The old exonuclease of bacteriophage P2

Abstract

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...

Polynucleotide phosphorylase of Escherichia coli is required for the establishment of bacteriophage P4 immunity

“…In addition, the mutation appeared to interfere with the lytic development of P4: both P4 wild-type and clear-plaque mutants, such as P4 cI405 and P4 vir1, formed smaller plaques and plated at slightly lower efficiencies on bfl-1 mutant strains than on the bfl ϩ isogenic strains, whereas P4 sut1 (see Materials and Methods and reference 44) could not form plaques on the (Table 2). These P4 growth defects appeared to be dependent on the Old function of the P2 helper prophage (25,31,40). In fact, both P4 ϩ and the above-described mutants, including P4 sut1, plated with equal efficiencies on bfl-1 and bfl ϩ strains lysogenic for the P2 old deletion mutant P2 del1, whereas the P4 ϩ clear-plaque phenotype (Table 2) and the lysogenization defect (see below) persisted.…”

“…In bfl-1, however, a severe growth defect is observed only with P4 sut1, and this is mediated by the helper phage P2 old gene. This gene codes for an exonuclease (31) and is responsible for the killing of E. coli recBC mutants by P2, the exclusion of phage by P2 prophage, and the increased X-ray sensitivity of the P2 lysogens (17,25,40). In the P4 sut1 mutant, the level of expression of the P4 primase-helicase gene ␣ might be increased, because of both suppression of transcription termination at t 151 and the fact that the cnr gene, a negative regulator of ␣, is fused to the upstream gene orf151 and might thus be not completely functional (43,44).…”

Bacteriophage P2

“…76 Purified Old protein has been reported to have 5 0 to 3 0 exonuclease activity on DNA, as well as an uncharacterized ribonuclease activity. 77 The P2 tin gene product inhibits the growth of T-even bacteriophages. 78 Tin blocks the action of their single-stranded binding proteins (gp32), thus interfering with T-even phage DNA replication.…”

Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems

“…A great boost for P2 studies was the discovery in 1966 by Erich Six (81) of the remarkable satellite phage P4 and its complex interactions ("transactivation") with P2 and P2-like phages (22,29,36,65). A number of very intriguing observations, peculiar to P2, remain incompletely understood and deserve further study: striking metabolic effects on the frequency of lysogenization (18,19); cell sensitization to a small molecular product (21); the complex interactions between P2 nonessential gene old, the host cell, and a coinfecting phage lambda (24,44,63,72,80); and others. Based on the results of screens of coliforms in Paris and Los Angeles (23,53) and the Dhillons' extensive work in Hong Kong (40), it became clear that-to the extent that modern notions of segmental evolution (57) allow it-P2 and lambda are representative of two main groups of temperate phages for such bacteria.…”