The bacteriophage P1 restriction endonuclease

The bacteriophage P1 restriction endonuclease has been purified from Escherichia coli lysogenic for P1. This restriction endonuclease P has a sedimentation coefficient of 9.3 S. Unlike the E. coli K restriction endonuclease, endonuclease P does not require S-adenosylmethionine for breakage of DNA. S-adenosylmethionine does, however, stimulate the rate of double-strand breakage of DNA by endonuclease P. Hydrolysis of ATP by endonuclease P could not be detected under conditions in which the K restriction endonuclease massively degrades ATP.The enzyme makes a limited number of double-strand breaks in unmodified or heterologously modified λ DNA. In the presence of S-adenosylmethionine, it does not cut every DNA molecule to the same extent. Incubation of λ DNA with excess amounts of enzyme in the presence of S-adenosylmethionine results in less breakage of the DNA than with smaller amounts of enzyme. This effect is not seen in the absence of S-adenosylmethionine. The maximum amount of cutting in the absence of S-adenosylmethionine appears to be greater than the maximum amount of cutting in its presence. This is most likely due to the modification methylase activity of P1 restriction endonuclease.     

Characterization of mutations of the bacteriophage P1 mod gene encoding the recognition subunit of the EcoP1 restriction and modification system.

This study characterized several mutations of the bacteriophage P1 mod gene. This gene codes for the subunit of the EcoP1 restriction enzyme that is responsible for DNA sequence recognition and for modification methylation. We cloned the mutant mod genes into expression vectors and purified the mutant proteins to near homogeneity. Two of the mutant mod genes studied were the c2 clear-plaque mutants described by Scott (Virology 41:66-71, 1970). These mutant proteins can recognize EcoP1 sites in DNA and direct restriction but are unable to modify DNA. Methylation assays as well as S-adenosylmethionine (SAM) binding studies showed that the c2 mutants are methylation deficient because they do not bind SAM, and we conclude that the mutations destroy the SAM-binding site. Both of the c2 mutations lie within a region of the EcoP1 mod gene that is not conserved when compared with the mod gene of the related EcoP15 system. EcoP15 and EcoP1 recognize different DNA sequences, and we believe that this region of the protein may code for the DNA-binding site of the enzyme. The other mutants characterized were made by site-directed mutagenesis at codon 240. Evidence is presented that one of them, Ser-240----Pro, simultaneously lost the capacity to bind SAM and may also have changed its DNA sequence specificity.     

Resistance of bacteriophage H1 to restriction and modification by Bacillus subtilis R

H1, a 5-hydroxymethyluracil (HMU)-containing Bacillus subtilis bacteriophage, was neither restricted nor modified upon infection of B. subtilis R cells. In vitro, H1 DNA was not restricted by BsuR under standard conditions (200 mM salt), although the expected frequency of -GGCC- cleavage sites was approximately 250. However, four specific sites were cleaved under nonstandard conditions (low salt or high pH) or in the presence of organic solvents, like dimethyl sulfoxide and glycerol. After the substitution of thymine for HMU by DNA cloning in B. subtilis, a BsuR cleavage site was restricted and modified under standard conditions. No additional sites were detected after shotgun-cloning of about 11% of the chromosome. The nucleotide sequence of a cleavage site was found to be 5'. .C-A-Hmu-A-A-C-Hmu-Hmu-Hmu-G-G-C-C-Hmu-A-G-. . .3', which shows the presence of a bona fide BsuR (GGCC) recognition sequence, flanked by (Hmu-A)-rich sequences. The results suggested that the resistance of H1 to restriction and modification by B. subtilis R was due to (i) a strong bias against the GGCC-recognition sequence and (ii) protection of the four remaining GGCC sites as a consequence of HMU-A base pairs flanking the sites.     

Vibrio cholerae bacteriophage CP-T1: characterization of bacteriophage DNA and restriction analysis

Temperature bacteriophage CP-T1 of Vibrio cholerae has a capsid that is 45 nm in diameter, a contractile tail 65 nm long and 9.5 nm wide, and a baseplate with several spikes or short tail fibers. The linear double-stranded DNA is 43.5 +/- 1.4 kilobases long, and the phage genome is both terminally redundant and partially circularly permuted. The extent of terminal redundancy is ca. 4%, and circular permutation is up to ca. 44%. Circular restriction maps have been constructed for the enzymes HindIII, EcoRI, BamHI, and PstI. By restriction endonuclease and heteroduplex analyses of phage DNA, the presence and location of a site (pac) at which packaging of phage DNA is initiated was established.     

Cloning type-II restriction and modification genes

We have cloned into Escherichia coli the genes for 38 type-II bacterial modification methyltransferases. The clones were isolated by selecting in vitro for protectively modified recombinants. Most of the clones modify their DNA fully but a substantial number modify only partially. In approximately one-half of the clones, the genes for the corresponding endonucleases are also present. Some of these clones restrict infecting phages and others do not. Clones carrying endonuclease genes but lacking methyltransferase genes have been found, in several instances, to be viable.     

The EcoDXX1 restriction and modification system: cloning the genes and homology to type I restriction and modification systems

The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1. A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system. The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and EcoB. Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems.     

Cloning and sequencing the HinfI restriction and modification genes

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).     

The EcoDXX1 restriction and modification system: Cloning the genes and homology to type I restriction and modification systems

The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1. A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system. The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and Ecob. Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems.     

Cloning and expression of the MspI restriction and modification genes

The genes for the MspI restriction (R) and modification enzymes (recognition sequence CCGG) have been cloned into Escherichia coli using the vector pBR322. Clones carrying both genes have been isolated from libraries prepared with EcoRI, HindIII and BamHI. The smallest fragment that encodes both activities is a 3.6-kb HindIII fragment. Plasmids purified from the clones are fully resistant to digestion by MspI, indicating that the modification gene is functional in E. coli. The clones remain sensitive to phage infection, however, indicating that the endonuclease is dysfunctional. When the R gene is brought under the control of the inducible leftward promoter from phage lambda, the level of endonuclease increases and the level of methylase decreases, suggesting that the genes are transcribed in opposite directions.     

The deoxyribonucleic acid modification enzyme of bacteriophage P1. Purification and properties

The stereochemistry of dl-glycerol 3-phosphate was studied by X-ray-crystallographic techniques. All the bond lengths and angles are within normally accepted limits except the ester bond, which is one of the largest yet noted, being 0.1637nm. The conformation of the molecule is such that an intramolecular hydrogen bond is formed between the hydroxyl group on the beta-carbon atom and the phosphate group. The crystal, which was grown by alcohol diffusion into an aqueous solution, is held together by sodium co-ordination and a complex system of hydrogen bonds. A table of the observed and calculated structure factors, F(obs.) and F(calc.), has been deposited as Supplementary Publication 50010 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.     

The deoxyribonucleic acid modification enzyme of bacteriophage P1. Subunit structure

The bacteriophage P1 modification enzyme was purified 1400-fold from induced lysogens of a thermoinducible mutant of bacteriophage P1. The most purified fraction, when analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate, showed two principal stained bands. The two bands co-sedimented in a glycerol gradient with the modification activity, at a rate which, when compared with the rate of sedimentation of marker proteins, corresponds to a sedimentation coefficient in water of 6S. The mobilities of the bands on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis corresponded to polypeptides of molecular weight 70000 and 45000 and they were present in equimolar amounts. It was concluded that the 6S species of the enzyme is a dimer of unlike subunits.     

A mutational analysis of the bacteriophage P1 recombinase Cre

Bacteriophage P1 encodes a 38,600 Mr site-specific recombinase, Cre, that is responsible for reciprocal recombination between sites on the P1 DNA called loxP. Using in vitro mutagenesis 67 cre mutants representing a total of 37 unique changes have been characterized. The mutations result in a wide variety of phenotypes as judged by the varying ability of each mutant Cre protein to excise a lacZ gene located between two loxP sites in vivo. Although the mutations are found throughout the entire cre gene, almost half are located near the carboxyl terminus of the protein, suggesting a region critical for recombinase function. DNA binding assays using partially purified mutant proteins indicate that mutations in two widely separated regions of the protein each result in loss of heparin-resistant complexes between Cre and a loxP site. These results suggest that Cre may contain two separate domains, both of which are involved in binding to loxP.     

A restriction endonuclease cleavage map of bacteriophage P2

Summary A restriction endonuclease cleavage map of phage P2 was constructed. The enzymes used and, within parenthesis, the number of their cleavage sites on the P2 lg cc DNA molecule were: AvaI(3), BalI(1), BamI(3), BglII(3), HaeIII (more than 40; only three were mapped), HindIII(0), HpaI(10), KpnI(3), PstI(3), SalI(2) and SmaI(1). The EcoRI cleavage sites (3), as determined earlier, were used as reference points for this study. The DNAs of a variety of P2 mutants carrying chromosomal aberrations (del1, del2, del3, del6, vir22, vir37(2), vir79 and vir94) were also similarly examined.     

Cloning and sequence analysis of the plasmid-borne genes encoding the Eco29kI restriction and modification enzymes

The Eco29kI restriction-modification system (RMS2) has been found to be localized on the plasmid pECO29 occurring naturally in the Escherichia coli strain 29k (Pertzev, A.V., Ruban, N.M., Zakharova, M.V., Beletskaya, I.V., Petrov, S.I., Kravetz, A.N., Solonin, A.S., 1992. Eco29kI, a novel plasmid encoded restriction endonuclease from Escherichia coli. Nucleic Acids Res. 20, 1991). The genes coding for this RMS2, a SacII isoschizomer recognizing the sequence CCGCGG have been cloned in Escherichia coli K802 and sequenced. The DNA sequence predicts the restriction endonuclease (ENase) of 214 amino acids (aa) (24 556 Da) and the DNA-methyltransferase (MTase) of 382 aa (43 007 Da) where the genes are separated by 2 bp and arranged in tandem with eco29kIR preceding eco29kIM. The recombinant plasmid with eco29kIR produces a protein of expected size. ṀEco29kI contains all the conserved aa sequence motifs characteristic of m⁵C-MTases. Remarkably, its variable region exhibits a significant similarity to the part of the specific target-recognition domain (TRD) from ṀBssHII—multispecific m⁵C-MTase (Schumann, J.J., Walter, J., Willert, J., Wild, C., Koch D., Trautner, T.A., 1996. ṀBssHII: a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties. J. Mol. Biol. 257, 949–959), which recognizes five different sites on DNA (HaeII, MluI, Cfr10I, SacII and BssHII), and the comparison of the nt sequences of its variable regions allowed us to determine the putative TRD of ṀEco29kI.     

Cloning and sequencing the HinfI restriction and modification genes

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).     

Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein

Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium Escherichia coli. A phylogenetic analysis indicated that the P1 ssb gene coexists with its E. coli counterpart as an independent unit and does not represent a recent acquisition of the phage. The P1 and E. coli SSB proteins are fully functionally interchangeable. SSB-P1 is nonessential for phage growth in an exponentially growing E. coli host, and it is sufficient to promote bacterial growth in the absence of the E. coli SSB protein. Expression studies showed that the P1 ssb gene is transcribed only, in an rpoS-independent fashion, during stationary-phase growth in E. coli. Mixed infection experiments demonstrated that a wild-type phage has a selective advantage over an ssb-null mutant when exposed to a bacterial host in the stationary phase. These results reconciled the observed evolutionary conservation with the seemingly redundant presence of ssb genes in many bacteriophages and conjugative plasmids.     

Transduction of chromosomal genes between enteric bacteria by bacteriophage P1.

We have used P1 transduction to create intergeneric hybrid strains of enteric bacteria by moving the genA and hut genes between Klebsiella aerogenes, Escherichia coli and Salmonella typhimurium. The use of E. coli as the recipient in such transductions permits the construction of episomes and specialized transducing phage containing non-E. coli material. The effect of host restriction modification and deoxyribonucleic acid homology on the frequency of intergeneric transduction of these loci has been examined.