Cloning the DdeI restriction-modification system using a two-step method.

DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.     

Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli

In Escherichia coli, cytosine DNA methylation is catalyzed by the DNA cytosine methyltransferase (Dcm) protein and occurs at the second cytosine in the sequence 5'CCWGG3'. Although the presence of cytosine DNA methylation was reported over 35?years ago, the biological role of 5-methylcytosine in E.?coli remains unclear. To gain insight into the role of cytosine DNA methylation in E.?coli, we (1) screened the 72 strains of the ECOR collection and 90 recently isolated environmental samples for the presence of the full-length dcm gene using the polymerase chain reaction; (2) examined the same strains for the presence of 5-methylcytosine at 5'CCWGG3' sites using a restriction enzyme isoschizomer digestion assay; and (3) quantified the levels of 5-methyl-2'-deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm-mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5-methylcytosine ranges from 0.86% to 1.30% of the cytosines. We also demonstrate that Dcm reduces the expression of ribosomal protein genes during stationary phase, and this may explain the highly conserved nature of this DNA modification pathway.     

Characterization of the mcrBC region of Escherichia coli K-12 wild-type and mutant strains.

We have carried out an analysis of the Escherichia coli K-12 mcrBC locus in order to (1) elucidate its genetic organization, (2) to identify the proteins encoded by this region, and (3) to characterize their involvement in the restriction of DNA containing methylated cytosine residues. In vitro expression of recombinant plasmids carrying all or portions of the mcrBC region revealed that the mcrB and mcrC genes are organized as an operon. The mcrBC operon specifies five proteins, as evident from parallel in vitro and in in vivo expression studies. Three proteins of 53, 35 and 34 kDa originate from mcrB expression, while two proteins of 37 and 16 kDa arise from mcrC expression. Products of both the mcrB and mcrC genes are required to restrict the methylated substrate DNA used in this study. We also determined the nature of mutant mcrBC loci in comparison to the E. coli K-12 wild-type mcrBC locus. A major goal of these studies was to clarify the nature of the mcrB-1 mutation, which is carried by some strains employed in previous analyses of the E. coli K-12 McrBC system. Based on our analyses the mutant strains investigated could be divided into different complementation groups. The mcrB-1 mutation is a nonsense or frameshift mutation located within mcrB. It causes premature termination of mcrB gene product synthesis and reduces the level of mcrC gene expression. This finding helps to understand an existing conflict in the literature. We also describe temperature-sensitive McrA activity in some of the strains analysed and its relationship to the previously defined differences in the tolerance levels of E. coli K-12 mcrBC mutants to cytosine methylation.     

Genetic Control of the Secondary Modification of Deoxyribonucleic Acid in Escherichia coli

The wild-type restriction and modification alleles of Escherichia coli K-12 and B were found to have no measurable effect on the patterns of methylated bases in the deoxyribonucleic acid (DNA) of these strains. The genetic region controlling the methylation of cytosine in E. coli K-12 was mapped close to his, and the presence or absence of this gene in E. coli B or E. coli K had no effect on the restriction and modification properties of these strains. Thus, only a few of the methylated bases in the DNA of these strains are involved in host modification, and the biological role of the remainder remains obscure.     

McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning.

The McrA and McrB (modified cytosine restriction) systems of E. coli interfere with incoming DNA containing methylcytosine. DNA from many organisms, including all mammalian and plant DNA, is expected to be sensitive, and this could interfere with cloning experiments. The McrA and B phenotypes of a few strains have been reported previously (1-4). The Mcr phenotypes of 94 strains, primarily derived from E. coli K12, are tabulated here. We briefly review some evidence suggesting that McrB restriction of mouse-modified DNA does occur in vivo and does in fact interfere with cloning of specific mouse sequences.     

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.     

Improved methods of cultivation and production of deuteriated proteins from E. coli strains grown on fully deuteriated minimal medium

AIMS: The aim was to develop reliable and economical protocols for the production of fully deuteriated biomolecules by bacteria. This required the preparation of deuterium-tolerant bacterial strains and an understanding of the physiological mechanisms of acquisition of deuterium tolerance. METHODS AND RESULTS: We report here improved methods for the cultivation of Escherichia coli on fully deuteriated minimal medium. A multi-stage adaptation protocol was developed; this included repeated plating and selection of colonies and resulted in highly deuterium-tolerant cell cultures. Three E. coli strains, JM109, MRE600 and MRE600Rif, were adapted to growth on deuteriated succinate medium. This is the first report of JM109 being adapted to deuteriated minimal media. The adapted strains showed good, consistent growth rates and were capable of being transformed with plasmids. Expression of heterologous proteins in these strains was reliable and yields were consistently high (100-200 mg l-1). We also show that all E. coli cells are inherently capable of growth on deuteriated media. CONCLUSIONS: We have developed a new adaptation protocol that resulted in three highly deuterium-tolerant E. coli strains. Deuterium-adapted cultures produced good yields of a deuteriated recombinant protein. We suggest that E. coli cells are inherently capable of growth on deuteriated media, but that non-specific mutations enhance deuterium tolerance. Thus plating and selection of colonies leads to highly deuterium-tolerant strains. SIGNIFICANCE AND IMPACT OF STUDY: An understanding of the mechanism of adaptation of E. coli to growth on deuteriated media allows strategies for the development of disabled deuterium-tolerant strains suitable for high-level production of deuteriated recombinant proteins and other biomolecules. This is of particular importance for nuclear magnetic resonance and neutron scattering studies of biomolecules.     

DNA protection with the DNA methylase M . BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII

BamHI fragments of the Bacillus brevis var. GB plasmid pAD1 have been cloned in Escherichia coli HB101 using pBR322 plasmid as a vector. The analysis of the recombinant plasmids showed that additional PstI sites had appeared in cloned fragments of pAD1. Methylation of the recombinant plasmids in vitro by enzymes from B. brevis GB cells blocks cleavage at these additional PstI sites of cloned pAD1 fragments and at the PstI site of pBR322. Among DNA methylases of B. brevis GB, the cytosine DNA methylase M . BbvI is the most likely agent modifying the recognition sequences of PstI. The methylase can modify cytosine residues in PstI or PvuII sites if these recognition sequences are linked to G at 5'- or to C at 3'-termini. In particular, in vitro methylation of the SV40 DNA by B. brevis GB methylases protects one of the two PstI sites and two of the three PvuII sites. The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.     

Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli.

An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.     

Genetic Analysis of the Cardiac Methylome at Single Nucleotide Resolution in a Model of Human Cardiovascular Disease

Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.     

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.     

A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integration

A novel cloning vector to aid in the construction of single copy β-galactosidase reporter systems for gene expression studies in lactose metabolizing Escherichia coli strains, including STEC, is described. The plasmid allows construction of translational fusions of cloned gene promoters to a short segment of E. coli lacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5' to the cloning site. PCR amplification using opposing primers complementary to the upstream lacI fragment and the downstream lacZ fragment generates a linear template suitable for integration using pRedET recombination. Integration of linear template derived from the recombinant plasmid into host strains replaces the entire native lacZ promoter and fuses the promoter of interest in-frame with the lacZ gene, thus simultaneously producing a single-copy, chromosomal reporter system and eliminating background lacZ expression. Studies comparing ahpC expression from a chromosomal fusion in the lac open with that on a plasmid in E. coli strain EDL933 are shown.     

Pathogenic Escherichia coli

Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.     

Genetic and physiological studies of an Escherichia coli locus that restricts polynucleotide kinase- and RNA ligase-deficient mutants of bacteriophage T4.

The RNA ligase and polynucleotide kinase of bacteriophage T4 are nonessential enzymes in most laboratory Escherichia coli strains. However, T4 mutants which do not induce the enzymes are severely restricted in E. coli CTr5X, a strain derived from a clinical E. coli isolate. We have mapped the restricting locus in E. coli CTr5X and have transduced it into other E. coli strains. The restrictive locus seems to be a gene, or genes, unique to CTr5X or to be an altered form of a nonessential gene, since deleting the locus seems to cause loss of the phenotypes. In addition to restricting RNA ligase- and polynucleotide kinase-deficient T4, the locus also restricts bacteriophages lambda and T4 with cytosine DNA. When lambda or T4 with cytosine DNA infect strains with the prr locus, the phage DNA is injected, but phage genes are not expressed and the host cells survive. These phenotypes are unlike anything yet described for a phage-host interaction.     

Methylation of 23S rRNA nucleotide G745 is a secondary function of the RlmAI methyltransferase

Several groups of Gram-negative bacteria possess an RlmA(I) methyltransferase that methylates 23S rRNA nucleotide G745 at the N1 position. Inactivation of rlmA(I) in Acinetobacter calcoaceticus and Escherichia coli reduces growth rates by at least 30%, supposedly due to ribosome malfunction. Wild-type phenotypes are restored by introduction of plasmid-encoded rlmA(I), but not by the orthologous Gram-positive gene rlmA(II) that methylates the neighboring nucleotide G748. Nucleotide G745 interacts with A752 in a manner that does not involve the guanine N1 position. When a cytosine is substituted at A752, a Watson-Crick G745-C752 pair is formed occluding the guanine N1 and greatly reducing RlmA(I) methylation. Methylation is completely abolished by substitution of the G745 base. Intriguingly, the absence of methylation in E. coli rRNA mutant strains causes no reduction in growth rate. Furthermore, the slow-growing rlmA(I) knockout strains of Acinetobacter and E. coli revert to the wild-type growth phenotype after serial passages on agar plates. All the cells tested were pseudorevertants, and none of them had recovered G745 methylation. Analyses of the pseudorevertants failed to reveal second-site mutations in the ribosomal components close to nucleotide G745. The results indicate that cell growth is not dependent on G745 methylation, and that the RlmA(I) methyltransferase therefore has another (as yet unidentified) primary function.     

Cloning and analysis of the Clostridium perfringens tetracycline resistance plasmid, pCW3

Clostridium perfringens strain CW92 carries pCW3, a conjugative 47-kb plasmid that confers inducible resistance to tetracycline. The plasmid was examined by restriction endonuclease analysis and by cloning each of the five ClaI fragments of pCW3 in Escherichia coli, using pBR322. Analysis of the recombinant plasmids allowed the deduction of a detailed restriction map of pCW3. The tetracycline resistance determinant of pCW3 was mapped by examining the phenotype of recombinant E. coli clones derived from the cloning, into pUC vector plasmids, of EcoRI fragments from pCW3. The C. perfringens tetracycline resistance determinant was expressed in E. coli and was shown to be located on two juxtaposed EcoRI fragments which together encompass a 4-kb region of pCW3. Deletion experiments showed that the tetracycline resistance gene, and/or its control regions, contained internal EcoRI and SphI sites. E. coli strains that carried recombinant plasmids with only the 4-kb region were found to express tetracycline resistance constitutively. In contrast, recombinant plasmids harboring a 10.5-kb ClaI fragment of pCW3, that included the 4-kb region, coded for an inducible tetracycline resistance phenotype. The existence of a negatively regulated resistance gene, similar to that proposed for several other bacteria is postulated.     

Cloning and constitutive expression of the N-acetylneuraminate lyase gene of Escherichia coli

The N-acetylneuraminate (NANA) lyase (EC 4.1.3.3) gene from Escherichia coli was self-cloned in E. coli. Transformants were selected by complementation of a NANA lyase-deficient E. coli strain. One clone was found to produce NANA lyase, and it contained a recombinant plasmid, pNAL1, with a 9.0-kilobase HindIII insert. The cloning of the NANA lyase gene resulted in the change from inducible to constitutive production of the enzyme. The level of expression of the NANA lyase gene in E. coli(pNAL1) clones was two- to three-fold higher than that in the fully induced wild-type strains.     

Cloning and constitutive expression of the N-acetylneuraminate lyase gene of Escherichia coli.

The N-acetylneuraminate (NANA) lyase (EC 4.1.3.3) gene from Escherichia coli was self-cloned in E. coli. Transformants were selected by complementation of a NANA lyase-deficient E. coli strain. One clone was found to produce NANA lyase, and it contained a recombinant plasmid, pNAL1, with a 9.0-kilobase HindIII insert. The cloning of the NANA lyase gene resulted in the change from inducible to constitutive production of the enzyme. The level of expression of the NANA lyase gene in E. coli(pNAL1) clones was two- to three-fold higher than that in the fully induced wild-type strains.     

A gene required for very short patch repair in Escherichia coli is adjacent to the DNA cytosine methylase gene.

Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mismatches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previously we have shown that a plasmid containing an 11-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa. The genes responsible for these phenotypes have been identified by DNA sequence analysis. The gene essential for cytosine methylation, dcm, is predicted to code for a 473-amino-acid protein and is not required for VSP repair. It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRII methylase. The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein. This gene, named vsr, is not essential for DNA methylation. Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm. The two genes appear to be transcribed from a common promoter but are in different translational registers. This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation.