a Functional Reverse Transcriptase within a Highly Variable Locus in the P2-Like Coliphages

2006

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“…A unifying hypothesis to explain these phenomena involves the postulate that phage X converts recB+ recC+ cells to a phenotype characteristic of recB-or recCmutants and that the gam gene of X is required for this phenotypic conversion (see ref. 4 and Discussion).…”

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Purification and Properties of the γ-Protein Specified by Bacteriophage λ: An Inhibitor of the Host RecBC Recombination Enzyme

Sakaki

Karu

Linn

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

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Previous experiments have indicated that the gam gene of bacteriophage X is responsible for an inhibition of the RecBC DNase-an enzyme that is essential for the major host pathway of genetic recombination. We report here experiments that define the inhibitor as the protein product of the gam gene ("1y-protein") This report presents evidence that the inhibitor is the yprotein itself and describes the purification of the y-protein and its effects on purified RecBC DNase. MATERIALS AND METHODSBacteriophage and Bacteria. The Escherichia coli strains used and their relevant genetic characteristics were: the Rec+ Endonuclease I-strain JC6724 and its related recB21 recC22 2215 double mutant JC6720; the Su recB21 mutant JC4695 (3, 7). The X phage mutations used were: redX314, a mutation in the structural gene for X-exonuclease (8); gam210 and gam37, nonsense and temperature-sensitive mutations, respectively, in the gam gene (1); biol, a deletion-addition mutation that has deleted most of the X recombination region including the gam gene (1); bio72, a deletion-addition mutation that has deleted most of the X recombination region, but not the gam gene (1). All phage except bio72gam37 carried the cd857 mutation, which renders the X cI protein thermolabile and thus allows thermal induction of X prophage (9). DNA and Enzymes. E. coli ['H]DNA (5500-7500 cpm/ nmol) was prepared by the methods of Oishi (10) The RecBC DNase used to monitor the purification of 7-protein ("Brij lysate preparation") was made as follows.A culture of E. coli JC6724 (6 to 8 X 108 cells per ml) was harvested, lysed with Brijj and fractionated with streptomycin sulfate and ammonium sulfate as described below for the purification of y-protein. The ammonium sulfate fraction(1-ml) was layered on a gradient (11-ml) of 10-30% glycerol [in 10 mM Tris * HOl (pH 8.0), 10 mM MgC12, 5 mM 2-mercaptoethanol, and 0. Assay for 7y-Proten measured its capacity to inhibit the activity of the RecBC enzyme. The preparation of 7-protein was "preincubated" with RecBC DNase, and then the substrate (usually double-stranded DNA) was added to measure remaining activity of the RecBC enzyme. For assay for exonuclease activity with double-stranded DNA and "Brij lysate preparation" of RecBC DNase, the following procedure was used. Preincubation mixtures contained: 10 mM Tris HCl (pH 8.0), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 0.5 mM ATP, and 0.2-0.6 units of "Brij lysate preparation" of RecBC

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“…Nicked circular molecules were also found, but they might have been derived from closed circular molecules. DNA replicated poorly in P2-lysogenic cells (15), and late mRNA was not found (23). During normal infections, DNA replicates in the theta form early after infection, ϪBovine serum albumin ................................................................ 90 ϪATP ............................................................................................... 68 ϪDithiothreitol .......................................................................... and rolling-circle intermediates appear in the late stages of infection (2).…”

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confidence: 99%

The old exonuclease of bacteriophage P2

Myung

Calendar

1995

J Bacteriol

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The Old protein of bacteriophage P2 is responsible for interference with the growth of phage and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5 to 3, and digestion initiates at either the 5-phosphoryl or 5-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5-phosphoryl mononucleotides.Temperate coliphage P2 encodes a nonessential gene called old that kills Escherichia coli recB and recC mutants (22) and interferes with the growth of temperate coliphage (15). On the basis of its amino acid sequence, Koonin and Gorbalenya (10) have suggested that the P2 Old protein may be a nuclease. We have purified the Old protein fused to MalE protein and found it to have both DNase and RNase activities. MATERIALS AND METHODS Bacteria and plasmids. E. coli JM105 carries lacIq for control of the lac promoter and lacZ⌬M15 for complementation analysis (26). pHM3W carries the P2 old gene under control of the lac promoter. It is a reconstruction of pDD3 (9), which was found to carry a mutation in the old gene. To create a fusion of the maltose-binding protein (MBP) and the P2 old gene, we cloned a 3.0-kb BamHIClaI fragment containing the old gene from pSF1 (3) into pUC119 that had been cleaved with BamHI and AccI. The resulting plasmid was called pHM1W. Using site-directed mutagenesis (11), we mutated the old gene so that the nucleotides from Ϫ3 to ϩ1 were changed from AATA (6) to CAGC, using the primer ATTTAAGGAAAACAGCTGACTGTACGTC. This transforms the methionine ATG start codon to a leucine CTG codon and creates a PvuII site, CAGCTG. We cut out the modified old gene with PvuII and HindIII and ligated it to pMAL-c2 (New England Biolabs) cleaved with XmnI and HindIII to give pHM6W.Media. LB broth (0.5% NaCl) and 2xYT are described by Sambrook et al. (21).Tritium-labeled DNAs.[ 3 H]thymine-labeled T7 phage DNA was prepared by the procedure of Joseph and Kolodner (7) at a specific activity of 11.2 cpm/pmol. Single-stranded DNA was produced by heat denaturation of duplex DNA at 100ЊC for 10 min and then promptly chilled on ice.3 H-labeled phage DNA was made by infecting E. coli JM105 at a density of 2 ϫ 10 8 cells per ml in LB broth at a multiplicity of infection of 5 in the presence of radioactive thymine (2 Ci/ml). Phage were precipitated with 8% polyethylene glycol with an average molecular weight of 8,000. They were further purified, and the DNA was extracted by using a preparation kit from Promega.3 H-labeled pBR322 and pEE470 were purified from E. coli JM105 grown in LB broth in the presence of 2 Ci of [ 3 H]thymine per ml. Nicked circular DNA was produced from 3 Hlabeled pEE470 (16) by treatment with pancreatic DNase at a concentration of...

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Bacteriophage P2: Interaction with phage lambda and with recombination-deficient bacteria

Cited by 53 publications

References 21 publications

Identification of a Gene Encoding a Functional Reverse Transcriptase within a Highly Variable Locus in the P2-Like Coliphages

Identification of a Gene Encoding a Functional Reverse Transcriptase within a Highly Variable Locus in the P2-Like Coliphages

Purification and Properties of the γ-Protein Specified by Bacteriophage λ: An Inhibitor of the Host RecBC Recombination Enzyme

The old exonuclease of bacteriophage P2

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