GATC Sites Research Articles

Plasmid expression of mutS, - L and/or - H gene in Escherichia colidam cells results in strains that display reduced mutation frequency

Escherichia coli dam cells have an active but non-directed mismatch repair system; therefore, assembly of MutSLH complex at a mismatched base pair can result in MutH-mediated cleavage of GATC sites in both DNA strands. Unpaired double-strand breaks on a fraction of the replication errors occurring in dam cells presumably cause cell death, selectively eliminating these putative mutants from the population. We show that E. coli dam cells transformed with plasmids containing either the mutS, mutL or mutH gene display a mutation frequency three to eight times lower than that of the parental dam strain, due to increased mismatch-stimulated cell killing. Transformed strains are also more susceptible to killing by the base analogue 2-aminopurine. However, dam and dam transformed cells have similar duplication time, proportion of live/dead cells and morphology.

An Assessment of the Role of DNA Adenine Methyltransferase on Gene Expression Regulation in E coli

N6-Adenine methylation is an important epigenetic signal, which regulates various processes, such as DNA replication and repair and transcription. In γ-proteobacteria, Dam is a stand-alone enzyme that methylates GATC sites, which are non-randomly distributed in the genome. Some of these overlap with transcription factor binding sites. This work describes a global computational analysis of a published Dam knockout microarray alongside other publicly available data to throw insights into the extent to which Dam regulates transcription by interfering with protein binding. The results indicate that DNA methylation by DAM may not globally affect gene transcription by physically blocking access of transcription factors to binding sites. Down-regulation of Dam during stationary phase correlates with the activity of TFs whose binding sites are enriched for GATC sites.

DNA adenine methylation changes dramatically during establishment of symbiosis

The DNA adenine methylation status on specific 5'-GANTC-3' sites and its change during the establishment of plant-microbe interactions was demonstrated in several species of alpha-proteobacteria. Restriction landmark genome scanning (RLGS), which is a high-resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free-living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.

Epigenetic gene regulation in the bacterial world.

Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions.

Identification and mapping of open chromatin regions within a 140 kb polygenic locus of human chromosome 19 using E. coli Dam methylase

Using transient expression of the E. coli Dam methylase gene and analysis of the distribution of methylated GATC sites, we studied the distribution of open chromatin regions within a 140 kb long human genome segment in HEK-293 cells. Dam methylated sites were found in gene introns, exons, and intergenic regions, and their distribution along DNA was uneven. There were regions of high and low density of Dam methylated GATC sites, presumably corresponding to "open" and "closed" chromatin regions, respectively, and to the functional profile of the genomic locus under study. The Dam methylation profile was also generally in agreement with transcriptional activity of genes in the locus. Moreover, DNA regions accessible to Dam methylase apparently coincided with those hypersensitive to DNase I.

The Synchrony Phenotype Persists after Elimination of Multiple GATC Sites from the dnaA Promoter of Escherichia coli

To examine whether methylation of the GATC sites present in the dnaA promoter region is responsible for the strict temporal coordination of initiation events at oriC as measured by the synchrony of initiation, we introduced point mutations eliminating three (TGW1) and five (TGW2) of the six GATC sites present in the dnaA promoter region. All of the strains containing these mutations, including the one with five GATC sites eliminated, initiated chromosomal replication synchronously.

Structure and Substrate Recognition of the Escherichia coli DNA Adenine Methyltransferase

The structure of the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase in complex with cognate DNA was determined at 1.89 A resolution in the presence of S-adenosyl-L-homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3') half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E.coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S-adenosyl-L-methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120.

GATC Flanking Sequences Regulate Dam Activity: Evidence for how Dam Specificity may Influence pap Expression

Escherichia coli DNA adenine methyltransferase (Dam) plays essential roles in DNA replication, mismatch repair and gene regulation. The differential methylation by Dam of the two GATC sequences in the pap promoter regulates the expression of pili genes necessary for uropathogenic E.coli cellular adhesion. Dam processively methylates GATC sites in various DNA substrates, yet the two pap GATC sites are not processively methylated. We previously proposed that the flanking sequences surrounding the two pap GATC sites contribute to the enzyme's distributive methylation. We show here that replacement of the poorly methylated pap GATC sites with sites predicted to be processively methylated indeed results in an increase in Dam processivity. The increased processivity is due to a change in the methyltransfer kinetics and not the binding efficiency of Dam. A competition experiment in which the flanking sequences of only one pap GATC site were altered demonstrates that the GATC flanking sequences directly regulate the enzyme's catalytic efficiency. The GATC flanking sequences in Dam-regulated promoters in E.coli and other bacteria are similar to those in the pap promoter. Gene regulation from some of these promoters involves mechanisms and proteins that are quite different from those in the pap operon. Further, GATC sequences previously identified to remain unmethylated within the E.coli genome, but whose function remains largely unassigned, are flanked by sequences predicted to be poorly methylated. We conclude that the GATC flanking sequences may be critical for expression of pap and other Dam-regulated genes by affecting the activity of Dam at such sites and, thus, its processivity. A model is proposed, illustrating how the sequences flanking the GATC sites in Dam-regulated promoters may contribute to this epigenetic mechanism of gene expression, and how flanking sequences contribute to the diverse biological roles of Dam.

Specific N‐terminal interactions of the Escherichia coli SeqA protein are required to form multimers that restrain negative supercoils and form foci

The Escherichia coli SeqA protein binds preferentially to hemimethylated DNA and is required for inactivation (sequestration) of newly formed origins. A mutant SeqA protein, SeqA4 (A25T), which is deficient in origin sequestration in vivo, was found here to have lost the ability to form multimers, but could bind as dimers with wild-type affinity to a pair of hemimethylated GATC sites. In vitro, binding of SeqA dimers to a plasmid first generates a topology change equivalent to a few positive supercoils, then the binding leads to a topology change in the "opposite" direction, resulting in a restraint of negative supercoils. Binding of SeqA4 mutant dimers produced the former effect, but not the latter, showing that a topology change equivalent to positive supercoiling is caused by the binding of single dimers, whereas restraint of negative supercoils requires multimerization via the N-terminus. In vivo, mutant SeqA4 protein was not capable of forming foci observed by immunofluorescence microscopy, showing that N-terminus-dependent multimerization is required for building SeqA foci. Overproduction of SeqA4 led to partially restored initiation synchrony, indicating that origin sequestration may not depend on efficient higher-order multimerization into foci, but do require a high local concentration of SeqA.

Regulation of traJ transcription in the Salmonella virulence plasmid by strand‐specific DNA adenine hemimethylation

The traJ gene of the virulence plasmid of Salmonella enterica serovar Typhimurium (pSLT) encodes a transcriptional activator of the transfer operon. The leucine-responsive regulatory protein (Lrp) is an activator of traJ transcription. The upstream-activating-sequence of the pSLT traJ promoter contains two Lrp binding sites (LRP-1 and LRP-2), both necessary for transcriptional activation. The promoter-proximal site (LRP-2) contains a GATC site (GATC-II) whose methylation state affects Lrp binding: GATC-II methylation in both DNA strands decreases the affinity of Lrp for the LRP-2 site, while efficient Lrp binding occurs to a non-methylated GATC-II site. The effect of GATC-II hemimethylation on Lrp binding is strand-specific: methylation of the traJ non-coding strand permits formation of the major Lrp-DNA retardation complex, but methylation of the coding strand does not. This asymmetry supports a model in which passage of the replication fork may permit Lrp-mediated activation of conjugal transfer in one daughter plasmid molecule but not in the other. A remarkable trait of this regulatory design is that hemimethylation of a single GATC site can generate distinct epigenetic signals in otherwise identical plasmid DNA molecules.

Regulation of finP transcription by DNA adenine methylation in the virulence plasmid of Salmonella enterica.

DNA adenine methylase (Dam(-)) mutants of Salmonella enterica serovar Typhimurium contain reduced levels of FinP RNA encoded on the virulence plasmid. Dam methylation appears to regulate finP transcription, rather than FinP RNA stability or turnover. The finP promoter includes canonical -10 and -35 modules and depends on the sigma(70) factor. Regulation of finP transcription by Dam methylation does not require DNA sequences upstream from the -35 module, indicating that Dam acts at the promoter itself or downstream. Unexpectedly, a GATC site overlapping with the -10 module is likewise dispensable for Dam-mediated regulation. These observations indicate that Dam methylation regulates finP transcription indirectly and suggest the involvement of a host factor(s) responsive to the Dam methylation state of the cell. We provide evidence that one such factor is the nucleoid protein H-NS, which acts as a repressor of finP transcription in a Dam(-) background. H-NS also restrains transcription of the overlapping traJ gene, albeit in a Dam-independent fashion. Hence, the decreased FinP RNA content found in Dam(-) hosts of S. enterica appears to result from H-NS-mediated repression of finP transcription.

An oriC-like Distribution of GATC Sites Mediates Full Sequestration of Non-origin Sequences in Escherichia coli

Sequestration of newly replicated origins is one of the mechanisms required to limit initiation of Escherichia coli chromosome replication to once per generation. Origin sequestration lasts for a considerably longer period of time than the sequestration of other newly replicated regions of the chromosome. The reason for this may be the high number of GATC sites present in the origin. Alternatively, other sequence elements in the origin region may be important for its prolonged sequestration. To distinguish between these possibilities we constructed a DNA fragment containing ten GATC sites distributed with the same spacing as the ten GATC sites in the left half of oriC, but with random sequence between the GATC sites, and inserted it at a non-sequestered chromosome location. Sequestration of this GATC-cluster lasted as long as that of oriC, or even longer. The result shows that the presence of ten GATC sites, distributed as in oriC, is sufficient to cause full sequestration, and that other sequence elements most likely do not contribute to sequestration.

Crystal structure of a SeqA–N filament: implications for DNA replication and chromosome organization

Escherichia coli SeqA binds clusters of transiently hemimethylated GATC sequences and sequesters the origin of replication, oriC, from methylation and premature reinitiation. Besides oriC, SeqA binds and organizes newly synthesized DNA at replication forks. Binding to multiple GATC sites is crucial for the formation of stable SeqA-DNA complexes. Here we report the crystal structure of the oligomerization domain of SeqA (SeqA-N). The structural unit of SeqA-N is a dimer, which oligomerizes to form a filament. Mutations that disrupt filament formation lead to asynchronous DNA replication, but the resulting SeqA dimer can still bind two GATC sites separated from 5 to 34 base pairs. Truncation of the linker between the oligomerization and DNA-binding domains restricts SeqA to bind two GATC sites separated by one or two full turns. We propose a model of a SeqA filament interacting with multiple GATC sites that accounts for both origin sequestration and chromosome organization.

GATC Methylation by Dam methylase in archaea: its roles and possible transcription regulation by an FFRP

Methylation of pairs of adenines at their N6 positions in GATC sites (GATC methylation) in archaeal genomic DNAs has been studied. Genomic DNAs in cells of three archaeal species were found as fully methylated, while those of four other archaeal species were free of such methylation. Consistently with this pattern of the presence or absence of GATC methylation, homologues of E. coli Dam methylase was found present or absent, but various other types of DNA methylases were not. A Dam homologue from Pyrococcus sp. OT3 was expressed and its expected function of methylating GATC was confirmed. By methylation of each adenine the DNA duplex was destabilized by 0.56 ± 0.10 Kcal/mol, and this effect was additive. For some archaea, transcription regulation of Dam methylase gene appears to be needed upon cell replication, and in regions upstream of three Dam methylase genes, nucleotide sequences close to TTTTCTTTGAAAA were present. This arrangement, five bases each at the ends, which are complementary to each other, sandwiching three T bases at the center, fits into a pattern the same as those recognized by dimers of transcription factors, feast/famine regulatory proteins (FFRPs). (Communicated by Masanori OTSUKA, M.J.A.)

Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis. Food contamination by Listeria monocytogenes has been implicated in numerous outbreaks and sporadic cases of human illness. Most commonly implicated in listeriosis are highly processed, ready-to-eat (RTE) foods that are kept refrigerated for various periods of time. At risk for listeriosis are people in the extremes of age, pregnant women and their fetuses, cancer patients, and others experiencing immunosuppression (13, 24, 35, 38). Listeriosis can have severe symptoms (septicemia, meningitis, and stillbirths) and a high mortality rate (20 to 30%). Hence, regulations exist in numerous nations concerning the density (e.g., 1 CFU/25 g) of cells of the etiologic agent permissible in RTE foods. Such regulations are based on the hypothesis that any L. monocytogenes strain that can be detected in RTE foods has the potential to pose serious hazards to human health. The potential hazard posed by listerial contamination of RTE foods can be influenced by the number of cells at the point of consumption, which would depend on conditions of storage, type of food matrix and its impact on growth, presence of competing microflora and antimicrobial agents, etc. In addition, the strain type of L. monocytogenes involved may be of importance. It is likely, based on studies with other bacterial pathogens, that some strains and strain clusters (clonal groups) within the species might be more pathogenic than others. Speculations have been formulated that only a fraction of the strains of L. monocytogenes found in foods may be capable of causing human illness (20). There is indeed evidence that the repertoire of strains ca

Functional Characterization of Escherichia coli DNA Adenine Methyltransferase, a Novel Target for Antibiotics

We have characterized Escherichia coli DNA adenine methyltransferase, a critical regulator of bacterial virulence. Steady-state kinetics, product inhibition, and isotope exchange studies are consistent with a kinetic mechanism in which the cofactor S-adenosylmethionine binds first, followed by sequence-specific DNA binding and catalysis. The enzyme has a fast methyl transfer step followed by slower product release steps, and we directly demonstrate the competence of the enzyme cofactor complex. Methylation of adjacent GATC sites is distributive with DNA derived from a genetic element that controls the transcription of the adjacent genes. This indicates that the first methylation event is followed by enzyme release. The affinity of the enzyme for both DNA and S-adenosylmethionine was determined. Our studies provide a basis for further structural and functional analysis of this important enzyme and for the identification of inhibitors for potential therapeutic applications.

Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods.

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.

Binding of SeqA Protein to Hemi-methylated GATC Sequences Enhances Their Interaction and Aggregation Properties

The SeqA protein regulates chromosome initiation and is involved in segregation in Escherichia coli. One SeqA protein binds to two hemi-methylated GATC sequences to form a stable SeqA-DNA complex. We found that binding induced DNA bending, which was pronounced when the two sequences were on the same face of the DNA. Two SeqA molecules bound cooperatively to each pair of hemi-methylated sites when the spacing between the sites was < or = 30 bp. This cooperative binding was able to stabilize the binding of a wild type to a single hemi-methylated site, or mutant form of SeqA protein to hemi-methylated sites, although such binding did not occur without cooperative interaction. Two cooperatively bound SeqA molecules interacted with another SeqA bound up to 185 bp away from the two bound SeqA proteins, and this was followed by aggregation of free SeqA proteins onto the bound proteins. These results suggest that the stepwise interaction of SeqA proteins with hemi-methylated GATC sites enhances their interaction and leads to the formation of SeqA aggregates. Cooperative interaction followed by aggregation may be the driving force for formation of the SeqA foci that appear to be located behind replication forks.

Genome of staphylococcal phage K: a new lineage of Myoviridae infecting gram-positive bacteria with a low G+C content.

Phage K is a polyvalent phage of the Myoviridae family which is active against a wide range of staphylococci. Phage genome sequencing revealed a linear DNA genome of 127,395 bp, which carries 118 putative open reading frames. The genome is organized in a modular form, encoding modules for lysis, structural proteins, DNA replication, and transcription. Interestingly, the structural module shows high homology to the structural module from Listeria phage A511, suggesting intergenus horizontal transfer. In addition, phage K exhibits the potential to encode proteins necessary for its own replisome, including DNA ligase, primase, helicase, polymerase, RNase H, and DNA binding proteins. Phage K has a complete absence of GATC sites, making it insensitive to restriction enzymes which cleave this sequence. Three introns (lys-I1, pol-I2, and pol-I3) encoding putative endonucleases were located in the genome. Two of these (pol-I2 and pol-I3) were found to interrupt the DNA polymerase gene, while the other (lys-I1) interrupts the lysin gene. Two of the introns encode putative proteins with homology to HNH endonucleases, whereas the other encodes a 270-amino-acid protein which contains two zinc fingers (CX(2)CX(22)CX(2)C and CX(2)CX(23)CX(2)C). The availability of the genome of this highly virulent phage, which is active against infective staphylococci, should provide new insights into the biology and evolution of large broad-spectrum polyvalent phages.

Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically.

In rapidly growing Escherichia coli cells replication cycles overlap and initiation occurs at multiple replication origins (oriCs). All origins within a cell are initiated essentially in synchrony and only once per cell cycle. Immediate re-initiation of new origins is avoided by sequestration, a mechanism dependent on the SeqA protein and Dam methylation of GATC sites in oriC. Here, GATC sites in oriC were changed to GTTC. This reduced the sequestration to essentially the level found in SeqA-less cells. The mutant origins underwent re-initiation, showing that the GATC sites in oriC are required for sequestration. Each re-initiation eventually gave rise to a cell containing an extra nucleoid. The three-nucleoid cells displayed one asymmetrically placed FtsZ-ring and divided into a two-nucleoid cell and a one-nucleoid cell. The three nucleoid-cells thus divided into three daughters by two consecutive divisions. The results show that extra rounds of replication cause extra daughter cells to be formed prematurely. The fairly normal mutant growth rate and size distribution show, however, that premature rounds of replication, chromosome segregation, and cell division are flexibly accommodated by the existing cell cycle controls.