Evidence for an evolutionary antagonism between Mrr and Type III modification systems

The Mrr protein of Escherichia coli is a laterally acquired Type IV restriction endonuclease with specificity for methylated DNA. While Mrr nuclease activity can be elicited by high-pressure stress in E. coli MG1655, its (over)expression per se does not confer any obvious toxicity. In this study, however, we discovered that Mrr of E. coli MG1655 causes distinct genotoxicity when expressed in Salmonella typhimurium LT2. Genetic screening enabled us to contribute this toxicity entirely to the presence of the endogenous Type III restriction modification system (StyLTI) of S. typhimurium LT2. The StyLTI system consists of the Mod DNA methyltransferase and the Res restriction endonuclease, and we revealed that expression of the LT2 mod gene was sufficient to trigger Mrr activity in E. coli MG1655. Moreover, we could demonstrate that horizontal acquisition of the MG1655 mrr locus can drive the loss of endogenous Mod functionality present in S. typhimurium LT2 and E. coli ED1a, and observed a strong anti-correlation between close homologues of MG1655 mrr and LT2 mod in the genome database. This apparent evolutionary antagonism is further discussed in the light of a possible role for Mrr as defense mechanism against the establishment of epigenetic regulation by foreign DNA methyltransferases.     

Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum.

In vitro-packaged cosmid libraries of DNA from the bacterium Xanthomonas campestris pv. malvacearum were restricted 200- to 1,000-fold when introduced into Mcr+ strains of Escherichia coli compared with restriction in the Mcr- strain HB101. Restriction was predominantly associated with the mcrBC+ gene in E. coli. A plasmid (pUFR052) encoding the XmaI and XmaIII DNA methylases was isolated from an X. campestris pv. malvacearum library by a screening procedure utilizing Mcr+ and Mcr- E. coli strains. Transfer of plasmids from E. coli strains to X. campestris pv. malvacearum by conjugation was enhanced by up to five orders of magnitude when the donor cells contained pUFR052 as well as the plasmid to be transferred. Subcloning of pUFR052 revealed that at least two regions of the plasmid were required for full modification activity. Use of such modifier plasmids is a simple, novel method that may allow the efficient introduction of genes into any organism in which restriction systems provide a potent barrier to such gene transfer.     

Highly efficient positive selection of recombinant plasmids using a novel rglB-based Escherichia coli K-12 vector system

We have developed pBR328-derived vectors which allow highly efficient positive selection of recombinant plasmids. The system is based on the rglB-coded restriction activity of Escherichia coli K-12 directed against 5-methylcytosine (5mC)-containing DNA. The vectors code for cytosine-specific, temperature-sensitive DNA methyltransferases (ts-Mtases), whose specificity elicits RglB restriction. 5mC-free vector DNA - a prerequisite to allow establishment of such plasmids in cells expressing the RglB nuclease activity - can be prepared from cultures grown at 42 degrees C. At 30 degrees C the vector plasmids are vulnerable to RglB restriction due to the expression of suicidal Mtase activity. Cloning a DNA fragment into the ts-Mtase-coding gene disrupts the lethal methylation and thus permits selection of such recombinant plasmids at 30 degrees C. The standard vector used, pBN73, contains unique recognition sites for nine restriction enzymes within the ts-Mtase-coding gene, which can be used independently or in combination for the construction of recombinant plasmids selectable by the rglB-coded activity. Plasmid pBN74, which carries the determinants for both the ts-Mtase and the RglB nuclease, contains seven unique sites within the ts-Mtase-coding gene. While selection of recombinant plasmids derived from pBN73 obligatorily requires the employment of rglB+ strains, selection of pBN74 derivatives can be performed independent of the E. coli-host genotype. It remains to be elucidated whether positive selection of pBN74-derived recombinant plasmids can also be achieved in hosts other than E. coli. Plasmids pBN73, pBN74 and the recombinants are structurally stable. Generally applicable procedures, as developed during the establishment of this vector system, are described; they allow the isolation of ts-Mtases and facilitate the cloning of genes coding for nucleases directed against 5mC-containing DNA.     

Expression of orthopoxvirus DNA sequences in Escherichia coli cells]

We observed the expression of recombinant plasmids genes containing ectromelia virus DNA fragments in E. coli minicells. Using plasmids with vaccinia or ectromelia viruses DNA fragments inserted upstream of lacZ gene we showed that certain orthopoxvirus genome fragments carry out a promoter-like function in bacterial cells.     

New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments

Two new shuttle vectors have been constructed by fusing the Escherichia coli plasmid pUC9 with the Staphylococcus aureus plasmids pU110 and pC194. The resulting hybrids replicate in both E. coli and Bacillus subtilis and contain seven restriction sites within a part of the lacZ gene. Insertion of foreign DNA into those sites can be easily detected in E. coli and hybrid plasmids can subsequently be transformed into B. subtilis.     

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum.     

Cloning the KpnI restriction-modification system in Escherichia coli

The genes encoding the KpnI restriction and modification (R-M) system from Klebsiella pneumoniae, recognizing the sequence, 5'-GGTAC decreases C-3', were cloned and expressed in Escherichia coli. Although the restriction endonuclease (ENase)- and methyltransferase (MTase)-encoding genes were closely linked, initial attempts to clone both genes as a single DNA fragment in a plasmid vector resulted in deletions spanning all or part of the gene coding for the ENase. Initial protection of the E. coli host with MTase expressed on a plasmid was required to stabilize a compatible plasmid carrying both the ENase- and the MTase-encoding genes on a single DNA fragment. However, once established, the MTase activity can be supplied in cis to the kpnIR gene, without an extra copy of kpnIM. A chromosomal map was generated localizing the kpnIR and kpnIM genes on 1.7-kb and 3.5-kb fragments, respectively. A final E. coli strain was constructed, AH29, which contained two compatible plasmids: an inducible plasmid carrying the kpnIR gene which amplifies copy number at elevated temperatures and a pBR322 derivative expressing M.KpnI. This strain produces approx. 10 million units of R.KpnI/g of wet-weight cells, which is several 1000-fold higher than the level of R.KpnI produced by K. pneumoniae. In addition, DNA methylated with M.KpnI in vivo does not appear to be restricted by the mcrA, mcrB or mrr systems of E. coli.     

The Corynebacterium glutamicum cglIM gene encoding a 5-cytosine methyltransferase enzyme confers a specific DNA methylation pattern in an McrBC-deficient Escherichia coli strain

The cglIM gene of the coryneform soil bacterium Corynebacterium glutamicum ATCC 13032 has been cloned and characterized. The coding region comprises 1092 nucleotides and specifies a protein of 363 amino acid residues with a deduced M_r of 40 700. The amino acid sequence showed striking similarities to methyltransferase enzymes generating 5-methylcytosine residues, especially to M·NgoVII from Neisseria gonorrhoeae recognizing the sequence GCSGC. The cglIM gene is organized in an unusual operon which contains, in addition, two genes encoding stress-sensitive restriction enzymes. Using PCR techniques the entire gene including the promoter region was amplified from the wild-type chromosome and cloned in Escherichia coli. Expression of the cglIM gene in E. coli under the control of its own promoter conferred the C. glutamicum-specific methylation pattern to co-resident shuttle plasmids and led to a 260-fold increase in the transformation rate of C. glutamicum. In addition, the methylation pattern produced by this methyltransferase enzyme is responsible for the sensitivity of DNA from C. glutamicum to the modified cytosine restriction (Mcr) system of E. coli.     

Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7

The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida. These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively. To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment. The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511. This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays. The expression of at least two of these genes in E. coli appeared to be regulated by the presence of the inducer salicylic acid. In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment. A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments.     

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.     

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.     

Characterization of DNA fragments encoding fimbriae of the uropathogenic Escherichia coli strain KS71

Recombinant plasmids were constructed that expressed the KS71A, KS71B and KS71C fimbrial antigens of the pyelonephritogenic Escherichia coli strain KS71 (O4:K12) in E. coli HB101. The KS71C-encoding genes were located on a 6.4 kb HindIII-XhoI fragment obtained from the recombinant cosmid pKTH145 that expresses this antigen. Spontaneous KS71C-mutants were isolated that contained a 0.8 kb insert in a specific restriction fragment of KS71C-encoding recombinant plasmids. The KS71B-encoding segment was located on a 11.5 kb deletable DNA fragment of recombinant cosmid pKTH144. A DNA fragment encoding the KS71A fimbria was obtained on a 12 kb EcoRI fragment of the recombinant cosmid expressing this antigen in E. coli HB101 and closely resembled the KS71B-encoding fragment. In the recombinant cosmid, the KS71B-expressing region was flanked by homologous DNA segments. A similar stretch of DNA was found close to the KS71A-expressing DNA region.     

A broad-host-range vector system for cloning and translational lacZ fusion analysis

A broad-host-range vector system for studying translational fusions was constructed. The region that retains the origin of replication, nic, mob, and rep genes of the broad-host-range plasmid RSF1010 was isolated as either an HincII or a PstI-PvuII restriction fragment. These restriction fragments were ligated to tetracycline, kanamycin, or streptomycin/spectinomycin resistance genes to generate plasmids pUI501, pUI511, pUI504, and pUI506. A functional lacZ gene lacking downstream lac operon sequences together with the lac promoter was constructed from plasmids pMC1871 and pUC18. This lacZ gene was inserted into pUI501 and pUI511 to generate plasmids pUI502, pUI503, pUI512, and pUI513. An oligodeoxynucleotide sequence that carries three unique blunt-end restriction sites was synthesized, annealed, and ligated in frame to the amino-terminal end of the lacZ gene in each of these plasmids. This multiple cloning sequence will allow translational fusions to the lacZ gene in all three reading frames. The stability of these plasmids and the expression of the lacZ gene in both Escherichia coli and Rhodobacter sphaeroides were studied.     

Restriction mapping of recombinant plasmids carrying the genes for arginine biosynthesis in Escherichia coli K-12

ArgA and argECBH genes of Escherichia coli K-12 were cloned on the pBR322 vector. Restriction maps of the recombinant plasmids were constructed. Deletion mutants of these recombinant plasmids, retaining the functional argA and argE genes, were obtained using different restriction enzymes. All of the recombinant derivatives have the replication properties of the pBR322 vector.     

表达大肠杆菌分子伴侣GroEL、GroES和GrpE载体的构建及其应用

大肠杆菌（Escherichia coli）共表达系统常要求质粒具有不同抗生素抗性以及不同的复制子。利用粘性末端PCR技术,以含有大肠杆菌分子伴侣基因GroEL、GroES和唧E的pR—GESP质粒为模板,设计两对引物,通过两次独立的PCR反应扩增3个基因的多顺反子,将形成粘性末端的PCR产物插入NcoI和Xho1酶切的pACY．CDuet-1质粒,构建的pA—GESP质粒具有p15A复制子及氯霉素抗性,和具有ColE1复制子及卡那霉素抗性表达载体pET28b相容。SDS—PAGE显示含有pA—GESP质粒的大肠杆菌细胞中3个分子伴侣蛋白的表达水平和含有pR—GESP质粒的大肠杆菌细胞没有明显差异,它们对玉米丝氨酸消旋酶的可溶性表达有部分促进作用,但对N端含有组氨酸标签的玉米铁氧还蛋白还原酶的表达没有作用,在三个含有不同抗生素基因的质粒中共表达分子伴侣、5-氨基乙酰丙酸合酶和尿卟啉原III甲基化酶,两个酶连续催化的荧光产物在细胞内积累量为562．13±3．17／OD600,而没有分子伴侣的积累量为457．66±4．98／OD600,表明分子伴侣改善部分蛋白在大肠杆菌的可溶性表达和催化功能。     

The unique FauI restriction-modification system: cloning and comparative analysis of protein structure

The nucleotide sequence was established for the full-length Flavobacterium aquatile operon coding for the FauI restriction-modification system. The operon is unusual in structure and has the gene order control protein gene-DNA methyltransferase A gene-restriction endonuclease gene-DNA methyltransferase B gene, other than in the known analogs. The genes are similarly oriented and overlap. On evidence of sequence analysis, both methyltransferases are C5 enzymes, the control protein is similar to that of other restriction-modification systems, and restriction endonuclease is low-homologous to other enzymes cleaving the DNA upper strand in position 4 or 5 relative to the recognition site.     

Characterization and expression of the Escherichia coli Mrr restriction system.

The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.     

The Unique FauI Restriction–Modification System: Cloning and Protein Sequence Comparisons

The nucleotide sequence was established for the full-length Flavobacterium aquatile operon coding for the FauI restriction–modification system. The operon is unusual in structure and has the gene order control protein / DNA methyltransferase A / restriction endonuclease / DNA methyltransferase B, other than in the known analogs; the genes are similarly oriented and overlap. On evidence of sequence analysis, both methyltransferases are C5 enzymes, the control protein is similar to that of other restriction–modification systems, and the restriction endonuclease shows low similarity to other enzymes cleaving the DNA upper strand in position 4 or 5 relative to the recognition site.