Microbes Environ. Vol. 26, No. 2, 172–180, 2011 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10203

Isolation and Genetic Characterization of Aurantimonas and Methylobacterium Strains from Stems of Hypernodulated Soybeans

MIZUE ANDA1, SEISHI IKEDA1,2, SHIMA EDA1, TAKASHI OKUBO1, SHUSEI SATO3, SATOSHI TABATA3, HISAYUKI MITSUI1, and KIWAMU MINAMISAWA1* 1Graduate School of Life Sciences, Tohoku University, 2–1–1 Katahira, Aoba-ku, Sendai 980–8577, Japan; 2Memuro Research Station, National Agricultural Research Center for Hokkaido Region, Shinsei, Memuro-cho, Kasaigun, Hokkaido 082–0081, Japan; and 3Kazusa DNA Research Institute, 2–6–7 Kazusa-kamatari, Kisarazu, Chiba 292–0818, Japan (Received November 30, 2010—Accepted March 24, 2011—Published online April 21, 2011)

The aims of this study were to isolate Aurantimonas and Methylobacterium strains that responded to soybean nodulation phenotypes and nitrogen fertilization rates in a previous culture-independent analysis (Ikeda et al. ISME J. 4:315–326, 2010). Two strategies were adopted for isolation from enriched bacterial cells prepared from stems of field-grown, hypernodulated soybeans: PCR-assisted isolation for Aurantimonas and selective cultivation for Methylobacterium. Thirteen of 768 isolates cultivated on Nutrient Agar medium were identified as Aurantimonas by colony PCR specific for Aurantimonas and 16S rRNA gene sequencing. Meanwhile, among 187 isolates on methanol- containing agar media, 126 were identified by 16S rRNA gene sequences as Methylobacterium. A clustering analysis (>99% identity) of the 16S rRNA gene sequences for the combined datasets of the present and previous studies revealed 4 and 8 operational taxonomic units (OTUs) for Aurantimonas and Methylobacterium, respectively, and showed the successful isolation of target bacteria for these two groups. ERIC- and BOX-PCR showed the genomic uniformity of the target isolates. In addition, phylogenetic analyses of Aurantimonas revealed a phyllosphere-specific cluster in the genus. The isolates obtained in the present study will be useful for revealing unknown legume-microbe interactions in relation to the autoregulation of nodulation. Key words: Aurantimonas, Methylobacterium, soybean, plant-associated bacteria, REP-PCR

Various microorganisms reside on plants (30, 57). idea is consistent with the fact that Aurantimonas and However, many questions about the driving forces and Methylobacterium are phylogenetically closely related to ecological rules underlying the relationships between these Bradyrhizobium (13). However, further investigations at microbes and their host plants remain unanswered (14, 42). the molecular, biochemical, or cellular level are required to Leguminous plants have established a mutualistic symbiosis obtain direct evidence to clarify the interactions between with rhizobia, and regulate the degree of nodulation of these bacterial groups and plants. their roots in response to the presence of certain symbiotic The recently established genus Aurantimonas consists of rhizobia (5, 48). This regulatory system, known as the auto- 4 species, which were isolated from diseased coral (8), a regulation of nodulation, involves long-distance signaling terrestrial cave wall (18), dust (56), and a water-cooling between the shoots and roots (28, 34). Nodulation is inhib- system (22). Although a few Aurantimonas species have ited through autoregulation by heavy supplies of nitrate been investigated as a manganese oxidizer and a coral to the roots of leguminous plants (5, 35). However, the pathogen (9, 41), common characteristics among this genus interactions between the autoregulation system(s) and non- are not known. With respect to the phyllosphere, only 2 rhizobial microbes in the phyllosphere remain unclear (38). strains of Aurantimonas sp. have been isolated from rice A recent culture-independent study demonstrated that plants by random isolation (29); the biology of this genus the community structures of stem-associated bacteria were in relation to plant symbiosis is largely unknown. An iso- significantly affected by nodulation phenotypes and nitro- lation strategy for Aurantimonas has not been established; gen fertilization levels (17). Among the communities, the rela- therefore, partial sequences of 16S rRNA genes are the only tive abundances of 2 operational taxonomic units (OTUs) information available for isolating Aurantimonas species. corresponding to Aurantimonas and Methylobacterium The genus Methylobacterium is known for its pink- were especially sensitive to nodulation phenotype and de- pigmented facultative methylotrophs (PPFMs); it is widely creased under heavy levels of N fertilization. These results distributed in soils, air, aquatic environments, and the phyllo- suggest that stem-associated bacteria, especially subpopula- sphere (6, 12). Methylobacterium species are isolated on tions of Alphaproteobacteria such as Aurantimonas and a selective medium containing methanol as the sole carbon Methylobacterium, are tightly controlled in a manner simi- source, with pink-pigmented colonies (39). lar to that of rhizobia through the autoregulation system Culture-independent molecular techniques have revealed and the nitrogen signaling pathway in plants (16, 17). This the large diversity of microbial communities and the roles they play in the environment (10, 20, 46). Although the * Corresponding author. E-mail: [email protected]; massive datasets have revealed novel uncultured phyla (59) Tel: +81–22–217–5684; Fax: +81–22–217–5684. and key microbes responding to environmental changes (17), Bacterial Isolation from Soybean Stem 173 it is crucial to isolate these microorganisms to better understand their real roles in the environment (20, 33, 49). The aims of the present study were to isolate and characterize the genetic variability of the Aurantimonas and Methylobacterium strains that responded to soybean nodu- lation phenotypes and nitrogen fertilization rates in previ- ous culture-independent analyses (17). Two strategies were employed for the isolations: PCR-assisted isolation with a specific primer set for Aurantimonas species and selective cultivation for Methylobacterium species. The target bac- terial groups were successfully isolated.

Materials and Methods Plant Materials Soybean seeds (hypernodulating mutant derived from cultivar Enrei, Glycine max. (L.) Merr. cv. Sakukei 4 (31)) were planted on 3 June 2009 in an experimental field at Tohoku University (Kashimadai, Miyagi, Japan). The plants were harvested on 11 August 2009, and immediately transported on ice to the laboratory. The leaves were removed manually and discarded, and the stems were washed well with tap water and stored at 4°C until homo- genization. To prepare stem-associated bacterial cells that included both epiphytes and endophytes, a composite sample of stems (approximately 20 g) was homogenized without surface steriliza- tion. The bacterial cells were then extracted and purified by using an enrichment method (15). The bacterial cell fraction was stored as glycerol stock at −80°C. Primer design and isolation of Aurantimonas species All 16S rRNA sequence data used for primer design are listed in Table S1. These consist of Aurantimonas species and reference sequences of 61 OTUs from Sakukei 4 grown under standard N Fig. 1. Outline of isolation strategies for Aurantimonas spp. and conditions reported by Ikeda et al. (17). The sequences of the 16S Methylobacterium spp. Media names are abbreviated as follows; Nutri- rRNA gene (corresponding to position 109–665 bp of Escherichia ent Agar (NA) medium, Minimal Medium (MM) agar (3), and Ammo- coli 16S rRNA gene) were aligned by CLUSTAL W (51). Sites nium Mineral Salts (AMS) agar (58). Culture plates used for the first specific to Aurantimonas species were searched, but only a region isolation step are shown in Fig. S1. corresponding to position 297 bp of the E. coli 16S rRNA gene which is common to Aurantimonas species and a Fulvimarina species (Accession No. AY371414) was found (data not shown). stored at 4°C until screening. Colonies were visually selected Thus, specific primers were designed by including the position 297 based on colony color (yellow or white) and subjected to colony bp at the 3' terminus of the forward primer AU297-2F (5'-CGAC PCR with an Aurantimonas-specific primer under the conditions GATCSRTAGCTGGTCCA-3') and the reverse primer AU297-2R described above. Positive colonies were subjected to single-colony (5'-CAATGTGGCTGATCATCCTCCT-3'). These primers were isolation on a NA medium with no antibiotic substances 3 times to designed by the alteration of the second base from the 3' terminus check the purity of isolates. into a mismatch among all sequences in order to suppress non- specific amplification. The effectiveness of the combination of the Isolation of methylotrophic bacteria specific primers and universal primers 27F and 1525R (26) was The strategy for isolating Methylobacterium species is shown in evaluated by using Aurantimonas sp. Pd-E-(l)-m-D-3(3) and Fig. 1. A portion of the enriched bacterial cells was also serially Aurantimonas sp. Pd-S-(l)-l-N-4(3) as positive controls and diluted and plated onto selective medium plates: Minimal Medium T Methylobacterium extorquens JCM2802 as a negative control. In (MM) agar (3) or Ammonium Mineral Salts (AMS) agar (58), each the case of primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') containing 0.5% (v/v) methanol and 10 μg mL−1 filter-sterilized and AU297-2R, 277 bp of DNA were amplified with no smear cycloheximide. After incubation at 28°C for 10 days, colonies were band (data not shown) from only the positive controls. Cycling chosen randomly from the highest-dilution plate and subcultured conditions were as follows: Initial denaturation for 2 min at 94°C; on a selective medium in order to confirm methylotrophy. Single- then 25 cycles of 30 s at 94°C, 30 s at 65°C and 2 min at 72°C; and colony isolation on a NA medium was done 2 or 3 times to check a final extension for 10 min at 72°C. The amplification reactions the purity of the isolates with no antibiotic substances. were performed in a 12.5-μL volume containing 1×Ex Taq buffer, 0.2 mM dNTP mixture, 1 μM each primer, 10 ng of template DNA, Sequencing of 16S rRNA genes and 0.25 U of Ex Taq DNA polymerase (Takara Bio, Otsu, Japan). After single-colony isolation, genomic DNA was prepared with Each colony was picked up with a sterilized 200-μL pipette tip and a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The transferred to the PCR tube as a DNA template. universal primers 27F and 1525R (5'-AAGGAGGTGWTCCAR The strategy for isolating Aurantimonas species is shown in CC-3') were used (26). Cycling conditions were as follows: initial Fig. 1. A portion of the enriched bacterial cells was serially diluted denaturation for 2 min at 94°C; then 25 cycles of 30 s at 94°C, 30 s and plated onto a Nutrient agar (NA) medium (Difco, Becton- at 55°C and 2 min at 72°C; and a final extension for 10 min at Dickinson, Sparks, USA) containing 10 μg mL−1 filter-sterilized 72°C. The amplification reactions were performed in a 12.5-μL cycloheximide. After incubation at 28°C for 10 days, plates were volume containing 1×Taq buffer, 0.2 mM dNTP mixture, 1 μM 174 ANDA et al. each primer, 10 ng of template DNA, and 0.25 U of Ex Taq DNA were carried out with primers ERIC 1R and ERIC 2, and BOX A1R, polymerase (Takara). respectively, according to the procedure of Versalovic et al. (52) as Sequencing was conducted with a Type 3730xl DNA Analyzer modified by Raja (40). The amplification reactions were performed (Applied Biosystems, Foster City, CA, USA) using a BigDye in a 20-μL volume containing 1×Ex Taq buffer, 0.25 mM dNTP Terminator Cycle Sequencing Reaction Kit (Applied Biosystems). mixture, 1 μM each primer, 50 ng of template DNA, and 1 U of Template DNAs were prepared using an ExoSAP-IT Kit (GE Ex Taq DNA polymerase (Takara). ERIC-PCR and BOX-PCR Healthcare, Uppsala, Sweden). A partial sequence of the 16S rRNA products were separated by electrophoresis on 1.25% SeaKem gene was obtained by using 27F as a sequencing primer. Sequences agarose gels (FMC BioProducts, Rockland, ME) for 210 min at were processed manually to eliminate low-quality regions. Then the 3.7 V cm−1. Fingerprints were analyzed using FPQuest Software region of the 16S rRNA gene (corresponding to position 109–665 (Bio-Rad). Dice’s coefficient and the unweighted pair group bp of the Escherichia coli 16S rRNA gene) was used for the method with arithmetic mean (UPGMA) algorithm were used to sequence analyses. perform cluster analyses and to construct dendrograms. Nearly complete 16S rRNA gene sequences were generated for representative strains of OTUs with different partial 16S rRNA Accession numbers gene sequences. PCR products were ligated into a plasmid vector Nucleotide sequences of the 16S rRNA genes obtained from the pGEM-T Easy (Promega, Madison, USA) and transformed into bacterial isolates in the present study have been deposited in the competent cells of Escherichia coli DH5α (Toyobo, Tokyo, Japan) DDBJ database under accession numbers AB600000 to AB600142 by using a pGEM-T Easy Vector System (Promega). After the (Table S2 and S3). transformants were cultured overnight at 37°C on Luria-Bertani μ −1 (LB) agar plates containing ampicillin (50 g mL ), positive trans- Results and Discussion formants (white-colony-morphotype) were cultured overnight on LB liquid medium. Plasmids were obtained by using QIAprep Miniprep (Qiagen). Sequences of the 16S rRNA genes were Isolation of Aurantimonas species obtained by using primers M13BWFw, M13BWRev (BioDynamics Of 768 colonies, 14 were successfully screened by colony Laboratory, Tokyo, Japan), 357f (32), 27f, 1100r, and 1525r (26). PCR using a primer specific to Aurantimonas species (Fig. After vector, primer, and low quality regions of sequences were 1). Among the 14 isolates, 13 sequences were classified as removed manually, edited sequences were assembled with the soft- Aurantimonas species by the RDP Classifier (Table S2). The ware ATGC (Genetyx, Tokyo, Japan). other isolate, AU25, was identified as an Agrobacterium sp. Sequence analysis which does not have a primer target site (Table S2). The sequences were aligned by using CLUSTAL W (51). On Interestingly, no isolate was obtained as a single colony in the basis of the alignment, a distance matrix was constructed using the first step of isolation. Thus, all colonies for Aurantimonas the DNADIST program from PHYLIP ver. 3.66 (http://evolution. sp. were co-isolated along with other bacteria (Fig. 1). genetics.washington.edu/phylip.html) with default parameters. In Moreover, all Aurantimonas isolates formed small colonies order to select a strain for inoculation testing, the default mothur (45) settings were used with threshold values of 99% sequence of less than 1 mm in diameter and showed white and yellow identity for the definition of OTUs for conducting the clustering colors on NA medium in the first isolation step (Table S2). analysis with the data set including both cultured and uncultured These features in the isolation of Aurantimonas could explain bacterial sequences in the present and previous studies, respec- why Aurantimonas species have not been widely investi- tively. The number of OTUs shared between libraries was calcu- gated as plant-associated bacteria by culture-dependent lated by using mothur. methods. Interestingly, isolated Aurantimonas strains devel- Phylogenetic analysis oped yellow or orange colonies on NA medium (Table S2) Phylogenetic analyses were carried out with the Classifier after the single colony isolation. All type strains in this genus program of RDP-II release 10 (55) with confidence levels of 80%. showed a yellow or orange color due to carotenoid pigments BLASTN (1) was also used to classify the isolates and to identify (8, 18, 22, 56). Aurantimonas strains isolated from rice also the closest relatives in the public databases. develop a yellow or orange color (Fig. S2), suggesting that For the phylogenetic analysis, sequences were aligned using the these colors are common to the genus Aurantimonas. CLUSTAL W program (51). The neighbor-joining method was used for building phylogenetic trees (43). The PHYLIP format tree Phylogenetic analysis of Aurantimonas isolates output was obtained by using the bootstrapping procedure (11); 1000 bootstrap trials were used. The trees were constructed using By using the RDP Classifier, 37 sequences of 16S rRNA TreeView software (37) and MEGA version 4.0 (50). gene cloned from stems of Sakukei 4 in a previous study (17) were classified as Aurantimonas. These 37 sequences were Genetic variability combined with 13 sequences of 16S rRNA gene of the Template DNA was prepared with a DNeasy Blood & Tissue Kit Aurantimonas isolates obtained in the present study, and a (Qiagen) modified as follows. Each colony grown on a plate was suspended in 1 mL of Buffer ATL in 2-mL screw-capped tubes total of 50 Aurantimonas sequences were used for the containing 0.5 g of silica beads (0.1 mm in diameter) and the tubes clustering analysis. The Aurantimonas isolates were classi- were processed in a bead beater (FastPrep FP100A; Thermo fied into 4 OTUs (Fig. 2). Among them, OTU A1 was shown Electron, Waltham, MA, USA) for 40 s at level 6. The tubes were to correspond to OTU AP6, whose abundance was shown to then centrifuged for 1 min at 16,000×g at room temperature. The fluctuate according to nodulation phenotype and nitrogen supernatant was collected and transferred to a fresh 1.5-mL levels in the previous study (17). Three isolates were microtube; then 200 μL of the supernatant was added to another 1.5-mL microtube with 0.3 μL of RNase (Bio-Rad Laboratories, included in OTU A1. Isolates corresponding to all OTUs Hercules, USA). After incubation for 30 min at room temperature, reported by Ikeda (OTUs AP6, AP7, and AP11) (17) were Buffer AL was added according to the manufacturer’s instructions. obtained in OTUs A1, A2 and A4, respectively, in the Enterobacterial repetitive intergenic consensus sequence-PCR present study. OTU A3 was dominant and unique to the (ERIC-PCR) and BOX repeat element-based PCR (BOX-PCR) isolate collection in the present study (Fig. 2). This may be Bacterial Isolation from Soybean Stem 175

Fig. 2. Phylogenetic distribution of Aurantimonas spp. based on 16S rRNA gene sequences for isolates in the present study and clones from a previous study (17). The dendrogram (left) indicates the phylogenetic relationships among the representative sequences of each OTU (defined by 99% identity). The table indicates the numbers and relative abun- dances (RA) of isolates or clones belonging to each OTU. The sequences discussed in the main text are highlighted in gray. caused by a culture bias, because differences in intra-genus genetic variability have been detected for Aurantimonas species between culture-dependent and -independent analyses (15). Phylogenetic analysis revealed that the representative isolate of OTU A1 belongs to the cluster containing Aurantimonas sp. strain Pd-E-(l)-m-D-3(3), Aurantimonas sp. strain Pd-S-(l)-l-N-4(3), and Aurantimonas ureilytica (Fig. 3A). Strains Pd-E-(l)-m-D-3(3) and Pd-S-(l)-l-N-4(3) were isolated from rice as an epiphyte and an endophyte, respectively (29), while A. ureilytica 5715S-12T was isolated from an air sample (56). Representative isolates of OTU A2, A3, and A4 were phylogenetically closely related to strains Pd-E-(l)-m-D-3(3) and Pd-S-(l)-l-N-4(3) (Fig. 3A). These results may suggest the presence of a plant-associated group in the genus Aurantimonas (shaded region of Fig. 3A). A phyllosphere cluster of Aurantimonas To examine precise phylogenetic positions for an isolate corresponding to the sequence, the nearly full-length sequence of the 16S rRNA gene was determined for isolates of Aurantimonas sp. (isolates AU4, AU12, AU20 and AU22 corresponding to the representative sequences of OTU A3, A4, A1, and A2, respectively), and used to construct a phylogenetic tree with reference sequences (Fig. 3B and Table S4). Some references were derived from plant materi- als such as alpine shoots (47) and bark of the tropic tree Mallotus nudiflorus (54). The phylogenetic analysis re- vealed that Cluster I mainly including phyllosphere- associated Aurantimonas strains was phylogenetically distantly related to Cluster II mainly consisting of ocean- Fig. 3. Phylogenetic tree of 16S rRNA genes for representative sequences of OTUs in the present study. (A) A region of the 16S rRNA associated Aurantimonas strains (Fig. 3B and Table S4). Escherichia coli gene (corresponding to bp 109–665 of the 16S rRNA Moreover,representative sequences of OTU A4 showed 99% gene) was used for the sequence analyses. The tree was constructed identity to the uncultured bacterium 9_C02 (FN421801) in by the neighbor-joining method. The scale represents 0.01 substitutions a clone library analysis of soybean by Delmotte et al. (7), per site. The numbers at nodes are the percentages of 1,000 bootstrap < and the isolates belonging to OTU A3 have only 95% replicates, and values of 50 are not shown. The positions of repre- T sentative sequences are indicated as OTU names in bold. Sources of identity to A. ureilytica 5715S-12 (DQ883810). These re- Aurantimonas spp. are abbreviated as follows: P, plant; A, air; S, soil; sults suggest the novelty of the Aurantimonas isolates ob- H, human; T, terrestrial cave wall; W, water cooling system; C, coral; F, tained in the present study. Although Aurantimonas members fjord; and SW, sea water. (B) Phylogenetic tree based on nearly full- length 16S rRNA. Reference strains (>1,200 bp) were obtained from have been found in the phyllosphere, and their wide distri- the RDP Browser, and a region of the 16S rRNA gene (corresponding bution on several plant species has been suggested by culture- to position 109–1406 in the Escherichia coli 16S rRNA gene) was dependent and independent methods (7, 17, 29), only two used for the sequence analysis. Reference strains were clustered by OTU (99%) as shown in Table S4. The values in parentheses show (1) isolates from rice phyllosphere have been reported (29). By the number of sequences belonging to the OTU, (2) the number of employing the semi-specific PCR primer, the present study isolates from the phyllosphere, and (3) the number of sequences of demonstrated the successful isolation of strains previously uncultured bacteria from the phyllosphere. 176 ANDA et al.

level of 0.70 as shown in Fig. 4. Isolation of Methylobacterium species Methylotrophic bacteria (187 isolates) were randomly selected on 2 selective media (AMS and MM media). Their 16S rRNA gene sequences were analyzed and 129 sequences were obtained (Fig. 1). Among these, 126 sequences were classified as Methylobacterium species by the RDP Classifier (Fig. 1). The colonies of the corresponding isolates exhibited the pink pigment characteristic of Methylobacterium spe- cies, with the exception of strain MM93, which was non- pigmented (Table S3). The other 3 sequences were classified as Rhodococcus sp. and Mycobacterium sp. (Table S3). Members of these genera have been isolated from the phyllosphere and are reported to be degraders of air pollutants (44, 60) and to be methyl- Fig. 4. Genetic variability of Aurantimonas isolates belonging to otrophic (4, 27), suggesting that they might also play a role OTU A1 (highlighted in gray in Fig. 2.) and related reference strains as scavengers of monocarbon wastes from plants similar to determined from (A) ERIC-PCR and (B) BOX-PCR profiles. Filled cir- cles indicate reference strains (Aurantimonas sp. strain Pd-E-(l)-m-D- the role played by Methylobacterium species in the phyllo- 3(3) (D3) and Aurantimonas sp. strain Pd-S-(l)-l-N-4(3) (N4)). Squares sphere (6). indicate groups defined with more than 70% similarity. Gray squares indicate groups consisting of isolates belonging to OTU A1; black Clustering analysis for Methylobacterium isolates squares indicate groups consisting of reference strains. The cluster analyses were performed based on Dice’s coefficients and UPGMA. In a previous study, 54 sequences in a clone library constructed from stems of Sakukei 4 were classified with the RDP Classifier as Methylobacterium species (17). These 54 sequences were combined with the 126 sequences from the Methylobacterium isolates obtained in the present study to give a total of 180 Methylobacterium species sequences for the clustering analysis. The clustering analysis revealed the presence of at least 8 OTUs in the stems of Sakukei 4 (Fig. 5). Among them, OTU M4 was identified as equivalent to OTU AP30, whose abundance was previously shown by Ikeda et al. (17) to fluctuate according to nodulation pheno- type and nitrogen level. Thirty-three isolates belonging to OTU M4 were obtained. Fig. 5. Phylogenetic distribution of Methylobacterium spp. based on Methylobacterium isolates corresponding to all OTUs 16S rRNA gene sequences for isolates in the present study and clones (AP30, AP32, AP34, AP35, and AP36) found in the pre- from a previous study (17). The dendrogram (left) indicates the phylo- vious culture-independent study (17) were obtained in the genetic relationships among the representative sequences of each OTU present study, although OTUs M3 and M6 were unique to the (defined by 99% identity). The table indicates the numbers and the rela- tive abundances (RA) of isolates or clones belonging to each OTU. The current isolate collection (Fig. 5). This result suggested sequences discussed in the main text are highlighted in gray. the high culturability of this bacterial group from the phyllo- sphere. Recently, Knief et al. (24, 25) have also reported that reported as uncultured bacteria (Figs. 2 and 3B). Therefore, Methylobacterium community members are present in our isolation strategy for Aurantimonas would be useful plants over years, and major groups detected by cultivation- to facilitate revealing their ecological functions in the phyllo- independent methods have been successfully isolated as sphere. The genome sequencing of these strains might be shown in the present study. Thus, Methylobacterium species helpful in providing an insight into plant-associated are stable community members in the phyllosphere. Aurantimonas members by comparing the draft genome of Aurantimonas sp. SI85-9A1 derived from ocean water (9). Phylogenetic analysis of Methylobacterium isolates Phylogenetic analysis also revealed that the representative The clustering analyses and subsequent phylogenetic isolate of OTU A1 belonged to the cluster containing analyses of 16S rRNA sequences allowed the genetic rela- Aurantimonas sp. strain Pd-E-(l)-m-D-3(3), Aurantimonas tionships among the isolates or OTUs to be determined sp. strain Pd-S-(l)-l-N-4(3), and Aurantimonas ureilytica (Fig. 6A). Methylobacterium species are divided into 2 major (Fig. 3B). ERIC-PCR and BOX-PCR were used to character- clusters (21), and the isolates of Methylobacterium in the ize genetic variability among Aurantimonas isolates in OTU present study were distributed between both of these clusters A1. Both ERIC- and BOX-PCR showed that soybean iso- (Fig. 6A). The only non-pigmented isolates belonged to OTU lates AU20, AU26 and AU40 belonging to OTU A1 were M6 and were shown to be 96% identical to M. jeotgali, clearly divided into 2 groups, and were evidently different which is a non-pigmented Methylobacterium species reported from the reference strains Aurantimonas sp. Pd-E-(l)-m-D- by Aslam et al. (2). Although ‘pink-pigment’ is a valuable 3(3) and Aurantimonas sp. Pd-S-(l)-l-N-4(3) at a similarity indication for the isolation of Methylobacterium species, this Bacterial Isolation from Soybean Stem 177

Fig. 6. Phylogenetic tree of 16S rRNA genes for representative sequences of OTUs in the present study. (A) A region of the 16S rRNA gene (corresponding to bp 109–665 of the Escherichia coli 16S rRNA gene) was used for the sequence analyses. Tree con- struction is the same as described in the legend to Fig. 3A. Sources of Methylobacterium isolates are abbreviated and shown after the strain names as follows: P, plant; A, air; WS, water sludge; S, soil; W, water; F, food. (B) Nearly full-length 16S rRNA (a region of the 16S rRNA gene corresponding to position 109–1406 in Escherichia coli) was used for the sequence analysis. 178 ANDA et al.

Fig. 7. Genetic variability of Methylobacterium isolates belonging to OTU M4 (highlighted in gray in Fig. 5.) and related reference strains deter- mined from (A) ERIC-PCR and (B) BOX-PCR profiles. Filled circles indicate reference strains (M. extorquens DM4, AM1, PA1, and JCM2802T). Squares indicate groups defined with more than 70% similarity. Gray squares indicate groups consisting of isolates belonging to OTU M4; black squares indicate groups consisting of reference strains. The cluster analyses were performed based on Dice’s coefficients and UPGMA. characteristic is not always appropriate for representing the and BOX-PCR were classified into 4 subgroups (groups 1, 2, whole genus Methylobacterium in nature. The representative 3 and 4) and showed that isolates in OTU M4 were distinctly sequence of OTU M4 was identical to M. extorquens DM4, dissimilar to the reference strains, M. extorquens AM1, PA1, AM1, and PA1, which are all well-known strains and whose and DM4 at a similarity level of 0.70 as shown in Fig. 7. genome sequencing have been completed (24, 53) (Fig. 6A). In particular, the group 1 members (Fig. 7) were frequently To examine the precise phylogenetic position for an isolate isolated, indicating that they are major Methylobacterium corresponding to the sequence, the nearly full-length within OTU M4 under the conditions examined (Fig. 7). sequence of 16S rRNA gene was determined for isolates of M. extorquens AM1, PA1, and DM4 were respectively Methylobacterium sp. AMS1, AMS5, AMS19, AMS21, isolated from an air sample (53), the phyllosphere of AMS49, AMS64, AMS84, and MM70 corresponding to the Arabidopsis (24), and contaminated soil (53). The results representative sequences of OTU M2, M4, M5, M1, M7, of ERIC- and BOX-PCR suggest considerable plasticity in M8, M3, and M6, respectively, and a phylogenetic tree was the genomic structures of M. extorquens (Fig. 7) derived constructed (Fig. 6B). A BLAST search showed that OTU from different environmental habitats. The genome M4 has high identity (99%) to M. extorquens DM4, AM1, sequencing of OTU M4 might be helpful in providing an in- and PA1 and phylogenetic analysis revealed that OTU M4 sight into soybean plant-associated Methylobaterium species was grouped into a cluster including M. extorquens DM4, by comparing the genomes of M. extorquens AM1, PA1, AM1, PA1, and CM4 was constructed (Fig. 6B). The 3 and DM4 derived from different environmental habitats. reference (DM4, AM1, and PA1) strains have an identical 16S rRNA gene sequence, although their biological traits, Isolation of target microbes based on culture-independent such as carbon substrate utilization patterns and competitive- analysis ness in the phyllosphere, are considerably different (24, 53). New isolation strategies have been proposed for previ- Therefore, ERIC- and BOX-PCR were carried out to charac- ously uncultured microbes (20, 49). Although random iso- terize genetic variability among the isolates in OTU M4 and lation from plant tissues has been carried out (19, 36), there the above reference strains (Fig. 7). The profiles of ERIC- are few reports of the isolation of targeted plant-associated Bacterial Isolation from Soybean Stem 179 bacteria based on previous cultivation-independent analysis 3. Attwood, M.M., and W. Harder. 1972. A rapid and specific (23–25) such as has been done in the present study. The enrichment procedure for Hyphomicrobium spp. Antonie Van Leeuwenhoek 38:369–377. target Aurantimonas were obtained by colony PCR and 4. Boden, R., E. Thomas, P. Savani, D.P. Kelly, and A.P. Wood. careful single-colony isolation (Fig. 1), because the physiol- 2008. Novel methylotrophic bacteria isolated from the River Thames ogy and biochemistry of the Aurantimonas species were still (London, UK). Environ. Microbiol. 10:3225–3236. unknown. On the other hand, the Methylobacterium species 5. Carroll, B.J., D.L. McNeil, and P.M. Gresshoff. 1985. Isolation and properties of soybean [Glycine max (L.) Merr.] mutants that nodulate targeted in the present study were isolated by using selective in the presence of high nitrate concentrations. Proc. Natl. Acad. Sci. media containing methanol as the sole carbon source (Fig. 1), USA 82:4162–4166. although they showed genetic variability different from that 6. Corpe, W.A. 1985. A method for detecting methylotrophic bacteria of authentic Methylobacterium strains (Fig. 7). on solid surfaces. J. Microbiol. Methods 3:215–221. 7. Delmotte, N., C. Knief, S. Chaffron, G. Innerebner, B. Roschitzki, The identity of the isolates thus obtained with the R. Schlapbach, C. von Mering, and J.A. Vorholt. 2009. Community target bacteria from a previous work (17) was supported proteogenomics reveals insights into the physiology of phyllosphere based on the 16S rRNA gene sequences. In addition, the bacteria. Proc. Natl. Acad. Sci. USA 106:16428–16433. Aurantimonas and Methylobacterium isolates obtained here 8. Denner, E.B., G.W. Smith, H.J. Busse, P. Schumann, T. Narzt, S.W. Polson, W. Lubitz, and L.L. Richardson. 2003. Aurantimonas showed unique phylogenetic positions (Figs. 3 and 6) and coralicida gen. nov., sp. nov., the causative agent of white plague genetic variability (Figs. 4 and 7). The utilization of these type II on Caribbean scleractinian corals. Int. J. Syst. 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Stackebrandt (ed.), beans, and the genetic variability of these strains was evalu- The Prokaryotes. Springer, New York. ated. Based on clustering analyses of 16S rRNA gene 13. Gupta, R.S., and A. Mok. 2007. Phylogenomics and signature proteins for the alpha proteobacteria and its main groups. BMC sequences, the isolates corresponding to OTUs AP6 and Microbiol. 7:106. AP30, which were shown in the previous culture-independent 14. Hardoim, P.R., L.S. van Overbeek, and J.D. van Elsas. 2008. study to be extremely sensitive to nodulation phenotypes Properties of bacterial endophytes and their proposed role in plant and nitrogen fertilization levels (17), were successfully ob- growth. Trends Microbiol. 16:463–471. 15. Ikeda, S., T. Kaneko, T. Okubo, et al. 2009. Development of a tained from soybean stems. These isolates will be a valuable bacterial cell enrichment method and its application to the community resource for investigating the functional roles of Auranti- analysis in soybean stems. Microbial Ecol. 58:703–714. monas and Methylobacterium in the phyllosphere and for 16. Ikeda, S., T. Okubo, M. Anda, et al. 2010. Community- and analyzing the interactions of these bacterial species with genome-based views of plant-associated bacteria: Plant-bacterial interactions in soybean and rice. Plant Cell Physiol. 51:1398–1410. plants by further studies such as inoculation tests and 17. Ikeda, S., T. Okubo, T. Kaneko, et al. 2010. Community shifts of genome sequencing. soybean stem-associated bacteria responding to different nodulation phenotypes and N levels. ISME J. 4:315–326. 18. Jurado, V., J.M. Gonzalez, L. Laiz, and C. Saiz-Jimenez. 2006. 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