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PubMed1991

Identification of the recognition sequence for the M.StyLTI methyltransferase of Salmonella typhimurium LT7: an asymmetric site typical of type-III enzymes.

The StyLTI restriction-modification (R-M) system is encoded by chromosomal genes of Salmonella typhimurium LT7. We report here the identification of the nucleotide (nt) sequence methylated by the StyLTI modification methyltransferase (M.StyLTI). This enzyme was partially purified from an Escherichia coli strain expressing the cloned M.StyLTI-encoding gene, but lacking StyLTI restriction activity, and used to methylate DNAs of known sequence, using S-adenosyl-[methyl-3H]-methionine as the methyl donor. The [3H]methylated DNA was then digested with various endonucleases. Examination of labelled and unlabelled restriction fragments allowed us to map the M.StyLTI sites in perfectly defined regions of the DNA. Comparison of the nt sequences of DNA segments with or without M.StyLTI sites permitted us to identify the asymmetric and nondegenerate pentanucleotide, 5'-CAGAG-3', 3'-GTCTC-5' as the StyLTI sequence. M.StyLTI was found to methylate only the 3' A (see asterisk) in the upper strand of this sequence. Thus, M.StyLTI recognises and methylates the DNA in a manner very similar to that of the three known type-III MTases, M.EcoPI, M.EcoP15, and M.HinfIII. This strongly suggests that StyLTI constitutes a fourth type-III R-M system.

Adenine↗
PubMed1991

Cloning, sequence and characterization of m5C-methyltransferase-encoding gene, hgiDIIM (GTCGAC), from Herpetosiphon giganteus strain Hpa2.

We have cloned the gene (hgiDIIM) encoding the methyltransferase (MTase) of the SalI isoschizomeric restriction-modification (R-M) system, HgiDII (GTCGAC), into Escherichia coli. The hgiDIIM gene has been isolated from the same plasmid library of Herpetosiphon giganteus strain Hpa2, as was the previously cloned R-M system, HgiDI [AcyI/GRCGYC; Düsterhöft et al., Nucleic Acids Res. 19 (1991) 1049-1056]. Sequencing and functional localization of hgiDIIM revealed an open reading frame (ORF) of 354 codons (39786 Da) with significant homologies to the group of m5C-, rather than the m4C-/m6A-, MTases. Subsequent cloning and analysis of adjacent chromosomal segments led to the identification of two additional ORFs upstream (ORF15, 139 codons) and downstream (ORF68, 611 codons) from hgiDIIM with the same transcriptional orientation as the hgiDIIM gene. However, the expected restriction enzyme function was not found in either of these ORFs.

Amino Acid Sequence↗
PubMed1992

The corrected nucleotide sequences of the TaqI restriction and modification enzymes reveal a thirteen-codon overlap.

The nucleotide sequence of the genes encoding methyltransferase TaqI (M.TaqI) and restriction endonuclease TaqI (R.TaqI) with the recognition sequence, TCGA, were analyzed in clones isolated from independent libraries. The genes, originally reported as 363 and 236 codons long [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] were redetermined as 421 and 263 codons long, respectively. The C terminus of the taqIM gene overlaps the N terminus of the taqIR gene by 13 codons, as observed with the isoschizomeric TthHB8I restriction-modification system [Barany et al., Gene 112 (1992) 13-20]. Removal of the overlapping codons did not interfere with in vivo M.TaqI activity. We postulate the overlap plays a role in regulating taqIR expression.

Amino Acid Sequence↗
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↗
PubMed1992

Cloning and sequence analysis of the StsI restriction-modification gene: presence of homology to FokI restriction-modification enzymes.

StsI endonuclease (R.StsI), a type IIs restriction endonuclease found in Streptococcus sanguis 54, recognizes the same sequence as FokI but cleaves at different positions. A DNA fragment that carried the genes for R.StsI and StsI methylase (M.StsI) was cloned from the chromosomal DNA of S.sanguis 54, and its nucleotide sequence was analyzed. The endonuclease gene was 1,806 bp long, corresponding to a protein of 602 amino acid residues (M(r) = 68,388), and the methylase gene was 1,959 bp long, corresponding to a protein of 653 amino acid residues (M(r) = 76,064). The assignment of the endonuclease gene was confirmed by analysis of the N-terminal amino acid sequence. Genes for the two proteins were in a tail-to-tail orientation, separated by a 131-nucleotide intercistronic region. The predicted amino acid sequences between the StsI system and the FokI system showed a 49% identity between the methylases and a 30% identity between the endonucleases. The sequence comparison of M.StsI with various methylases showed that the N-terminal half of M.StsI matches M.NIaIII, and the C-terminal half matches adenine methylases that recognize GATC and GATATC.

Amino Acid Sequence↗
PubMed1992

Identification of the CTAG-recognizing restriction-modification systems MthZI and MthFI from Methanobacterium thermoformicicum and characterization of the plasmid-encoded mthZIM gene.

Two CTAG-recognizing restriction and modification (R/M) systems, designated MthZI and MthFI, were identified in the thermophilic archaeon Methanobacterium thermoformicicum strains Z-245 and FTF, respectively. Further analysis revealed that the methyltransferase (MTase) genes are plasmid-located in both strains. The plasmid pFZ1-encoded mthZIM gene of strain Z-245 was further characterized by subcloning and expression studies in Escherichia coli followed by nucleotide sequence analysis. The mthZIM gene is 1065 bp in size and may code for a protein of 355 amino acids (M(r) 42,476 Da). The deduced amino acid sequence of the M.MthZI enzyme shares substantial similarity with four distinct regions from several m4C- and m6A-MTases, and contains the TSPPY motif that is so far only found in m4C-MTases. Partially overlapping with the mthZIM gene and in reverse orientation, an additional ORF was identified with a size of 606 bp potentially coding for a protein of 202 amino acids (M(r) 23.710 Da). This ORF is suggested to encode the corresponding endonuclease R.MthZI.

Amino Acid Sequence↗
PubMed1992

Modular organization of related Archaeal plasmids encoding different restriction-modification systems in Methanobacterium thermoformicicum.

Nucleotide sequence comparison of the related 13513-bp plasmid pFV1 and the 11014-bp plasmid pFZ1 from the thermophilic archaeon Methanobacterium thermoformicicum THF and Z-245, respectively, revealed a homologous, approximately 8.2 kb backbone structure that is interrupted by plasmid-specific elements. Various highly conserved palindromic structures and an ORF that could code for a NTP-binding protein were identified within the backbone structure and may be involved in plasmid maintenance and replication. Each plasmid contains at comparable locations a module which specifies components of different restriction-modification (R/M) systems. The R/M module of pFV1 contained, in addition to the genes of the GGCC-recognizing R/M system MthTI, an ORF which may be involved in repair of G-T mismatches generated by deamination of m5C at high temperatures.

Amino Acid Sequence↗
PubMed1976

DNA restriction and modification systems in Salmonella. SQ, a new system derived by recombination between the SB system of Salmonella typhimurium and the SP system of Salmonella potsdam.

As the result of P1-mediated cotransduction with serB from Salmonella potsdam to the Escherichia coli/Salmonella typhimurium hybird 4617, one recombinant, L4004, was isolated which had a restriction-modification (R--M) system different from the SB and SP systems of its parents, and was designated SQ. The genes of SQ were allelic to those of the SB system of S. typhimurium and were shown by complementation experiments to be functionally related to those of the K system of E. coli. Evidence that the SQ system in L4004 arose as the result of a recombination event within the hsdS genes of SB and SP is discussed.

Alleles↗
PubMed1978

Biochemical characterization of the restriction-modification system of Bacillus sphaericus.

A type II restriction endonuclease (endo R . Bsp) has been purified from Bacillus sphaericus to electrophoretic homogeneity. The enzyme appears to be a single polypeptide chain with a molecular weight of 35000. Its pH optimum is around 8.2, it requires 20 mM Mg2+ for optimal activity and it is inhibited by Zn2+. The yield of the enzyme is higher than that of any type II restriction endonuclease so far reported. The enzyme also cleaves single-stranded DNA, albeit at a slower rate. It seems likely that single-stranded DNA is cleaved at the same sequences as double-stranded DNA. Bacillus sphaericus also contains a modification methylase (meth M . Bsp) which completely protects the cell's own DNA against cleavage by its restriction endonuclease. The methylase activity has been partially purified, it copurifies with the nuclease until the next to the last step. The enzyme does not require ATP or Mg2+, it transfers the methyl group of S-adenosyl-methionine to cytosine residues of DNA. As the action of this methylase completely protects any DNA from endo R . Bsp cleavage, it seems likely that the methylase recognizes and methylates the same sequence (dG-dG-dC-dC) as the nuclease.

Bacillus↗
PubMed1992

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.

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↗
PubMed1991

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.

Cloning, Molecular↗
PubMed1991

Transfer of the genes for the StyLTI restriction-modification system of Salmonella typhimurium to strains lacking modification ability results in death of the recipient cells and degradation of their DNA.

The genes encoding the restriction-modification system StyLTI of Salmonella typhimurium were inserted in vivo into the conjugative plasmid pULB21. This allowed us to transfer the StyLTI genes at a very high frequency and to monitor the fate of recipient cells after mating. Transfer of the StyLTI restriction and modification genes into a modificationless recipient was lethal and resulted in degradation of the cell's DNA. This indicates that, in contrast to any other known restriction-modification systems, StyLTI cannot be established after horizontal transfer into a naive host.

Conjugation, Genetic↗
PubMedDate not supplied

[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.

Citrobacter freundii↗
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↗
PubMedDate not supplied

[Properties of the replicator region of the natural plasmid pLG13 containing genes of the EcoRV restriction-modification system].

The previously constructed plasmid pILRV8 that induces endonuclease EcoRV gene overexpression kills cells of some E. coli strains under the induction of this enzyme synthesis. Cell transformation by natural plasmid pLG13 carrying genes of the EcoRV restriction--modification system was found to appreciably enhance cell viability ("survival") under endonuclease overproduction. A plasmid pLG13 region located in immediate proximity to the methylase gene was shown to be responsible for the above effect. This region was also capable for autonomous replication. The analysis of the DNA primary structure in the found replicator region allowed to refer the pLG13 to ColE1 family plasmids. Perturbations in the region lead to loss of the "survival" effect and change of the plasmid replicative properties. A relationship between the replicon elements, the EcoRV genes region and "survival" effect is discussed. Based on the replicon found multicopy vector molecules have been constructed.

Base Sequence↗