Nucleoside triphosphate-dependent restriction enzymes

The known nucleoside triphosphate-dependent restriction enzymes are hetero-oligomeric proteins that behave as molecular machines in response to their target sequences. They translocate DNA in a process dependent on the hydrolysis of a nucleoside triphosphate. For the ATP-dependent type I and type III restriction and modification systems, the collision of translocating complexes triggers hydrolysis of phosphodiester bonds in unmodified DNA to generate double-strand breaks. Type I endonucleases break the DNA at unspecified sequences remote from the target sequence, type III endonucleases at a fixed position close to the target sequence. Type I and type III restriction and modification (R-M) systems are notable for effective post-translational control of their endonuclease activity. For some type I enzymes, this control is mediated by proteolytic degradation of that subunit of the complex which is essential for DNA translocation and breakage. This control, lacking in the well-studied type II R-M systems, provides extraordinarily effective protection of resident DNA should it acquire unmodified target sequences. The only well-documented GTP-dependent restriction enzyme, McrBC, requires methylated target sequences for the initiation of phosphodiester bond cleavage.     

Different restriction and modification phenotypes in ruminal lactate-utilizing bacteria

Analysis of restriction and modification activities in lactate-utilizing bacteria belonging to the Megasphaera elsdenii and Mitsuokella multiacida species revealed the presence of GATC-specific, MboI isospecific, restriction-modification (R-M) systems in all strains tested. While restriction endonucleases isolated from M. elsdenii strains were found to be sensitive to Dam methylation, enzymes from M. multiacida cleaved DNA irrespective of Dam methylation. The comparison of type II R-M systems specificities in three closely related lactate-utilizing ruminal bacterial species indicated complete lack of restriction and/or modification enzymes previously characterized from Selenomonas ruminantium in tested M. elsdenii and M. multiacida strains. R-M systems are believed to represent the main defense tool against phage infection. Based on the results of our experiments it could be assumed that M. elsdenii and M. multiacida use the different strategy for bacteriophage protection compared to S. ruminantium.     

Hypervariation and phase variation in the bacteriophage 'resistome'

Most bacteria encode proteins for defence against infection by bacteriophages. The mechanisms that bring about phage defence are extremely diverse, suggesting frequent independent evolution of novel processes. Phage defence determinants are often plasmid or phage-encoded and many that are chromosomal show evidence of lateral transfer. Recent studies on restriction-modification (R-M) systems show that these genes are amongst the most rapidly evolving. Some bacteria have contingency genes that encode alternative target specificity determinants for Type I or Type III R-M systems, thus expanding the range of phages against which the host population is immune. The most counter-intuitive observation, however, is the prevalence of phase variation in many restriction systems, but recent arguments suggest that switching off expression of R-M systems can aid phage defence.     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

Bacteriophage Orphan DNA Methyltransferases: Insights from Their Bacterial Origin,Function, and Occurrence

Type II DNA methyltransferases (MTases) are enzymes found ubiquitously in the prokaryotic world, where they play important roles in several cellular processes, such as host protection and epigenetic regulation. Three classes of type II MTases have been identified thus far in bacteria which function in transferring a methyl group from S-adenosyl-l-methionine (SAM) to a target nucleotide base, forming N-6-methyladenine (class I), N-4-methylcytosine (class II), or C-5-methylcytosine (class III). Often, these MTases are associated with a cognate restriction endonuclease (REase) to form a restriction-modification (R-M) system protecting bacterial cells from invasion by foreign DNA. When MTases exist alone, which are then termed orphan MTases, they are believed to be mainly involved in regulatory activities in the bacterial cell. Genomes of various lytic and lysogenic phages have been shown to encode multi- and mono-specific orphan MTases that have the ability to confer protection from restriction endonucleases of their bacterial host(s). The ability of a phage to overcome R-M and other phage-targeting resistance systems can be detrimental to particular biotechnological processes such as dairy fermentations. Conversely, as phages may also be beneficial in certain areas such as phage therapy, phages with additional resistance to host defenses may prolong the effectiveness of the therapy. This minireview will focus on bacteriophage-encoded MTases, their prevalence and diversity, as well as their potential origin and function.     

Evidence of Horizontal Transfer of the EcoO109I Restriction-Modification Gene to Escherichia coli Chromosomal DNA

A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5'-(A/G)GGNCC(C/T)-3', was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.     

A motif conserved among the type I restriction-modification enzymes and antirestriction proteins: a possible basis for mechanism of action of plasmid-encoded antirestriction functions.

Antirestriction proteins Ard encoded by some self-transmissible plasmids specifically inhibit restriction by members of all three families of type I restriction-modification (R-M) systems in E.coli. Recently, we have identified the amino acid region, 'antirestriction' domain, that is conserved within different plasmid and phage T7-encoded antirestriction proteins and may be involved in interaction with the type I R-M systems. In this paper we demonstrate that this amino acid sequence shares considerable similarity with a well-known conserved sequence (the Argos repeat) found in the DNA sequence specificity (S) polypeptides of type I systems. We suggest that the presence of these similar motifs in restriction and antirestriction proteins may give a structural basis for their interaction and that the antirestriction action of Ard proteins may be a result of the competition between the 'antirestriction' domains of Ard proteins and the similar conserved domains of the S subunits that are believed to play a role in the subunit assembly of type I R-M systems.     

The effect ofrecA mutation on the expression ofEcoKI andEcoR124Ihsd genes cloned in a multicopy plasmid

Type I restriction-modification (R-M) endonucleases are composed of three subunits—HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of clonedEcoKI andEcoR124Ihsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wthsd genes in a multicopy plasmid inEscherichia coli recA ⁺ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the clonedhsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation inE. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.     

A rapid and efficient method for cloning genes of type II restriction-modification systems by use of a killer plasmid

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.     

New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method

The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test. To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I). Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10(-1)-10(-2)). When multiple recognition sites were present, greater reductions in EOT values were observed. Once established in the cell, the plasmids were subjected to modification (EOT = 1.0). We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells. Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively. Eco1158I, an isoschizomer of EcoBI, was also found in this study.     

A Rapid and Efficient Method for Cloning Genes of Type II Restriction-Modification Systems by Use of a Killer Plasmid

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.     

DNA restriction-modification systems mediate plasmid maintenance.

Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease. Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome. Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells. Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.     

Characterization of AloI, a restriction-modification system of a new type.

We report the properties of the new AloI restriction and modification enzyme from Acinetobacter lwoffi Ks 4-8 that recognizes the DNA target 5' GGA(N)6GTTC3' (complementary strand 5' GAAC(N)6TCC3'), and the nucleotide sequence of the gene encoding this enzyme. AloI is a bifunctional large polypeptide (deduced M(r) 143 kDa) revealing both DNA endonuclease and methyltransferase activities. Depending on reaction cofactors, AloI cleaves double-stranded DNA on both strands, seven bases on the 5' side, and 12-13 bases on the 3' side of its recognition sequence, and modifies adenine residues in both DNA strands in the target sequence yielding N6-methyladenine. For cleavage activity AloI maintains an absolute requirement for Mg(2+) and does not depend on or is stimulated by either ATP or S-adenosyl-L-methionine. Modification function requires the presence of S-adenosyl-L-methionine and is stimulated by metal ions (Ca(2+)). The C-terminal and central parts of the protein were found to be homologous to certain specificity (HsdS) and modification (HsdM) subunits of type I R-M systems, respectively. The N-terminal part of the protein possesses the putative endonucleolytic motif DXnEXK of restriction endonucleases. The deduced amino acid sequence of AloI shares significant homology with polypeptides encoding HaeIV and CjeI restriction-modification proteins at the N-terminal and central, but not at the C-terminal domains. The organization of AloI implies that its evolution involved fusion of an endonuclease and the two subunits, HsdM and HsdS, of type I restriction enzymes. According to the structure and function properties AloI may be regarded as one more representative of a newly emerging group of HaeIV-like restriction endonucleases. Discovery of these enzymes opens new opportunities for constructing restriction endonucleases with a new specificity.     

Various characteristics of the anti-restriction mechanism in bacteriophage T5

Bacteriophage T5 is not confined by the restriction systems of the second type EcoRII and EcoRV. Bacteriophage T5 DNA is not modified by EcoRII and EcoRV methylases in vivo. The sites of recognition for restriction endonuclease EcoRV are mapped at 24.4; 57.6; 68.5; 70.2% of T5 DNA, while the sites at 5.1; 7.6% are recognized by EcoRII, the sites at 5.75; 6.0 and 6.5% are recognized by HpaI in FST. A high activity of restriction endonucleases EcoRI and EcoRV is demonstrated in crude extracts of E. coli B834 (RI) and E. coli B834 (RV) cells infected by bacteriophage T5. The simultaneous infection of E. coli B834 (RI) or E. coli B834 (RV) cells by the amber mutants of bacteriophage T5 and the suppressing phage lambda NM761 does not result in the protection of lambda DNA by the T5 anti-restriction mechanism. The presented data support the hypothesis that the anti-restriction mechanism of bacteriophage T5 is based on prevention of T5 DNA contacts with restriction enzymes by a specific phage protein.     

Isolation and characterization of a HpyC1I restriction-modification system in Helicobacter pylori

Using transposon shuttle mutagenesis, we identified six Helicobacter pylori mutants from the NTUH-C1 strain that exhibited decreased adherence and cell elongation. Inverse polymerase chain reaction and DNA sequencing revealed that the same locus was interrupted in these six mutants. Nucleotide and amino acid sequences showed no homologies with H. pylori 26695 and J99 strains. This novel open reading frame contained 1617 base pairs. The amino acid sequence shared 24% identity with a putative nicking enzyme in Bacillus halodurans and 23 and 20% identity with type IIS restriction endonucleases PleI and MlyI, respectively. The purified protein, HpyC1I, showed endonuclease activity with the recognition and cleavage site 5'-CCATC(4/5)-3'. Two open reading frames were located upstream of the gene encoding HpyC1I. Together, HpyC1I and these two putative methyltransferases (M1.HpyC1I and M2.HpyC1I) function as a restriction-modification (R-M) system. The HpyC1I R-M genes were found in 9 of the 15 H. pylori strains tested. When compared with the full genome, significantly lower G + C content of HpyC1I R-M genes implied that these genes might have been acquired by horizontal gene transfer. Plasmid DNA transformation efficiencies and chromosomal DNA digestion assays demonstrated protection from HpyC1I digestion by the R-M system. In conclusion, we have identified a novel R-M system present in approximately 60% of H. pylori strains. Disruption of this R-M system results in cell elongation and susceptibility to HpyC1I digestion.     

MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5′-TCCRAC-3′. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5′-GTYGGA-3′. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.     

Plasmid-encoded antirestriction protein ArdA can discriminate between type I methyltransferase and complete restriction-modification system

Many promiscuous plasmids encode the antirestriction proteins ArdA (alleviation of restriction of DNA) that specifically affect the restriction activity of heterooligomeric type I restriction-modification (R-M) systems in Escherichia coli cells. In addition, a lot of the putative ardA genes encoded by plasmids and bacterial chromosomes are found as a result of sequencing of complete genomic sequences, suggesting that ArdA proteins and type I R-M systems that seem to be widespread among bacteria may be involved in the regulation of gene transfer among bacterial genomes. Here, the mechanism of antirestriction action of ArdA encoded by IncI plasmid ColIb-P9 has been investigated in comparison with that of well-studied T7 phage-encoded antirestriction protein Ocr using the mutational analysis, retardation assay and His-tag affinity chromatography. Like Ocr, ArdA protein was shown to be able to efficiently interact with EcoKI R-M complex and affect its in vivo and in vitro restriction activity by preventing its interaction with specific DNA. However, unlike Ocr, ArdA protein has a low binding affinity to EcoKI Mtase and the additional C-terminal tail region (VF-motif) is needed for ArdA to efficiently interact with the type I R-M enzymes. It seems likely that this ArdA feature is a basis for its ability to discriminate between activities of EcoKI Mtase (modification) and complete R-M system (restriction) which may interact with unmodified DNA in the cells independently. These findings suggest that ArdA may provide a very effective and delicate control for the restriction and modification activities of type I systems and its ability to discriminate against DNA restriction in favour of the specific modification of DNA may give some advantage for efficient transmission of the ardA-encoding promiscuous plasmids among different bacterial populations.     

RloC: a wobble nucleotide-excising and zinc-responsive bacterial tRNase

The conserved bacterial protein RloC, a distant homologue of the tRNA^Lys anticodon nuclease (ACNase) PrrC, is shown here to act as a wobble nucleotide-excising and Zn⁺⁺-responsive tRNase. The more familiar PrrC is silenced by a genetically linked type I DNA restriction-modification (R-M) enzyme, activated by a phage anti-DNA restriction factor and counteracted by phage tRNA repair enzymes. RloC shares PrrC's ABC ATPase motifs and catalytic ACNase triad but features a distinct zinc-hook/coiled-coil insert that renders its ATPase domain similar to Rad50 and related DNA repair proteins. Geobacillus kaustophilus RloC expressed in Escherichia coli exhibited ACNase activity that differed from PrrC's in substrate preference and ability to excise the wobble nucleotide. The latter specificity could impede reversal by phage tRNA repair enzymes and account perhaps for RloC's more frequent occurrence. Mutagenesis and functional assays confirmed RloC's catalytic triad assignment and implicated its zinc hook in regulating the ACNase function. Unlike PrrC, RloC is rarely linked to a type I R-M system but other genomic attributes suggest their possible interaction in trans . As DNA damage alleviates type I DNA restriction, we further propose that these related perturbations prompt RloC to disable translation and thus ward off phage escaping DNA restriction during the recovery from DNA damage.     

Molecular Properties of Streptococcus thermophilus Plasmid pER35 Encoding a Restriction Modification System

Bacteriophage attack on lactic fermentation bacteria (LFB) is costly to the dairy industry because it results in product loss. One mechanism used by LFB to protect themselves from bacteriophage attack is restriction of foreign DNA. Three plasmids, pER16, pER35, and pER36, from three different strains of the thermotolerant dairy fermentation bacterium Streptococcus thermophilus were sequenced. One of these plasmids, pER35, isolated from S. thermophilus ST135, encoded a type IC restriction-modification (R-M) system very similar to those encoded on plasmids pIL2614 in Lactococcus lactis subsp. lactis and pND861 in Lactococcus lactis biovar diacetylactis. The high degree of identity between the R-M systems encoded on pER35, pIL2614, and pND861 indicated the potential for horizontal transfer of these genes between different species of lactic fermentation bacteria. Similar to the functional R-M system encoded on pIL2614 that protects the mesophilic L. lactis subsp. lactis against phage attack, the R-M system on pER35 most likely functions in the same role in S. thermophilus ST135. The plasmid pER16 was found to encode the specificity subunit of the R-M system, but not the R or M subunits. In addition, all three plasmids encoded proteins that are present on other S. thermophilus plasmids, including a protein for rolling-circle replication (RepA) and a low-molecular-weight stress protein (Hsp). The presence of a complete R-M system encoded on a plasmid in S. thermophilus, a species that often lacks plasmids, is novel and may be beneficial for protecting S. thermophilus from bacteriophage attack under dairy fermentation conditions.