The restriction-modification system in Streptomyces flavopersicus

To clone bifunctional vectors in streptomycetes, it was necessary to define the restriction-modification system ofStreptomyces flavopersicus. Plasmid DNA from bifunctional vectors pIJ699 and pXED3-13, isolated fromE. coli strains with different methylation systems:E. coli DH5α (dam ⁺ dcm ⁺),E. coli MB5386(dam dcm), E. coli CB51 (dam dcm ⁺),E. coli NM544 (dam ⁺ dcm), was used for transformation of protoplasts from strainS. flavopersicus. Restriction ofdcm-methylated DNA fromS. flavopersicus was established. As a host in the intermediate cloning strainE. coli NM544 (dam ⁺ dcm) should be used, as thedcm-transmethylase-dependent strainS. flavopersicus does not process DNA from this strain.     

Establishment of a novel sensitive method for detecting methylation modification on DNA of escherichia coli cell

This study focused on finding a novel sensitive method to determine the methylation modification at DNA dam (GATC) sites in Escherichia coli. A new plasmid which contained three GATC sites recognized by restriction enzyme BclI and one GAATTC site recognized by EcoRI was transformed into E. coli stains AB1157(dam ⁺) and GM2929(dam ⁻) respectively. Then the plasmid DNA was digested by restriction enzyme BclI(T*GATCA), which was sensitive to methylation. The results showed that the plasmid derived from AB1157 was not digested while that from GM2929 was, for the methylation level of the former was high while the latter was low. So by detecting the methylation of plasmid transferred into the strain, we could determine whether methylaion existed at DNA dam (GATC) site in E. coli. This method was effective and rapid; moreover, the digested fragments were not dispersive. It also made a basis for the detection of whether methylation occurred in mode beings by low-energy ion beam. The article is published in the original.     

Establishment,counts, and identification of the fibrolytic microflora in the digestive tract of rabbit. Influence of feed cellulose content

Dam-mediated adenine methylation at GATC sites can interfere with gene expression. By use oflacZ fusion technology, the efficiency oftrpR andtrpS promoters (which contain a GATC site) and oftrp (the target of TrpR repressor) was analyzed indam ⁺ anddam ^– backgrounds. In exponentially growing cells, thedam mutation leads to an increased activity oftrpR promoter but does not affecttrpS ortrp promoters. The Dam-mediated induction oftrpR was, however, found to be repressed bytrpR-mediated autoregulation. In contrast,trp-lacZ directed-galactosidase activity was increased at least sixfold indam ^– cells in late logarithmic growth phase. Indam ⁺ cells, expression oftrp-lacZ was similarly late-growth-phase induced, albeit to a reduced extent. Hence, we propose that enhancement of growth phase-dependent gene induction constitutes a previously unidentified trait ofdam mutation. This finding is discussed in the context of the pleiotropic phenotype ofdam mutation.     

Methyl directed DNA mismatch repair inVibrio cholerae

Mismatches in DNA occur either due to replication error or during recombination between homologous but non-identical DNA sequences or due to chemical modification of bases. The mismatch in DNA, if not repaired, result in high spontaneous mutation frequency. The repair has to be in the newly synthesized strand of the DNA molecule, otherwise the error will be fixed permanently. Three distinct mechanisms have been proposed for the repair of mismatches in DNA in prokaryotic cells and gene functions involved in these repair processes have been identified. The methyl-directed DNA mismatch repair has been examined inVibrio cholerae, a highly pathogenic gram negative bacterium and the causative agent of the diarrhoeal disease cholera. The DNA adenine methyltransferase encoding gene (dam) of this organism which is involved in strand discrimination during the repair process has been cloned and the complete nucleotide sequence has been determined.Vibrio cholerae dam gene codes for a 21.5 kDa protein and can substitute for theEscherichia coli enzyme. Overproduction ofVibrio cholerae Dam protein is neither hypermutable nor lethal both in Escherichia coli andVibrio cholerae. WhileEscherichia coli dam mutants are sensitive to 2-aminopurine,Vibrio cholerae 2-aminopurine sensitive mutants have been isolated with intact GATC methylation activity. The mutator genesmutS andmutL involved in the recognition of mismatch have been cloned, nucleotide sequence determined and their products characterized. Mutants ofmutS andmutL ofVibrio cholerae have been isolated and show high rate of spontaneous mutation frequency. ThemutU gene ofVibrio cholerae, the product of which is a DNA helicase II, codes for a 70 kDa protein. The deduced amino acid sequence of themutU gene hs all the consensus helicase motifs. The DNA cytosine methyltransferase encoding gene (dam) ofVibrio cholerae has also been cloned. Thedcm gene codes for a 53 kDa protein. This gene product might be involved in very short patch (VSP) repair of DNA mismatches. The vsr gene which is directly involved in VSP repair process codes for a 23 kDa protein. Using these information, the status of DNA mismatch repair inVibrio cholerae will be discussed.     

Viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants requires increased expression of specific genes in the SOS regulon

Summary We have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam ^-). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD and dinF) were found to express higher levels of -galactosidase in dam ^- strains than in isogenic dam ⁺ strains. The attempted construction of dam ^- strains that were also mutant in one of several SOS genes indicated that the viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for dam ^- strain viability.     

DNA methylation in the Alcohol dehydrogenase-1 gene of maize

Using a battery of methylation-sensitive restriction enzymes, cytosine methylation at 23 sites in a 7.6 kb region surrounding the Alcohol dehydrogenase-1 (Adh1) gene was measured in DNA prepared from immature maize cobs. Both the 5 upstream region and the entire coding region were hypomethylated in the two alleles examined. Methylation in Adh1 is independent of changes in Mutator transposable element methylation. The role of DNA methylation in Adh1 gene regulation is discussed.     

A Non-Restricting and Non-Methylating <Emphasis Type="Italic">Escherichia coli</Emphasis> Strain for DNA Cloning and High-Throughput Conjugation to <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Escherichia coli strains are used in secondary metabolism research for DNA cloning and transferring plasmids by intergeneric conjugation. Non-restricting strains are desirable for DNA cloning and non-methylating strains are beneficial for transferring DNA to methyl-restricting hosts, like Streptomyces coelicolor. We have constructed a non-methylating E. coli strain, JTU007, by deleting the DNA methylation genes dcm and dam from the widely used non-restricting cloning host DH10B. JTU007 was tested as donor for the conjugative transfer of a plasmid containing the 39 kb actinorhodin biosynthesis gene cluster to S. lividans and S. coelicolor. The Dcm⁻ Dam⁻ strain JTU007 transferred DNA into S. coelicolor A(3)2 derivatives at high frequency. To demonstrate the usefulness of E. coli JTU007 for gene cloning, we constructed a comprehensive S. toxytricini genomic cosmid library, and transferred it using high-throughput conjugation to the methyl-restricting S. coelicolor. One of the cosmid clones produced a brown pigment, and the clone was revealed to carry a tyrosinase operon. JTU007 is more useful than ET12567 because it does not restrict methylated DNA in primary cloning, and gives higher transformation and cosmid infection frequencies.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Plasmid DNA fermentation strain and process‐specific effects on vector yield,quality, and transgene expression

Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication. We also investigated the impact of differences in epigenetic dcm methylase‐directed cytosine methylation on plasmid production, transgene expression, and immunogenicity. While Escherichia coli plasmid production yield and quality are unaffected, dcm− versions of CMV and CMV‐HTLV‐I R promoter plasmids had increased transgene expression in human cells. Surprisingly, despite improved expression, dcm− plasmid is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e., production strain) early in product development and that dcm− strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial. Biotechnol. Bioeng. 2011;108: 354–363. © 2010 Wiley Periodicals, Inc.     

Counterselection of GATC sequences in enterobacteriophages by the components of the methyl-directed mismatch repair system

Summary Weak to severe deficit of GATC sequences in the DNA of enterobacteriophages appears to be correlated with their undermethylation during growth indam ⁺ (GATC ade-methylase) bacteria. This observation is corroborated by the sequence analysis showing no evidence for site-specific mutagenicity of 6meAde. The MutH protein of the methyl-directed mismatch repair system recognizes and cleaves the undermethylated GATC sequences in the course of mismatch repair. To enquire whether the MutH function of the methyldirected mismatch repair system participates in counterselection of GATC sequences in enterobacteriophages, we have studied the yield of bacteriophage X174 containing either 0, 1, or 2 GATC sequences, in wild type,dam, andmut (H, L, S, U) Escherichia coli. Following transfection with unmethylated DNA containing two GATC sequences, a net decrease in the yield of infective particles was observed in all bacterialmutH ⁺ dam^– strains, whereas no detectable decrease was observed in bacteria infected by DNA without GATC sequence. This effect of the MutH function is maximum in wild type andmutL andmutS bacteria whereas the effect is not significant inmutU bacteria, suggesting an interaction of the, helicase II with the MutH protein.However, indam ⁺ bacteria, the presence of GATC sequences leads to an increased yield of infective particles. The effect of GATC sequence and its Dam methylation system on phage yield inmutH ^– bacteria reveals that methylated GATC sequences are advantageous to the phage. These results suggest that the methyl-directed mismatch repair system, and in particular its MutH protein, may have participated in severe counterselection of GATC sequences from enterobacteriophages, presumably, by DNA cleavage or by interfering with DNA replication or packaging when GATC sequences are undermethylated. Coevolution of the Dam and MutH proteins could then account for the loss of GATC sequences from DNA of bacteriophages growing indam ⁺ hosts.     

In vitro methylation of bacteriophage lambda DNA by wild type (dam+) and mutant (damh) forms of the phage T2 DNA adenine methylase.

The wild-type (dam⁺) and mutant (dam^h) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam⁺ and dam^h enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 dam^h enzyme methylated λ DNA to a higher level than did the dam⁺ enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 dam^h, but not T2 dam⁺ protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R·EcoP1. (3) T2 dam⁺ and dam^h were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ·dam₃ DNA (contains no N⁶-methyladonine) methylated with either enzyme was made resistant to cleavage by R·DpnII. In contrast, only the T2 dam^h modified DNA was resistant to further methylation by M·EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978b). (4) λ·dam₃ DNA was partially methylated to the same level with T2 dam⁺ or T2 dam^h; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam⁺ enzyme methylates G-A-C sequences less efficiently than the T2 dam^h methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes.     

The Escherichia coli dam gene is expressed as a distal gene of a new operon

Summary DNA containing the Escherichia coli dam gene and sequences upstream from this gene were cloned from the Clarke-Carbon plasmids pLC29-47 and pLC13-42. Promoter activity was localized using pKO expression vectors and galactokinase assays to two regions, one 1650–2100 bp and the other beyon 2400 bp upstream of the dam gene. No promoter activity was detected immediately in front of this gene; plasmid pDam118, from which the nucleotide sequence of the dam gene was determined, is shown to contain the pBR322 promoter for the primer RNA from the pBR322 rep region present on a 76 bp Sau3A fragment inserted upstream of the dam gene in the correct orientation for dam expression. The nucleotide sequence upstream of dam has been determined. An open reading frame (ORF) is present between the nearest promoter region and the dam gene. Codon usage and base frequency analysis indicate that this is expressed as a protein of predicted size 46 kDa. A protein of size close to 46 kDa is expressed from this region, detected using minicell analysis. No function has been determined for this protein, and no significant homology exist between it and sequences in the PIR protein or GenBank DNA databases. This unidentified reading frame (URF) is termed urf-74.3, since it is an URF located at 74.3 min on the E. coli chromosome. Sequence comparisons between the regions upstream of urf-74.3 and the aroB gene show that the aroB gene is located immediately upstream of urf-74.3, and that the promoter activity nearest to dam is found within the aroB structural gene. This activity is relatively weak (about 15% of that of the E. coli gal operon promoter). The promoter activity detected beyond 2400 bp upstream of dam is likely to be that of the aroB gene, and is 3 to 4 times stronger than that found within the aroB gene. Three potential DnaA binding sites, each with homology of 8 of 9 bp, are present, two in the aroB promoter region and one just upstream of the dam gene. Expression through the site adjacent to the dam gene is enhanced 2-to 4-fold in dnaA mutants at 38°C. Restriction site comparisons map these regions precisely on the Clarke-Carbon plasmids pLC13-42 and pLC29-47, and show that the E. coli ponA (mrcA) gene resides about 6 kb upstream of aroB.     

Characterisation of a complex restriction/modification system detected in a Bifidobacterium longum strain

 Two type-II restriction endonucleases, BloI and BloII, have been detected in a Bifidobacterium longum strain. BloI is influenced by dam methylation: it cleaves dam ^- but not dam ⁺ DNA. It shows a temperature and pH optimum of 45°C and pH 7.5. Restriction analysis and cloning experiments showed that the recognition sequence is RGATCY and that the enzyme cuts 5′ to the guanine residue. It is an isoschizomer of commercial enzymes, BstYI and XhoII. The second activity is not inhibited by dam methylation. It has a temperature optimum between 25°C and 30°C and shows a broad pH optimum between 4.5 and 7.0. The activity is thermolabile and can be heat-killed by a 5 min incubation at 60°C. Cloning and sequencing experiments revealed that its recognition sequence is CTGCAG and that it cuts 5′ to the second guanine residue in the sequence. This enzyme is the first described isoschizomer of PstI. Received: 22 May 1995/Accepted: 26 July 1995     

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

Summary The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 by away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam ^– mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.     

Alleviation of type I restriction in adenine methylase (dam) mutants of Escherichia coli

Summary The host-controlled EcoK-restriction of unmodified phage .O is alleviated in dam mutants of Escherichia coli by 100- to 300-fold. In addition, the EcoK modification activity is substantially decreased in dam ^- strains. We show that type I restriction (EcoB, EcoD and EcoK) is detectably alleviated in dam mutants. However, no relief of EcoRI restriction (Type II) occurs in dam ^- strains and only a slight effect of dam mutation on EcoP1 restriction (Type III) is observed. We interpret the alleviation of the type I restriction in dam ^- strains to be a consequence of induction of the function which interferes with type I restriction systems.     

Dam inactivation in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>: prevalence among diverse hyperinvasive lineages

Background  

DNA adenine methyltransferase (Dam) activity is absent in many, but not all, disease isolates of Neisseria meningitidis, as a consequence of the insertion of a restriction endonuclease-encoding gene, the 'dam replacing gene' (drg) at the dam locus. Here, we report the results of a survey to assess the prevalence of drg in a globally representative panel of disease-associated meningococci.     

Expression of a DNA methylation (dam) gene inEscherichia coli K-12

Plasmid pMQ3, carrying thedam gene ofEscherichia coli on a 6.1 Kb fragment, shows a tenfold increase in relative DNA adenine methylase activity, while plasmid pdam118, with a 1.14 Kbdam insert, shows only a twofold increase, although both plasmids were derived from plasmid pLC13–42. Since a copy number effect did not seem to be the cause of this difference, we have subcloned pMQ3 in order to determine whether the additional chromosomal DNA present in this plasmid is responsible for the enhancement of methylase activity. We show that the 346 base pairs upstream ofdam contain sequences necessary for expression. DNA sequence analysis has revealed that in pdam118 only the 118 bases 5-prime to thedam gene are present in other constructs and that the additional upstream material is pBR322 DNA. This shows that pdam118 carries a DNA duplication.