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.     

Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV).

The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.     

Cloning and characterization of restriction fragments of phage Mu DNA

Abstract The Eco RI-A and B, the Bam HI-C and the two Eco RI- Bam HI restriction fragments of transposing phage Mu DNA were inserted into vector plasmids pRSF2124 and pBR322. Quantitative marker rescue experiments for five genes located on the Eco RI-A fragment revealed complementation of phages carrying amber mutations in genes C , E , H , F and L . The in vitro coding capacity of the recombinant plasmids was assayed in a DNA-directed protein synthesizing system.     

Circular permutation of DNA cytosine-N4 methyltransferases: in vivo coexistence in the BcnI system and in vitro probing by hybrid formation

Sequence analysis of the BcnI restriction-modification system from Bacillus centrosporus revealed four open reading frames (bcnIC, bcnIR, bcnIB and bcnIA) that are arranged as two converging collinear pairs. One pair encodes a putative small regulatory protein, C.BcnI, and the restriction endonuclease R.BcnI. The other two gene products are the DNA cytosine-N4 methyltransferases M.BcnIA and M.BcnIB, which differ by circular permutation of conserved sequence motifs. The BcnI methyltransferases are isospecific on double-stranded DNA [methylation specificity CC(C/G)GG], but M.BcnIA can also methylate the target sites in single-stranded DNA. Functional analysis shows that bcnIA is dispensable (bcnIB is capable of protecting the DNA against the in vivo activity of bcnIR); in contrast, no stable clones were obtained if bcnIB alone was deleted from the system. By analogy with the DpnII system, the second methylase M.BcnIA may play a role in the transformation proficiency of its gram-positive host. The interchangeability of homologous elements in the beta class of cytosine-N4 methylases was probed by hybrid formation between M.BcnIB and its closest homolog M.Cfr9I (CCCGGG) employing a novel semi-random strategy combined with selection for catalytic activity. The fusion points in the active hybrids mapped in a narrow region located between sequence motifs X and I. Our data illustrate that recombination of two related sequences by circular permutation may serve as an evolutionary mechanism for creating new specificities of amino MTases.     

Physical map of the bacteriophage L (Salmonella typhimurium)

Abstract A circular restriction map of the genome of the phage L ( Salmonella typhimurium ) has been constructed with five restriction endonucleases, Eca I, Eco RI, Bam HI, Bgl I, and Pst I. The Eco RI fragments of phage-L DNA were cloned into pACYC184, and the resulting recombinant plasmids pL1, pL2,…,pL7 were introduced into Salmonella typhimurium . The genes present on the fragments cloned were identified by the marker rescue experiments with the L-phage amber mutants. A physical gene map of the L genome obtained in this way was compared with that of P22.     

Cloning and mapping of fourteen different DNA fragments containing Aspergillus nidulans 5S rRNA genes

Fourteen different plasmids hybridizing to Aspergillus nidulans 5S rRNA were isolated from a gene bank obtained after cloning Sau3A partial digests of A. nidulans DNA in a yeast--Escherichia coli vector, pBB29. The restriction maps of these plasmids were determined. The size of the cloned fragments was 2.7-9.5 kb, 12 of the plasmids were found to code for single 5S rRNA genes and 2 coded for 2 genes. No similarity of the sequences surrounding the 5S rRNA genes was found by restriction mapping.     

DNA restriction dependent on two recognition sites: activities of the SfiI restriction-modification system in Escherichia coli

In contrast to many type II restriction enzymes, dimeric proteins that cleave DNA at individual recognition sites 4-6 bp long, the SfiI endonuclease is a tetrameric protein that binds to two copies of an elongated sequence before cutting the DNA at both sites. The mode of action of the SfiI endonuclease thus seems more appropriate for DNA rearrangements than for restriction. To elucidate its biological function, strains of Escherichia coli expressing the SfiI restriction-modification system were transformed with plasmids carrying SfiI sites. The SfiI system often failed to restrict the survival of a plasmid with one SfiI site, but plasmids with two or more sites were restricted efficiently. Plasmids containing methylated SfI sites were not restricted. No rearrangements of the plasmids carrying SfiI sites were detected among the transformants. Hence, provided the target DNA contains at least two recognition sites, SfiI displays all of the hallmarks of a restriction-modification system as opposed to a recombination system in E. coli cells. The properties of the system in vivo match those of the enzyme in vitro. For both restriction in vivo and DNA cleavage in vitro, SfiI operates best with two recognition sites on the same DNA.     

The type IC hsd loci of the enterobacteria are flanked by DNA with high homology to the phage P1 genome: implications for the evolution and spread of DNA restriction systems

Eco R124I, Eco DXXI and Eco prrI are the known members of the type IC family of DNA restriction and modification systems. The first three are carried on large, conjugative plasmids, while Eco prrI is chromosomally encoded. The enzymes are coded by three genes, hsdR , hsdM and hsdS . Analysis of the DNA sequences upstream and downstream of the type IC hsd loci shows that all are highly homologous to each other and also to sequences present in the bacteriophage P1 genome. The upstream sequences include functional phd and doc genes, which encode an addiction system that stabilizes the P1 prophage state, and extend to and beyond pac , the site at which phage DNA packaging begins. Downstream of the hsd loci, P1 DNA sequences begin at exactly the same place for all of the systems. For Eco DXXI and Eco prrI the P1 homology extends for thousands of base pairs while for Eco R124I an IS 1 insertion and an associated deletion have removed most of the P1-homologous sequences. The significance of these results for the evolution of DNA restriction and modification systems is discussed.     

Genetic structure of the IncN plasmid N3

N3, a plasmid of incompatibility group N (IncN) was mapped by cleavage with restriction endonucleases. The restriction fragments were cloned into vector plasmids. All of the genes unique to IncN plasmids such as specific replication machinery, a restriction-modification system, and repair functions were located on a large portion which had no cleavage sites for many of the site-specific six base-identifying restriction endonucleases tested.     

Plasmid localization and cloning of restriction modification genes from Citrobacter freundii 4111 strain]

Over 60 producing strains of restriction endonucleases type II have been found among 500 different strains, mostly Enterobacteriaceae. The strain Citrobacter freundii 4111 produces restriction endonuclease CfrBI, a new isoschisomer of StyI. The genes of the restriction-modification system CfrBI were located on the multicopy plasmid pZE8 containing the Co1E1-type replicon and cloned to E. coli K802. The deletion variant of 3.2-kb pZE8 which contains intact restriction-modification and a DNA fragment responsible for autonomous plasmid replication was selected among the recombinant plasmids. The strain with higher R. CfrBI production (at least 10,000,000 U/g cells, which is 500-fold higher than the wild strain) was constructed.     

Positions of sea urchin (Strongylocentrotus purpuratus) histone genes relative to restriction endonuclease sites on the chimeric plasmids pSp2 and pSp17.

The positions of the several sea urchin histone genes on the eukaryotic fragments of the chimeric plasmids pSp2 and pSp17 have been mapped relative to the Eco RI and Hind III restriction endonuclease sites on the plasmids. Two principal mapping methods using the electron microscope have been used: (a) the R-loop procedure and a new modification thereof to map the genes on duplex DNA; (b) the gene 32-ethidium bromide technique to visualize RNA-DNA hybrids on single strands of DNA. It is known that there are two histone genes, H3 and H2A, on pSp17. There are two Eco RI sites at the two junctions of the procaryotic segment with the eucaryotic segment on the plasmid. We show, by an electron microscope method, that for H2A, with a length of 0.52 kilobases (kb), one end of the gene is situated 0.02 to 0.03 kb from one RI site, and that there is a Hind III site within this gene at about 0.13 kb from the end phe other RI site of this plasmid. The H4 gene lies between H2B and H1. The ms the incubation temperature is raised up to a temperature just below that at which strand dissociation of the duplex DNA occurs.     

Plasmids of the cyanobacterium Synechocystis 6701

Abstract Plasmids were obtained from Synechocystis 6701 using a lysis method that employed a hemicellulase digestion procedure. Eight major bands were observed in the initial preparation. Four of the smaller plasmids were isolated using preparative agarose electrophoresis gels and identified by restriction endonuclease analysis. Chromosomal DNA was screened with 15 restriction enzymes and 6 ( Eco RI, Sst I, Hpa I, Bst EII, Acc I, and Bgl II) were effective. Analysis of DNA fragments from plasmids pSCY 1–4 indicated that each plasmid was unique and that their approximate sizes were 5, 7.5, 13.5 and 15 kb, respectively. Digestion of pSCY 1 and pSCY 4 with Bgl II produced DNA fragments that may be used to construct a conjugation vector for this unicellular cyanobacterium.     

Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG X dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 X 10(3) bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.     

Molecular cloning and expression in E. coli of a yeast gene coding for beta-galactosidase.

The yeast Kluyveromyces lactis synthesizes a beta-galactosidase (EC 3.2.1.32) which is inducible by lactose. We have isolated the gene that codes for this enzyme using recombinant DNA techniques. K. lactis DNA was partially digested with the restriction endonuclease Eco R1 and joined to Eco R1-digested pBR322 plasmid DNA using DNA ligase. ligase. A lac-mutant of Escherichia coli lacking the structural gene for beta-galactosidase was transformed with ligated DNA. Three lac+ transformants containing recombinant plasmids were selected. Two of the plasmids (pK15 and pK17) contain four Eco R1-K. lactis DNA fragments having molecular weights of 2.2, 1.4, 0.55 and 0.5 x 10(6) daltons. The other plasmid (pK16) lacks the smallest fragment. E. coli carrying any of these plasmids produce beta-galactosidase activity that has a sedimentation coefficient and immunological determinants that are nearly identical to K. lactis beta-galactosidase and distinctly different from E. coli beta-galactosidase. DNA-DNA hybridization studies show that the four Eco R1 fragments in pK15 hybridize to K. lactis but not to E. coli DNA.     

A novel plasmid-mediated DNA restriction-modification system in E. coli

R plasmids from 101 clinical isolates were transferred to E. coli J62 by conjugation and tested for the presence of R plasmid-mediated restriction-modification DNA systems. Thirty R plasmids were found to inhibit phage λ. vir development. Ten plasmids determined restriction modification system; nine of them proved identical with R.M. EcoRII. One transconjugant, E. coli J62 pLG74, was shown to have a restriction-modification system different from all the known R plasmid-mediated systems. Site-specific endonuclease has been isolated from E. coli J62 pLG74 which differed from all the known restriction endonucleases in the number of cleavage sites on phages λ, φX 174, virus SV40, plasmid pBR322 DNA molecules.     

Streptomyces lipmanii expresses two restriction systems that inhibit plasmid transformation and bacteriophage plaque formation

Bacteriophage host range studies suggested that several beta-lactam-producing streptomycetes express similar restriction-modification systems. Streptomyces lipmanii LE32 expressed two restriction-modification systems, designated SliI and SliII. A mutant strain, PM87, was defective only in SliI restriction but expressed both SliI and SliII modification. Streptomyces sp. strain A57986, a natural isolate partially deficient in the expression of SliI and SliII restriction, nevertheless modified bacteriophage DNA for both SliI and SliII specificities. Protoplasts of PM87 and A57986 were transformed by several plasmids, and the modified plasmids isolated from these strains transformed wild-type S. lipmanii efficiently.     

Monomeric restriction endonuclease BcnI in the apo form and in an asymmetric complex with target DNA

Restriction endonuclease BcnI cleaves duplex DNA containing the sequence CC/SGG (S stands for C or G, / designates a cleavage position) to generate staggered products with single nucleotide 5'-overhangs. Here, we show that BcnI functions as a monomer that interacts with its target DNA in 1:1 molar ratio and report crystal structures of BcnI in the absence and in the presence of DNA. In the complex with DNA, BcnI makes specific contacts with all five bases of the target sequence and not just with a half-site, as the protomer of a typical dimeric restriction endonuclease. Our data are inconsistent with BcnI dimerization and suggest that the enzyme introduces double-strand breaks by sequentially nicking individual DNA strands, although this remains to be confirmed by kinetic experiments. BcnI is remotely similar to the DNA repair protein MutH and shares approximately 20% sequence identity with the restriction endonuclease MvaI, which is specific for the related sequence CC/WGG (W stands for A or T). As expected, BcnI is structurally similar to MvaI and recognizes conserved bases in the target sequence similarly but not identically. BcnI has a unique machinery for the recognition of the central base-pair.