Cloning and expression of the MspI restriction and modification genes

The genes for the MspI restriction (R) and modification enzymes (recognition sequence CCGG) have been cloned into Escherichia coli using the vector pBR322. Clones carrying both genes have been isolated from libraries prepared with EcoRI, HindIII and BamHI. The smallest fragment that encodes both activities is a 3.6-kb HindIII fragment. Plasmids purified from the clones are fully resistant to digestion by MspI, indicating that the modification gene is functional in E. coli. The clones remain sensitive to phage infection, however, indicating that the endonuclease is dysfunctional. When the R gene is brought under the control of the inducible leftward promoter from phage lambda, the level of endonuclease increases and the level of methylase decreases, suggesting that the genes are transcribed in opposite directions.     

Molecular cloning and expression in Escherichia coli of two modification methylase genes of Bacillus subtilis

Two modification methylase genes of Bacillus subtilis R were cloned in Escherichia coli by using a selection procedure which is based on the expression of these genes. Both genes code for DNA-methyltransferases which render the DNA of the cloning host E. coli HB101 insensitive to the BspRI (5'-GGCC) endonuclease of Bacillus sphaericus R. One of the cloned genes is part of the restriction-modification (RM) system BsuRI of B. subtilis R with specificity for 5'-GGCC. The other one is associated with the lysogenizing phage SP beta B and produces the methylase M.BsuP beta BI with specificity for 5'-GGCC. The fragment carrying the SP beta B-derived gene also directs the synthesis in E. coli of a third methylase activity (M.BsuP beta BII), which protects the host DNA against HpaII and MspI cleavage within the sequence 5'-CCGG. Indirect evidence suggests that the two SP beta B modification activities are encoded by the same gene. No cross-hybridization was detected either between the M.BsuRI and M.BsuP beta B genes or between these and the modification methylase gene of B. sphaericus R, which codes for the enzyme M.BspRI with 5'-GGCC specificity.     

The HgaI restriction-modification system contains two cytosine methylase genes responsible for modification of different DNA strands.

A DNA fragment of about 3.4 kilobase pairs that expressed the HgaI modification activity was cloned from the chromosomal DNA of Haemophilus gallinarum, and its nucleotide sequence was determined. Two open reading frames (ORF) which could code for structurally similar proteins were identified in the upstream and middle regions and a truncated ORF in the downstream region in the same orientation. When the respective ORFs were separately cloned, the clones carrying the upstream and middle ORFs both expressed the modification activity, indicating that the two genes are involved in modification of the HgaI restriction-modification system. In order to determine the sites of modification precisely, the respective genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the HgaI recognition site was treated with each of these enzymes, and, after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to HgaI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by each gene product. Analysis of the species and positions of modified bases by the Maxam-Gilbert method further demonstrated that the gene products from the upstream and middle ORFs participated in methylation of the internal cytosine residues of the strands carrying 3'-CTGCG-5' and 5'-GACGC-3', respectively. We concluded that the HgaI modification system consisted of two cytosine methylase genes responsible for modification of different strands in the target DNA.     

Cloning and expression in Escherichia coli of the BanI and BanIII restriction-modification systems from Bacillus aneurinolyticus

The genes coding for the GGPyPuCC-specific (BanI) and ATCGAT-specific (BanIII) restriction-modification systems of Bacillus aneurinolyticus IAM1077 were cloned and expressed in Escherichia coli using pBR322 as a vector. The plasmids carrying the BanI and BanIII restriction-modification genes were designated pBanIRM8 and pBanIIIRM12, respectively. The restriction maps of these recombinant plasmids were constructed. These two plasmids were stably maintained in E. coli HB101. However, when E. coli JM109 was used as a host, pBanIIIRM12 was efficiently propagated but pBanIRM8 was not. The HB101 cells carrying only the restriction gene of BanIII were viable, but the BanI restriction gene carrier could not form colonies on agar plates. The growth of bacteriophage λ was strongly restricted only in the F. coli HB101 cells harboring pBanIRM8. These facts indicate that the BanI restriction enzyme is expressed and functions more efficiently than BanI modification enzyme in E. coli.     

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.     

Localization of Separate Genetic Loci for Reduced Sensitivity towards Small Isometric-Headed Bacteriophage sk1 and Prolate-Headed Bacteriophage c2 on pGBK17 from Lactococcus lactis subsp. lactis KR2

The mechanism of reduced sensitivity to the small isometric-headed bacteriophage sk1 encoded on a 19-kilobase (kb) HpaII fragment subcloned from pKR223 of Lactococcus lactis subsp. lactis KR2 was examined. The reduced sensitivity to phage sk1 was due to a modest restriction/modification (R/M) system that was not active against prolate-headed phage c2. The genetic loci for the R/M system against sk1 and the abortive phage infection (Abi) mechanism effective against phage c2 were then localized by restriction mapping, subcloning, and deletion analysis. The restriction gene was localized to a region of a 2.7-kb EcoRV fragment and included an EcoRI site within that fragment. The modification gene was found to be physically separable from the restriction gene and was present on a 1.75-kb BstEII-XbaI fragment. The genetic locus for the Abi phenotype against phage c2 was localized to a region containing a 1.3-kb EcoRI fragment. Attempts to clone the c2 Abi mechanism independent of the sk1 R/M system were unsuccessful, suggesting that expression of the abi genes required sequences upstream of the modification gene. Some pGBK17 (vector pGB301 plus a 19-kb HpaII insert fragment) transformants exhibited the R/M system against phage sk1 but lost the Abi mechanism against phage c2. These transformants contained a 1.2- to 1.3-kb insertion in the Abi region. The data identified genetic loci on a cloned 19-kb HpaII fragment responsible for restriction activity and for modification activity against a small isometric-headed phage and for Abi activity against prolate-headed phage c2. A putative insertion element was also found to inactivate the abi gene(s).     

Overproduction of the EcoRII endonuclease and methylase by Escherichia coli strains carrying recombinant plasmids constructed in vitro

Recombinant DNA molecules were constructed from the plasmid pIL203 and the EcoRI-fragment of N3 plasmid containing EcoRII endonuclease and methylase genes and also a gene for resistance to sulfanilamide. The pIL203 plasmid, used as a vector, consisted of the Bam HI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the Bam HI-EcoRI-fragment of lambda phage containing promoters, a thermosensitive mutation in the cI gene and a suppressible amber mutation in the cro gene. Ampicillin-sulfanilamide-resistant clones were selected and tested for their restriction and modification phenotype. The recombinant plasmid DNA, isolated from ApRSuR-resistant clones, which restricted and modified phage lambda imm21 with EcoRII specificity, had the EcoRI-fragment with EcoRII genes in a single orientation. The recombinant plasmid pSK323 was transferred into E. coli strains with su-, su1, su2 or su3 phenotypes. The synthesis of products of EcoRII genes by these strains grown at 37 degrees C is increased by 10--50-fold.     

Cloning of the restriction-modification genes of Bacillus centrosporus in Escherichia coli

A genomic library of Bacillus centrosporus was obtained using pBR327 as a vector. The total plasmid DNA of the library was cleaved by the BcnI restriction endonuclease and then transformed in Escherichia coli RR1. Two clones possessing restriction and DNA modification profiles of BcnI were identified among the transformants. Their respective plasmids were 13.3 and 9.05 kbp in size. Restriction mapping of both plasmids showed each of them to contain two sites for HindIII and one for both Eco31I and Eco47III, located at the same distance. This was assumed to be the location region of the BcnI restriction-modification genes. Confirmation of the assumption was obtained by deletion mapping of the recombinant plasmids. Special features concerning cloning of the restriction-modification genes are discussed on the basis of the results obtained.     

Cloning type-II restriction and modification genes

We have cloned into Escherichia coli the genes for 38 type-II bacterial modification methyltransferases. The clones were isolated by selecting in vitro for protectively modified recombinants. Most of the clones modify their DNA fully but a substantial number modify only partially. In approximately one-half of the clones, the genes for the corresponding endonucleases are also present. Some of these clones restrict infecting phages and others do not. Clones carrying endonuclease genes but lacking methyltransferase genes have been found, in several instances, to be viable.     

Novel molecular features of the fibrolytic intestinal bacterium Fibrobacter intestinalis not shared with Fibrobacter succinogenes as determined by suppressive subtractive hybridization

Suppressive subtractive hybridization was conducted to identify unique genes coding for plant cell wall hydrolytic enzymes and other properties of the gastrointestinal bacterium Fibrobacter intestinalis DR7 not shared by Fibrobacter succinogenes S85. Subtractive clones from F. intestinalis were sequenced and assembled to form 712 nonredundant contigs with an average length of 525 bp. Of these, 55 sequences were unique to F. intestinalis. The remaining contigs contained 764 genes with BLASTX similarities to other proteins; of these, 80% had the highest similarities to proteins in F. succinogenes, including 30 that coded for carbohydrate active enzymes. The expression of 17 of these genes was verified by Northern dot blot analysis. Of genes not exhibiting BLASTX similarity to F. succinogenes, 30 encoded putative transposases, 6 encoded restriction modification genes, and 45% had highest similarities to proteins in other species of gastrointestinal bacteria, a finding suggestive of either horizontal gene transfer to F. intestinalis or gene loss from F. succinogenes. Analysis of contigs containing segments of two or more adjacent genes revealed that only 35% exhibited BLASTX similarity and were in the same orientation as those of F. succinogenes, indicating extensive chromosomal rearrangement. The expression of eight transposases, and three restriction-modification genes was confirmed by Northern dot blot analysis. These data clearly document the maintenance of carbohydrate active enzymes in F. intestinalis necessitated by the preponderance of polysaccharide substrates available in the ruminal environment. It also documents substantive changes in the genome from that of F. succinogenes, which may be related to the introduction of the array of transposase and restriction-modification genes.     

Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII

The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI. Both chromosomal and plasmid DNA were modified in the selected clones. None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked. Crude cell extracts prepared from the recombinant strains, but not the host (E. coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG. Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain. 5-Methylcytosine was identified as the product of the reaction chromatographically. The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods. This modification blocks cleavage by both MspI and its isoschizomer HpaII. HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate. The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.     

Overproduction of the hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype

The geneshsdM andhsdS for M.EcoKI modification methyltrasferase and the complete set ofhsdR,hsdM andhsdS genes coding for R.EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation inhsdS gene, were cloned in pBR322 plasmid and introduced intoE. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. ThehsdS _ts-1 mutation, mapped previously in the distal variable region of thehsdS gene with C^{1 245}-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42°C. In clones transformed with the wholehsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutanthsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of thehsdS _ts-1 mutation on restriction and modification.     

Cloning the BamHI restriction modification system.

BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.     

Experimental genome evolution: large-scale genome rearrangements associated with resistance to replacement of a chromosomal restriction-modification gene complex

Type II restriction enzymes are paired with modification enzymes that protect type II restriction sites from cleavage by methylating them. A plasmid carrying a type II restriction-modification gene complex is not easily replaced by an incompatible plasmid because loss of the former leads to cell death through chromosome cleavage. In the present work, we looked to see whether a chromosomally located restriction-modification gene complex could be replaced by a homologous stretch of DNA. We tried to replace the PaeR7I gene complex on the Escherichia coli chromosome by transducing a homologous stretch of PaeR7I-modified DNA. The replacement efficiency of the restriction-modification complex was lower than expected. Some of the resulting recombinant clones retained the recipient restriction-modification gene complex as well as the homologous DNA (donor allele), and slowly lost the donor allele in the absence of selection. Analysis of their genome-wide rearrangements by Southern hybridization, inverse polymerase chain reaction (iPCR) and sequence determination demonstrated the occurrence of unequal homologous recombination between copies of the transposon IS3. It was strongly suggested that multiple rounds of unequal IS3-IS3 recombination caused large-scale duplication and inversion of the chromosome, and that only one of the duplicated copies of the recipient PaeR7I was replaced.     

The location of a temperature-sensitive trans-dominant mutation and its effect on restriction and modification in Escherichia coli K12

An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4 alpha gt57 beta gt14 and lambda vir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage lambda carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdXts+d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the lambda phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the Xts+d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of Xts+d mutation was studied. The results of complementation experiments, using F'-merodiploids or plasmid pBR322 with an inserted Xts+d mutation, support the idea that the HsdSts+d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.     

Cloning and regulation of gene expression of EcoRV restriction- modification system

A number of recombinant plasmids, containing EcoRV restriction-modification genes have been constructed. Individual genes of this system were introduced into plasmids of various incompatibility groups. Promoter regions of genes encoding methylase and restrictase have been cloned and studied. With the use of specialized vector pVE8 it was shown that the efficiency of the endonuclease gene promoter is comparable with early lambda phage promoters and produced about 70% of PL efficiency. The efficiency of the methylase gene promoter region was twice less than the efficiency of the restriction endonuclease gene promoter. Plasmid with restriction endonuclease gene promoter located downstream in relation to the additional regulatable phage lambda promoter PL has been obtained. It enabled us to construct strains 30-40 fold overproducing this enzyme under conditions of inactivation of the temperature sensitive phage repressor c1857. This construction directs the production of a high level (10%) of the total cellular soluble proteins) of the EcoRV restriction enzyme. The factors that influenced the level of enzyme synthesis under induction are discussed.