IJSEM Papers in Press. Published September 21, 2012 as doi:10.1099/ijs.0.041368-0

1 Endobacter medicaginis gen. nov. sp. nov. isolated from alfalfa nodules in an acidic 2 soil in Spain 3 4 Martha Helena Ramírez-Bahena1,2, Carmen Tejedor3, Isidro Martín3, Encarna 5 Velázquez2, 3, Alvaro Peix1,2* 6 7 8 1: Instituto de Recursos Naturales y Agrobiología de Salamanca. Consejo Superior de 9 Investigaciones Científicas. (IRNASA-CSIC). Salamanca. Spain. 10 2: Unidad Asociada Grupo de Interacciones planta-microorganismo Universidad de 11 Salamanca-IRNASA (CSIC). Salamanca. Spain 12 3: Departamento de Microbiología y Genética. Universidad de Salamanca. Salamanca. 13 Spain 14 15 16 17 18 Running title: Endobacter medicaginis gen. nov. sp. nov. 19 20 Abstract 21 A bacterial strain designed M1MS02T was isolated from a surface sterilised nodule of 22 Medicago sativa in Zamora (Spain). The 16S rRNA gene sequence of this strain showed 23 96.5 and 96.2% identities, respectively, with respect to Gluconacetobacter liquefaciens 24 IFO 12388T and Granulibacter bethesdensis CGDNIH1T from the Family 25 . The isolate was Gram negative, non-sporulated aerobic motile by a

26 subpolar flagellum coccoid to rod-shaped bacterium. Major fatty acid is C18:1 7c 27 (39.94%) and major ubiquinone is Q-10. The lipid profile consisted of 28 diphosphatidylglycerol, phosphatidylethanolamine, two aminophospholipids, three 29 aminolipids, four glycolipids, two phospholipids and a lipid. Catalase positive and 30 oxidase and urease negative. Acetate and lactate are not oxydized. Acetic acid is

31 produced from ethanol in culture media supplemented with 2% CaCO3. Ammonium 32 sulphate is assimilated in glucose medium. It produces dihydroxyacetone from glycerol. 33 The phylogenetic and phenotypic analyses commonly used to differentiate the family 34 Acetobacteraceae genera showed that the strain M1MS02T should be classified into a 35 new genus within this family for which the name Endobacter medicaginis gen nov., sp. 36 nov. is proposed (type strain M1MS02T, LMG 26838T, CECT 8088T). To our 37 knowledge, this is the first report of Acetobacteraceae occurrence as legume 38 nodules endophytes. 39 40 41 42 Keywords: endophytes, Medicago, nodules, Acetobacteraceae, Endobacter 43 44 45 * Corresponding author: 46 Alvaro Peix. Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, c/Cordel 47 de Merinas 40-52, 37008 Salamanca, Spain. Tel. +34923219606; Fax. +34923219609; 48 E-mail: [email protected] 49 50 51 Genebank Accession numbers for strain M1MS02 gene sequences are: JQ436923 for 52 16S rRNA, JX424585 for recA and JX424586 for 16S-23S rRNA ITS 53 54 The family Acetobacteraceae currently includes 30 validly described genera recorded in 55 the List of Prokaryotic Names with Standing in Nomenclature by Dr. Euzeby (http:// 56 www.bacterio.cict.fr) mainly differentiated on the basis of rrs gene sequences (Sievers 57 & Swings, 2005). Some of these genera contain that are plant endophytes such 58 as Gluconacetobacter diazotrophicus which is endophytic in sugar cane (Saravanan et 59 al., 2008) but other species such as Gluconacetobacter azotocaptans, 60 Gluconacetobacter johannae and Swaminathania salitolerans are endophytic of coffee, 61 corn and rice, respectively (Dong et al., 1994, Loganathan & Nair, 2004). Nevertheless 62 no member of family Acetobacteraceae have been reported up to date as endophytes of 63 legume nodules. These structures are induced by rhizobia, but commonly other non- 64 nodulating endophytic bacteria are present inside the nodules sharing niche with the 65 bacterial endosymbionts. In the case of Medicago, several strains of the former genus 66 Agrobacterium have been isolated from nodules of different species of this legume (Kan 67 et al., 2007, Djedidi et al., 2011) and the presence of the actinobacteria 68 Micromonospora in nodules of Medicago sativa has been reported by Trujillo et al. 69 (2010). However no data are available up to date about other endophytes from alpha- 70 present in Medicago sativa nodules, and to our knowledge this is the first 71 report of Acetobacteriaceae occurring in legume nodules. In this work we isolated an 72 endophytic strain named M1MS02T from a root nodule of this host that belongs to a 73 new genus and species within family Acetobacteraceae for which the name Endobacter 74 medicaginis gen. nov. sp. nov. is proposed. 75 76 The strain M1MS02T was isolated from a nodule of Medicago sativa growing in Matilla 77 la Seca (Zamora, Spain) during a study of rhizobia and nodular endophytes present in 78 different legumes. To sterilize the root nodules they were washed several times with

79 sterile distilled water and were then surface sterilized in HgCl2 2,5% (w/v) for 2 min. 80 The nodules were rinsed five times with sterile distilled water and then crushed using a 81 sterile pestle. The homogenized nodule tissue was inoculated on yeast extract mannitol -1 -1 -1 82 agar (Vincent, 1970) modified (10 gl mannitol, 1 gl yeast extract, 0.2 gl K2HPO4, -1 -1 -1 83 0.2 gl MgSO4.7H2O, 0.5 gl NaCl, 20 gl agar) and the plates were incubated at 28 ºC 84 for 4 days. In parallel, some of the disinfected entire nodules were incubated in the same 85 medium in order to ensure their complete external disinfection and no growth was 86 observed around these nodules. The cultures used in further phenotypic and molecular 87 studies were purified from a single colony after 2 days incubation at 28 ºC on YMA. 88 The colonies were white, mucoid, translucent and convex on this medium. 89 The strain was grown in nutrient broth (Difco, Becton Dickinson, BBL) for 48h at 22°C 90 to check for motility by phase-contrast microscopy using the hanging drop method. 91 Gram staining was carried out by the procedure described by Doetsch (1981) after 24h 92 incubation at 28°C. The flagellation type was determined by electron microscopy after 93 48h incubation in TSA at 22°C as was previously described (Rivas et al., 2007). Cells 94 of strain M1MS02T were Gram-negative, rod-shaped, non-sporulating, and motile by 95 means of a subpolar flagellum (figure S1). 96 The 16S rRNA gene of the strain M1MS02T was analyzed as described by Rivas et al., 97 2007), the recA gene as described by Gaunt et al. (2001) and the ITS fragment as 98 described by Peix et al. (2005). The sequence obtained was compared with those from 99 the GenBank using the BLASTN (Altschul et al., 1990) and EzTaxon (Chun et al., 100 2007) programs. Sequences were aligned using the Clustal_X software (Thompson et 101 al., 1997) and distances were calculated according to Kimura´s two-parameter model 102 (Kimura, 1980). The phylogenetic tree was inferred using the neighbor joining and 103 maximum likelihood models (Saitou & Nei, 1987, Rogers & Swofford, 1998). MEGA5 104 package (Tamura et al., 2011) was used for all analyses. 105 The comparison of the 16S rRNA gene sequence of strain M1MS02T against those of 106 EzTaxon database showed that it is phylogenetically equidistant to two genera of family 107 Acetobacteraceae. The closest related species are Gluconacetobacter liquefaciens IFO 108 12388T, which is the type species of this genus, and Granulibacter bethesdensis 109 CGDNIH1T with 96.3 and 96.2% identities, respectively. The remaining species of 110 genus Gluconacetobacter showed identities similar or lower according to the results of 111 the mentioned database. The results of the maximum likelihood phylogenetic analysis 112 (figure 1) confirmed the clustering of M1MS02T within family Acetobacteraceae where 113 this strain forms a separated branch. Congruent results were obtained when the 114 phylogenetic tree was constructed using the neighbor joining method (data not shown). 115 Therefore M1MS02T represents a new genus within this family in which several 116 recently described genera have identity values in their 16S rRNA genes higher than 117 96% as occurs in the case of Granulibacter bethesdensis CGDNIH1T that presented 118 96.1% identity with Gluconacetobacter liquefaciens IFO 12388T. There are some 119 genera that presented even higher identity values as occurs between Kozakia and the 120 genera Gluconacetobacter, Asaia and Swaminathania, between Tanticharoenia and 121 Ameyamaea, between Ameyamaea and Neoasaia, between Acetobacter and Neoasaia, 122 between Asaia and Neoasaia with identities higher than 97% and even some genera 123 have more than 98% identity such as Swaminathania and Asaia. 124 The results of the recA gene analysis confirmed that strain M1MS02T is placed in an 125 independent branch phylogenetically very divergent to G. bethesdensis and to the 126 cluster formed by species of genus Gluconobacter (figure S2) with identity values lower 127 than 83% with respect to the remaining analysed species. The distances found in the 128 recA gene with respect to their closest relatives (species from genus Gluconacetobacter 129 and Acidiphilum) were similar to those found for example between the type species of 130 genus Acetobacter, A. aceti, and several species of genus Gluconobacter. These results 131 support the classification of strain M1MS02T in a new genus within family 132 Acetobacteraceae. 133 The 16S-23S rRNA internal transcribed spacer (ITS) analysis also showed the 134 phylogenetic divergence of the strain M1MS02T with respect to other species of family 135 Acetobacteraceae (figure S3) with identity values lower than 60% with respect to the 136 remaining analyzed species. These values were much lower than those found among 137 other genera of family Acetobacteraceae, as occurs for example between the type 138 species of Acetobacter and Gluconobacter that showed 84% identity. 139 Therefore, the results of recA gene and ITS fragment of strain M1MS02T agree with 140 those of the 16S rRNA gene analysis supporting the proposal that it represents a new 141 genus within the family Acetobacteraceae. 142 The cellular fatty acids were analysed by using the Microbial Identification System 143 (MIDI Inc Sherlock MIS software 6.1) using an Agilent 6890N gas chromatograph at 144 DSMZ. The strains were grown on TSB (Becton Dikinson, BBL) for 24h at 28°C and

145 160 rpm. The major fatty acids are C18:1 7c (39.94%), C19:0 cyclo 8c (12.15%) and

146 C16:0 (13.40%). Other fatty acids identified were C18:1 2OH (6.88%), C18:0 (4.58%), C16:0

147 2OH (2.82%), C16:0 3OH (2.73%), C17:1 6c (2.02%), C14:0 3OH/ C16:1 ISO I in summed

148 feature 2 (2.58%), C18:0 3OH (1.87%) C17:0 (1.65%), C14:0 (1.46%), C15:0 ISO (1.31%),

149 C16:1 6c/C16:1 7c in summed feature 3 (1.04%), and C13:0 ISO, C13:0 anteiso, C13:1,

150 C14:0 ISO, C15:0 anteiso, C16:0 ISO, C17:0 ISO, 11methyl C18:1 7c, C17:0 2OH in amounts 151 lower than 1%. These results are in agreement with those obtained for other members of

152 family Acetobacteraceae that have C18:1 7c as major fatty acid (Sievers & Swings, 153 2005). 154 Analysis of respiratory quinones and polar lipids was carried out by Dr. Brian Tindall 155 and the Identification Service and at DSMZ, from freeze dried cells using the methods 156 described by Tindall (1990a; 1990b). The strain M1MS02 has Q-10 as major 157 ubiquinone (75%) and Q-9 as secondary ubiquinone (25%). This result agrees with 158 those of the remaining genera of family Acetobacteraceae except in the case of genus 159 Acetobacter that has Q-9 as major respiratory quinone (Table 1). The strain M1MS02T 160 displayed a lipid profile (figure S4) consisting of diphosphatidylglycerol (DPG), 161 phosphatidylethanolamine (PE), two aminophospholipids (PLN1-PLN2), three 162 aminolipids (AL1-AL3), four glycolipids (GL1-GL4), two phospholipids (PL1-PL4) 163 and a lipid (L1). 164 DNA for analysis of DNA base composition was prepared according to Chun & 165 Goodfellow (1995). The mol % G+C content of DNA was determined using the thermal 166 denaturation method (Mandel & Marmur, 1968). The G+C content of strain M1MS02T 167 was 60.3%. This value is within the range of family Acetobacteraceae members 168 (Sievers & Swings, 2005; Greenberg et al., 2006). 169 The phenotypic characterization was performed using the media and methods 170 commonly used in family Acetobacteraceae described in Greenberg et al., (2006) and 171 Sievers & Swings (2005). Phenotypic characteristics of the new species are reported 172 below in the species description and the differences with respect to the closest 173 phylogenetically related genera are recorded in Table 1. The new genus differed from 174 its closest relative genera Granulibacter and Gluconacetobacter in the flagellation and 175 in the oxidation of acetate and lactate. From Granulibacter it also differs in the absence 176 of pigmentation, in the production of dihydroxyacetone from glycerol, in the 177 assimilation of methanol and in the production of acid from glycerol. 178 179 Therefore on the basis of phylogenetic and phenotypic tests usually performed in the 180 family Acetobacteraceae we propose the strain M1MS02T to be classified into a new 181 genus within this family for which the name Endobacter medicaginis gen. nov. sp. nov. 182 is proposed (type strain M1MS02T, LMG 26838T, CECT 8088T). 183 184 Description of Endobacter gen. nov. 185 Endobacter (En.do.bac'ter. Gr. pref. endo, within; N.L. masc. n. bacter, a rod; N.L. 186 masc. n. Endobacter, a rod isolated from the inner of a root nodule of Medicago sativa). 187 Cells are Gram-negative, motile by a subpolar flagellum and coccoid to rod-shaped. 188 Strictly aerobic. Catalase positive. Oxidase negative. Urease negative. Colonies are 189 white and mucoid on YMA medium. It can grow from 20ºC to 37ºC with an optimum 190 temperature for growth of 28ºC. Optimum pH for growth is 5.0–7 but it can grow to pH 191 3.5. Acetate and lactate are not oxydized. Acetic acid is produced from ethanol in

192 presence of 2% CaCO3 in the medium. The lipid profile consists of 193 diphosphatidylglycerol, phosphatidylethanolamine, two aminophospholipids, three 194 aminolipids, four glycolipids, two phospholipids and a lipid. The major fatty acids are

195 C18:1 7c, C19:0 cyclo 8c and C16:0. Other fatty acids identified are C18:1 2OH, C18:0,

196 C16:0 2OH, C16:0 3OH, C17:1 6c, C14:0 3OH/ C16:1 ISO I in summed feature 2, C18:0 3OH

197 C17:0, C14:0, C15:0 ISO, C16:1 6c/C16:1 7c in summed feature 3, and in lower amounts

198 C13:0 ISO, C13:0 anteiso, C13:1, C14:0 ISO, C15:0 anteiso, C16:0 ISO, C17:0 ISO, 11methyl

199 C18:1 7c, C17:0 2OH. Major ubiquinone is Q-10. Ammonium sulphate is assimilated on 200 glucose medium. It produces dihydroxyacetone from glycerol. The type species is 201 Endobacter medicaginis. 202 203 Description of Endobacter medicaginis sp. nov. 204 Endobacter medicaginis (me.di.ca'gi.nis. N.L. gen. n. medicaginis, of Medicago, 205 isolated from Medicago sativa). 206 Characteristics are the same as those described for the genus. Characteristics at species 207 level are the following: Methanol as a sole carbon source is not used. It grows on 208 glutamate agar and mannitol agar. Acid is produced from glucose, xylose, glycerol and 209 ethanol, but not from mannitol, sorbitol, dulcitol, lactose, sucrose or maltose. DNA base 210 composition is 60.3% mol% G+C. The type strain is M1MS02T (LMG 26838T, CECT 211 8088T), which was isolated from a nodule of Medicago sativa. 212 213 Acknowledgments 214 This research was funded by Junta de Castilla y León (Regional Government, Spain) 215 under research project CSI02A09 and by MICINN (Central Government, Spain) under 216 research project AGL2010-17380. MHRB is recipient of a JAE-Doc researcher contract 217 from CSIC. We thank Dr. J. Euzeby for his valuable help in providing the correct 218 etymology for the name of the new taxon, and M. Ortiz for technical assistance in 219 electron microscopy. 220 221 References 222 223 Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D.J. (1990). Basic 224 local alignment search tool. J Mol Biol 215, 403-410. 225 226 Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia 227 with 16S rRNA sequences. Int J Syst Bacteriol 45, 240-245. 228 229 Chun, J., Lee, J.H., Jung, Y., Kim, M., Kim, S., Kim, B.K. & Lim, Y. W. 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In: A 324 Manual for the Practical Study of Root-Nodule, pp. 1-13. Edited by J. M. Vincent. 325 Oxford. Blackwell Scientific Publications Press. 326 327 Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T., 328 Uchimura, T. & Komagata, K. (2000). Asaia bogorensis gen. nov., sp. nov., an 329 unusual acetic acid bacterium in the -Proteobacteria. Int J Syst Evol Microbiol 50, 330 823–829. 331 332 Yamashita, S., Uchimura, T. & Komagata, K. (2004). Emendation of the genus 333 Acidomonas Urakami, Tamaoka, Suzuki and Komagata 1989. Int J Syst Evol Microbiol 334 54: 865-870. 335 336 Yukphan, P., Malimas, T., Potacharoen, W., Tanasupawat, S., Tanticharoen, M. & 337 Yamada, Y. (2005). Neoasaia chiangmaiensis gen. nov., sp. nov., a novel osmotolerant 338 acetic acid bacterium in the alpha-Proteobacteria. J Gen Appl Microbiol 51, 301–311. 339 340 Figure legend: 341 342 Figure 1. Maximum likelihood phylogenetic tree based on nearly complete 16S rRNA 343 gene sequence (1492 nucleotides) of Endobacter medicaginis M1MS02T and the 344 remaining genera of family Acetobacteraceae. The significance of each branch is 345 indicated by a bootstrap value (%) calculated for 1000 subsets. Bar, 2 nt substitutions 346 per 100 nt. 347 69 Neokomagataea thailandica BCC25710T (AB513363) 82 Saccharibacter floricola S‐877T(AB110421) 36 Gluconobacter oxydans NBRC14819T (AB178433) Acetobacter aceti NCIMB 8621T (X74066) 20 31 Commensalibacter intestini A911T (EU409601) Ameyamaea chiangmaiensis AC04T (AB303366) 25 83 Tanticharoenia sakaeratensis NBRC103193T (AB304087)

T 45 Asaia bogorensis 71 (AB025928) 92 Swaminathania salitolerans PA51T (AF459454) 68 Kozakia baliensis Yo‐3T (AB056321) 87 Neoasaia chiangmaiensis NBRC101099T (AB208549) 94 Acidomonas methanolica LMG1668T (X77468) Gluconacetobacter liquefaciens IFO12388T (X75617)

50 71 Gluconacetobacter diazotrophicus PAl 5T(CP001189) 99 Gluconacetobacter johannae CFN‐Cf55T(AF111841) 91 99 Gluconacetobacter azotocaptans CFN‐Ca54T(AF192761) Endobacter medicaginis M1MS02T (JQ436923) 59 Granulibacter bethesdensis CGDNIH1T(CP000394)

40 Acidiphilium cryptum ATCC 33463T (D30773) 88 Acidocella facilis ATCC 35904T (D30774) 15 Acidisoma tundrae WM1T (AM947652) 35 Rhodopila globiformis DSM 161T (D86513) 28 Acidisphaera rubrifaciens HS‐AP3T (D86512) 72 Acidicaldus organivorans Y008T (AY140238) 45 Rhodovastum atsumiense G2‐11T (AB381935) 21 Rhodovarius lipocyclicus CCUG 44693T (AJ633644)

T 17 Roseococcus suduntuyensis SHET (EU012448) 98 Rubritepida flocculans DSM 14296T (AF465832) Belnapia rosea CPCC 100156T (HQ641379)

66 Paracraurococcus ruber NS89T (D85827) Craurococcus roseus NS130T (D85828) Roseomonas gilardii subsp. gilardii ATCC49956T (AY150045)

T 17 Stella humosa DSM5900 (AJ535710) 100 Burkholderia cepacia ATCC25416T (U96927)

0,02 Table 1. Characteristics that differentiate Endobacter gen. nov. from phylogenetically related and relevant genera within family Acetobacteraceae Genera: 1, Endobacter; 2; Granulibacter (Greenberg et al., 2006); 3, Gluconacetobacter (Sievers & Swings, 2005); 4, Acetobacter (Sievers & Swings, 2005); 5, Gluconobacter (Sievers & Swings, 2005); 6, Kozakia (Lisdiyanti et al., 2002); 7, Acidomonas (Yamashita et al., 2004; Sievers & Swings, 2005); 8, Asaia (Yamada et al., 2000); 9, Neoasaia (Yukphan et al., 2005); 10, Swaminathania (Loganathan & Nair, 2004). +, positive; 2, negative; W, weakly positive; d, delayed; nd, no data. §Data recorded by Yamada et al. (2000). For the type strains. ‡This result was weak in Urakami et al. (1989). ¥This result was weak according to Lisdiyanti et al. (2002). ∫This result is recorded in Loganathan & Nair (2004) and Yukphan et al. (2005). ¶This result is recorded by Sievers & Swings (2005) for the type species of the genus. £These results are from Lisdiyanti et al. (2002). †These results are recorded in Lisdiyanti et al. (2002). ‡These results are from Yupkhan et al. (2009).

Characteristic 1 2 3 4 5 6 7 8 9 10 Flagellation Subpolar Nonmotile Peritrichous Peritrichous Polar Nonmotile Polar or Peritrichous Nonmotile Peritrichous or non-motile or non-motile or non-motile non-motile or non-motile Pigmentation - + +/- +/- +/- - +/- +/- + + Oxidation of: Acetate - w + + - w +‡ + - w Lactate - + +¶ + - w - + - w Assimilation of ammonium + w§ w§ w§ -§ w/-§ + + w nd sulfate on glucose medium* Growth on: Glutamate agar + + +£ +£ -£ - -∫ + + + Mannitol agar + w +/- +/- + + w + + + Dihydroxyacetone from + - +/- +/- + + w +/- w + glycerol Utilization of methanol - + - -/W - - + - - - Acetic acid from ethanol- + w/- + + + + + w/- + + 2.0% CaCO3 agar Acid production from: Mannitol - - +/- +/- + - - +/- w - Sorbitol - - -£ -£ + - - +/- +(d) + Dulcitol - - -£ -£ -£ - - + w +/- Glycerol + w/- + - + + - + + + D-Xylose + - +/-† +† +† + - + + +/- Sucrose - - -† w† +† +/- - +† + nd Major isoprenoid quinone Q-10 Q-10 Q-10 Q-9 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 DNA G+C content (mol%) 60.3 59 55–67 50.5–60.3 52–64 56.8–57.2 63–66 59.3–61 63.1 57.6-59.9

Figure S1. Electron micrograph of strain M1MS02T showing the subpolar flagellum. Bar (1cm), 1 m Granulibacter bethesdensis CGDNIH1T (DQ340305) 58 54 Commensalibacter intestini A911T (NZ AGFR01000012)

T 40 Acetobacter pasteurianus IFO 3191 (BACG01000009) 70 Acetobacter tropicalis NBRC 101654 (NZ_BABS01000010) 50 Acetobacter aceti NBRC 14818T (NZ_BABW01000646) Gluconacetobacter hansenii ATCC 23769 (NZ_ADTV01000010) Gluconacetobacter xylinus NBRC 3288 (AP012159) 46 72 Gluconacetobacter oboediens 174Bp2 (NZ_CADT01000007) 100 Gluconacetobacter europaeus LMG 18494 (NZ_CADR01000002) Gluconacetobacter diazotrophicus PAl 5T (NC_011365) Endobacter medicaginis M1MS02T (JX424585)

T 88 Acidiphilium multivorum AIU301 (AP012035) 56 Acidocella facilis ATCC 35904T (D16538)

0.05

Figure S2. Neighbour joining phylogenetic tree based on partial recA gene sequences (527 nucleotides) of Endobacter medicaginis M1MS02T and other genera of family Acetobacteraceae. The significance of each branch is indicated by a bootstrap value (%) calculated for 1000 subsets. Bar, 5 nt substitutions per 100 nt. T 100 Asaia bogorensis BCC 12264 (AB208551) 67 Swaminathania salitolerans LMG 21291T (AB220163)

T 89 Kozakia baliensis BCC 12275 (AB208554) Tanticharoenia sakaeratensis BCC 15772T (AB524879) Neoasaia chiangmaiensis BCC 15763T (AB208550) 55 67 54 Acidomonas methanolica BCC 12263T (AB210135) Saccharibacter floricola JCM 12116T (AB210092)

53 T 96 Gluconobacter oxydans NBRC14819 (AB163869) 86 Neokomagataea thailandica BCC25710T (AB513365) 34 Acetobacter aceti DSM3508T (AJ007831) 99 Gluconacetobacter liquefaciens LMG 1382T (AJ007833) Endobacter medicaginis M1MS02T (JX424586) Granulibacter bethesdensis CGDNIH1T (DQ340304) Commensalibacter intestini A911T(AGFR01000021)

0.05

Figure S3. Neighbour joining phylogenetic tree based on complete ITS fragment sequences (797 nucleotides) of Endobacter medicaginis M1MS02T and other genera of family Acetobacteraceae. The significance of each branch is indicated by a bootstrap value (%) calculated for 1000 subsets. Bar, 5 nt substitutions per 100 nt. Figure S4. Polar lipids profile of strain Endobacter medicaginis M1MS02. DPG: Diphosphatidylglycerol; PN1-PN2: Aminophospholipids; PE: Phosphatidylethanolamine; AL1-AL3: Aminolipids; GL1-GL4: Glycolipids; L1: Lipids; PL1-PL2: Phospholipid (PL1 overlaps with the amino lipids AL1and AL2, for which its possible identity as phosphatidylglycerol could not be confirmed).