Cloning and nucleotide sequences of the BanI restriction-modification genes in Bacillus aneurinolyticus

The genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39,841 and 42,637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21 bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.     

BsuBI--an isospecific restriction and modification system of PstI: characterization of the BsuBI genes and enzymes.

The enzymes of the Bacillus subtilis BsuBI restriction/modification (R/M) system recognize the target sequence 5'CTGCAG. The genes of the BsuBI R/M system have been cloned and sequenced and their products have been characterized following overexpression and purification. The gene of the BsuBI DNA methyltransferase (M.BsuBI) consists of 1503 bp, encoding a protein of 501 amino acids with a calculated M(r) of 57.2 kD. The gene of the restriction endonuclease (R.BsuBI), comprising 948 bp, codes for a protein of 316 amino acids with a predicted M(r) of 36.2 kD. M.BsuBI modifies the adenine (A) residue of the BsuBI target site, thus representing the first A-N6-DNA methyltransferase identified in B. subtilis. Like R.PstI, R.BsuBI cleaves between the A residue and the 3' terminal G of the target site. Both enzymes of the BsuBI R/M system are, therefore, functionally identical with those of the PstI R/M system, encoded by the Gram negative species Providencia stuartii. This functional equivalence coincides with a pronounced similarity of the BsuBI/PstI DNA methyltransferases (41% amino acid identity) and restriction endonucleases (46% amino acid identity). Since the genes are also very similar (58% nucleotide identity), the BsuBI and PstI R/M systems apparently have a common evolutionary origin. In spite of the sequence conservation the gene organization is strikingly different in the two R/M systems. While the genes of the PstI R/M system are separated and transcribed divergently, the genes of the BsuBI R/M system are transcribed in the same direction, with the 3' end of the M gene overlapping the 5' end of the R gene by 17 bp.     

Cloning and expression of the BalI restriction-modification system.

BalI, a type II restriction-modification (R-M) system from the bacterium, Brevibacterium albidum, recognizes the DNA sequence 5'-TGGCCA-3'. We cloned the genes encoding the BalI restriction endonuclease and methyltransferase and expressed them in Escherichia coli. The two genes were aligned tail-to-tail and their termination codons overlapped. BalI restriction endonuclease and methyltransferase comprise 260 and 280 amino acids, respectively, and have molecular weights of 29 043 and 31 999 Da. The amino acid sequence of BalI methyltransferase is similar to that of other m6A MTases, although it has been categorized as a m5C methyltransferase. A high expression system for the BalI restriction endonuclease was constructed in E. coli for the production of large quantities of enzyme.     

Cloning of the BssHII restriction-modification system in Escherichia coli : BssHII methyltransferase contains circularly permuted cytosine-5 methyltransferase motifs.

BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA between the first and second bases to generate a four base 5'overhang. BssHII restriction endonuclease was purified from the native Bacillus stearothermophilus H3 cells and its N-terminal amino acid sequence was determined. Degenerate PCR primers were used to amplify the first 20 codons of the BssHII restriction endonuclease gene. The BssHII restriction endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene (bssHIIM) were cloned in Escherichia coli by amplification of Bacillus stearothermophilus genomic DNA using PCR and inverse PCR. BssHII methyltransferase (M.BssHII) contains all 10 conserved cytosine-5 methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus, the conserved motifs of M. BssHII are circularly permuted relative to the motif organizations of other cytosine-5 methyltransferases. M.BssHII and the non-cognate multi-specific phiBssHII methyltransferase, M.phiBss HII [Schumann,J. et al . (1995) Gene, 157, 103-104] share 34% identity in amino acid sequences from motifs I-VIII, and 40% identity in motifs IX-X. A conserved arginine is located upstream of a TV dipeptide in the N-terminus of M.BssHII that may be responsible for the recognition of the guanine 5' of the target cytosine. The BssHII restriction endonuclease gene was expressed in E.coli via a T7 expression vector.     

Cloning and nucleotide sequence of the genes coding for the Sau96I restriction and modification enzymes.

The genes coding for the GGNCC specific Sau96I restriction and modification enzymes were cloned and expressed in E. coli. The DNA sequence predicts a 430 amino acid protein (Mr: 49,252) for the methyltransferase and a 261 amino acid protein (Mr: 30,486) for the endonuclease. No protein sequence similarity was detected between the Sau96I methyltransferase and endonuclease. The methyltransferase contains the sequence elements characteristic for m5C-methyltransferases. In addition to this, M.Sau96I shows similarity, also in the variable region, with one m5C-methyltransferase (M.SinI) which has closely related recognition specificity (GGA/TCC). M.Sau96I methylates the internal cytosine within the GGNCC recognition sequence. The Sau96I endonuclease appears to act as a monomer.     

Purification, cloning and sequence analysis of RsrI DNA methyltransferase: lack of homology between two enzymes, RsrI and EcoRI, that methylate the same nucleotide in identical recognition sequences.

RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced. This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI. The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da. A fragment of R. sphaeroides chromosomal DNA exhibited M.RsrI activity in E. coli and was used to sequence the rsrIM gene. The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15. In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology. Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes. The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl. Acids Res. (1989) 17, this issue).     

Stepwise cloning and molecular characterization of the HgiDI restriction-modification system from Herpetosiphon giganteus Hpa2.

The restriction-modification system HgiDI from Herpetosiphon giganteus strain Hpa2 has been cloned in E. coli in a two-step procedure. Selection of the methyltransferase (M.HgiDI) gene in vitro was performed using the heterologous restriction endonuclease AhaII, an isoschizomer of Acyl and HgiDI (GRCGYC). Cloning of the complete HgiDI endonuclease (R.HgiDI) gene could only be achieved in recipient cells harbouring a recombinant plasmid, which was expressing the corresponding methyltransferase and could thereby prevent the host from self-destruction of its genetic material. The HgiDI restriction-modification system was sequenced and functionally correlated with two open reading frames of 309 (M) and 359 (R) codons. In homology studies M.HgiDI showed significant similarities to 20 other m5C-methyltransferases and turned out to be the most compact enzyme of this group described so far. Initial attempts for overexpression of M.HgiDI and partial purification of R.HgiDI have been successful.     

Characterization of Chlorella virus PBCV-1 CviAII restriction and modification system.

A second DNA site-specific (restriction) endonuclease (R.CviAII) and its cognate adenine DNA methyltransferase (M.CviAII) were isolated from virus PBCV-1 infected Chlorella strain NC64A cells. R.CviAII, a heteroschizomer of the bacterial restriction endonuclease NlaIII, recognizes the sequence CATG, and does not cleave CmATG sequences. However, unlike NlaIII, which cleaves after the G and does not cleave either CmATG or mCATG sequences, CviAII cleaves between the C and A and is unaffected by mCATG methylation. The M.CviAII and R.CviAII genes were cloned and their DNA sequences were determined. These genes are tandemly arranged head-to-tail such that the TAA termination codon of the M.CviAII methyltransferase gene overlaps the ATG translational start site of R.CviAII endonuclease. R.CviAII is the first chlorella virus site-specific endonuclease gene to be cloned and sequenced.     

The NIaIV restriction and modification genes of Neisseria lactamica are flanked by leucine biosynthesis genes

The genes encoding the Neisseria lactamica restriction endonuclease IV (R.NIaIV) and its cognate DNA methyltransferase (M.NlaIV), both of which recognize the sequence GGNNCC, have been cloned in Escherichia coli and overexpressed using the T7 polymerase/promoter system. Analysis of a sequenced 3.58 kb fragment established the gene order, leuD-M.NlaIV-R.NlaIV-leuB. The predicted primary sequence of M.NlaIV (423 amino acids) shows the highest degree of identity to a pair of cytosine-specific methyltransferases, M.BanI (44.9%) and M.HgiCI (44.3%), which recognize the sequence GGYRCC (Y, pyrimidines; R, purines). In contrast, the R.NlaIV protein sequence (243 amino acids) is unique in the existing database, a situation that holds for most endonucleases. Flanking the NlaIV modification and restriction genes are homologues of the leuD and leuB genes of enteric bacteria, which code for enzymes in the leucine biosynthesis pathway. This gene context implies a possible new mode of gene regulation for the RM.NlaIV system, which would involve a mechanism similar to the recently discovered leucine/Lrp regulon in E. coli.     

Identification of an isoschizomer of the HhaI DNA methyltransferase in Mycoplasma arthritidis

The genome of Mycoplasma arthritidis strain 158 has modified cytosine residues at AGCT sequences that render the DNA resistant to digestion with the AluI restriction endonuclease. The DNA methyltransferase responsible for the base modification has previously been designated MarI. From the complete genome sequence of M. arthritidis , we identify Marth_orf138 as a candidate marI gene. Marth_orf138 was cloned in Escherichia coli and its TGA codons converted to TGG. DNA isolated from E. coli cells expressing the modified Marth_orf138 gene was degraded by the AluI nuclease, indicating that Marth_orf138 does not code for MarI. However, the DNA from E. coli was found to have acquired resistance to the restriction endonuclease HhaI. Genomic DNA from M. arthritidis was also found to be resistant to HhaI (recognizes GCGC). The M. arthritidis isoschizomer of the HhaI DNA methyltransferase, coded by Marth_orf138, is designated MarII. Transformation of M. arthritidis was not significantly affected by modification of plasmid at HhaI sites, indicating that the mycoplasma lacks a restriction endonuclease that recognizes GCGC sites.     

Nucleotide sequence of the EcoRII restriction endonuclease gene

The nucleotide sequence of a 1394 basepair (bp) DNA fragment containing the EcoRII restriction endonuclease (R.EcoRII) gene was determined. The endonuclease gene is 1206 bp in length (predicted 402 amino acids (aa) and Mr = 45 178) and is separated by 33 bp from the EcoRII modification methylase (M.EcoRII) gene. The EcoRII restriction-modification system has a tail-to-tail organization of the two genes.     

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.     

Cloning and characterization of the MboII restriction-modification system

The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.     

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.     

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.