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Results for “DNA Restriction-Modification Enzymes”

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PubMed2025

The early injected genomic region determines sensitivity to Type I restriction-modification defence against Autographiviridae phages.

Bacteriophages must evade bacterial defences to establish successful infections. Type I restriction-modification (RM) systems recognize specific DNA motifs and degrade unmethylated foreign DNA, restricting phage replication. In this study, we detected that Marinomonas mediterranea MMB-2 uses a Type I RM system (Mme2I) to protect against several new phages in the Murciavirus genus within the Autographiviridae family. Whole-genome sequencing and methylation analysis revealed a DNA sequence motif methylated in M. mediterranea MMB-2, which is also present in the phages. Phages lacking the motif within the leading, first injected, region of their genomes, either natural isolates or escape mutants of sensitive phages, successfully infect M. mediterranea MMB-2, despite the presence of the recognition motif elsewhere in their genomes. These results highlight the importance of considering RM motif locations when predicting avoidance of restriction sites as escape mechanisms from RM systems. Additionally, our findings indicate an important role for RM systems in specifically influencing the organization of the leading injected regions of phage genomes, which are highly variable and often encode diverse anti-defence systems.

Genome, Viral↗
PubMed2025

Lactococcal mobile genetic elements harbour a diverse phage defensome rich in restriction-modification systems.

The genomes of 43 distinct lactococcal strains were reconstructed by a combination of long- and short-read sequencing, resolving the plasmid complement and methylome of these strains. The genomes comprised 43 chromosomes of approximately 2.5 Mb each and 269 plasmids ranging from 2 to 211 kb (at an average occurrence of 6 per strain). A total of 953 antiphage genes representing 538 phage defence systems were identified in the 43 strains and were catalogued and cross-correlated with co-occurrent mobile elements, which indicated that almost 60% of these systems are predicted to be mobile. Detailed analysis established that restriction-modification (R-M) systems form a significant portion of this mobile phage defensome. As such, all detected Type I, II, and III-associated methylated motifs (46 of which were unique to this study) were matched to their corresponding methylating enzymes by homology detection or molecular cloning. The cumulative antiphage activity of selected systems and the ability of truncated R-M genes to contribute to methylation were demonstrated. This study reveals, for the first time, the dairy lactococcal plasmidome to be a rich reservoir of orphan HsdS-encoding genes, in a comprehensive survey of (mobile) phage defence systems in lactic acid bacteria.

Bacteriophages↗
PubMed1979

Genetic recombination during transformation in Bacillus subtilis: appearance of a deoxyribonucleic acid methylase.

In Bacillus subtilis the ability to take up deoxyribonucleic acid (DNA) and undergo genetic transformation may coincide with the induction of defective phage(s) and the expression of possibly related cryptic genes. A restriction-modification enzyme system appears to be expressed. Targets of the restriction activity on the DNA can be blocked my methylation catalyzed by the methyl transferase. It is shown that cellular DNA becomes progressively methylated and reaches the maxium level during the peak of competency. Deoxycytidine residues of both incoming donor and resident DNA are methylated. The possible participation of these enzymes in recombination and the general role of cryptic genes in inducible functions are discussed.

Bacillus subtilis↗
PubMed1978

Cloning of restriction and modification genes in E. coli: the HbaII system from Haemophilus haemolyticus.

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R-PstI site, (formula: see text), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3'-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3'-OH termini, thus regenerating the PstI cleavage site sequences. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r-m-recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage lambdacI-0). A single phage-resistant clone was found. The recombinant plasmid, pD110, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.

DNA Restriction Enzymes↗
PubMed1978

Nucleotide sequence of the recognition site for the restriction-modification enzyme of Escherichia coli B.

The nucleotide sequence of the recognition site for the restriction-modification enzyme of Escherichia coli B (SB site) has been determined. The recognition site is a 15-nucleotide sequence consisting of the trimer 5'TGA3', followed by an 8-nucleotide domain of variable sequence, which in turn is followed by tetramer 5'TGCT3'. The sequence has no 2-fold rotational symmetry. Single base changes in the constant nucleotide domains result in the loss of sensitivity to both restriction and modification. Our data are also consistent with modification occurring by methylation of two adenine residues per SB site: one on the adenine of the trimer 5'TGA3' and the other on the complementary strand on the adenine complementary to the first thymine of the tetramer 5'TGCT3'. All nine independently isolated spontaneous mutants at the SB1 site of bacteriophage f1 are caused by a G-to-T transversion. Mutations at the SB2 site are caused by various single base changes.

Adenine↗
PubMed1978

Biological characteristics of a type I restriction-modification system in Staphylococcus aureus.

Two restriction-modification systems, S1 and S2, are present in Staphylococcus aureus RN450 (S. Iordanescu and M. Surdeanu, J. Gen. Microbiol., 96:277-281, 1976). System S2 affects phage multiplication after both infection and transfection. Unmodified plasmid and chromosomal DNAs are also not expressed following transduction and transformation into a restrictive host. Restricted phages are, however, capable of conferring phage-mediated competence, although the state of competence does not affect the restriction-modification system. The restricting activity of system S2 is inactivated by heat treatment of the cells. An enzymatic activity that restricts unmodified phage DNA in the presence of ATP, Mg2+, and S-adenosylmethionine was recovered from cell-free extracts of a strain RN450 derivative.

DNA Restriction Enzymes↗
PubMed1979

[Characteristics of bacteriophage lambda and P1 modification-restriction in Escherichia coli strains controlled by factor R124].

The specifities of restriction of bacteriophages P1 and lambda controlled by R plasmids in Escherichia coli have been investigated. The isogenic strains harbouring the plasmids pAS26 coding for restriction endonuclease R.EcoRI, R245 coding for restriction endonuclease R.EcoRII and and R124 have been investigated in the present work. Modification-restriction controlled by R124 has been found to differ in specificity from those controlled by R245 and pAS26. Frequencies of restriction of bacteriophages P1vir and lambdavir specified by R124 pasmid differ from the frequencies in the strains harbouring pAS26 and R245 plasmids as well. The difference is due to the specifity of restriction-modification controlled by R124 plasmid. The data obtained are consistent with the determination of R124 specified restriction-modification activity as a novel one designated R.EcoRIII.

Anti-Bacterial Agents↗