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

C5-methyl-cytosine, N4-methyl-cytosine, and N6-methyl-adenine are found Keywords cell cycle-regulated methyltransferase; DNA adenine methylation; in silico restriction landmark genome sc

establishment of symbiosis

Hiroyuki Ichida1,2, Tomoki Matsuyama3, Tomoko Abe2and Takato Koba1

1 Graduate School of Science and Technology, Chiba University, Matsudo, Japan

2 Accelerator Application Research Group, Nishina Center for Accelerator-Based Science, RIKEN, Hirosawa, Wako, Saitama, Japan

3 Cellular Biochemistry Laboratory, Discovery Research Institute, RIKEN, Hirosawa, Wako, Saitama, Japan

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

sequen-cing of entire genomes in various organisms, and the

resulting data allow a comprehensive analysis of

gen-ome 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 dicotyledo-nous 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

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 7

December 2006, accepted 11 December

2006)

doi:10.1111/j.1742-4658.2007.05643.x

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 bacter-oids 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 mainten-ance of symbiotic and parasitic relationships

Abbreviations

CcrM, cell cycle-regulated methyltransferase; Dam, deoxyadenosine methyltransferase; RLGS, restriction landmark genome scanning; SUM, specifically unmethylated region; Vi, virtual image.

in microbial genomes In bacteria, these methylated

bases are best known as important agents for

restric-tion-modification systems, which distinguish self and

nonself DNA to protect bacteria from invaders In this

system, the host DNA is methylated and only

unmeth-ylated DNA is digested by cognate restriction

endo-nucleases [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

can-cer [6,7] CpG methylation is also known to participate

in genomic imprinting and X-chromosome inactivation

[8,9]

Cell-cycle-regulated methyltransferase (CcrM),

ori-ginally cloned from Caulobacter crescentus [10], is

the best-characterized solitary methyltransferase that

is not associated with restriction-modification

sys-tems, 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

methyltrans-ferase groups due to their domain structures and

tar-get 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

mam-mals, including humans, was slightly reduced when

bac-terial strains over-expressing CcrM were inoculated

into murine macrophages The necessity for CcrM

methylation in diverse bacteria suggests its

import-ance 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

genome-wide 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

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 import-ant 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 combi-nation with MboI as a second dimension fragmenta-tion 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 ana-lysis of whole-genome sequences (virtual RLGS pat-terns) 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 cor-responding to the changed region would be in different locations or absent on the real RLGS pattern com-pared to the virtual RLGS pattern Thus, we can obtain their sequences by comparing the real and vir-tual RLGS patterns, without cloning

The ability of in silico RLGS analysis to comprehen-sively visualize genome changes was examined (Fig 2 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%, respect-ively 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

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

methyl-ated 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 3 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

Fig 1 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).

Fig 2 Map of visualized regions on the RLGS patterns and specifically unmethyl-ated 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 combina-tions are AscI–MboI, BspEI–MboI, and NotI–MboI, from inner to outer, respect-ively 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

1 Mb.

Table 1 RLGS coverage (%) in the three bacteria 1D, Percentage of genome regions that visualize in first dimensional (agarose gel) electro-phoresis 2D, Percentage of genome regions that visualize in second dimensional (polyacrylamide gel) electroelectro-phoresis.

Enzyme combination

and dimension

Agrobacterium tumefaciens C58 Circular chr.

Agrobacterium tumefaciens C58 Linear chr.

Bradyrhizobium japonicum USDA110

Mesorhizobium loti MAFF303099

Fig 3 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 pat-tern, 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.

A B

H G

lengths were the same in the first and second

dimen-sions To clarify whether the formation of clustered

spots was due to insufficient cleavage activity of HinfI,

we obtained real and virtual RLGS images of

Pseudo-monas 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 lotiMAFF303099 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

tech-nique to determine cytosine methylation levels in base

pair resolution, demonstrated that the cytosine

nucleo-tides, including HinfI sites, were completely

unmethyl-ated (data not shown) Therefore, the blocking of

HinfI cleavage was caused by DNA adenine

methyla-tion 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

unmethyl-ated, and the methylation status had been inherited

stably We refer to these regions as specifically

un-methylated regions (SUMs)

Comprehensive catalog of specifically

unmethylated regions

Real and virtual RLGS images obtained with HinfI

clearly demonstrated the occurrence of SUMs;

how-ever, their nucleotide sequences could not be obtained

by in silico RLGS profiling because most of the spots

were methylated and the number of informative

land-mark 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

per-formed (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 1 to 15; this reflects, at least partly, the

levels of unmethylation in the genome The distribu-tion of SUMs on the main chromosome (50 sequences

in total) is shown in Fig 2 (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 chro-mosome, 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) Nod-ules 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 intensi-ties derived from SUMs were decreased distinctly in bacteroids (Fig 3E,F, and data not shown; arrow-heads indicate some examples of spots that disap-peared); these patterns were obtained under the same conditions, i.e., the amount of DNA, reagent lots, elec-trophoresis, autoradiography, and film development and digitization These results suggest that the bacter-ial 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 nod-ules 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 4 and Table 2) The other nine loci were un-methylated in both nodules and free-living bacteria

These results clearly demonstrate that the CcrM

methylation status changes during the establishment of

plant–microbe interactions It is possible that the

dif-ference 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

ana-lysis 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

origin-ated from a change in the DNA methylation state

during the establishment of symbiosis

SUMs are ubiquitous in a variety of

plant-associated bacteria

To investigate whether the formation of SUMs is a

common phenomenon in bacteria, we obtained real

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 dis-persed 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 tumefac-iens 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

Discussion

We showed that in silico RLGS profiling, which is based on a comparison between real RLGS patterns

Table 2 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.

Identifier

Length

Corresponding genome region Unmethylated band intensity

a Located in symbiosis island b Contained repeat core unit of nod box c Contained CtrA binding motif.

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 3 and Table 1)

The average first dimension coverage of the main

chro-mosome 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

chan-ges 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

essen-tial 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

inhi-bited by CcrM methylation [13] Over-expression of

CcrM decreases the replication ability of B abortus

[14]; thus, this methylase might contribute to the

regu-lation 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

We obtained 145 nonredundant SUMs from 339 indi-vidual clones using adapter-mediated PCR (Table S2), which may comprise  85% of all SUMs in the gen-ome of free-living M loti MAFF303099 Sequencing and mapping results suggest that the SUMs are loca-lized in low-GC regions on the genome, and their aver-age GC content was much lower than those of the main chromosome and two plasmids (Fig 2 and Table 2) Horizontal gene transfer is thought to be a major force in genome plasticity and may play a cru-cial role in evolution [16] Historically, fitness-enhan-cing 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 pro-karyotes and from propro-karyotes to eupro-karyotes are iden-tified via comparative genomic analysis [17] The relationship between SUMs and the GC content indi-cates 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 methyl-transferases 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 chro-mosome contains 23 stably unmethylated GATC sites and that all of them are located in the 5¢ noncoding

Fig 4 DNA methylation status before and after establishing symbiosis (A) Hybridization pattern of ARM-AH1-B07 The ratio of methylated and unmethylated signals differed before (living bacteria) and after (nodule) the symbiotic relationship was established Lane 1, free-living M loti MAFFF303099 DNA digested with NotI and HinfI; lane 2, nodule DNA digested with NotI and HinfI; lane 3, free-free-living M loti MAFFF303099 DNA digested with NotI; lane 4, nodule DNA digested with NotI (B) Signal quantification results Methylated and

unmethylat-ed signal intensities were normalizunmethylat-ed by NotI-derivunmethylat-ed signals (lanes 3 and 4) The data were calculatunmethylat-ed from three individual hybridization experiments.

region of putative open reading frames They also

des-cribed several independent lines of evidence supporting

protein binding at these sites [18] Therefore,

competi-tion 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-bind-ing motifs in the cloned SUM sequences The bindprotein-bind-ing

motif for CtrA, a global response regulator

(TTAA-N7-TTAA [19]), was identified in the SUM clone

ARM-MH1-B05 The motif was located 5 bp upstream

of the unmethylated HinfI site The unmethylated

HinfI site of ARM-MH1-B05 was located between

positions 6 514 955 and 6 514 959 on the main

chro-mosome This region is 149 and 298 bp upstream of

mll7872 (position 6 514 807–6 513 713; encodes an

unknown protein) and mlr7873 (position 6 515 256–

6 517 130; 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 2

and S2) The number of nucleotides between the nod

repeat core unit and the unmethylated HinfI site varied

from 1 to 681, and averaged 283 Although most

NodD proteins bind to the promoter when specific

flavonoids are present, some are activated

independ-ently 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

competi-tively 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

demon-strated that in silico RLGS analysis is an effective

methodology for bacterial genomes We used it to

visu-alize 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,

par-ticularly for protein–DNA interactions Although we

focused on plant–microbe symbiotic interactions, para-sitic 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 japoni-cum 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 hydrogen-phosphate, 0.2 g magnesium sulfate, 0.1 g sodium chloride,

5 g mannitol, 5 g sodium gluconate, and 0.5 g yeast extract per litre, pH 6.9) at 30C [24] A tumefaciens was cultured

in YEP medium (10 g yeast extract, 10 g peptone, and 5 g sodium chloride per litre, pH 7.0) at 28C Genomic DNA

of Pseudomonas syringae pv tomato DC3000 was pur-chased 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⁄ 8 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 sus-pension for 1 min 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 accord-ing to standard procedures, unless otherwise specified Bac-terial DNA was extracted from fresh log-phase cultures (optical density at 600 nm, 0.4–0.6) using the cetyltrimethyl-ammonium 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

Restriction landmark genome scanning

RLGS was performed according to published protocols

with minor modifications [27] Briefly, DNA was digested

with a cohesive end-producing (landmark) enzyme

Sequen-ase 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 min at

37C 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 37C for 2 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

Laborator-ies, Tokyo, Japan) The reproducibility of real RLGS

ima-ges was confirmed by at least three individual experiments

The restriction enzyme combinations for each RLGS

pat-tern are specified in the Results and figure legends

In silico identification of the visualized spots was

per-formed 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

repor-ted 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:

7 036 071)· 100 The first and second dimension coverages

were calculated computationally from the in silico

simula-tion of RLGS reacsimula-tions using the genome sequences The

spots located between 500 and 15 000 bp in the first

dimen-sion and 100 and 1000 bp in the second dimendimen-sion 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

Methylation profiling

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 3 min, followed by gradual cooling to room

tempera-ture (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 3 h and recovered by

ethanol precipitation The precipitated DNA was dissolved

in 3 lL of water and added into the ligation mixture

[100 ng each of landmark- and HinfI-adapter and 5 lL of Ligation High solution (Toyobo, Tokyo, Japan) in 8.5 lL] The mixture was incubated at 16C for 16 h and subjected

to PCR amplification without purification PCR was per-formed in a 50-lL reaction volume containing 5 lL of 10· PCR buffer (Takara, Tokyo, Japan), 4 lL of 2.5 mm each dNTP mixture, 10 pmol each of CasA- and CasB-specific primers, 1 lL of ligation solution, and 1.25 U of Taq DNA polymerase (Takara) Amplification was per-formed with 30 cycles of 94C for 30 s, 55 C for 60 s, and

72C for 90 s Successful amplification was confirmed by separation of the products in a 7% nondenaturing poly-acrylamide 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 Bio-systems, Foster City, CA, USA) at the Research Resource Center, RIKEN-BSI Base identification, assembly, and mapping to whole-genome sequences were performed auto-matically 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,

1 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+ mem-branes (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 Bio-Sciences) according to the manufacturer’s instructions Hybridization was performed overnight in the supplied hybridization buffer containing 0.1 m sodium chloride at

42C

Quantification of plasmid copy numbers Copy numbers of the two plasmids, pMLa and pMLb, of

M loti MAFF303099 under free-living and bacteroid con-ditions were determined by real-time PCR Three primer pairs, which amplify evenly distributed regions on the tar-get, 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 per-formed with 40 cycles of 94C for 30 s, 60 C for 30 s, and