The most common modification of the genome is the methylation of adenine at the n6 position. Dna adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock. Dna methylation provides a mechanism by which additional information is imparted to dna, and such epigenetic information can alter the timing and targeting. Unlike eukaryotes, however, bacteria use dna adenine methylation rather than dna cytosine methylation as an epigenetic signal. Request pdf dna adenine methylation and bacterial pathogenesis methylation of dna by the dna adenine methyltransferase dam provides an epigenetic. Two wellstudied dna methyltransferases that do not involve such restrictionmodification pairs are dam dna. Roles of dna adenine methylation in regulating bacterial gene. Although salmonellacontain a dna cytosine methylase dcm, the role of cytosine methylation in this organism is. The chemical diversity of dna modifications expanded with our discovery of phosphorothioates pts as physiological modifications of the dna sugarphosphate backbone in which a nonbridging oxygen is replaced by sulfur 12. An essential role for dna adenine methylation in bacterial virulence. Epigenomic characterization of clostridioides difficile finds. Cloning, sequence analysis and heterologous expression of.
The yhdj gene was cloned, and the enzyme was overexpressed and purified. Initial studies with dam mutants showed that dam regulates the expression of certain genes in e. In mammals dna methylation is essential for normal development and is associated. Analysis can be genespecific or global depending on downstream applications 1. Here, we describe how two diverse modifications, adenine methylation 6ma and phosphorothioation pt, have evolved to occupy the same genomic sites in bacteria. In higher organisms, where only 5methylcytosine is found.
This study focused on a type i restrictionmodification system designated yrmi of y. Bacteria produce a variety of enzymes capable of methylating dna. Dna sequence analysis revealed that the putative yhdj dna methyltransferase gene of escherichia coli is 55% identical to the nostoc sp. Methylation and restriction analysis showed that the dna. Dna adenine methylation is required to replicate both. In escherichia coli k12 and many gammaproteobacteria, this modification is catalyzed by dna adenine methyltransferase dam at the gatc consensus sequence and is known to modulate cellular processes including transcriptional regulation of gene expression. Dam, the most described bacterial dnamethyltransferase, is widespread in gammaproteobacteria.
Methylation can change the activity of a dna segment without changing the sequence. A dna adenine methylase mutant of shigella flexneri shows. While adenine methylation has been largely studied in many bacterial systems and it was shown to influence crucial vital processes, such as bacterial cell cycle, only a few studies have so far. Although bacterial dna methyltransferases are generally associated with restriction modification systems to protect against phage infection, dna methylation also regulates dna replication, repair, and transcription. Original article dna adenine methylation modulates pathogenicity of klebsiella pneumoniae genotype k1 chitai fang a,b,c, wenching yi b,c, chiatung shun d, shihfeng tsai e a institute of epidemiology and preventive medicine, college of public health, national taiwan. Cytosine methylation in dna is catalyzed by dna methyltransferases mtases 1 that transfer methyl groups from the ubiquitous donorsadenosyllmethionine adomet producing modified cytosines with a methyl group at either c5 or n4. Dam is a wellestablished example of epigenetic regulation in bacteria. Dna adenine methylase dam mutants of salmonella enterica serovar typhimurium grown under laboratory conditions express the std fimbrial operon, which is tightly repressed in the wild type. Dna adenosine methylation dam methylation of the 5. Methylation of gatc sites is required for precise timing between rounds of dna replication in escherichia coli. Restriction digestion shows that the enzyme methylates adenine in the sequence gatc. Physiological dna methylation the only known covalent modification of dna molecule is accomplished by transfer of the methyl group from sadenosyl methionin to 5 position of the pyrimidine ring of cytosine. Dam plays an essential role in methyldirected mismatch repair mdmr because it allows discrimination between parental and daughter dna strands 1. Genomewide mapping of methylated adenine residues in.
Dna methylation of cytosine residues appears to be important for the regulation of biological processes in both plants and animals. The establishment and maintenance of dna methylation is generated by specific enzymes known as dna methyltransferases. Selective inhibitors of bacterial dna adenine methyltransferases neda mashhoon, 1cynthia pruss, michael carroll, paul h. Dna adenine methylase is essential for viability and plays. Selective inhibitors of bacterial dna adenine methyltransferases. The methyl group may be incorporated on the n6 position of the adenine or at different molecular positions of the cytosine n4 reflections on the significance of dna methylation in living organisms.
Polyphosphate polyp is an important metabolite and signaling molecule in prokaryotes and eukaryotes. Roles of dna adenine methylation in hostpathogen interactions. Increasing evidence in last years suggests that dna rna methylation can influence biological function, including regulation of dna rna replication and repair, and gene expression 3336. A dna adenine methylase mutant of shigella flexneri shows no. The pt system is more complicated than methylation and entails sequenceselective and r p. Reich2 the authors describe the discovery and characterization of several structural classes of smallmolecule inhibitors of bacterial. This enzymebased technology minimizes damage to dna and produces high quality libraries that enable superior detection of 5mc and 5hmc from fewer sequencing reads. The authors describe the discovery and characterization of several structural classes of smallmolecule inhibitors of bacterial dna adenine methyltransferases.
Using mass spectrometry, singlemolecule realtime sequencing, and. Regulation of the salmonella enterica std fimbrial operon by. Methylation of cytosine residues in dna occurs in diverse organisms from bacteria to humans. New technologies have fostered renewed interest in bacterial epigenetics, with dna modifications defending against other microbes and controlling gene expression. In prokaryotes, dna methylation is involved in determination of dna host specificity, virulence, dna repair, chromosome replication and segregation, cell cycle regulation and gene expression.
In escherichia coli the dam methylase plays roles in the initiation of replication, mismatch repair, and gene regulation. Apr 11, 2017 new technologies have fostered renewed interest in bacterial epigenetics, with dna modifications defending against other microbes and controlling gene expression. The dna adenine methyltransferase dam methylase of. In both alphaproteobacteria and gammaproteobacteria, the roles of dna base methylation are especially well known for n6methyladenine, including control of chromosome replication, nucleoid segregation, postreplicative correction of dna mismatches, cell cyclecoupled transcription, formation of bacterial cell lineages, and regulation of. Dna methylation is a process by which methyl groups are added to the dna molecule. In phase off cells, the global regulator lrp is bound to pap sites proximal to the pilin promoter, whereas in phase on cells, lrp is bound to promoter distal sites. It is an orphan methyltransferase that is not part of a restrictionmodification system and regulates gene expression. Dam dna methylation can play a role in various genes expression and is involved in pathogenicity of several bacterial species. Dna adenine methylation and bacterial pathogenesis. We have found that the local regulator papi increases the affinity of lrp for. All virulencerelated alterations associated with dam methylase overproduction disappeared when critical amino acids within its dna methylation motif were eliminated, thereby confirming that dna adenine methylation is involved in a. When located in a gene promoter, dna methylation typically acts to repress gene transcription. Immediately after dna synthesis, the daughter strand remains unmethylated for a short time. We cloned and sequenced the dna adeninen 6 methyltransferase gene of the human pathogen actinobacillus actinomycetemcomitans m.
The expression of pyelonephritisassociated pili pap in uropathogenic escherichia coli is epigenetically controlled by a reversible off to on switch. Ccrm for cell cycleregulated methyltransferase, originally identified as the enzyme responsible for methylating gantc sites in the caulobacter genome, is a 39kda protein with 49% identity to the haemophilus influenzae hinfi. Regulation of the salmonella enterica std fimbrial operon. Additionally, in salmonella, dam is a global regulator of bacterial gene expression and plays a critical role in virulence, and mutants with altered levels of dam activity elicit. Dna adenine methylation modulates pathogenicity of klebsiella. In a number of other bacterial species, mutation or overexpression of dam leads to attenuation of virulence. Effects of dna methylation on expression of virulence. Convergence of dna methylation and phosphorothioation. Dna adenine methylation is required to replicate both vibrio. At the turn of the century, dna adenine methylation was found to be essential for the pathogenesis of salmonella enterica subsp.
Profiling of human gut virome with oxford nanopore technology. Here, we show that uncontrolled production of std fimbriae in s. A dna adenine methyltransferase of escherichia coli that. Dna adenine methylation and bacterial pathogenesis request pdf. Request pdf dna adenine methylation and bacterial pathogenesis methylation of dna by the dna adenine methyltransferase dam provides an epigenetic signal that influences and regulates. In a number of other bacterial species, mutation or overexpression of dam leads to attenuation of. Reflections on the significance of dna methylation in. Methylation profiles of epigenome are used for disease identification and for research and therapeutic development. Methods to analyze dna methylation, dna demethylation, and their functional effects are critical to epigenetics researchers. M, ccrm appears to lack a cognate restriction enzyme. Phyletic patterns of dna adenine methylases, demethylases, and potential modified dna. Genomewide dam methylation in escherichia coli during long.
These methylated residues andor the level of dammt can influence. In many species, the majority of adenine methylation is accomplished by the dna adenine methylase dam. In escherichia coli, methylation serves to silence newly synthesized hemimethylated sister origins. To test the hypothesis that the reduction in virulence of dam salmonella. An essential role for dna adenine methylation in bacterial. Inactivation of dna adenine methyltransferase alters virulence factors in actinobacillus actinomycetemcomitans. Damdependent dna methylation has previously been shown to modulate chromosome replication, dna repair and transcription in e. Methylation at adenine residues by dam controls the timing and targeting of important biological processes such as dna replication, methyldirected mismatch repair, and transposition. A new method for dna methylation analysis at the single base level, nebnext enzymatic methylseq emseq, is now available. Dna methyltransferases and epigenetic regulation in bacteria. Cloning, sequence analysis and heterologous expression of the. Adenine methylation can alter the interactions of regulatory proteins with dna, either by a direct steric effect or by an indirect effect on dna structure 18, 61, 62. Inactivation of dna adenine methyltransferase alters. Dedonc, and shi chena,d,2 akey laboratory of combinatorial biosynthesis and drug discovery, ministry of education, school of pharmaceutical sciences, wuhan university, wuhan.
Although both adenine and cytosine methylations are found in both bacteria and eukaryotes, cytosine methylation has been. Dna adenine methylation is not present in higher eukaryotes including humans. The purpose of this study was to determine the role played by the dam ortholog identified in the entomopathogenic bacterium photorhabdus luminescens. Regulation of salmonella enterica pathogenicity island 1 by. Dna methylation and cancer dna methylation plays a critical role in the control of gene activity. Methylation of dna has multiple consequences for bacterial physiology including the regulation of chromosome replication, dna segregation, mismatch repair, transposition, and transcriptional regulation. Dna adenine methylation modulates pathogenicity of.
Dna adenine methylase, dam methylase is an enzyme that adds a methyl group to the adenine of the sequence 5gatc3 in newly synthesized dna. Phasevarions of bacterial pathogens microbiology society. A putative dna adenine methyltransferase is involved in. There is strong evidence that dam regulates genes by. Dna methylation has been shown to affect a range of processes including. Of all of the dna methylation systems known in bacteria, gatc methylation appears to have the highest impact on gene expression barras. Dam methylation contributes to the control of the initiation of dna replication in e. To determine whether these observations could be extended to shigella, a dam mutant of shigella flexneri 2a was characterized and examined for the role of dam in. The results discussed above show that adenine methylation is critical for salmonella pathogenesis. A dna adenine methyltransferase of escherichia coli that is. Dna methylation is a biological process by which methyl groups are added to the dna molecule. Transcriptional regulation by dna adenine methyltransferases dam has been investigated for escherichia coli, and for plasmidencoded fimbriae in salmonella.
Thus, in the absence of dam, bacteria show an increased mutation rate. Regulation of salmonella enterica pathogenicity island 1. Methylation of dna by the dna adenine methyltransferase dam provides an epigenetic signal that influences and regulates numerous physiological processes in the bacterial cell including chromosome replication, mismatch repair, transposition, and transcription. Epigenomic characterization of clostridioides difficile. In escherichia coli k12 and many gammaproteobacteria, this modification is catalyzed by dna adenine methyltransferase dam at the gatc consensus sequence and is known to modulate cellular processes including transcriptional. Ecodam is an adeninen6 dna methyltransferase that methylates the gatc sites in the escherichia coli genome.
Dna adenine methylase plays a pivotal role in many bacterial functions, including the replication, repair, transposition, and segregation of chromosomal dna 26, 28. Dam, the most described bacterial dna methyltransferase, is widespread in gammaproteobacteria. Some adenine methyltransferases have been shown not only to protect specific dna restriction sites from cleavage by a restriction endonuclease, but also to play a role in various bacterial processes and sometimes in bacterial virulence. Summary like many eukaryotes, bacteria make widespread use of postreplicative dna methylation for the epigenetic control of dnaprotein interactions. Dna adenine methylation is widely used to control many dna transactions, including replication. To determine whether these observations could be extended to shigella, a dam mutant of shigella flexneri 2a was characterized and examined for the role of dam in pathogenesis. These observations raise the possibility that dam inhibitors may be used as antimicrobial agents. Epigenetic regulation by dna methylation is a wellcharacterized process in. Mutants of salmonella defective in dna adenine methylase dam have been reported to be attenuated for virulence and to provide protective immunity when used as vaccine strains.
Whether its pathogenicity is controlled by dna adenine methylase dam, an epigenetic regulator of bacterial virulence gene expression, is yet unknown. The dna adenine methyltransferase dam methylase of gammaproteobacteria and the cell cycleregulated methyltransferase ccrm methylase of alphaproteobacteria catalyze an identical reaction methylation of adenosine moieties using sadenosylmethionine as a methyl donor at similar dna targets gatc and gantc, respectively. Roles of dna adenine methylation in regulating bacterial. These enzymes are essential for bacterial virulence dna adenine methyltransferase dam and cell viability cell cycleregulated methyltransferase ccrm. In addition, dam regulates the expression of operons such as pyelonephritisassociated pili pap, which are an important virulence determinant in upper urinary tract infections 2, 3. This methylation almost exclusively involves the addition of a methyl group to carbon 5 of cytosine nucleotides, and specifically those cytosines that precede guanines i. Dnaadenine methylation is not present in higher eukaryotes including humans. A growing number of reports describe a role for dna adenine methylation in regulating the expression of various bacterial genes related to virulence in diverse pathogens, suggesting that dna methylation may be a widespread and versatile regulator of virulence gene expression. Effects of dna methylation on expression of virulence genes. Bacterial dna methylation and methylomes springerlink. Flow cytometry analysis of wildtype caulobacter cultures reveals two distinct dna peaks, indicative of cell populations with either one or two chromosomes 36, 37. In mammals, dna methylation is essential for normal development and is associated with a number of key processes.