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PubMed1984

Methylation dependent expression of the mom gene of bacteriophage Mu: deletions downstream from the methylation sites affect expression.

The expression of the DNA modification gene (mom) of bacteriophage Mu requires the cellular deoxyadenosine methylase (dam) and a transactivation factor from the phage. By hypothesis, the transcription of mom is activated by methylation of three GATC sequences upstream from the mom gene. We have introduced small deletions at a fourth GATC site located about 140 base pairs downstream from the primary methylation region. Some of the deletions severely affect the mom gene expression. We propose from this analysis that (1) some important elements, possibly the promoter, concerned with the expression of mom are located between nucleotides 840 and 880 from the right end of Mu and (2) the mom protein starts with the codon GTG located at position 810. We favor the hypothesis that methylation turns off transcription upstream, thereby allowing the main mom promoter to function.

Base Sequence↗
PubMed1986

Methylation of spore DNA in Bacillus coagulans strain 26.

The modification status of DNA throughout the life cycle of Bacillus coagulans strain 26 was analysed by restriction analysis with methylation-sensitive enzymes. A significant fraction of the GATC sequences (dam target) in spore DNA contain N6-methyladenine, a modification that is lacking during the vegetative phase. From the modulation of the modification pattern of GATC sites, the existence of a de novo methylase may be inferred. Spore DNA was more sensitive than vegetative cell DNA to BamHI, HpaI, SalI and XhoI, indicating that the sites for these enzymes are modified during the vegetative growth phase.

Bacillus↗
PubMed1990

[Genetic determinants of restriction-modification systems in bacteria].

The recent data on the molecular arrangement and functioning of the genetical determinants for the enzymes of restriction-modification systems are discussed. The problems of restriction endonucleases and methylases genes localization, regulation of activity of the genes for enzyme systems of the II type restriction-modification, characteristics of the primary structure of the genes and phylogeny of restriction endonucleases and methylases are reviewed.

Bacteria↗
PubMed1993

Natural variation of the NgoII restriction-modification system of Neisseria gonorrhoeae.

The NgoII restriction-modification (R-M) system of Neisseria gonorrhoeae recognizes the sequence 5'-GGCC-3'. This system is encoded by two separate genes, dcmB for the methyltransferase (MTase) and dcrB for the restriction endonuclease (ENase). Three strains that vary in their NgoII phenotype were examined. Strain Pgh3-2 produced detectable levels of both enzymes, strain F62 lacked detectable levels of the dcrB gene product, and strain WR302 failed to produce either gene product. Strains that lacked either enzyme activity still possessed the genes that encode them. Transcriptional fusions of dcrB in strains F62 and Pgh3-2 indicate that this gene is transcribed at nearly identical levels in each strain. The DNA encoding the NgoII R-M system was cloned from the three strains, and the nucleotide sequence was determined. The dcrB genes of WR302 and F62 possess the same frameshift mutation (base position 1435) which would result in a truncated protein. The WR302 dcmB was found to have a point mutation that changed Arg288 (a residue that is conserved in all prokaryotic and phage cytosine MTases sequenced to date) to Trp.

Amino Acid Sequence↗
PubMed1999

Cell to cell transmission of donor DNA overcomes differential incorporation of non-homologous and homologous markers in Neisseria gonorrhoeae.

The neisseriae are naturally competent for DNA transformation. This genetic study examines whether the modification status of chromosomal donor DNA affects transformation of Neisseria gonorrhoeae to drug resistance. When a single modification system was inactivated, unmodified chromosomal donor DNA was not restricted when used to transform the cognate restriction+ host, irrespective of whether the donor DNA carried a point mutation (homologous marker) or a drug-resistance gene cassette (non-homologous marker). These observations contrasted transformations performed with unmodified plasmid donor DNAs, where the incoming DNA was excluded. However, during the study, it became apparent that certain strains of gonococci showed differential incorporation of non-homologous markers when compared with the incorporation of the homologous marker, even when the donor DNAs were prepared from parental strains. Differential incorporation of markers could be rescued either through cell to cell transmission of donor DNA, or by performing in vitro transformations with donor DNA preparations that were obtained from spent culture supernatants. Overall, the data indicate that, in addition to the exclusion of foreign DNA through the requirement for a genus-specific uptake sequence, gonococci appear capable of excluding DNA on the basis of homology.

Conjugation, Genetic↗
PubMed1997

The Neisseria gonorrhoeae S.NgoVIII restriction/modification system: a type IIs system homologous to the Haemophilus parahaemolyticus HphI restriction/modification system.

Strains of Neisseria gonorrhoeae possess numerous restriction-modification (R-M) systems. One of these systems, which has been found in all strains tested, encodes the S. NgoVIII specificity (5'TCACC 3') R-M system. We cloned two adjacent methyltransferase genes (dcmH and damH), each encoding proteins whose actions protect DNA from digestion by R.HphI or R.Ngo BI (5'TCACC 3'). The damH gene product is a N 6-methyladenine methyltransferase that recognizes this sequence. We constructed a plasmid containing multiple copies of the S.NgoVIII sequence, grew it in the presence of damH and used the HPLC to demonstrate the presence of N 6-methyladenine in the DNA. A second plasmid, containing overlapping damH and Escherichia coli dam recognition sequences in combination with various restriction digests, was used to identify which adenine in the recognition sequence was modified by damH. The predicted dcmH gene product is homologous to 5-methylcytosine methyltransferases. The products of both the dcmH and damH genes, as well as an open reading frame downstream of the damH gene are highly similar to the Haemophilus parahaemolyticus hphIMC , hphIMA and hphIR gene products, encoding the Hph I Type IIs R-M system. The S.NgoVIII R-M genes are flanked by a 97 bp direct repeat that may be involved in the mobility of this R-M system.

Amino Acid Sequence↗
PubMed2001

Characterization of the type IV restriction modification system BspLU11III from Bacillus sp. LU11.

We report the characterization and cloning of the genes for an unusual type IV restriction-modification system, BspLU11III, from Bacillus sp. LU11. The system consists of two methyltransferases and one endonuclease, which also possesses methyltransferase activity. The three genes of the restriction-modification system, bsplu11IIIMa, bsplu11IIIMb and bsplu11IIIR, are closely linked and tandemly arranged. The corresponding enzymes recognize the dsDNA sequence 5'-GGGAC-3'/5'-GTCCC-3', with M.BspLU11IIIa modifying the A (underlined) of one strand and M.BspLU11IIIb the inner C (underlined) of the other strand. R.BspLU11III has both endonuclease and adenine-specific methyltransferase activities and is able to protect the DNA against cleavage by itself. In contrast to all type IV restriction-modification systems described so far, which have only one adenine-specific methyltransferase, BspLU11III is the first type IV restriction-modification system that includes two methyltransferases, one of them being cytosine specific.

Amino Acid Sequence↗
PubMed2005

A dichotomous epigenetic mechanism governs expression of the LlaJI restriction/modification system.

The LlaJI restriction/modification (R/M) system is comprised of two 5mC MTase-encoding genes, llaJIM1 and llaJIM2, and two genes required for restriction activity, llaJIR1 and llaJIR2. Here, we report the molecular mechanism by which this R/M system is transcriptionally regulated. The recognition sequence for the LlaJI MTases was deduced to be 5'GACGC'3 for M1.LlaJI and 5'GCGTC'3 for M2.LlaJI, thus together constituting an asymmetric complementary recognition site. Two recognition sequences for both LlaJI MTases are present within the LlaJI promoter region, indicative of an epigenetic role. Following in vivo analysis of expression of the LlaJI promoter, we established that both LlaJI MTases were required for complete transcriptional repression. A mutational analysis and DNA binding studies of this promoter revealed that the methylation of two specific cytosines by M2.LlaJI within this region was required to trigger the specific and high affinity binding of M1.LlaJI, which serves to regulate expression of the LlaJI operon. This regulatory system therefore represents the amalgamation of an epigenetic stimulation coupled to the formation of a MTase/repressor:promoter complex.

Base Sequence↗
PubMed1989

The fokI restriction-modification system. I. Organization and nucleotide sequences of the restriction and modification genes.

A DNA fragment that carried the genes coding for FokI endonuclease and methylase was cloned from the chromosomal DNA of Flavobacterium okeanokoites, and the coding regions were assigned to the nucleotide sequence by deletion analysis. The methylase gene was 1,941 base pairs (bp) long, corresponding to a protein of 647 amino acid residues (Mr = 75,622), and the endonuclease gene was 1,749 bp long, corresponding to a protein of 583 amino acid residues (Mr = 66,216). The assignment of the methylase gene was further confirmed by analysis of the N-terminal amino acid sequence. The endonuclease gene was downstream from the methylase gene in the same orientation, separated by 69 bp. The promoter site, which could be recognized by Escherichia coli RNA polymerase, was upstream from the methylase gene, and the sequences adhering to the ribosome-binding sequence were identified in front of the respective genes. Analysis of the gene products expressed in E. coli cells by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weights of both enzymes coincided well with the values estimated from the nucleotide sequences, and that the monomeric forms were catalytically active. No significant similarity was found between the sequences of the two enzymes. Sequence comparison with other related enzymes indicated that FokI methylase contained two copies of a segment of tetra-amino acids which is characteristic of adenine-specific methylase.

Amino Acid Sequence↗
PubMed1979

Modification of DNA in chromatin with methyltransferase from Haemophilus influenzae Rd.

The accessibility of DNA in nucleosome dimers (as a model of the chromosomal chain of nucleosomes) was determined by means of modification methylases from Haemophilus influenzae Rd. Using these enzymes, the rate of modification of nucleosome dimers is about one fifth the rate observed with protein-free DNA from chromatin subunit dimers. Methylated DNA sites in nucleosome dimers are readily accessible to micrococcal nuclease. The analysis of the fragment pattern of nucleosomes after methylation and mild nuclease treatment reveals that the methylated sites are predominantly located in the internucleosomal linker DNA. Polylysine binding experiments further support this interpretation. This compound preferentially interacts with the nucleosomal core DNA and protects it against internal cleavage. It neither affects the degradation of methylated sites drastically nor does it inhibit the methylation of nucleosome dimers. Thus, a combination of protection, cleavage and modification is proposed as a useful tool for the analysis of the structure of chromatin.

Adenine↗
PubMed1975

Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes.

Deoxyribonucleic acid (DNA)-cytosine methylation specified by the wild-type Escherichia coli K 12 mec+ gene and by the N-3 drug resistance (R) factor was studied in vivo and in vitro. Phage lambda and fd were propagated in the presence of L-[methyl-3H]methionine in various host bacteria. The in vivo labeled DNA was isolated from purified phage and depurinated by formic acid-diphenylamine treatment. The resulting pyrimidine oligonucleotide tracts were separated according to size and base composition by chromatography on diethylaminoethyl-cellulose in 7 M urea at pH 5.5 and 3.5, respectively. The distribution of labeled 5-methylcytosine in DNA pyrimidine tracts was identical for phage grown in mec+ and mec minus (N-3) cells. For phage lambda the major 5-methylcytosine containing tract was the tripyrimidine, C2T; for both fd-mec minus (N-3) DNA and fd-mec+DNA, C2T was the sole 5-methylcytosine-containing tract. When various lambda DNAs were methylated to saturation in vitro by crude extracts from mec+ and mec minus (N-3) cells, the extent of cytosine methylation was the same. This is in contrast to in vivo methylation where lambda-mec minus (N-3) DNA contains twice as many 5-methylcytosines per genome as lambda-mec+ DNA. Therefore, we suggest that the K12 met+ cytosine methylase and the N-3 plasmid modification methylase are capable of recognizing the same nucleotide sequences, but that the in vivo methylation rate is lower in mec+ cells.

Animals↗
PubMed1997

The in vitro assembly of the EcoKI type I DNA restriction/modification enzyme and its in vivo implications.

Type I DNA restriction/modification enzymes protect the bacterial cell from viral infection by cleaving foreign DNA which lacks N6-adenine methylation within a target sequence and maintaining the methylation of the targets on the host chromosome. It has been noted that the genes specifying type I systems can be transferred to a new host lacking the appropriate, protective methylation without any adverse effect. The modification phenotype apparently appears before the restriction phenotype, but no evidence for transcriptional or translational control of the genes and the resultant phenotypes has been found. Type I enzymes contain three types of subunit, S for sequence recognition, M for DNA modification (methylation), and R for DNA restriction(cleavage), and can function solely as a M2S1 methylase or as a R2M2S1 bifunctional methylase/nuclease. We show that the methylase is not stable at the concentrations expected to exist in vivo, dissociating into free M subunit and M1S1, whereas the complete nuclease is a stable structure. The M1S1 form can bind the R subunit as effectively as the M2S1 methylase but possesses no activity; therefore, upon establishment of the system in a new host, we propose that most of the R subunit will initially be trapped in an inactive complex until the methylase has been able to modify and protect the host chromosome. We believe that the in vitro assembly pathway will reflect the in vivo situation, thus allowing the assembly process to at least partially explain the observations that the modification phenotype appears before the restriction phenotype upon establishment of a type I system in a new host cell.

Chromatography, Gel↗
PubMed1988

[Cytosine DNA-methylase of a SsoII-type from Shigella sonnei 47 cells].

Shigella sonnei 47 cells were found to contain DNA-methylase SsoII which is a modifying component of the system of host specificity of SsoII. The recognition sequence (RS) of methylase SsoII is represented by a five-member palyndromic structure--5'...CCNGG...3'--with a degenerated central nucleotide. Modification of SsoII affords protection of acceptor DNA not only from SsoII type restriction, but also from other restrictases, e. g., Eco RII having an analogous RS but with a less degenerated central nucleotide pair. A simple and rapid procedure for isolation and purification of DNA-methylase ScoII, which employs hydrophobic chromatography on phenyl-Sepharose, has been developed. The enzyme preparation does not contain trace amounts of specific and nonspecific endonucleases and keeps stable on storage in 30% glycerol over a period of one year.

DNA-Cytosine Methylases↗
PubMed1996

Gene transfer in Mycoplasma arthritidis: transformation, conjugal transfer of Tn916, and evidence for a restriction system recognizing AGCT.

Mycoplasma arthritidis is a rat pathogen causing a severe polyarthritis. The study of its pathogenic mechanisms has been hampered by the lack of genetic systems for use with M. arthritidis. Described here are procedures for genetic transformation of M. arthritidis and conjugal transfer of Tn916 from an enterococcal donor to M. arthritidis. The location of Tn916 insertion sites in the mycoplasmal chromosome was random, suggesting that Tn916 may be useful as an insertional mutagen in this organism. Additionally, a restriction and modification system was identified which presented a strong barrier to gene transfer. For transformation, the restriction system was circumvented by using DNA that was modified in vitro with the appropriate site-specific methylase (AluI).

Conjugation, Genetic↗
PubMed1984

The organization and complete nucleotide sequence of the PstI restriction-modification system.

We have determined the nucleotide sequence of a 4.0-kilobase DNA fragment containing the genes of the PstI restriction-modification system. Two large open reading frames were identified within the sequence and were ascribed to the restriction enzyme and methylase by the analysis of a series of deletion mutants. The two genes are encoded on opposite DNA strands, and hence must be transcribed from separate promoters rather than as a polycistronic message. The sequence of the first 10 amino acids of the restriction endonuclease was determined by sequential Edman degradation of the purified protein, permitting the alignment of the polypeptide with the DNA sequence. The NH2 terminus of the modification enzyme was established by sequential Edman degradation of the protein synthesized in bacterial minicells with different radiolabeled amino acids. The initiation codons of the two genes are separated by 130 base pairs. The deduced amino acid sequences indicate that the restriction endonuclease contains 326 amino acids with a calculated Mr = 37,370; the modification enzyme is composed of 507 amino acids with a calculated Mr = 56,830. There is no significant homology between the two proteins at the level of the primary structure. Antibody raised against the purified restriction endonuclease did not immunoprecipitate the modification enzyme. The transcription initiation sites were mapped using mung bean nuclease. Both of the transcripts begin with adenosine. The initiation sites are separated by only 70 base pairs. This close proximity suggests that the promoters for the two divergent genes overlap. DNase I protection experiments show that Escherichia coli RNA polymerase has a higher affinity for the methylase promoter than for the restriction enzyme promoter.

Amino Acid Sequence↗
PubMed1986

Modification enhancement by the restriction alleviation protein (Ral) of bacteriophage lambda.

The product of the lambda ral gene alleviates restriction and enhances modification by the Escherichia coli K-12 restriction and modification system. An open reading frame (orf) located between genes N and Ea10 has been assigned to the ral gene. We have cloned this orf in a plasmid where its transcription is controlled by a thermolabile lambda repressor. Inactivation of the lambda repressor caused a 1000-fold reduction in K-specific restriction of unmodified lambda phage and a 100-fold increase in modification. In minicells transformed with ral+ plasmids, derepression resulted in the appearance of a polypeptide with a lower mobility than that predicted for a protein encoded by the orf attributed to ral; in a transcription and translation system in vitro DNA from a ral+ plasmid encoded a polypeptide with the same mobility. This polypeptide was absent when the plasmid DNA carried a mutant ral gene. The nucleotide sequence of this mutant gene defined two base changes, one of which inactivates the initiation codon of the orf. The K restriction endonuclease, which is also a K-specific methylase, is encoded by three genes designated hsdR, hsdM and hsdS, although the hsdR polypeptide is not essential for the methylase activity. We show that Ral enhances modification in a host strain lacking the entire hsdR gene, and lambda phages carrying the hsdM and S genes modify their own DNA inefficiently in the absence of Ral, despite the fact that derivatives of these phages provide efficient amplification of the K-specific methylase. Our data support a model in which, as a consequence of the interaction of Ral with either the hsdM or the hsdS polypeptide, the conformation of the enzyme is changed and the efficiency of methylation of unmodified target sites is enhanced. It has been postulated that Ral counteracts Rho, but in our experiments Ral did not relieve transcriptional polarity.

Bacteriophage lambda↗
PubMed1987

Cloning of two type II methylase genes that recognise asymmetric nucleotide sequences: FokI and HgaI.

The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells. FokI methylase gene is contained on a 3.80 kb piece of F. okeanokoites DNA. Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI. Unmodified lambda DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI. The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H. galinarum DNA. This plasmid is resistant to digestion by HgaI. Neither the FokI nor the HgaI restriction endonuclease was detected in either clone. This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.

Cloning, Molecular↗