Dam Methylation Sites Research Articles

Complete Sequence of Plasmid pMP1 from the Marine Environmental Vibrio vulnificus and Location of Its Replication Origin

A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine-cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.

The interaction between dam methylation sites and Xba1 restriction digest sites in Escherichia coli O157:H7 EDL933

The aim of this study was to gain a better understanding of the reason for the predicted pulsed-field gel electrophoresis (PFGE) pattern for the sequenced Escherichia coli O157:H7 EDL933 (EDL933) being different from that observed in practice, using the restriction enzyme Xba1. Primers were designed that flanked either side of each of the predicted Xba1 restriction sites, and the resultant PCR products were sequenced. No sequencing errors were found in the published genome. The distribution of dam methylation sites within the genome was investigated, and a new PFGE pattern was predicted by assuming that any Xba1 restriction site that coincided with a dam methylation site would not be cut. The estimated mean band sizes were obtained from six replicate gels. It was found that the observed and predicted PFGE patterns were in good agreement. The difference between the observed and the predicted PFGE patterns for EDL933, using Xba1, could be accounted for by assuming that the methylated restriction sites were not cut. PFGE is commonly used as a subtyping method. This study provides additional information about the basic technique that could enhance the interpretation of PFGE patterns in comparative studies of the E. coli isolates.

Integrated regulatory responses of fimB to N -acetylneuraminic (sialic) acid and GlcNAc in Escherichia coli K-12

Bacterial-host attachment by means of bacterial adhesins is a key step in host colonization. Phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (mainly on-to-off switching). fimB is separated from the divergent yjhATS operon by a large (1.4 kbp) intergenic region. Short ( approximately 28 bp) cis-active elements (regions 1 and 2) close to yjhA stimulate fimB expression and are required for sialic acid (Neu(5)Ac) sensitivity of its expression [El-Labany, S., Sohanpal, B. K., Lahooti, M., Akerman, R. & Blomfield, I. C. (2003) Mol. Microbiol. 49, 1109-1118]. Here, we show that whereas NanR, a sialic acid-response regulator, binds to region 1, NagC, a GlcNAc-6P-responsive protein, binds to region 2 instead. The NanR- and NagC-binding sites lie adjacent to deoxyadenosine methylase (Dam) methylation sites (5'-GATC) that are protected from modification, and the two regulators are shown to be required for methylation protection at regions 1 and 2, respectively. Mutations in nanR and nagC diminish fimB expression, and both fimB expression and FimB recombination are inhibited by GlcNAc (3- and >35-fold, respectively). Sialic acid catabolism generates GlcNAc-6-P, and whereas GlcNAc disrupts methylation protection by NagC alone, Neu(5)Ac inhibits the protection mediated by both NanR and NagC as expected. Type 1 fimbriae are proinflammatory, and host defenses enhance the release of both Neu(5)Ac and GlcNAc by a variety of mechanisms. Inhibition of type 1 fimbriation by these amino sugars may thus help balance the interaction between E. coli and its hosts.

The regulation of pap and type 1 fimbriation in escherichia cola

The ability of bacterial pathogens to bind to the host mucosa is a critical step in the pathogenesis of many bacterial infections and, for Escherichia coli, a large number of different fimbrial adhesins have been implicated as virulence factors. In this chapter, our current understanding of the regulatory mechanisms that control the expression of two of the best characterized fimbrial adhesins, pyelonephritis-associated pilus (encoded by pap) and the type 1 fimbria (encoded by fim), will be described. The expression of both fimbrial adhesins is controlled by phase variation (the reversible and apparently random switching between expressing ('on') and non-expressing ('off') states), and is regulated in response to environmental conditions. The phase variation of pap (and of some other fimbriae in Escherichia coli) is determined by the formation of alternative nucleoprotein complexes that either activate (phase 'on') or suppress (phase 'off') transcription of the fimbria genes. Formation of each complex protects a single Dam methylation site (5' GATC) from modification (GATCdist in phase 'on' cells and GATCprox in phase 'off' cells). Furthermore, complex formation is inhibited by methylation of the two 5' GATC sites. Both the phase variation of pap and the transcription of the pap genes in phase 'on' cells, are regulated and expression is subject to both positive and negative feedback control. In contrast to pap, the phase variation of fim is determined by the site-specific inversion of a short element of DNA (the fim switch). In phase 'on' cells, a promoter within the invertible element directs the transcription of the fim structural genes, whereas in phase 'off' cells transcription of the fimbrial genes is silenced. Despite the very different molecular mechanisms controlling the expression of pap and fim, the two systems share many features in common and have probably evolved to fulfill the same function. In addition to details about the molecular mechanisms that control pap and fim, the possible physiological significance of the observed regulation will be discussed.

Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro.

Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo. We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region. SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.

Analysis of the Replication Elements of the pMJ101 Plasmid from the Fish Pathogen Vibrio ordalii

Vibrio ordalii is a major cause of vibriosis in wild and cultured marine salmonids and carries pMJ101, a 30-kb cryptic plasmid that replicates in the absence of DNA polymerase I without producing single-stranded intermediates. A recombinant derivative harboring the pMJ101 replication region proved to be compatible with pJM1, a plasmid containing the iron acquisition system required for the virulence of V. anguillarum 775, another important pathogen that causes vibriosis. Sequence analysis of a 1.56-kb fragment harboring the pMJ101 replication region revealed the presence of typical features found in DNA origins including an AT-rich region, 11 dam-methylation sites of which 5 are within the putative ori region, and five copies of the 9-bp consensus sequence for DnaA binding. Gel retardation assays demonstrated that the latter replication element indeed binds DnaA purified from Escherichia coli. A potential open reading frame encoding a hydrophilic protein with a predicted pI of 10.3 and an Mr of 33,826 was found adjacent to the ori region. Although these properties are typical of DNA-binding proteins, no significant homology was found between this predicted protein, named RepM, and other previously characterized proteins. Reverse transcriptase–polymerase chain reaction analysis of total RNA demonstrated the presence of repM mRNA in V. ordalii. The major initiation site of this mRNA was located 187 nucleotides upstream of the GTG initiation codon as determined by nuclease S1 protection assays. This transcription initiation site is preceded by putative −10 and −35 promoter sequences that control the expression of the repM replication gene. These results demonstrate that the replication region of pMJ101 shares some structural and sequence similarities with other DNA replication regions, which include DnaA binding and methylation sites and an open reading frame encoding a distinct protein required for its replication.

Molecular mechanism of the inhibitory effect of methylation outside the recognition sequence of restriction endonuclease Pvu II on its cleavage activity

In 1991, we found that methylation outside the Pvu II recognition sequence could partially inhibit its cleavage activity. To clarify the molecular mechanism, three plasmids with different methylation states were constructed. Then, together with the original one, four plasmids were digested with different amounts of Pvu II. Results show that methylation on both sites results in 90 % inhibition; moving the methylated site one base further away decreases the inhibitory effect to about 30 %; with the adjacent dam methylation site eliminated, the inhibitory effect disappears. The data suggest that the inhibition of cleavage activity caused by outside methylation is not “all or none”, and the degree of inhibition is dependent on the position and the number of methylated bases.

GATC motifs may alter the conformation of DNA depending on sequence context and N6-adenine methylation status: possible implications for DNA-protein recognition.

As part of our analysis of the role of a uniquely clustered set of dam methylation sites (the motif GATC) within the origin of DNA replication in Escherichia coli, we have studied the effect of GATCs in various methylation states on the intrinsic curvature of DNA. We have designed a set of DNA linkers and used commercially available linkers containing GATC motifs. The linkers were ligated and the electrophoretic mobility of the resulting multimers in different states of methylation was tested relative to reference fragments. We report that properly phased GATCs in certain sequence environments modulate DNA curvature and that these effects may be enhanced by N6-adenine methylation of the GATCs. These structural alterations may in turn affect DNA-protein interactions, especially those involving proteins that rely on both primary sequence and structure for recognition. We present an example, where introduction of a GATC within an integration host factor (IHF) binding site, which does not alter the consensus sequence, reduces the binding affinity of the protein for the modified site.

Parental strand recognition of the DNA replication origin by the outer membrane in Escherichia coli.

The outer membrane of Escherichia coli binds the origin of DNA replication (oriC) only when it is hemimethylated. We report here the results of a footprinting analysis with the outer membrane which demonstrate that its interaction with oriC occurs mainly at the left moiety of the minimal oriC, where 10 out of 11 Dam methylation sites are concentrated. Two regions, flanking the Integration Host Factor (IHF) sites, are preferentially recognized at the minimum membrane concentration at which oriC plasmid replication is inhibited in vitro. We have identified the putative proteins involved in hemimethylated oriC binding and cloned one of the corresponding genes (hobH). The purified LacZ-HobH fusion protein specifically binds oriC DNA at the same preferential sites as the membrane. A mutant of the hobH gene reveals partial asynchronous initiation of DNA replication.

On the role of the Escherichia coli integration host factor (IHF) in repression at a distance of the pyrimidine specific promoter P1 of the carAB operon

Binding of integration host factor to its target site, centered around nucleotide — 305 upstream of the transcription startpoint, exerts antagonistic effects on the expression of P1, the upstream pyrimidine specific promoter of the E coli and S typhimurium carAB operons. IHF stimulates P1 promoter activity in minimal medium, but also increases the repressibility of this promoter by pyrimidines. We present evidence strongly suggesting that IHF exerts these effects by modulating the binding of another pyrimidine specific regulatory molecule, probably the product of gene carP. The carAB control region contains a GATC Dam methylation site, 106 bp upstream of the P1 transcription startpoint, which can be protected in vivo against methylation. This protection requires at least the regulatory carP gene product and a high pyrimidine nucleotide pool and, as shown here, the integration host factor. Whether CarP directly binds this site or exerts its protective effect indirectly is not yet known. In the absence of IHF ( himA) or in mutants affected in the IHF target site this protection is strongly impaired, suggesting that IHF positively influences the formation or the stability of the protective protein-DNA complex some 200 bp downstream. Furthermore, we have demonstrated that the distance separating the IHF and GATC Dam methylase target sites is crucial for the in vivo protection and for pyrimidine mediated regulation of P1 promoter expression. Indeed, shortening this distance by 6 bp, and more surprisingly also by 11 bp, results in a severe reduction of the degree of in vivo protection of the GATC site against methylation and concomitantly of the repressibility by pyrimidines of P1 promoter activity. The absence of both these efefcts in a double, deletion-duplication, mutant resulting in a net increase of the intervening sequence by 1 bp, clearly demonstrates that these effects are not due to the disruption of an important regulatory site, but must be attributed to variations in the distance separating different protein binding sites.

Partial purification and characterization of Ral8I, a class-IIS restriction endonuclease from Ruminococcus albus 8 which recognizes 5′-GGATC

Heparin-agarose chromatography was used to isolate a restriction endonuclease (ENase) from the cellulolytic Gram + anaerobe, Ruminococcus albus 8. The enzyme, Ral8I, was eluted from the column using 230–310 mM Na +. However, the preparation was active only with DNA subsrates that were not Dam-methylated. Moreover, the restriction fragment pattern generated from simian virus 40 (SV40) DNA was not consistent with the expected number of Dam-methylation sites. Alignment of the Dam-methylation sites in SV40 DNA indicated that Ral8I may actually recognize the asymmetric sequence, GGATC. This was confirmed by nucleotide (nt) sequence analysis and, further, Ral8I was found to cause cleavage of the DNA approx. 5 nt downstream from the recognition sequence. Ral8I can therefore be classified as a type-IIS restriction endonuclease and is an isoschizomer of AlwI, BinI and BthII.

Assessment of inhomogeneities in anE.coliphysical map

A statistical method based on r-fragments, sums of distances between (r + 1) consecutive restriction enzyme sites, is introduced for detecting nonrandomness in the distribution or too markers in sequence data. The technique is applicable whenever large numbers of markers are available and will detect clumping, excessive dispersion or too much evenness of spacing of the markers. It is particularly adapted to varying the scale on which inhomogeneities can be detected, from nearest neighbor interactions to more distant interactions. The r-fragment procedure is applied primarily to the Kohara et al. (1) physical map of E. coli. Other applications to DAM methylation sites in E. coli and NotI sites in human chromosome 21 are presented. Restriction sites for the eight enzymes used in (1) appear to be randomly distributed, although at widely differing densities. These conclusions are substantially in agreement with the analysis of Churchill et al. (3). Extreme variability in the density of the eight restriction enzyme sites cannot be explained by variability in mono-, di- or trinucleotide frequencies.

Unique sequence requirements for the P1 plasmid replication origin

We have carried out a detailed genetic analysis of the P1 plasmid replication origin and shown that it has four major structural requirements: the DnaA box, a series of five 7-base pair (bp) repeats, a GC-rich spacer and five 19-bp repeats that bind the P1 RepA protein. The origin requires the DnaA protein and its recognition sequence (the DnaA box). However, although give boxes are present in two separate blocks in the wild type, just one, placed either to the left or right of the core origin sequences, is sufficient for function as long as the box conforms exactly to the proposed consensus. Each of the five 7-bp repeats that constitute the core of the origin is required; mutations within any of the first six base pairs can block origin function. The required bases include, but are not limited to, those constituting dam methylation sites. Also essential is a 39-bp GC-rich sequence. We show this to be a spacer of critical length that separates the 7-bp repeats from the last required region; a series of 19-bp repeats that bind the P1 RepA initiator protein.

The absence of adenine methylation increases the ligation efficiency of partially filled staggered DNA ends

colonies, while the addition of the methylated BamHl fragment resulted in 22 transformants. The presence of an equal amount of nonmethylated BamHl fragment stimulated the number of transformants to 335. This 15-fold stimulation of transformatio n frequency is attributed to the absence of the methyl group at the A residues of the BamHl ends, since neither of the BamHl fragments in the absence of vector contributed to the number of transformants. Mini DNA preps were performed on six of the damr and six of the dam+ transformants. Restriction digests demonstrated that all of the damr transformants contained plasmids that acquired the BamHl fragments, while 2 of the 6 damr clones had recombined to form aberrant plasmids. The inhibition of ligation of 2 base overhangs by methyl groups has been reported previously and was found to be the reason that Pvul ends ligate to each other at low frequencies (New England Biolabs 1988-1989 catalog, p. 27). We speculate that ligation of the partially filled cohesive ends created by any restriction enzyme that cuts at dam or dcm sites will be inhibited by the methyl groups. Because many enzymes that can be used for partial fill-in ligations, such as Bell and Bgll, cut at dam methylation sites, we recommend that DNA for these purposes be isolated from dam~ strains.

Dam methyltransferase sites located within the loop region of the oligopurine-oligopyrimidine sequences capable of forming H-DNA are undermethylatedin vivo

Several derivatives of pUC18 plasmid were constructed that contained oligopurine-oligopyrimidine (pur-pyr) motifs surrounded by Dam methylation sites. Inserts of two of the molecules (pPP1 and pPP2) were able to adopt the triple-stranded conformation in vitro and show in vivo a remarkable undermethylation of specific sites when grown in JM105 dam+ strain. Mapping experiments revealed that undermethylated GATC sequences were located exclusively within the single-stranded loop region of the sequence involved in H-DNA formation. Control molecules which either contained the pur-pyr tracts (pPPK and pKK42) or not (pUC18) and were not able to form the triple-stranded conformation were found to be normally methylated by the dam gene product in vivo. Location of GATC within the triplex forming sequence seems to be a prerequisite for achieving its in vivo undermethylation. E.coli host factors are involved in the observed phenomenon. This has been deduced from the fact that the undermethylated state of pPP1 and pPP2 does not depend on the phase of growth of host cells and is steadily maintained up to 50 hours, whereas the kinetics of Dam methylation in vitro of sites located within the triplex loop does not differ substantially from the kinetics of methylation of other sites on the vector. Full methylation can be readily achieved in vitro. Additional factor(s) that operate in vivo to control the undermethylated state are most likely proteins since the observed effect can be suppressed by chloramphenicol administration to the cell cultures.

The effect of dam methylation on the expression of glnS in E. coli

The wild type glnS promoter contains a dam methylation site. In dam strains, the expression of glnS is enhanced 2.6-fold. A mutated form of the promoter has been isolated in which the dam methylation site is lost. Expression of this promoter is insensitive to dam methylation.

The effect of codon usage on the oligonucleotide composition of the E. coli genome and identification of over- and underrepresented sequences by Markov chain analysis.

As shown in the accompanying paper (5), the oligonucleotide composition of the E. coli genome is highly asymmetric for sequences up to 6 bp in length when ranked from highest to lowest abundance. We show here that this largely reflects codon usage because heavily used codons were found in the highly abundant oligomers whereas rarely used codons, with some exceptions, occurred in sequences in low abundance. Furthermore, linear regression analysis revealed a strong correlation between the frequencies of each trinucleotide and its usage as a codon. Dinucleotides are also not randomly distributed across each codon position and the dinucleotide composition of genes that are transcribed but not translated (rRNA and tRNA genes) was highly related to that seen in genes encoding polypeptides. However, 45 tetra-, 8 penta-, and 6 hexanucleotides were significantly over- or underabundant by Markov chain analysis and could not be accounted for by codon usage. Of these underrepresented sequences, many were palindromes, including the Dam methylation site.

DNA methylation differentially enhances the expression of one of the two E. coli dnaA promoters in vivo and in vitro.

The promoter/regulatory region of the dnaA gene, whose gene product is required for the initiation of DNA replication in Escherichia coli K-12, contains an unusually large number of Dam methylation sites. In this paper we report that the expression of the dnaA gene is decreased in Dam- strains of E. coli. The decrease in the expression of dnaA was measured in vivo using a dnaA-lacZ gene fusion. In vivo S1 nuclease mapping demonstrated that the decrease was due to a differential decrease in expression from the more proximal of the two dnaA promoters, dnaA2P. Comparison of the strengths of the two dnaA promoters in an in vitro transcription system using methylated and unmethylated DNA templates suggests that the effect of methylation on dnaA2P is probably at the level of RNA polymerase/DNA interaction. We suggest that this effect of methylation may be important in controlling the expression of dnaA during the E. coli cell cycle.

Bacteriophage P1 cre gene and its regulatory region : Evidence for multiple promoters and for regulation by DNA methylation

The bacteriophage P1 site-specific recombination system consists of two components, a site, loxP, at which recombination occurs, and a recombinase protein, Cre. In this paper, we present the DNA sequence of the cre structural gene and its upstream regulatory region. Analysis of the sequence indicates: (1) that cre encodes a protein of 343 amino acids; (2) that cre and loxP are separated by a 434 base-pair region that contains a 73 amino acid open reading frame, orf1; and (3) that cre and orf1 are oriented with their amino-terminal ends proximal to loxP. We have identified three promoters that are located upstream of the cre structural gene. Their activities range from 7 to 10% of the activity of the galactose operon promoter. The promoter furthest from cre, p R1, contains two Dam methylation sites (5′-G-A-T-C-3′) in its −35 region, and is sensitive to Dam methylation. Its transcription is three- to fourfold higher in a dam − host than it is in a dam + host. The promoter closest to cre, p R3, signals the production of an RNA transcript that functions inefficiently for Cre protein synthesis because it lacks a ribosome recognition site. None of the three cre promoters is sensitive to proteins expressed by the P1 prophage, including the c1 repressor protein. To assess the role of cre in the P1 life-cycle, we isolated cre mutants and studied their behavior in recA + and recA − hosts. Those studies indicate that Cre is dispensable for viral vegetative growth and lysogeny in a recA + host, but is required for both processes in a recA − host. The cre requirement for lysogeny suggests that the protein is essential for the cyclization of newly injected terminally redundant virion DNA. The requirement for vegetative growth suggests that Cre also has a role to play in the viral lytic cycle after the viral DNA has been cyclized.

IS10 transposition is regulated by DNA adenine methylation.

We show that dam- mutants are a major class of E. coli mutants with increased IS10 activity. IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds. Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains. Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons. Transposition is also increased when IS10 is hemimethylated. One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element. Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time. Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated. We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork.