Genetic basis of the complementary DpnI and DpnII restriction systems of S. pneumoniae: an intercellular cassette mechanism

Cells of S. pneumoniae contain either DpnI, a restriction endonuclease that cleaves only the methylated DNA sequence 5'-GmeATC-3', or DpnII, which cleaves the same sequence when not methylated. A chromosomal DNA segment containing DpnII genes was cloned in S. pneumoniae. Nucleotide sequencing of this segment revealed genes encoding the methylase and endonuclease and a third protein of unknown function. When the plasmid was introduced into DpnI cells, recombination during chromosomal facilitation of its establishment substituted genes encoding the DpnI endonuclease and another protein in place of the DpnII genes. DNA hybridization and sequencing showed that the DpnI and DpnII segments share homology on either side but not between themselves or with other regions of the chromosome. Thus, the complementary restriction systems are found on nonhomologous and mutually exclusive cassettes that can be inserted into a particular point in the chromosome of S. pneumoniae on the basis of neighboring homology.     

Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase.

The gene coding for the pneumococcal DNA adenine methylase that recognizes the sequence 5'-GATC-3' was cloned in a strain of Streptococcus pneumoniae that lacked both restriction endonucleases DpnI and DpnII. The gene was cloned as a 3.7-kilobase fragment of chromosomal DNA from a DpnII-containing strain inserted in both possible orientations in the multicopy plasmid vector pMP5 to give recombinant plasmids pMP8 and pMP10. Recombinant plasmids were selected by their resistance to DpnII cleavage. Cells carrying the recombinant plasmids modified phage in vivo so that it was restricted by DpnI- but not DpnII-containing hosts. They also showed levels of DNA methylase activity five times higher than that in cells of the original DpnII strain. No DpnII activity was observed in the clones; therefore, it was concluded that the insert did not contain an intact DpnII endonuclease gene and that methylation of host DNA did not turn on a latent form of the gene.     

Transfer of recombinant plasmids containing the gene for DpnII DNA methylase into strains of Streptococcus pneumoniae that produce DpnI or DpnII restriction endonucleases.

Plasmid transfer via the transformation pathway of Streptococcus pneumoniae was weakly restricted by the DpnI or DpnII restriction endonuclease, either of which gave a reduction only to 0.4, compared with phage infection, which was restricted to 10(-5). The greater sensitivity of plasmid transfer compared with chromosomal transformation, which was not at all restricted, can be attributed to partially double-stranded intermediates formed from two complementary donor fragments. However, clustering of potential restriction sites in the plasmids increased the probability of escape from restriction. The recombinant plasmid pMP10 , in which the gene for the DpnII DNA methylase was cloned, can be transferred to strains that contain neither restriction enzyme or that contain DpnII as readily as can the vector pMP5 . Introduction of pMP10 raised the level of methylase by five times the level normally present in DpnII strains. Transfer of pMP10 to DpnI -containing strains was infrequent, presumably owing to the suicidal methylation of DNA which rendered it susceptible to the host endonuclease. The few clones in which pMP10 was established had lost DpnI . Loss of the plasmid after curing of the cell eliminated the methylase but did not restore DpnI . Although this loss of DpnI could result from spontaneous mutation, its relatively high frequency, 0.1% suggested that the loss was due to a regulatory shift.     

Identification and sequence analysis of a methylase gene in Porphyromonas gingivalis.

A gene from the periodontal organism Porphyromonas gingivalis has been identified as encoding a DNA methylase. The gene, referred to as pgiIM, has been sequenced and found to contain a reading frame of 864 basepairs. The putative amino acid sequence of the encoded methylase was 288 amino acids, and shared 47% and 31% homology with the Streptococcus pneumoniae DpnII and E. coli Dam methylases, respectively. The activity and specificity of the pgi methylase (M.PgiI) was confirmed by cloning the gene into a dam- strain of E. coli (JM110) and performing a restriction analysis on the isolated DNA with enzymes whose activities depended upon the methylation state of the DNA. The data indicated that M.PgiI, like DpnII and Dam, methylated the adenine residue within the sequence 5'-GATC-3'.     

Hypermutation in pathogenic bacteria: frequent phase variation in meningococci is a phenotypic trait of a specialized mutator biotype

Expression of serogroup B meningococcal capsular polysaccharide undergoes frequent phase variation involving reversible frameshift mutations within a homopolymeric repeat in the siaD gene. A high rate of phase variation is the consequence of a biochemical defect in methyl-directed mismatch repair. The mutator phenotype is associated to the absence of DNA adenine methyltransferase (Dam) activity in all pathogenic isolates and in 50% of commensal strains. Analysis of the meningococcal dam gene region revealed that in all Dam- strains a gene encoding a putative restriction endonuclease (drg) that cleaves only the methylated DNA sequence 5'-GmeATC-3' replaced the dam gene. Insertional inactivation of the dam and/or drg genes indicated that high rates of phase variation and hypermutator phenotype are caused by absence of a functional dam gene.     

Cloning and sequencing of LlaDCHI [corrected] restriction/modification genes from Lactococcus lactis and relatedness of this system to the Streptococcus pneumoniae DpnII system.

The natural 7.8-kb plasmid pSRQ700 was isolated from Lactococcus lactis subsp. cremoris DCH-4. It encodes a restriction/modification system named LlaDCHI [corrected]. When introduced into a phage-sensitive L. lactis strain, pSRQ700 confers strong phage resistance against the three most common lactococcal phage species, namely, 936, c2, and P335. The LlaDCHI [corrected] endonuclease was purified and found to cleave the palindromic sequence 5'-GATC-3'. It is an isoschizomer of Streptococcus pneumoniae DpnII. The plasmid pSRQ700 was mapped, and the genetic organization of LlaDCHI [corrected] was localized. Cloning and sequencing of the entire LlaDCHI [corrected] system allowed the identification of three open reading frames. The three genes (llaIIA, llaIIB, and llaIIC) overlapped and are under one putative promoter. A putative terminator was found at the end of llaIIC. The genes llaIIA and llaIIB coded for m6A methyltransferases, and llaIIC coded for an endonuclease. The LlaDCHI [corrected] system shares strong genetic similarities with the DpnII system. The deduced amino acid sequence of M.LlaIIA was 75% identical with that of M.DpnII, whereas M.LlaIIB was 88% identical with M.DpnA. However, R.LlalII shared only 31% identity with R.DpnII.     

Characterization of a novel type II restriction-modification system, Sth368I, encoded by the integrative element ICESt1 of Streptococcus thermophilus CNRZ368

A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to phiST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5'-GATC-3'. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5'-GATC-3' was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5'-GATC-3'. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5'-GATC-3' and is related to the Sau3AI and LlaKR2I restriction systems.     

Site specific endonuclease from Fusobacterium nucleatum.

Four different isolates of Fusobacterium nucleatum (A,C,D and E) contain restriction endonucleases of differing specificity. Whilst many of the endonucleases are isochizomers of known enzymes, two novel activities are Fnu DII which recognizes and cleaves the sequence 5'-CGCT-3'/3'-GCGC-5' AND Fnu EI which recognizes and cleaves the sequence 5'-GATC-3'/3'-CTAG-5' irrespective of the extent of methylation of the adenine residues.     

Evolution and function of the neisserial dam-replacing gene

Phase variation through slippage-like mechanisms involving homopolymeric tracts depends in part on the absence of Dam-methylase in several pathogenic isolates of Neisseria meningitidis. In Dam-defective strains drg (dam-replacing gene), flanked by pseudo-transposable small repeated elements (SREs), replaced dam. We demonstrate that drg encodes a restriction endonuclease (NmeBII) that cleaves 5'-GmeATC-3'. drg is also present in 50% of Neisseria lactamica strains, but in most of them it is inactive because of the absence of an SRE-providing promoter. This is associated with the presence of GATmeC, suggesting an alternative restriction-modification system (RM) specific for 5'-GATC-3', similar to Sau3AI-RM of Staphylococcus aureus 3A, Lactococcus lactis KR2 and Listeria monocytogenes.     

Cleavage at the twelve-base-pair sequence 5''-TCTAGATCTAGA-3'' using M.Xbal (TCTAGm6A) methylation and DpnI (Gm6A/TC) cleavage.

The DNA methylase M.Xbal was isolated from an E. coli recombinant clone. We deduce that the enzyme methylates at the sequence 5'-TCTAGm6A-3'. In combination with the methylation-dependent restriction endonuclease, DpnI (5'-Gm6A/TC-3'), DNA cleavage occurs at the sequence 5'-TCTAGA/TCTAGA-3'. This twelve-base-pair site should occur once every 16,000,000 base pairs in a random sequence of DNA. The exceptional rarity of the M.XbaI/DpnI sequence makes it an ideal candidate for transpositional integration of a unique cleavage site into bacterial genomes. Retrotransposition into mammalian genomes is also an attractive possibility.     

Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections.

Methylations of highly specific sites in the promoter and 5' regions of eucaryotic genes have been shown to shut off gene activity and thus play a role in the long-term regulation of gene expression. It was therefore of interest to investigate whether site-specific DNA methylations could also play a role in adenovirus DNA in productive infections. It has been reported earlier that adenovirion DNA is not detectably methylated (U. Günthert, M. Schweiger, M. Stupp, and W. Doerfler, Proc. Natl. Acad. Sci. USA 73:3923-3927, 1976). In the present study, evidence for the methylation of cytidine residues in 5'-CCGG-3' and 5'-GCGC-3' sequences or the methylation of adenine residues in 5'-GATC-3' and 5'-TCGA-3' sequences in intranuclear adenovirus type 2(Ad2) DNA isolated and analyzed early (5 h) or late (24 h) after infection could not be obtained. In Ad2 DNA, 22.5% of all 5'-CG-3' dinucleotides reside in 5'-CCGG-3' and 5'-GCGC-3' sequences. Intranuclear viral DNA was examined by restriction endonuclease cleavage by using HpaII, MspI, HhaI, DpnI, or TaqI and Southern blot hybridizations. The HindIII fragments of Ad2 DNA served as hybridization probes. The data rendered it very unlikely that free intracellular adenovirus DNA in productively infected cells was extensively methylated. Thus, DNA methylation was not a likely element in the regulation of free adenovirus DNA expression in productively infected cells.     

Restriction endonucleases in Azospirillum

Azospirillum brasilense, A. amazonense, and A. lipoferum strains were screened for restriction endonucleases using phage lambda DNA. The extract of A. brasilense 29711 cleaved lambda DNA into specific fragments. It was concluded that this strain possesses a class II restriction endonuclease which was named AbrI. AbrI has a single recognition site on lambda DNA at position of approx. 33 500 bp. AbrI was characterized as an isoschizomer of XhoI, which cuts lambda DNA at 33 498 bp and cleaves double-stranded DNA at the sequence 5'-C TCGAG-3'. From other Azospirilla strains only A. amazonense QRZ42 extracts (AamI activity) cleaved DNA into specific fragments under certain conditions.     

Lack of DNA methylation in Schistosoma mansoni

Schistosoma mansoni genomic DNA from male and female adult worms was subjected to restriction by the isoschizomeric endonucleases HpaII and MspI, which display different sensitivities with respect to cytosine methylation. The digested DNA was hybridized with 13 S. mansoni probes. Southern blot analysis showed that there were no observable differences in the restriction patterns of the two isoschizomers and that the patterns were identical in male and female parasites. Adenine methylation was also ruled out since no differences were observed with DpnI, Sau3A1, or MboI restriction enzymes. The methylation-dependent restriction endonuclease McrBC, which cleaves DNA containing methylcytosine and will not cleave unmethylated DNA, did not digest S. mansoni genomic DNA. These results demonstrate that the genome of adult S. mansoni is not methylated.     

Proteins encoded by the DpnI restriction gene cassette. Hyperproduction and characterization of the DpnI endonuclease

Insertion mutations in the DpnI gene cassette of Streptococcus pneumoniae indicated that the two genes it contains, dpnC and dpnD, were transcribed from an adjacent promoter and that only dpnC was necessary for expression of the DpnI endonuclease. Large amounts of the DpnI endonuclease were produced from the cloned cassette in an Escherichia coli expression system, and the enzyme was purified to homogeneity. The DpnI endonuclease is composed of a single polypeptide of 30 kDa, which, as shown by NH2-terminal sequencing of the protein, is encoded by the entire dpnC open reading frame. The native protein sedimented as a monomer of 30 kDa in 0.5 M NaCl. A protein composed of a 20-kDa polypeptide, which is presumably encoded by dpnD, was also produced in large amounts. It was partially purified, but its function is unknown. Examination of the predicted amino acid sequence of DpnI revealed a potential metal-containing, DNA-binding finger structure. It is suggested that this structure provides the specificity for recognition of the methylated DNA sequence, 5'-GmATC-3', that is cleaved by the DpnI endonuclease.     

Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease.

The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme. The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine. This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease. Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification.     

Identification of a nuclease and host restriction-modification in the unicellular, aerobic nitrogen-fixing cyanobacterium Cyanothece sp.

In the process of developing a gene transfer system for the marine, unicellular, nitrogen-fixing cyanobacterium Cyanothece sp. strain BH68K, two major restriction barriers have been identified. A cell wall-associated nuclease exhibited non-site-specific degradation of covalently closed circular and linear double-stranded DNA molecules, including Cyanothece sp. strain BH68K chromosomal DNA. The nuclease is easily released from intact cells by using water or buffer containing Triton X-100. Nuclease activity was undetectable in cell extracts prepared from water-washed cells. Comparison of the restriction endonuclease susceptibility of Cyanothece sp. strain BH68K DNA to that of Anabaena sp. strain PCC 7120 revealed that these organisms have a nearly identical pattern of restriction and therefore may contain similar systems for DNA methylation. Restriction by DpnI, MboI, and Sau3AI indicated the presence of adenine methylation. Cyanothece sp. strain BH68K cell extracts contain a type II restriction endonuclease, Csp68KI. The activity of Csp68KI was easily detected in cell extracts without extensive purification. Csp68KI is an isoschizomer of AvaII and recognizes the nucleotide sequence 5'-GG(A/T)CC-3'. Cleavage occurs between the guanosine nucleotides producing 3-bp 5' overhang ends.     

Crystal structure and function of C-terminal Sau3AI domain

Sau3AI is a type II restriction enzyme that recognizes the 5'-GATC-3' sequence in double-strand DNA and cleaves at 5' to the G residue. The C-terminal domain of Sau3AI (Sau3AI-C), which contains amino acids from 233 to 489, was crystallized and its structure was solved by using the Multi-wavelength Anomalous Diffraction method. The Sau3AI-C structure at 1.9 A resolution is similar to the structure of MutH, a DNA mismatch repair protein that shares high sequence similarity with the N-terminal Sau3AI domain. The functional analysis shows that Sau3AI-C can bind DNA with one recognition sequence but has no cleavage activity. These results indicate that Sau3AI is a pseudo-dimer belonging to the type IIe restriction enzymes and the Sau3AI-C is the allosteric effector domain that assists DNA binding and cleavage.     

Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains.     

Purification of restriction endonuclease XcyI from Xanthomonas cyanopsidis

A new Type II restriction endonuclease XcyI, purified from Xanthomonas cyanopsidis 13D5, is an isoschizomer of SmaI and XmaI that cleaves at the nucleotide sequence 5'-C decreases CCGGG-3' of double-stranded DNA. The single restriction activity present in this strain permits rapid purification of 8000 units of cleavage activity from 10 g of freshly harvested cells. The resulting XcyI preparation is free of contaminating nuclease activities that interfere with in vitro manipulation of DNA.     

In silico restriction landmark genome scanning analysis of Xanthomonas oryzae pathovar oryzae MAFF 311018

We have developed a restriction landmark genome scanning (RLGS) system in silico, involving two-dimensional electrophoretic analysis of DNA by computer simulation that is based on the availability of whole-genome sequences for specific organisms. We applied the technique to the analysis of the Xanthomonas oryzae pathovar oryzae (Xoo) MAFF 311018, which causes bacterial blight in rice. The coverage that was found to be achievable using RLGS in silico, as a percentage of the genomic regions that could be detected, ranged from 44.5% to 72.7% per image. However, this reached a value of 96.7% using four images that were obtained with different combinations of landmark restriction enzymes. Interestingly, the signal intensity of some of the specific spots obtained was significantly lower than that of other surrounding spots when MboI, which cleaves unmethylated 5'-GATC-3' sites, was used. DNA gel blot analysis with both DNA adenine methylase (Dam)-sensitive and -insensitive isoschizomers (MboI and Sau3AI) revealed that Dam-mediated DNA adenine methylation had indeed occurred at these particular sites. These results suggest that a significant portion of the 5'-GATC-3' sites within the Xoo genome is stably methylated by Dam.