Tài liệu Báo cáo khoa học: DNA adenine methylation changes dramatically during establishment of symbiosis pdf

DNA adenine methylation changes dramatically during establishment of symbiosis Hiroyuki Ichida1,2, Tomoki Matsuyama3, Tomoko Abe2 and Takato Koba1 Graduate School of Science and Technology, Chiba University, Matsudo, Japan Accelerator Application Research Group, Nishina Center for Accelerator-Based Science, RIKEN, Hirosawa, Wako, Saitama, Japan Cellular Biochemistry Laboratory, Discovery Research Institute, RIKEN, Hirosawa, Wako, Saitama, Japan Keywords cell cycle-regulated methyltransferase; DNA adenine methylation; in silico restriction landmark genome scanning; plant–microbe interactions; specifically unmethylated region Correspondence H Ichida, Accelerator Application Research Group, Nishina Center for Accelerator-Based Science, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan Fax: +81 48 4624674 Tel: +81 48 4621111 Ext 5432 E-mail: ichida@riken.jp (Received 31 October 2006, revised December 2006, accepted 11 December 2006) 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 a-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 doi:10.1111/j.1742-4658.2007.05643.x Restriction landmark genome scanning (RLGS) is a method for the two dimensional display of end-labeled DNA restriction fragments and is an unbiased method for DNA methylation scanning in higher eukaryotes [1] Virtual image (Vi-) RLGS software simulates two dimensional DNA electrophoresis patterns based on whole-genome sequence data, allowing the rapid matching of DNA spots to their sequences without the time and effort of cloning [2] Recent advances in DNA sequencing technology have enabled the sequencing of entire genomes in various organisms, and the resulting data allow a comprehensive analysis of genome dynamics during host–microbe interactions Bradyrhizobium japonicum and Mesorhizobium loti are symbiotic bacteria that perform nitrogen fixation in host plant roots Their principal hosts are soybean and Lotus japonicus, which are an important grain crop and a model legume, respectively Biological nitrogen fixation is an important source of nitrogen in agricultural production, particularly for fabaceae crops, including soybean; for example,  90% of nitrogen is biologically fixed in well-nodulated soybean plants [3] Agrobacterium tumefaciens was originally isolated as a causal agent of crown gall disease in plants It infects more than 90 families of dicotyledonous plants, resulting in major agronomic losses worldwide [4] DNA methylation regulates critical functions in prokaryotic and eukaryotic cells C5-methyl-cytosine, N4-methyl-cytosine, and N6-methyl-adenine are found Abbreviations CcrM, cell cycle-regulated methyltransferase; Dam, deoxyadenosine methyltransferase; RLGS, restriction landmark genome scanning; SUM, specifically unmethylated region; Vi, virtual image FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS 951 Genomewide analysis of DNA adenine methylation H Ichida et al in microbial genomes In bacteria, these methylated bases are best known as important agents for restriction-modification systems, which distinguish self and nonself DNA to protect bacteria from invaders In this system, the host DNA is methylated and only unmethylated DNA is digested by cognate restriction endonucleases [5] In mammalian genomes, DNA is methylated at the C5 position of cytosine within CpG dinucleotide sequences In the human genome, almost half of the genes have short CpG-rich regions, which are called CpG islands A significant proportion of all CpG islands become methylated during development; when this happens, the associated promoter is stably silent Aberrant CpG hypermethylation and resulting transcriptional silencing is sometimes observed in cancer [6,7] CpG methylation is also known to participate in genomic imprinting and X-chromosome inactivation [8,9] Cell-cycle-regulated methyltransferase (CcrM), originally cloned from Caulobacter crescentus [10], is the best-characterized solitary methyltransferase that is not associated with restriction-modification systems, aside from deoxyadenosine methyltransferase (Dam) CcrM is thought to be a common regulatory component of a-proteobacteria [11] Both CcrM and Dam catalyze the transfer of a methyl group from S-adenosylmethionine to the N6 position of adenine However, they are classified in different methyltransferase groups due to their domain structures and target sequences CcrM transfers a methyl group to adenine embedded in 5¢-GANTC-3¢ sites and its homologues are considered essential for cell viability because of their involvement in the regulation of cell division, gene expression, and virulence [10,12,13] Robertson et al [14] showed that the replication ability of Brucella abortus, which infects many mammals, including humans, was slightly reduced when bacterial strains over-expressing CcrM were inoculated into murine macrophages The necessity for CcrM methylation in diverse bacteria suggests its importance in the regulation of gene expression However, the genomewide methylation status has not yet been elucidated due to the lack of an appropriate analysis tool We used in silico RLGS analysis to achieve genomewide monitoring of bacterial DNA methylation status, successfully demonstrated the existence of stably unmethlyated regions on several bacterial genomes, and demonstrated a dramatic change in methylation during plant–microbe interactions This approach may provide novel insights into a variety of symbiotic and parasitic relationships, including human diseases 952 Results In silico RLGS visualized the genomes efficiently We obtained RLGS patterns of A tumefaciens C58, B japonicum NBRC14792, and M loti MAFF303099 with several enzyme combinations The most important step in RLGS analysis is selecting landmark enzymes that produce well-focused and informative spot patterns We used AscI, BspEI, MluI, and NotI as landmark enzymes These four enzymes cleave specific GC-rich sequences and therefore gave satisfactory resolution of a sufficient number of spots because the genomes of all three bacterial strains have high GC content (data not shown) For example, approximately 1071, 979, and 1025 spots were visualized in the RLGS analysis of M loti MAFF303099 with AscI, BspEI, and NotI as landmark enzymes, respectively, in combination with MboI as a second dimension fragmentation enzyme (Fig 1A and data not shown) The reproducibility of spots was confirmed with at least triplicate analyses The RLGS patterns obtained experimentally (real RLGS patterns) and those simulated by computer analysis of whole-genome sequences (virtual RLGS patterns) matched almost perfectly (Fig 1) The validity of spot assignments was confirmed by PCR and sequencing with eluted spot DNA and its assigned sequence-specific primers (data not shown) If genome changes such as length mutations, point mutations at the restriction enzyme recognition sequences, and a change of methylation status occurred, the spots corresponding to the changed region would be in different locations or absent on the real RLGS pattern compared to the virtual RLGS pattern Thus, we can obtain their sequences by comparing the real and virtual RLGS patterns, without cloning The ability of in silico RLGS analysis to comprehensively visualize genome changes was examined (Fig and Table 1) We found that the spots located between 500 and 15 000 bp in the first dimension and between 100 and 1000 bp in the second dimension always showed sufficient resolution and reproducibility with every enzyme combination used (data not shown) The first dimension coverage of M loti MAFF303099 with the enzyme combinations of AscI–MboI, BspEI–MboI, and NotI–MboI was 42.3, 61.8, and 49.2%, respectively Overall, the first dimension coverage of the three enzyme combinations without duplication was 87.3% This result clearly demonstrates that RLGS analysis combined with in silico profiling enables efficient and high density scanning for mutations over the entire genome with good resolution FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS H Ichida et al Genomewide analysis of DNA adenine methylation Fig Comparison between real and virtual restriction landmark genome scanning (RLGS) patterns of M loti MAFF303099 (A) Real RLGS pattern of M loti MAFF303099 with NotI as the landmark enzyme and MboI as the second dimension fragmentation enzyme There are  1071 informative spots in the pattern (B) Virtual RLGS pattern calculated based on the whole-genome sequence The numbers to the right of the spots correspond to the sequence numbers listed separately The supplemental material contains a high-resolution version of the real and virtual RLGS patterns and the sequence list (C) Spot sequence identification by in silico RLGS profiling Boxed regions in (A) and (B) were compared, and the information was merged onto the real RLGS pattern Using this profile, the sequence of the mutated spots can be obtained immediately The colors of spots (B) and numbers (C) indicate their replication origin (blue, main chromosome; red, pMLa; green, pMLb) Adenine methylation status in M loti MAFF303099 We scanned for the adenine methylation status of M loti MAFF303099 using in silico RLGS profiling The a-proteobacteria, including M loti MAFF303099, are thought to have CcrM, which transfers a methyl group from S-adenosylmethionine to the amino group of the adenine moiety embedded in the sequence 5¢-GANTC-3¢ The restriction endonuclease HinfI cleaves unmethylated GANTC sites, but not methylated sites The cleavage by the landmark enzymes (AscI, MluI, and NotI) is not affected by CcrM methylation because of the lack of GANTC sequences on their recognition sequences; therefore, the spot intensity directly reflected the CcrM methylation status at the HinfI site of the corresponding genome region The deduced total coverage with these three enzyme combinations was 91.0% (Table 1) The real and virtual RLGS patterns of M loti MAFF303099 obtained with NotI–HinfI are shown in Fig Similar results were obtained with AscI–HinfI and MluI–HinfI (data not shown) Most of spots on the real RLGS patterns clustered on the top (Fig 3A; arrowhead); this feature was never observed with MboI Clustered spots were formed when the fragment FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS 953 Genomewide analysis of DNA adenine methylation H Ichida et al Fig Map of visualized regions on the RLGS patterns and specifically unmethylated regions (SUMs) of M loti MAFF303099 The innermost yellow line indicates the average GC content of the corresponding region The window size is 10 kb and is plotted for each 1-kb shift The white lines on the next three concentric circles indicate the positions of the landmark enzyme recognition sites, and the green lines indicate visualized genome regions in the RLGS pattern The enzyme combinations are AscI–MboI, BspEI–MboI, and NotI–MboI, from inner to outer, respectively The magenta lines indicate the genome regions visualized with at least one enzyme combination The blue bars indicate SUMs identified by adapter-mediated PCR The large divisions on the scale indicate Mb Table RLGS coverage (%) in the three bacteria 1D, Percentage of genome regions that visualize in first dimensional (agarose gel) electrophoresis 2D, Percentage of genome regions that visualize in second dimensional (polyacrylamide gel) electrophoresis Agrobacterium tumefaciens C58 Circular chr Agrobacterium tumefaciens C58 Linear chr Bradyrhizobium japonicum USDA110 Mesorhizobium loti MAFF303099 Enzyme combination and dimension 1D 2D 1D 2D 1D 2D 1D 2D AscI-Mbol BspEl-Mbol Notl-Mbol Total coverage Ascl-Hinfl Mlul-Hinfl Notl-Hinfl Total coverage 27.4 70.0 34.2 85.9 26.9 51.3 36.5 77.0 1.3 5.8 2.0 8.7 2.7 6.5 4.1 12.5 23.8 68.2 41.8 86.7 21.6 48.9 42.6 80.0 1.0 5.8 2.2 8.6 2.0 6.2 4.5 12.3 39.3 56.4 48.8 86.1 58.5 68.5 70.8 95.6 2.0 4.4 3.2 8.9 7.8 9.2 12.1 20.1 42.4 61.8 49.2 87.3 52.6 55.9 61.0 91.0 2.4 4.5 3.2 9.5 7.2 7.0 8.9 21.0 Fig Real and virtual RLGS patterns obtained using NotI–HinfI (A) Real RLGS pattern of M loti MAFF303099 The image was obtained using NotI as the landmark enzyme and HinfI as the second dimension fragmentation enzyme Although most of the spots are located on the top left (short in the first dimension and long in the second dimension), 104 apparent spots are visualized below the cluster (B) Virtual RLGS pattern calculated based on the whole-genome sequence and conditions corresponding to those in (A) Unlike in the real RLGS pattern, these spots are randomly dispersed, suggesting that specific genome regions of M loti MAFF303099 are unmethylated and that methylation status is stably heritable (C, D) Real (C) and virtual (D) RLGS patterns of P syringae DC3000 This strain does not have CcrM homologues; therefore, most of the spots are randomly dispersed on the image and are well matched with the virtual RLGS pattern (E, F) Real RLGS patterns of free-living M loti MAFF303099 (E) and bacteroids (F) obtained using AscI–HinfI Although these patterns were obtained under the same procedural conditions, the signal intensity of the spots that were located outside of the clustered region was slightly reduced in the bacteroids (G, H) Real RLGS patterns of B japonicum NBRC14792 (G) and A tumefaciens C58 (H) obtained using NotI–HinfI As observed in (A), most of the spots are located on the top, but some apparent spots appear below the cluster These results demonstrate the generality of SUMs in a variety of bacteria 954 FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS H Ichida et al Genomewide analysis of DNA adenine methylation A B C D E F G H FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS 955 Genomewide analysis of DNA adenine methylation H Ichida et al lengths were the same in the first and second dimensions To clarify whether the formation of clustered spots was due to insufficient cleavage activity of HinfI, we obtained real and virtual RLGS images of Pseudomonas syringae pv tomato DC3000 DNA, which is classified as a c-proteobacteria and does not have CcrM homologues The real and virtual images of P syringae pv tomato DC3000 with MluI–HinfI matched almost perfectly (Fig 3C,D) These results clearly showed that most of the GANTC sites in the M loti MAFF303099 genome were strictly methylated, and cleavage by HinfI was blocked due to methylation on adenine and ⁄ or cytosine residues on the GANTC sites Bisulfite sequencing, which is a widely used technique to determine cytosine methylation levels in base pair resolution, demonstrated that the cytosine nucleotides, including HinfI sites, were completely unmethylated (data not shown) Therefore, the blocking of HinfI cleavage was caused by DNA adenine methylation at GANTC sites Interestingly, 82, 94, and 104 spots were observed in the correct second dimension position in the images obtained with AscI–HinfI, MluI–HinfI, and NotI–HinfI, respectively (Fig 3A,B, and data not shown) These spots suggest that the M loti MAFF303099 genome was partly unmethylated, and the methylation status had been inherited stably We refer to these regions as specifically unmethylated regions (SUMs) Comprehensive catalog of specifically unmethylated regions Real and virtual RLGS images obtained with HinfI clearly demonstrated the occurrence of SUMs; however, their nucleotide sequences could not be obtained by in silico RLGS profiling because most of the spots were methylated and the number of informative landmark spots was insufficient for matching the real and virtual RLGS patterns There were 104 spots on the real NotI–HinfI RLGS pattern and its coverage was 61.0% (Fig 3A and Table 1) Therefore, we estimated that there were  170 nonredundant SUMs in the free-living M loti MAFF303099 genome Adapter-mediated PCR was used to amplify the SUMs in the genome Approximately 30 major bands were observed on 5% polyacrylamide gels when NotI and HinfI adapter-mediated amplification were performed (data not shown) We obtained 339 clones amplified with AscI, MluI, or NotI and HinfI Sequence analysis showed that these clones originated from 145 individual genome regions (Table S2) The number of times a sequence appeared in the 339 clones ranged from to 15; this reflects, at least partly, the 956 levels of unmethylation in the genome The distribution of SUMs on the main chromosome (50 sequences in total) is shown in Fig (blue bars) Fourteen of the SUMs (28%) were located in the symbiosis island, which is the best-characterized extraneous region on rhizobium genomes The average GC content of the 50 sequences was 56.8%, which is much lower than that of the main chromosome (62.7%) The overall average GC content of the 145 individual SUM sequences was also lower (57.3%) than that of the main chromosome Although the two plasmids, designated pMLa and pMLb, have a lower GC content than the main chromosome, the GC content of the SUMs was lower These results suggest that the formation of SUMs may be correlated with GC content Adenine methylation changes during nodule development To investigate the biological significance of SUMs, we monitored the change in adenine methylation status before and after establishing nodules (Fig 3E,F) Nodules consist of a mixture of plant and bacterial cells; therefore, uninfected plant root DNA was used as a negative control for the nodule pattern (data not shown) Comparisons between free-living M loti MAFF303099 and bacteroids with AscI–HinfI, MluI– HinfI, and NotI–HinfI revealed that the signal intensities derived from SUMs were decreased distinctly in bacteroids (Fig 3E,F, and data not shown; arrowheads indicate some examples of spots that disappeared); these patterns were obtained under the same conditions, i.e., the amount of DNA, reagent lots, electrophoresis, autoradiography, and film development and digitization These results suggest that the bacterial DNA adenine methylation status changes during the establishment of the symbiotic relationship and may contribute to the regulation of plant–microbe interactions To confirm the change in methylation during nodule development, DNA from free-living bacteria and nodules was probed with the cloned SUMs described above (Table 2) Although we collected 145 individual SUMs, fragments less than 200 bp in length did not provide sufficient sensitivity Therefore, the 29 SUMs that were longer than 200 bp in length and appeared twice or more were chosen as probes Of these, 27 (93.1%) gave significant signals in the NotI–HinfI digestion, but not in the NotI digestion The signal intensity of 20 loci was decreased in nodules; therefore, these loci are methylated during nodule development (Fig and Table 2) The other nine loci were unmethylated in both nodules and free-living bacteria FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS H Ichida et al Genomewide analysis of DNA adenine methylation Table DNA methylation levels at the SUMs in free-living and bacteroids, determined by DNA gel blot analysis The unmethylated HinfI is underlined Unmethylated band intensity was expressed as – (unmethylated signal not detected) and + to + + + + + (weakest to strongest signal) Asterisks indicate two or more unmethylated bands were detected Corresponding genome region Identifier ARM-AH1-A01 ARM-AH1-A05 ARM-AH1-B07b ARM-AH1-B09b ARM-AH1-C07b ARM-AH1-D02 ARM-AH1-G03 ARM-MH1-B05c ARM-MH2-A05 ARM-MH2-A12 ARM-MH2-B02 ARM-MH2-B04 ARM-NH1-A08 ARM-NH1-A11 ARM-NH1-B09 ARM-NH1-B10 ARM-NH1-C09 ARM-NH1-D07 ARM-NH1-F05 ARM-NH1-H12 ARM-NH2-B02 ARM-NH2-D07 ARM-MH1-D03 ARM-MH1-D05 ARM-NH1-A10 ARM-NH1-E12b ARM-NH1-F08 ARM-AH1-A06 ARM-NH1-B05b ARM-NH1-F06 a Length (bp) 747 310 600 315 613 578 278 265 400 224 226 268 222 563 405 226 528 314 246 288 393 586 246 297 528 555 583 434 566 261 GC (%) 52.0 63.6 54.5 53.1 55.8 63.4 59.9 50.4 63.3 60.4 59.4 55.6 64.9 60.1 53.1 54.5 61.6 60.4 54.9 51.2 53.0 57.1 55.3 54.6 55.3 57.6 58.1 57.6 56.2 58.1 Located in symbiosis island b Replicon Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr pMLa pMLa pMLa pMLa pMLa pMLb pMLb pMLb Unmethylated band intensity Position HinfI flanking sequence a 832 908–4 833 663 203 593–6 203 911 243 365–5 243 973a 028 568–5 028 891a 575 187–6 575 776 949 372–5 949 958 263 864–3 264 150 514 690–6 514 955 201 209–3 201 614 568 019–3 568 443 263 864–3 264 093 833 387–4 833 663a 854 311–4 854 541a 473 862–6 474 433 515 191–1 515 604 597 327–2 597 561 735 980–6 736 516 011 098–5 011 420a 720 571–3 720 825 916 344–4 916 637a 334 423–5 334 780 443 356–5 443 950 153 845–154 098 56 482–56 786 105 514–106 050 310 685–311 238 154 241–154 803 113 426–113 868 184 911–185 482 46 255–46 524 Contained repeat core unit of nod box These results clearly demonstrate that the CcrM methylation status changes during the establishment of plant–microbe interactions It is possible that the difference in signal intensity between free-living cells and bacteroids is attributable to changes in the number of plasmid copies DNA gel blot and real-time PCR analysis demonstrates that the number of copies of the pMLa and pMLb plasmids did not differ between before and after establishing symbiosis (Table S3) Therefore, the reduction in SUMs in nodules originated from a change in the DNA methylation state during the establishment of symbiosis c CCATTTCA TTTCGCGG GCCAAGAA CAAATTGC ACAGGTTC GGTTAGGG CCATTTGT TTAATCAT TCGTTAAC TAGGTGAC CCATTTGT TGTCGACG ACCCAGTC TCTTAATT CTGAAAAG TTCTCCAA ATATAGTT AGAGAATA AGGACCAT ATAATGAA TCAGTCAT CGTCGAGC GTCCCCTA TTCCCGGA TTTATATC TACGTTTT GCTTGTAG AATTCCCT GTCTGTTC TTGATTAA GAGTC GATTC GATTC GACTC GAATC GACTC GAATC GATTC GAATC GATTC GAATC GAATC GAGTC GAGTC GATTC GATTC GACTC GAGTC GATTC GAGTC GAGTC GATTC GATTC GAATC GAGTC GAGTC GATTC GACTC GAGTC GATTC Free-living GATGGGAC TATGGTGA TGTGGTCG AGGACGTT CTGTCGGG GAAGGAGA TATTTCAC GCTGGGCA ATTAACCA AGCCTTGT TATTTCAC GAGCATAT CGGGCGGA AAAAAAAT CGGACTCT AAATCTCC CGTCAAGT GTGTATTA GGACTTGG GTTCATCC ACTCCGAA CCAAGTTT CACTTTAT GTCAAATT TGTTACGG TGCAACAT ACTTCAAA CCGTCGAA AGCAAACC CTAATTTA Nodule + +++ ++++ + +++ + ++++ +++++ +++ + + +, * + + + +, * +, * ++++ +++++ + +, * + + + +, * +++ + +, * + +, * + +, * ++++ +++ ++ +++++ +++++ + + + + +, * + + + + +, * ++++ + + + +, * + – +++ – +++ + +++ +++ +++ – +, * +, * – +++ + – + + + +, * – + +, * + + +, * ++ + ++ +++ + +, * + +, * + +, * + Contained CtrA binding motif and virtual RLGS patterns of B japonicum NBRC14792 (genetically equivalent to USDA110) and A tumefaciens C58, which establish symbiotic and parasitic relationships with plants, respectively As in M loti MAFF303099, the spots were randomly dispersed in the virtual RLGS patterns; however, most of the spots were localized on the top, and only 67 and 32 spots were detected at the predicted positions in B japonicum NBRC14792 (Fig 3G) and A tumefaciens C58 (Fig 3H), respectively, with the NotI–HinfI combination These results suggest that SUMs are widely distributed in a variety of bacteria and may play a significant role in regulatory mechanisms SUMs are ubiquitous in a variety of plant-associated bacteria Discussion To investigate whether the formation of SUMs is a common phenomenon in bacteria, we obtained real We showed that in silico RLGS profiling, which is based on a comparison between real RLGS patterns FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS 957 Genomewide analysis of DNA adenine methylation A H Ichida et al B Fig DNA methylation status before and after establishing symbiosis (A) Hybridization pattern of ARM-AH1-B07 The ratio of methylated and unmethylated signals differed before (free-living bacteria) and after (nodule) the symbiotic relationship was established Lane 1, freeliving M loti MAFFF303099 DNA digested with NotI and HinfI; lane 2, nodule DNA digested with NotI and HinfI; lane 3, free-living M loti MAFFF303099 DNA digested with NotI; lane 4, nodule DNA digested with NotI (B) Signal quantification results Methylated and unmethylated signal intensities were normalized by NotI-derived signals (lanes and 4) The data were calculated from three individual hybridization experiments obtained experimentally and virtual RLGS patterns obtained by computer simulation of whole-genome sequences, is an effective method for monitoring genomic dynamism in bacteria (Fig and Table 1) The average first dimension coverage of the main chromosome using three enzyme combinations was 86.5% for the three bacterial strains examined These results clearly demonstrate that in silico RLGS profiling can provide rapid and accurate scanning for genomic changes in bacteria In silico RLGS analysis was used to monitor genomewide CcrM methylation during plant–microbe interactions CcrM, which is a member of the b group of methyltransferases, was originally discovered as a cell-cycle-regulated methyltransferase [10] and is essential for cell viability in a variety of a-proteobacteria [15] It is a global regulator of gene expression, in which the transcription of the ccrM gene itself is inhibited by CcrM methylation [13] Over-expression of CcrM decreases the replication ability of B abortus [14]; thus, this methylase might contribute to the regulation of host–parasite interactions We demonstrated that most of the GANTC sites, which correspond to CcrM target sequences, are usually methylated in the M loti MAFF303099, B japonicum NBRC14792, and A tumefaciens C58 genomes (Fig 3) However, some GANTC sites in these genomes are specifically unmethylated, and the methylation status is heritable 958 We obtained 145 nonredundant SUMs from 339 individual clones using adapter-mediated PCR (Table S2), which may comprise  85% of all SUMs in the genome of free-living M loti MAFF303099 Sequencing and mapping results suggest that the SUMs are localized in low-GC regions on the genome, and their average GC content was much lower than those of the main chromosome and two plasmids (Fig and Table 2) Horizontal gene transfer is thought to be a major force in genome plasticity and may play a crucial role in evolution [16] Historically, fitness-enhancing traits such as antibiotic resistance, virulence, organic solvent degradability, and symbiotic nitrogen fixation ability were transmitted by this mechanism Now, many gene candidates transferred among prokaryotes and from prokaryotes to eukaryotes are identified via comparative genomic analysis [17] The relationship between SUMs and the GC content indicates an association with genome evolution Why and how are some genome regions specifically unmethylated? The pioneering work conducted with Dam of Escherichia coli hints at the answers to these questions Dam belongs to the a group of methyltransferases and transfers a methyl group to the N6 position of the adenine in 5¢-GATC-3¢ sites Tavazoie and Church [18] found that the Escherichia coli chromosome contains 23 stably unmethylated GATC sites and that all of them are located in the 5Â noncoding FEBS Journal 274 (2007) 951962 ê 2007 The Authors Journal compilation ª 2007 FEBS H Ichida et al region of putative open reading frames They also described several independent lines of evidence supporting protein binding at these sites [18] Therefore, competition between methyltransferase and these proteins may be the mechanism behind stable unmethylation In this manner, SUMs in a-proteobacteria may be formed by competition between DNA-binding proteins and CcrM Our preliminary search found two protein-binding motifs in the cloned SUM sequences The binding motif for CtrA, a global response regulator (TTAAN7-TTAA [19]), was identified in the SUM clone ARM-MH1-B05 The motif was located bp upstream of the unmethylated HinfI site The unmethylated HinfI site of ARM-MH1-B05 was located between positions 514 955 and 514 959 on the main chromosome This region is 149 and 298 bp upstream of mll7872 (position 514 807–6 513 713; encodes an unknown protein) and mlr7873 (position 515 256– 517 130; encodes a cellulose synthase-like protein), respectively The other motif was a repeat core unit of nod box (ATC-N9-GAT [20]), which is the binding site of NodD, a LysR-type transcriptional regulator that directs specific flavonoid-dependent nodulation gene expression [21] Of the 145 nonredundant SUM sequences, 16 contained this core motif and seven of these were located on the symbiosis island (Table and S2) The number of nucleotides between the nod repeat core unit and the unmethylated HinfI site varied from to 681, and averaged 283 Although most NodD proteins bind to the promoter when specific flavonoids are present, some are activated independently of flavonoids and have greater transcriptional activity than the flavonoid-dependent proteins [22] In addition, the flavonoid-dependent NodD proteins also exhibit relatively weaker, but detectable, DNA-binding activity in the absence of inducers [23] Therefore, at least some type of NodD protein can competitively inhibit CcrM methylation, even in the free-living condition It is likely that SUMs are formed by competition between CcrM and DNA-binding proteins Biochemical analysis with purified CcrM and various DNA-binding proteins will be a key for further analysis This is the first report of specific unmethylation of GANTC sites and of methylation status changes in response to environmental conditions We also demonstrated that in silico RLGS analysis is an effective methodology for bacterial genomes We used it to visualize the dramatic change in DNA adenine methylation, but it is also applicable for genomewide scanning of insertions and deletions The dramatic change in the CcrM methylation state may reflect the cell status, particularly for protein–DNA interactions Although we Genomewide analysis of DNA adenine methylation focused on plant–microbe symbiotic interactions, parasitic interactions between plants or animals and microbes are also important for further studies DNA adenine methylation may provide novel insights into the regulation of bacterial gene expression Experimental procedures Bacterial strains, growth conditions, and plant inoculation Mesorhizobium loti MAFF303099 was obtained from the NIAS GenBank (Ibaraki, Japan) Bradyrhizobium japonicum NBRC14792 (genetically equivalent to USDA110) was obtained from the NITE Biological Resource Center (Chiba, Japan) Agrobacterium tumefaciens C58 was from our laboratory stock M loti and B japonicum were cultured in YEM medium (0.5 g dipotassium hydrogenphosphate, 0.2 g magnesium sulfate, 0.1 g sodium chloride, g mannitol, g sodium gluconate, and 0.5 g yeast extract per litre, pH 6.9) at 30 °C [24] A tumefaciens was cultured in YEP medium (10 g yeast extract, 10 g peptone, and g sodium chloride per litre, pH 7.0) at 28 °C Genomic DNA of Pseudomonas syringae pv tomato DC3000 was purchased from ATCC via an official local distributor (Summit Pharmaceuticals International, Tokyo, Japan) Lotus japonicus MG-20 seeds were a gift from the National Bio Resource Project (Miyazaki University, Miyazaki, Japan) Surface-sterilized L japonicus MG-20 seeds were germinated on B & D nitrogen-free plates [25] under a photoperiod of 16 h light ⁄ h dark at 22 °C A log-phase culture of M loti MAFF303099 (optical density at 600 nm, 0.4–0.6) was washed three times with sterilized distilled water L japonicus MG-20 seedlings with roots 15– 20 mm long were soaked in the washed bacterial cell suspension for The inoculated plants were placed on new B & D nitrogen-free plates and grown for 45 days Using this method, one to three nodules usually developed on each plant The nodules on green plants with elongated shoots were harvested as nitrogen-fixing nodules and used for the following experiments DNA protocols General molecular manipulations were carried out according to standard procedures, unless otherwise specified Bacterial DNA was extracted from fresh log-phase cultures (optical density at 600 nm, 0.4–0.6) using the cetyltrimethylammonium bromide procedure [26] Nodule DNA and plant root DNA were extracted from 100 mg of tissue using a Nucleon PhytoPure DNA extraction kit according to the manufacturer’s instructions (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) All oligonucleotide sequences used are listed in Table S1 FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS 959 Genomewide analysis of DNA adenine methylation H Ichida et al RLGS was performed according to published protocols with minor modifications [27] Briefly, DNA was digested with a cohesive end-producing (landmark) enzyme Sequenase version 2.0 (USB, Cleveland, OH, USA) was used to fill in the cohesive ends with [32P]dGTP[aP] and [32P]dCTP[aP] (GE Healthcare Bio-Sciences) by incubating for 30 at 37 °C The labeled DNA was separated by electrophoresis through a 60 cm, 0.8% agarose tube gel (first dimension separation) The agarose tube gel was treated with a second enzyme at 37 °C for h Second dimension separation was performed using nondenaturing 4% (w ⁄ v) polyacrylamide gels After overnight electrophoresis, the gels were dried and exposed to X-ray film in the presence of intensifying screens (Hi-SCREEN B-2, Fuji Film Medical, Tokyo, Japan) for 12–48 h All developed films were digitized using a laser film digitizer (Model 2905; Array Corp., Tokyo, Japan) Comparisons between two or more patterns were made using pdquest basic version 8.0 (Bio-Rad Laboratories, Tokyo, Japan) The reproducibility of real RLGS images was confirmed by at least three individual experiments The restriction enzyme combinations for each RLGS pattern are specified in the Results and figure legends In silico identification of the visualized spots was performed using a modified version of vi-rlgs software [2] with the whole-genome sequences of A tumefaciens C58, B japonicum USDA110, and M loti MAFF303099 reported previously [28–30] ‘Coverage’ was defined as the percentage of the total length of visualized spots in the RLGS images relative to the genome size For example, ‘first dimension coverage’ of an RLGS image of M loti MAFF303099 was calculated as (sum of first dimension length on the image ⁄ size of the main chromosome: 036 071) · 100 The first and second dimension coverages were calculated computationally from the in silico simulation of RLGS reactions using the genome sequences The spots located between 500 and 15 000 bp in the first dimension and 100 and 1000 bp in the second dimension were counted as ‘visualized’ spots Genome regions visualized with two or more combinations of enzymes were counted only once when calculating the total coverage from plural RLGS images [100 ng each of landmark- and HinfI-adapter and lL of Ligation High solution (Toyobo, Tokyo, Japan) in 8.5 lL] The mixture was incubated at 16 °C for 16 h and subjected to PCR amplification without purification PCR was performed in a 50-lL reaction volume containing lL of 10· PCR buffer (Takara, Tokyo, Japan), lL of 2.5 mm each dNTP mixture, 10 pmol each of CasA- and CasBspecific primers, lL of ligation solution, and 1.25 U of Taq DNA polymerase (Takara) Amplification was performed with 30 cycles of 94 °C for 30 s, 55 °C for 60 s, and 72 °C for 90 s Successful amplification was confirmed by separation of the products in a 7% nondenaturing polyacrylamide gel with appropriate size markers, and the PCR products were cloned using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) The insert of each clone was sequenced using a BigDye Terminator version 3.1 cycle sequencing kit and a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) at the Research Resource Center, RIKEN-BSI Base identification, assembly, and mapping to whole-genome sequences were performed automatically by an in-house integrated analysis environment based on phred ⁄ phrap [31] and A ⁄ G BLAST 2.2.10 (Apple Computer, Cupertino, CA, USA) All DNA sequences were submitted to the DNA Data Bank of Japan (DDBJ), with accession numbers AB264801 to AB265139 DNA methylation levels at the cloned putative SUMs were determined by DNA gel blot analysis Nodule DNA (5 lg) was digested overnight with or without 10 U of HinfI in a 30-lL reaction volume, which consisted of 50 mm Tris ⁄ HCl (pH 7.5), 10 mm magnesium chloride, mm dithiothreitol, 100 mm sodium chloride, 0.01% bovine serum albumin, 0.01% Triton X-100, and 10 U of NotI (Takara) The digested DNA was separated in 1% (w ⁄ v) agarose gels and transferred to Hybond N+ membranes (GE Healthcare Bio-Sciences) Probes were prepared by PCR amplification of the insert regions of each clone using the primers CasA-specific and CasB-specific Labeling and detection were performed using an ECL direct nucleic acid labeling and detection system (GE Healthcare BioSciences) according to the manufacturer’s instructions Hybridization was performed overnight in the supplied hybridization buffer containing 0.1 m sodium chloride at 42 °C Methylation profiling Quantification of plasmid copy numbers SUMs of the M loti MAFF303099 genome were amplified using adapter-mediated PCR The adapter was synthesized as two individual oligonucleotides and annealed by boiling for min, followed by gradual cooling to room temperature (Table S1) One microgram of M loti MAFF303099 DNA was digested with 10 units (U) of a landmark enzyme (NotI, AscI, or MluI) and HinfI for h and recovered by ethanol precipitation The precipitated DNA was dissolved in lL of water and added into the ligation mixture Copy numbers of the two plasmids, pMLa and pMLb, of M loti MAFF303099 under free-living and bacteroid conditions were determined by real-time PCR Three primer pairs, which amplify evenly distributed regions on the target, were designed for each replicon The reaction mixture consisted of 25 lL of SYBR premix Ex Taq (Takara), 10 pmol of primers, and 10 lL of diluted template DNA in 50 lL Amplification and real-time quantification were performed with 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and Restriction landmark genome scanning 960 FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS H Ichida et al 72 °C for 20 s using an ABI 7900HT (Applied Biosystems) Specific amplification of the targets was confirmed by melting curve analysis and 10% (w ⁄ v) polyacrylamide gel electrophoresis Acknowledgements The authors thank Sumie Ohbu (Nishina Center for Accelerator-Based Science, RIKEN) for her technical assistance H.I was supported by the Junior Research Associate Program of RIKEN This work was partially supported by a research grant for the study on genesis of matter from Ministry of Education, Culture, Sports, Science and Technology of Japan References Akama TO, Okazaki Y, Ito M, Okuizumi H, Konno H, Muramatsu M, Plass C, Held WA & Hayashizaki Y (1997) Restriction landmark genomic scanning (RLGSM)-based genome-wide scanning of mouse liver tumors for alterations in DNA methylation status Cancer Res 57, 3294–3299 Matsuyama T, Kimura MT, Koike K, Abe T, Nakano T, Asami T, Ebisuzaki T, Held WA, Yoshida S & Nagase H (2003) Global methylation screening in the Arabidopsis thaliana and Mus musculus genome: applications of virtual image restriction landmark genomic scanning (Vi-RLGS) Nucleic Acids Res 31, 4490–4496 Bergersen FJ, Turner GL, Gault RR, Chase DL & Brockwell J (1985) The natural abundance of 15N in an irrigated soybean crop and its use for the calculation of nitrogen fixation Aust J Agric Res 36, 411–423 DeCleene M & DeLay J (1976) The host range of crown gall Bot Gaz 42, 389–466 Wion D & Casadesus J (2006) N6-methyl-adenine: an ´ epigenetic signal for DNA–protein interactions Nat Rev Microbiol 4, 183–192 Bird A (2002) DNA methylation patterns and epigenetic memory Genes Dev 16, 6–21 Jones PA & Baylin SB (2002) The fundamental role of epigenetic events in cancer Nat Rev Genet 3, 415–428 Riggs AD & Pfeifer GP (1992) X-chromosome inactivation and cell memory Trends Genet 8, 169–174 Swain JL, Stewart TA & Leder P (1987) Parental legacy determines methylation and expression of an autosomal transgene: a molecular mechanism for parental imprinting Cell 50, 719–727 10 Zweiger G, Marczynski G & Shapiro L (1994) A Caulobacter DNA methyltransferase that functions only in the predivisional cell J Mol Biol 235, 472–485 11 Ratel D, Ravanat J, Berger F & Wion D (2006) N6-methyladenine: the other methylated base of DNA Bio Essays 28, 309–315 Genomewide analysis of DNA adenine methylation 12 Low DA, Weyend NJ & Mahan MJ (2001) Roles of DNA adenine methylation in regulating bacterial gene expression and virulence Infect Immun 69, 7197–7204 13 Stephens CM, Zweiger G & Shapiro L (1995) Coordinate cell cycle control of a Caulobacter DNA methyltransferase and the flagellar genetic hierarchy J Bacteriol 177, 1662–1669 14 Robertson GT, Reisenauer A, Wright R, Jensen RB, Jensen A, Shapiro L & Roop RMI (2000) The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages J Bacteriol 182, 3482–3489 15 Reisenauer A, Kahng LS, McCollum S & Shapiro L (1999) Bacterial DNA methylation: a cell cycle regulator? J Bacteriol 181, 5135–5139 16 Schwarzenlander C & Averhoff B (2006) Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27 FEBS J 273, 4210–4218 17 de la Cruz F & Davies J (2000) Horizontal gene transfer and the origin of species: lessons from bacteria Trends Microbiol 8, 128–133 18 Tavazoie S & Church GM (1998) Quantitive wholegenome analysis of DNA–protein interactions by in vivo methylase protection in E coli Nat Biotechnol 16, 566– 571 19 Siam R & Marczynski GT (2000) Cell cycle regulator phosphorylation stimulates two distinct modes of binding at a chromosome replication origin EMBO J 19, 1138–1147 20 Goethals K, Montagu MV & Holsters M (1992) Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins Proc Natl Acad Sci USA 89, 1646–1650 21 Perret X, Staehelin C & Broughton WJ (2000) Molecular basis of symbiotic promiscuity Microbiol Mol Biol Rev 64, 180–201 22 Mulligan JT & Long SR (1989) A family of activator genes regulates expression of Rhizobium meliloti nodulation genes Genetics 122, 7–18 23 Yeh KC, Peck MC & Long SR (2002) Luteolin and GroESL modulate in vitro activity of NodD J Bacteriol 184, 525–530 24 Taurian T, Aguilar PM & Fabra A (2002) Characterization of nodulating peanut rhizobia isolated from native soil population in Cordoba, Argentina Symbiosis 33, 59–72 25 Broughton WJ & Dilworth MJ (1971) Control of leghemoglobin synthesis in snake beans Biochem J 125, 1075–1080 26 Wilson K (1994) Preparation of genomic DNA from bacteria In Current Protocols in Molecular Biology, 4th edn (Ausubel FM, Brent R, Kingston RE, Moore DD, FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS 961 Genomewide analysis of DNA adenine methylation 27 28 29 30 31 H Ichida et al Seidman JG, Smith J A & Strauhl K, eds), pp 2.4.1– 2.4.2, John Willy & Sons Inc, Hoboken, NJ Hatada I, Hayashizaki Y, Hirotsune S, Komatsubara H & Mukai T (1991) A genomic scanning method for higher organisms using restriction sites as landmarks Proc Natl Acad Sci USA 8, 9523–9527 Goodner B, Hinkle G, Gattung S, Miller N, Blanchard M, Qurollo B, Goldman BS, Cao Y, Askenazi M, Halling C et al (2001) Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58 Science 294, 2323–2328 Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S, Watanabe A, Idesawa K, Ishikawa A, Kawashima K et al (2000) Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti DNA Res 7, 331–338 Kaneko T, Nakamura Y, Sato S, Minamisawa K, Uchiumi T, Sasamoto S, Watanabe A, Idesawa K, Iriguchi M, Kawashima K et al (2002) Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110 DNA Res 9, 189– 197 Ewing B, Hillier L, Wendl MC & Green P (1998) Basecalling of automated sequencer traces using Phred I Accuracy Assessment Genome Res 8, 175–185 962 Supplementary material The following supplementary material is available online: Fig S1 Real RLGS pattern of M loti MAFF303099 with NotI as the landmark endzyme and MboI as the second-dimension fragmentation enzyme Fig S2 Virtual RLGS pattern of M loti MAFF303099 with NotI–MboI calculated based on the whole-genome sequence Table S1 Oligonucleotides used in this study Table S2 BLAST summary of the 339 stably unmethylated region sequences Table S3 Plasmid copy numbers of M loti MAFF 303099, quantified by real-time PCR This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS ... N6-methyladenine: the other methylated base of DNA Bio Essays 28, 309–315 Genomewide analysis of DNA adenine methylation 12 Low DA, Weyend NJ & Mahan MJ (2001) Roles of DNA adenine methylation. .. Genomewide analysis of DNA adenine methylation A H Ichida et al B Fig DNA methylation status before and after establishing symbiosis (A) Hybridization pattern of ARM-AH1-B07 The ratio of methylated... bacterial DNA adenine methylation status changes during the establishment of the symbiotic relationship and may contribute to the regulation of plant–microbe interactions To confirm the change in methylation