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The major phase-variable outer membrane protein of Escherichia coli structurally resembles the immunoglobulin A1 protease class of exported protein and is regulated by a novel mechanism involving Dam and oxyR
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Here we report the characterization of an Escherichia coli gene (agn43) which encodes the principal phase-variable outer membrane protein termed antigen 43 (Ag43). The agn43 gene encodes a precursor protein of 107 kDa containing a 52-amino-acid signal sequence. Posttranslational processing generates an alpha43 subunit (predicted Mr of 49,789) and a C-terminal domain (beta43) with features typical of a bacterial integral outer membrane protein (predicted Mr of 51, 642). Secondary structure analysis predicts that beta43 exists as an 18-stranded beta barrel and that Ag43 shows structural organization closely resembling that of immunoglobulin A1 protease type of exoprotein produced by pathogenic Neisseria and Haemophilus spp. The correct processing of the polyprotein to alpha43 and beta43 in OmpT, OmpP, and DegP protease-deficient E. coli strains points to an autocatalytic cleavage mechanism, a hypothesis supported by the occurrence of an aspartyl protease active site within alpha43. Ag43, a species-specific antigen, possesses two RGD motifs of the type implicated in binding to human integrins. The mechanism of reversible phase variation was studied by immunochemical analysis of a panel of well-defined regulatory mutants and by analysis of DNA sequences upstream of agn43. Evidence strongly suggests that phase variation is regulated by both deoxyadenosine methylase (Dam) and by OxyR. Thus, oxyR mutants are locked on for Ag43 expression, whereas dam mutants are locked off for Ag43 expression. We propose a novel mechanism for the regulation of phase switching in which OxyR competes with Dam for unmethylated GATC sites in the regulatory region of the agn43 gene. 相似文献
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Spontaneous Mutations Occur near Dam Recognition Sites in a dam-Escherichia coli Host 总被引:4,自引:0,他引:4
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The mismatch repair system of Escherichia coli K12 removes mispaired bases from DNA. Mismatch repair can occur on either strand of DNA if it lacks N6-methyladenines within 5'-GATC-3' sequences. In hemimethylated heteroduplexes, repair occurs preferentially on the unmethylated strand. If both strands are fully methylated, repair is inhibited. Mutant (dam-) strains of E. coli defective in the adenine methylase that recognizes 5'-GATC-3' sequences (Dam), and therefore defective in mismatch repair, show increased spontaneous mutation rates compared to otherwise isogenic dam+ hosts. We have isolated and characterized 91 independent mutations that arise as a consequence of the Dam- defect in a plasmid-borne phage P22 repressor gene, mnt. The majority of these mutations are A:T----G:C transitions that occur within six base pairs of the two 5'-GATC-3' sequences in the mnt gene. In contrast, the spectrum of mnt- mutations in a dam+ host is comprised of a majority of insertions of IS elements and deletions that do not cluster near Dam recognition sites. These results show that Dam-directed post-replicative mismatch repair plays a significant role in the rectification of potential transition mutations in vivo, and suggest that sequences associated with Dam recognition sites are particularly prone to replication or repair errors. 相似文献
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The methylation of adenine in palindromic 5'-GATC-3' sites by Escherichia coli Dam supports diverse roles, including the essential regulation of virulence genes in several human pathogens. As a result of a unique hopping mechanism, Dam methylates both strands of the same site prior to fully dissociating from the DNA, a process referred to as intrasite processivity. The application of a DpnI restriction endonuclease-based assay allowed the direct interrogation of this mechanism with a variety of DNA substrates. Intrasite processivity is disrupted when the DNA flanking a single GATC site is longer than 400 bp on either side. Interestingly, the introduction of a second GATC site within this flanking DNA reinstates intrasite methylation of both sites. Our results show that intrasite methylation occurs only when GATC sites are clustered, as is found in gene segments both known and postulated to undergo in vivo epigenetic regulation by Dam methylation. We propose a model for intrasite methylation in which Dam bound to flanking DNA is an obligate intermediate. Our results provide insights into how intrasite processivity, which appears to be context-dependent, may contribute to the diverse biological roles that are carried out by Dam. 相似文献
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Chlamydia trachomatis is a Gram-negative eubacterium with a dimorphic developmental cycle and obligate intracellular growth in the eucaryotic host. The Dam transmethylase of Escherichia coli methylates at the N6 position of adenine in the sequence 5'-GATC-3' and the Dcm transmethylase adds methyl groups to the C5 position of the internal cytosines in the sequences 5'-CCWGG-3'. In contrast to E. coli, C. trachomatis DNA appears to have unmethylated Dam sites and only low level Dcm methylation. 相似文献
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Ichida H Maeda K Ichise H Matsuyama T Abe T Yoneyama K Koba T 《Biochemical and biophysical research communications》2007,363(3):852-856
We have developed a restriction landmark genome scanning (RLGS) system in silico, involving two-dimensional electrophoretic analysis of DNA by computer simulation that is based on the availability of whole-genome sequences for specific organisms. We applied the technique to the analysis of the Xanthomonas oryzae pathovar oryzae (Xoo) MAFF 311018, which causes bacterial blight in rice. The coverage that was found to be achievable using RLGS in silico, as a percentage of the genomic regions that could be detected, ranged from 44.5% to 72.7% per image. However, this reached a value of 96.7% using four images that were obtained with different combinations of landmark restriction enzymes. Interestingly, the signal intensity of some of the specific spots obtained was significantly lower than that of other surrounding spots when MboI, which cleaves unmethylated 5'-GATC-3' sites, was used. DNA gel blot analysis with both DNA adenine methylase (Dam)-sensitive and -insensitive isoschizomers (MboI and Sau3AI) revealed that Dam-mediated DNA adenine methylation had indeed occurred at these particular sites. These results suggest that a significant portion of the 5'-GATC-3' sites within the Xoo genome is stably methylated by Dam. 相似文献
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Escherichia coli DNA adenine methyltransferase (EcoDam) methylates the N-6 position of the adenine in the sequence 5'-GATC-3' and plays vital roles in gene regulation, mismatch repair, and DNA replication. It remains unclear how the small number of critical GATC sites involved in the regulation of replication and gene expression are differentially methylated, whereas the approximately 20,000 GATCs important for mismatch repair and dispersed throughout the genome are extensively methylated. Our prior work, limited to the pap regulon, showed that methylation efficiency is controlled by sequences immediately flanking the GATC sites. We extend these studies to include GATC sites involved in diverse gene regulatory and DNA replication pathways as well as sites previously shown to undergo differential in vivo methylation but whose function remains to be assigned. EcoDam shows no change in affinity with variations in flanking sequences derived from these sources, but methylation kinetics varied 12-fold. A-tracts immediately adjacent to the GATC site contribute significantly to these differences in methylation kinetics. Interestingly, only when the poly(A) is located 5' of the GATC are the changes in methylation kinetics revealed. Preferential methylation is obscured when two GATC sites are positioned on the same DNA molecule, unless both sites are surrounded by large amounts of nonspecific DNA. Thus, facilitated diffusion and sequences immediately flanking target sites contribute to higher order specificity for EcoDam; we suggest that the diverse biological roles of the enzyme are in part regulated by these two factors, which may be important for other enzymes that sequence-specifically modify DNA. 相似文献
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The O‐antigen of Salmonella lipopolysaccharide is a major antigenic determinant and its chemical composition forms the basis for Salmonella serotyping. Modifications of the O‐antigen that can affect the serotype include those carried out by the products of glycosyltransferase operons (gtr), which are present on specific Salmonella and phage genomes. Here we show that expression of the gtr genes encoded by phage P22 that confers the O1 serotype is under the control of phase variation. This phase variation occurs by a novel epigenetic mechanism requiring OxyR in conjunction with the DNA methyltransferase Dam. OxyR is an activator or a repressor of the system depending on which of its two binding sites in the gtr regulatory region is occupied. Binding is decreased by methylation at Dam target sequences in either site, and this confers heritability of the expression state to the system. Most Salmonella gtr operons share the key regulatory elements that are identified here as essential for this epigenetic phase variation. 相似文献
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Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections. 总被引:3,自引:0,他引:3
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Methylations of highly specific sites in the promoter and 5' regions of eucaryotic genes have been shown to shut off gene activity and thus play a role in the long-term regulation of gene expression. It was therefore of interest to investigate whether site-specific DNA methylations could also play a role in adenovirus DNA in productive infections. It has been reported earlier that adenovirion DNA is not detectably methylated (U. Günthert, M. Schweiger, M. Stupp, and W. Doerfler, Proc. Natl. Acad. Sci. USA 73:3923-3927, 1976). In the present study, evidence for the methylation of cytidine residues in 5'-CCGG-3' and 5'-GCGC-3' sequences or the methylation of adenine residues in 5'-GATC-3' and 5'-TCGA-3' sequences in intranuclear adenovirus type 2(Ad2) DNA isolated and analyzed early (5 h) or late (24 h) after infection could not be obtained. In Ad2 DNA, 22.5% of all 5'-CG-3' dinucleotides reside in 5'-CCGG-3' and 5'-GCGC-3' sequences. Intranuclear viral DNA was examined by restriction endonuclease cleavage by using HpaII, MspI, HhaI, DpnI, or TaqI and Southern blot hybridizations. The HindIII fragments of Ad2 DNA served as hybridization probes. The data rendered it very unlikely that free intracellular adenovirus DNA in productively infected cells was extensively methylated. Thus, DNA methylation was not a likely element in the regulation of free adenovirus DNA expression in productively infected cells. 相似文献
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Bacteria produce a variety of enzymes capable of methylating DNA. In many species, the majority of adenine methylation is accomplished by the DNA adenine methylase Dam. In Escherichia coli the Dam methylase plays roles in the initiation of replication, mismatch repair, and gene regulation. In a number of other bacterial species, mutation or overexpression of Dam leads to attenuation of virulence. Homologues of the dam gene exist in some members of the Firmicutes, including Streptococcus mutans, a dental pathogen. An S. mutans strain inactivated in the dam gene (SMU.504; here designated damA) was engineered, and phenotypes linked to cariogenicity were examined. A prominent observation was that the damA mutant produced greater amounts of glucan than the parental strain. Real-time PCR confirmed upregulation of gtfB. To determine whether other loci were affected by the damA mutation, a microarray analysis was carried out. Seventy genes were upregulated at least 2-fold in the damA mutant, and 33 genes were downregulated at least 2-fold. In addition to gtfB (upregulated 2.6-fold; 1.7-fold when measured by real-time PCR), other upregulated virulence factors included gbpC (upregulated 2.1-fold) and loci predicted to encode bacteriocins (upregulated 2- to 7-fold). Various sugar transport operons were also upregulated, the most extreme being the cellobiose operon (upregulated nearly 40-fold). Expression of sacB, encoding fructosyltransferase, was downregulated 2.4-fold. The sequence 5'-GATC-3' appeared to constitute the recognition sequence for methylation. These results provide evidence that DNA methylation in S. mutans has a global effect on gene expression, including that of genes associated with cariogenic potential. 相似文献