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Algimonas porphyrae gen. nov., sp. nov., a member of the family , isolated from a red alga Porphyra yezoensis.

Article in International Journal of Systematic and Evolutionary Microbiology · March 2012 DOI: 10.1099/ijs.0.040485-0 · Source: PubMed

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1 Algimonas porphyrae gen. nov., sp. nov., a member of the family Hyphomonadaceae,

2 isolated from a red alga Porphyra yezoensis.

3

4 Youhei Fukui1, Mahiko Abe2, Masahiro Kobayashi3, Hiroaki Saito1, Hiroshi Oikawa4,

5 Yutaka Yano 5, and Masataka Satomi1*.

6

7 1 National Research Institute of Fisheries Science, Fisheries Research Agency,

8 Yokohama, 236-8648, Japan

9 2 National Fisheries University, Shimonoseki, 759-6595, Japan

10 3 Seikai National Fisheries Research Institute, Nagasaki, 851-2213, Japan

11 4National Research Institute of Fisheries and Environment of Inland Sea, Fisheries

12 Research Agency, Hiroshima, 739-0452, Japan

13 5National Salmon Resources Center, Fisheries Research Agency, Sapporo, 062-0922,

14 Japan

15 *Author for correspondence: Masataka Satomi. Tel. & Fax: +81-45-788-7669

16 e-mail: [email protected].

17 National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4,

18 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan

19 Subjective category:

20 Running title: Algimonas porphyrae gen. nov., sp. nov.

21

22 The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of

23 strains 0C-2-2T, 0C-17, and LNM-3 are AB689189, AB689190, and AB689191,

24 respectively. 25 Summary

26 Three Gram-negative, stalked, motile , designated 0C-2-2T, 0C-17, and LNM-3,

27 were isolated from a red alga Porphyra yezoensis. 16S rRNA gene sequence analysis

28 revealed that the three novel strains belonged in the family Hyphomonadaceae, and are

29 closely related to Litorimonas taeanensis G5T (96.5 % 16S rRNA gene sequence

30 similarity) and Hellea balneolensis 26III/A02/215T (94.3 % similarity). The DNA G+C

31 contents of the new isolates (58.5-60.2 mol%) were clearly distinguished from L.

32 taeanensis G5 T (47.1 mol%) and H. balneolensis DSM 19091T (47.9 mol%). The G+C

33 content of L. taeanensis G5 T obtained in this study was distinct from a previous report

34 (63.6 mol%). DNA-DNA hybridization experiments showed that the new strains

35 constituted a single . Eleven phenotypic features of the three isolates differed

36 from those of both related genera. The predominant respiratory quinone was

37 ubiquinone-10 and the major fatty acid was C18:1ω7c. On the basis of polyphasic

38 taxonomic analysis, the novel strains represent a novel and species for which the

39 name Algimonas porphyrae gen. nov., sp. nov. is proposed, with the type strain 0C-2-2T

40 (= LMG 26424T = NBRC 108216T).

41

42

43

44

45

46

47

48 49 The families Caulobacteraceae and Rhodobacteraceae have been classified into the

50 order and Rhodobacterales within the class ,

51 respectively (Garrity et al., 2005). However, the family Rhodobacteraceae was

52 integrated into the order Caulobacterales on the basis of 16S rRNA gene sequences, and

53 the family Hyphomonadaceae was newly founded in the order Caulobacterales (Lee et

54 al., 2005). The family Hyphomonadaceae included four genera of ,

55 , Maricaulis, and Oceanicaulis (Lee et al., 2005) which were previously

56 classified as the family Rhodobacteraceae (Strömpl et al., 2003; Garrity et al., 2005).

57 Subsequently, additional genera, including Hellea, , Litorimonas, Ponticaulis,

58 Robiginitomaculum, and Woodsholea have been classified as members of the family

59 Hyphomonadaceae (http://www.bacterio.cict.fr). Most bacteria of the family

60 Hyphomonadaceae are characterized by having a single or two stalks. These type strains

61 have been isolated from marine habitats such as seawater (Lee et al., 2007; Alain et al.,

62 2008), brackish water (Schlesner et al., 1990), hydrothermal vent (Weiner et al., 2000),

63 beach sand (Jung et al., 2011), shellfish beds (Weiner et al., 1985), and dinoflagellates

64 (Strömpl et al., 2003). In this study, three strains designed as 0C-2-2T, 0C-17, and

65 LNM-3, were isolated from a red alga Porphyra yezoensis. We describe the

66 phylogenetic, genetic, phenotypic, and chemotaxonomic characters of the three novel

67 strains.

68

69 Thalli of P. yezoensis were cultured in a sterile modified half-strength SWM-III medium

70 (Ogata, 1970) at 17 °C. The samples of the culture medium and the thalli were

71 incubated on Marine agar 2216 (MA; Difco) at 20 °C for 2 weeks. Strains 0C-2-2T and

72 0C-17 were isolated from each culture medium in separate lots. Strain LNM-3 was 73 isolated from a thallus surface. Litorimonas taeanensis G5T and Hellea balneolensis

74 DSM 19091T were used as reference strains in all following tests. MA and Marine broth

75 2216 (MB; Difco) were used for the routine culture of all strains. Novel strains were

76 maintained at 20 °C. L. taeanensis G5T and H. balneolensis DSM 19091T were

77 maintained at 25 °C. All strains were stored at -80 °C in 20 % (v/v) glycerol.

78

79 DNA was prepared by the method of Johnson (1981). The 16S rRNA genes of the three

80 strains were amplified using universal primers of 27F and 1492R (Weisburg et al.,

81 1991). The almost-complete 16S rRNA gene sequences of strains 0C-2-2T (1331 bp),

82 0C-17 (1349 bp), and LNM-3 (1331 bp) were determined using six sequencing primers

83 27F, 519F, 1190F, 530R, 760R, and 1492R (Satomi et al., 1997) using an automated

84 DNA sequencer (model 3100; Applied Biosystems). The sequences close to the novel

85 strains were obtained from the BLAST-N algorithm (Altschul et al., 1990) in the

86 GenBank, EMBL, and DDBJ databases. The 16S rRNA gene sequences of novel strains

87 and the related strains were aligned using Clustal X programs (Thompson et al., 1997)

88 and the sequence similarities were calculated except for alignment gaps and ambiguous

89 bases, using MEGA version 4 (Tamura et al., 2007). Phylogenetic trees were

90 constructed according to three methods of neighbour-joining, maximum-likelihood, and

91 maximum-parsimony analyses. Neighbour-joining tree was constructed with a bootstrap

92 through 1000 replications based on Kimura’s 2 parameter model (Kimura, 1980) using

93 MEGA version 4 (Tamura et al., 2007). Maximum-likelihood and maximum-parsimony

94 trees were constructed using PHYLIP version 3.69 (Felsenstein, 2009). The similarities

95 of 16S rRNA gene sequences among the three strains were 100 %. The 16S rRNA gene

96 sequence of strain 0C-2-2T had the similarities of 96.5 % and 94.3 % with L. taeanensis 97 G5T and H. balneolensis 26III/A02/215T, respectively. The phylogenetic tree based on

98 neighbour-joining analysis showed that the three strains belonged in the family

99 Hyphomonadaceae, and formed an independent group with a high bootstrap value of

100 100 % (Fig. 1). The clustering formation was also recovered in the maximum-likelihood

101 and the maximum-parsimony analyses.

102

103 DNA G+C content was determined using a HPLC with a reversed-phase column

104 (COSMOSIL 5C18-MS-II, Nacalai Tesque) according to the method of Tamaoka &

105 Komagata (1984). DNA of Tetragenococcus halophilus industrial strain (Bio’c) was

106 used as a control in the experiment. DNA-DNA hybridization was performed by a

107 microplate hybridization method (Ezaki et al., 1989). Each DNA of strain 0C-2-2T, L.

108 taeanensis G5T, and H. balneolensis DSM 19091T was labeled with a photobiotin. The

109 DNA G+C contents and the DNA-DNA relatedness among the three strains, L.

110 taeanensis G5T, and H. balneolensis DSM 19091T are shown in Table 1. The G+C

111 contents of the three isolates ranged from 58.5 to 60.2 mol%. The values of L.

112 taeanensis G5T and H. balneolensis DSM 19091T were 47.1 and 47.9 mol%,

113 respectively. The value of H. balneolensis DSM 19091Twas similar to 46.8 mol%

114 (original value, Alain et al., 2008), but the value of L. taeanensis G5T measured in this

115 study was distinct from 63.6 mol% (original value, Jung et al., 2011). The content of T.

116 halophilus industrial strain was 36.1 mol%, which was similar to a previous value by

117 Justé et al. (2012), and our method yield accurate measurements in a previous study

118 (Fukui et al., 2012). Furthermore, we examined a partial sequence of the 16S rRNA gene of

119 L. taeanensis G5T, and confirmed that the sequence had 100 % similarity with that in database.

120 There were significant differences of > 10 mol% in the G+C contents between the three 121 strains (58.5-60.2 mol%) and the closest phylogenic neighbours (47.1-47.9 mol%),

122 which indicates the three strains belong to a different genus (Stackebrandt & Liesack,

123 1993). DNA-DNA hybridization relatedness among strains 0C-2-2T, 0C-17, and LNM-3

124 were 99.0-101.6 %, suggested that the three isolates belonged to the same species

125 (Wayne et al., 1987). On the other hand, strain 0C-2-2T was a different species from L.

126 taeanensis G5T (1.2-1.8 %) and H. balneolensis DSM 19091T (1.2-2.5 %).

127

128 Cell morphology and presences of a flagellum and a stalk were observed by a

129 transmission electron microscope (JEM-1200EX II, JEOL). Motility and Gram-stain

130 reaction (Merck) was ascertained by use of a light microscope (BX50, Olympus).

131 Colony morphology and pigmentation were observed on MA. Pigments of the cells

132 were extracted with acetone/methanol (7:2, v/v) (Biebl et al., 2005) and the absorption

133 spectrum between 350 and 900 nm was determined using a U-2000A spectrophotometer

134 (Hitachi). The following phenotypic tests were carried out using inoculated samples

135 over a period of 2 weeks. Growth under an anaerobic condition was tested on MA using

136 an Anaero Pack (Mitsubishi Gas Chemical). Temperature for growth was determined on

137 MA at 5-40 °C (at intervals of 5 °C). pH range for growth was determined in MB

138 adjusted to pH 4.0-10.0 (at intervals of one pH unit), using 1M NaOH and 1M HCl

139 solutions. After autoclaving, the pH values of MB were again measured and adjusted

140 with 1M NaOH and 1M HCl solutions. NaCl concentration for growth was tested in MB

141 prepared with various concentrations of 0.02, 0.5, 1-10 % (at intervals of 1 %, w/v)

142 NaCl (Alain et al., 2008). Phenotypic tests performed included the following: oxidase

143 (Oxidase reagent; bioMérieux) and catalase activities (3 % v/v H2O2), methyl red and

144 Voges-Proskauer reactions (Nissui Pharmaceutical), H2S production (Eiken Chemical), 145 hydrolysis of agar, alginate, DNA, starch, Tween 20, 40, and 80 (Sawabe et al., 1995),

146 and oxidation tests of various 10 % (w/v) carbon sources using OF basal medium

147 (Eiken Chemical). Other biochemical features were determined by using API 20NE,

148 API ZYM, and API 50CH (bioMérieux) following the manufacturer’s instructions

149 except that cells were suspended in 75 % artificial seawater (ASW; containing l-1

150 distilled water: 30 g NaCl, 0.7 g KCl, 5.3 g MgSO4. 7H2O, 10.8 g MgCl2. 6H2O,1.3 g

151 CaSO4. 2H2O). The test strips of API 20 and API ZYM were inoculated for 2 weeks and

152 1 day, respectively. Carbon utilization tests (API 50CH) were performed with basal

153 medium broth (Baumann et al., 1972) supplemented with 0.1 % (w/v) yeast extract for 3

154 weeks. Antibiotic susceptibility was determined using a disc diffusion method with

155 commercial disks (Eiken Chemical, Becton Dickinson) or filter paper disks impregnated

156 with different antibiotics. Antibiotic susceptibility as scored was positive when a

157 diameter of the inhibitory zone was above 10 mm (Alain et al., 2008). The phenotypic

158 characteristics of the three isolates are given in the species description and Table 2. The

159 main phenotypic features of the three isolates were rod-shaped cells, possession of a

160 polar flagellum or a prostheca (Fig. 2), Gram-negative staining reaction, and aerobic

161 growth, features that are frequently observed for members of the family

162 Hyphomonadaceae. The three isolates produced orange pigments with a maximum

163 absorption at 483-486 nm which was identical to a carotenoid (Biebl et al., 2005). The

164 absorption peak around 770 nm characterized by the presence of bacteriochlorophyll a

165 (Biebl et al., 2005) was not observed. The differences in phenotypic traits among the

166 three strains, L. taeanensis G5T, and H. balneolensis DSM 19091T are shown in Table 2.

167 In particular, eleven phenotypic features (mode of division, growth at 35 °C, nitrate

168 reduction, hydrolysis of gelatin, lipase and cysteine arylamidase activities, utilizations 169 of sucrose, starch, glycogen, and 2-keto-gluconate, and susceptibility to streptomycin)

170 were different between the three strains and the related species.

171

172 Isoprenoid quinones were extracted with chloroform/methanol (2:1, v/v) after

173 incubation in MB, and the type and length were determined by a HPLC method

174 according to the method described by Akagawa-Matsushita et al. (1992). Polar lipids

175 and methyl esters of cellular fatty acids (FAMEs) were extracted according to the

176 method described by Minnikin et al. (1984) and Ikemoto et al. (1978), respectively,

177 after the three isolates were cultured on MA at 20 °C for 10 days in stationary phase of

178 growth. The spots of the polar lipids on two-dimensional TLC were identified by

179 spraying with the appropriate detection reagents (Minnikin et al, 1984; Komagata &

180 Suzuki, 1987). The FAMEs was examined using a GC equipped with a flame-ionization

181 detecter (GC-2010; Shimadzu) and a capillary column (Omegawax 320; Supelco) and

182 were identified by comparing the retention times of a sample to those of a standard

183 (Supelco, USA). Furthermore, for the fatty acids (>1 % of the total), the FAMEs and the

184 DMOX derivatization (Luthria & Sprecher, 1993) were identified by a GC-MS (HP

185 5973N; Agilent Technologies). The predominant isoprenoid quinone of all isolates was

186 ubiquinone-10 (Q-10) which was observed in many members of the class

187 Alphaproteobacteria (Martens et al., 2006). TLC profiles of the polar lipids of the five

188 strains are shown in Fig. 3. The common polar lipids in all five isolates were

189 phosphatidylglycerol, glucuronopyranosyldiglyceride, mono-glycosyldiglyceride, three

190 unidentified phospholipids, and an unidentified glycolipid (GL1). Another unidentified

191 glycolipid (GL2) was detected in only L. taeanensis G5T. The presence of

192 phosphatidylglycerol, glucuronopyranosyldiglyceride, and mono-glycosyldiglyceride 193 are characteristic of members of the family Hyphomonadaceae (Alain et al., 2008). The

194 total cellular fatty acid compositions of all isolates are shown in Table 3. The major

195 fatty acids in the three strains were C18:1ω7c (34.9-71.6 %), which is a typical feature of

196 the class Alphaproteobacteria (Alain et al., 2008), followed by C18:1 2-OH, C18:0, and

197 C19:1ω8c. The compositions of C17:0, C18:1ω7c, C19:1ω8c, and C19:1 2-OH (>5 %) in strain

198 0C-17 were distinctly different from those in strains 0C-2-2T and LNM-3. In order to

199 ascertain these differences among three strains, we also examined FAMEs at early

200 period of incubation (8 days), but these results were similar to those in 10 days of

T 201 incubation. The three isolates specifically had C19:0, whereas either L. taeanensis G5 or

T 202 H. balneolensis DSM 19091 specifically had C18:2ω7c, 13c and C19:2ω7c, 14c. In

203 particular, the part of unsaturated fatty acids of nonadecanoate and 2-hydroxy fatty acids

204 were firstly detected in the family Hyphomonadaceae. The differences between this

205 study and previous studies (Alain et al., 2008; Jung et al., 2011) can be attributed to the

206 extraction method of FAMEs and culture temperatures.

207

208 In conclusion, strains 0C-2-2T, 0C-17, and LNM-3 are a novel genus and species

209 Algimonas porphyrae gen. nov., sp. nov on the basis of phylogenetic, genetic,

210 phenotypic, and chemotaxonomic features. In particular, A. porphyrae strains had

211 higher G+C contents (58.5-60.2 mol%) than related genera (47.1-47.9 mol%). The

212 eleven phenotypic features and the compositions of several fatty acids in the three

213 strains were also distinguished from those of the related genera.

214

215 Description of Algimonas gen. nov.

216 Algimonas (Al.gi.mo'nas. L. n. alga, seaweed; L. fem. n. monas, a monad, unit; N.L. 217 fem. n. Algimonas, a unit (bacterium) isolated from seaweed).

218 Cells are Gram-negative and straight to slight curved rods. Many cells are non-stalked

219 and possess a polar flagellum for motility. Some cells possess a prostheca.

220 Multiplication occurs by binary fission. Cells produce orange carotenoid pigments.

221 Bacteriochlorophyll a is not found. Aerobic condition and NaCl are required for growth.

222 The predominant isoprenoid quinone is Q-10. Polar lipids are phosphatidylglycerol,

223 glucuronopyranosyldiglyceride, mono-glycosyldiglyceride, three unidentified

224 phospholipids, and an unidentified glycolipid. The major cellular fatty acid is C18:1ω7c.

225 The genus belongs phylogenetically to the family Hyphomonadaceae in the class

226 Alphaproteobacteria. The G+C contents of the DNA are 58.5 to 60.2 mol%. The type

227 species of the genus is Algimonas porphyrae.

228

229 Description of Algimonas porphyrae sp. nov.

230 Algimonas porphyrae (por.phy'rae. N.L. gen. n. porphyrae, of Porphyra, isolated from

231 Porphyra yezoensis).

232 In addition to the description of the genus, the following properties are exhibited. The

233 cells are 2.13 μm long and 0.37 μm in diameter (n= 25) in MB after 5 days incubation.

234 Colonies on MA are circular, convex, entire, and 0.22 mm in diameter (n= 25) after 10

235 days incubation. Growth of the type strain occurs at 10-30 °C (optimum, 20 °C), pH

236 6.0-9.0 (optimum, pH 7.0-8.0), and 1-5 % of NaCl (optimum, 2-3 %). Oxidase is

237 negative and catalase is positive. Methyl red and Voges-Proskauer reactions are negative.

238 H2S and indole are not produced. Aesculin, Tween 20, 40, and 80 are hydrolysed, but

239 agar, alginate, DNA, gelatin, and starch are not. No acid is produced from carbohydrates.

240 Nitrate reduction activities are present. With API ZYM system, the type strain is 241 positive in alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, valine

242 arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, but negative in

243 lipase, cystine arylamidase, trypsin, chymotrypsin, α-galactosidase, β-galactosidase,

244 β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase,

245 α-mannosidase, and α-fucosidase. With API 50CH system, the type strain utilizes

246 galactose, glucose, mannose, esculin ferric citrate, maltose, and starch are utilized, but

247 not glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol,

248 methyl-β-D-xylopyranoside, fructose, sorbose, rhamnose, dulcitol, inositol, mannitol,

249 sorbitol, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside,

250 N-acetyl-glucosamine, amygdalin, arbutin, salicin, cellobiose, lactose, melibiose,

251 sucrose, trehalose, inulin, melezitose, raffinose, glycogen, xylitol, gentiobiose,

252 D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate,

253 2-keto-gluconate, or 5-keto-gluconate. The type strain is susceptible to (μg per disc

254 unless otherwise stated) ampicillin (10), carbenicillin (100), ciprofloxacin (5),

255 chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), nalidixic

256 acid (30), neomycin (30), norfloxacin (10), novobiocin (30), penicillin G (10U),

257 rifampicin (5), streptomycin (10), and vancomycin (30) but resistance to polymyxin B

258 (300 U) and tetracycline (30).

259 The type strain is 0C-2-2T (LMG 26424T = NBRC 108216T) isolated from the red alga

260 Porphyra yezoensis. Strains 0C-17 (LMG 26425 = NBRC 108217) and LNM-3 (LMG

261 26426 = NBRC 108218) are reference strains.

262

263 Acknowledgements

264 We acknowledge Dr. J. P. Euzéby for support in the Latin etymologies of the genus 265 and species name. We are grateful to Professor C. O. Jeon of Chung-Ang

266 University for kindly providing Litorimonas taeanensis G5T. This study was supported

267 by Research Fellowships of the Japan Society for the Promotion of Science for Young

268 Scientists (Project No. 23·4176).

269

270 References

271 Akagawa-Matsushita, M., Itoh, T., Katayama, Y., Kuraishi, H. & Yamasato, K.

272 (1992). Isoprenoid quinone composition of some marine Alteromonas, Marinomonas,

273 Deleya, Pseudomonas and Shewanella species. J Gen Microbiol 138, 2275-2281.

274 Alain, K., Tindall, B. J., Intertaglia, L., Catala, P. & Lebaron, P. (2008). Hellea

275 balneolensis gen. nov., sp. nov., a prosthecate alphaproteobacterium from the

276 Mediterranean Sea. Int J Syst Evol Microbiol 58, 2511-2519.

277 Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic

278 local alignment search tool. J Mol Biol 215, 403-410.

279 Baumann, L., Baumann, P., Mandel, M. & Allen, R. D. (1972). of aerobic

280 marine eubacteria. J Bacteriol 110, 402-429.

281 Biebl, H., Allgaier, M., Tindall, B. J., Koblizek, M., Lünsdorf, H., Pukall, R. &

282 Wagner-Döbler, I. (2005). Dinoroseobacter shibae gen. nov., sp. nov., a new aerobic

283 phototrophic bacterium isolated from dinoflagellates. Int J Syst Evol Microbiol 55,

284 1089-1096.

285 Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic

286 acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to

287 membrane filter hybridization in which radioisotopes are used to determine genetic

288 relatedness among bacterial strains. Int J Syst Bacteriol 39, 224-229. 289 Felsenstein, J. (2009). PHYLIP (phylogeny inference package), version 3.69.

290 Distributed by the author. Department of Genome Sciences, University of Washington,

291 Seattle, USA.

292 Fukui, Y., Abe, M., Kobayashi, M., Ishihara, K., Oikawa, H., Yano, Y. & Satomi, M.

293 (2012). Maritalea porphyrae sp. nov., isolated from a red alga (Porphyra yezoensis),

294 and transfer of Zhangella mobilis to Maritalea mobilis comb. nov. Int J Syst Evol

295 Microbiol 62, 43-48.

296 Garrity, G. M., Bell, J. A. & Lilburn, T. (2005). Order III. Rhodobacterales ord. nov.

297 In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2C, pp. 161. Edited by D.

298 J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer.

299 Ikemoto, S., Katoh, K. & Komagata, K. (1978). Cellular fatty acid composition in

300 methanol-utilizing bacteria. J Gen Appl Microbiol 24, 41-49.

301 Johnson, J. L. (1981). Genetic characterization. In Manual of Methods for General

302 Bacteriology, pp. 450-472. Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W.

303 Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, D.C.: American Society

304 for Microbiology.

305 Jung, J. Y., Kim, J. M., Jin, H. M., Kim, S. Y., Park, W. & Jeon, C. O. (2011).

306 Litorimonas taeanensis gen. nov., sp. nov., isolated from a sandy beach. Int J Syst

307 Evol Microbiol 61, 1534-1538.

308 Justé, A., Trappen, S. V. Verreth, C., Cleenwerck, I., Vos, P. D., Lievens, B. &

309 Willems, K. A. (2012). Characterization of Tetragenococcus strains from sugar thick

310 juice reveals a new species, Tetragenococcus osmophilus sp. nov., and divides

311 Tetragenococcus halophilus into two subspecies, T. halophilus subsp. halophilus subsp.

312 nov. and T. halophilus subsp. flandriensis subsp. nov. Int J Syst Evol Microbiol 313 62, 129-137.

314 Kimura, M. (1980). A simple method for estimating evolutionary rates of base

315 substitutions through comparative studies of nucleotide sequences. J Mol Evol 16,

316 111-120.

317 Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial

318 systematics. Methods Microbiol 19, 161-207.

319 Lee, K.-B., Liu, C.-T., Anzai, Y., Kim, H., Aono, T. & Oyaizu, H. (2005). The

320 hierarchical system of the ‘Alphaproteobacteria’: description of Hyphomonadaceae

321 fam. nov., Xanthobacteraceae fam. nov. and Erythrobacteraceae fam. nov. Int J Syst

322 Evol Microbiol 55, 1907-1919.

323 Lee, K., Lee, H. K., Choi, T.-H. & Cho, J.-C. (2007). Robiginitomaculum antarcticum

324 gen. nov., sp. nov., a member of the family Hyphomonadaceae, from Antarctic seawater.

325 Int J Syst Evol Microbiol 57, 2595-2599.

326 Luthria, D. L. & Sprecher, H. (1993). 2-Alkenyl-4,4-dimethyloxazolines as

327 derivatives for the structural elucidation of isomeric unsaturated fatty acids. Lipids 28,

328 561-564.

329 Martens, T., Heidorn, T., Pukall, R., Simon, M., Tindall, B. J. & Brinkhoff, T.

330 (2006). Reclassification of Roseobacter gallaeciensis Ruiz-Ponte et al. 1998 as

331 Phaeobacter gallaeciensis gen. nov., comb. nov., description of Phaeobacter inhibens

332 sp. nov., reclassification of Ruegeria algicola (Lafay et al. 1995) Uchino et al. 1999 as

333 Marinovum algicola gen. nov., comb. nov., and emended descriptions of the genera

334 Roseobacter, Ruegeria and Leisingera. Int J Syst Evol Microbiol 56, 1293-1304.

335 Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M.,

336 Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of 337 bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233-241.

338 Ogata, E. (1970). On a new algal culture medium SWM-III. Bull Jpn Soc Phycol 18,

339 171-173 (in Japanese).

340 Satomi, M., Kimura, B., Mizoi, M., Sato, T. & Fujii, T. (1997). Tetragenococcus

341 muriaticus sp. nov., a new moderately halophilic lactic acid bacterium isolated from

342 fermented squid liver sauce. Int J Syst Bacteriol 47, 832-836.

343 Sawabe, T., Oda, Y., Shiomi, Y. & Ezura, Y. (1995). Alginate degradation by bacteria

344 isolated from the gut of sea urchins and abalones. Microb Ecol 30, 193-202.

345 Schlesner, H., Bartels, C., Sittig, M., Dorsch, M. & Stackebrandt, E. (1990).

346 Taxonomic and phylogenetic studies on a new taxon of budding, hyphal Proteobacteria,

347 Hirschia baltica gen. nov., sp. nov. Int J Syst Bacteriol 40, 443-451.

348 Stackebrandt, E. & Liesack, W. (1993). Nucleic acids and classification. In Handbook

349 of New Bacterial Systematics, pp. 151–194. Edited by M. Goodfellow & A. G.

350 O'Donnell. London: Academic Press.

351 Strömpl, C., Hold, G. L., Lünsdorf, H., Graham, J., Gallacher, S., Abraham, W-R.,

352 Moore, E. R. B. & Timmis K. N. (2003). Oceanicaulis alexandrii gen. nov., sp. nov., a

353 novel stalked bacterium isolated from a culture of the dinoflagellate Alexandrium

354 tamarense (Lebour) Balech. Int J Syst Evol Microbiol 53, 1901-1906.

355 Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by

356 reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25,

357 125-128.

358 Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular

359 evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24,

360 1596-1599. 361 Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.

362 (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence

363 alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882.

364 Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O.,

365 Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other

366 authors (1987). Report of the ad hoc committee on reconciliation of approaches to

367 bacterial systematics. Int J Syst Bacteriol 37, 463-464.

368 Weiner, R. M., Devine, R. A., Powell, D. M., Dagasan, L. & Moore, R. L. (1985).

369 Hyphomonas oceanitis sp. nov., Hyphomonas hirschiana sp. nov., and Hyphomonas

370 jannaschiana sp. nov. Int J Syst Bacteriol 35, 237-243.

371 Weiner, R. M., Melick, M., O'Neill, K. & Quintero, E. (2000). Hyphomonas

372 adhaerens sp. nov., Hyphomonas johnsonii sp. nov. and Hyphomonas rosenbergii sp.

373 nov., marine budding and prosthecate bacteria. Int J Syst Evol Microbiol 50, 459-469.

374 Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal

375 DNA amplification for phylogenetic study. J Bacteriol 173, 697-703.

376

377 Figure legends

378 Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences

379 showing the relationships among strains 0C-2-2T, 0C-17, and LNM-3, and

380 other type strains in the family Hyphomonadaceae. Bootstrap values (>50 %) based on

381 1000 replications are listed as percentages at branch nodes. Filled circles indicate that

382 the corresponding nodes were recovered by all algorithms. Open circles indicate that

383 the corresponding nodes were recovered by two algorithms. Escherichia coli ATCC

384 11775T was used as an outgroup. Bar, 0.02 % sequence divergence. 385

386 Fig. 2. Transmission electron micrographs of strain 0C-2-2T negatively stained. (a) A

387 cell with a polar flagellum. Bar = 0.5 μm. (b) A cell with a prostheca in a state of binary

388 fission. Bar = 0.5 μm.

389

390 Fig. 3. Polar lipid profiles of (a) strain 0C-2-2T, (b) strain 0C-17, (c) strain LNM-3, (d)

391 L. taeanensis G5T, and (e) H. balneolensis DSM 19091T. Two-dimensional TLC was

392 performed by chloroform-methanol-water 65: 25: 4 (vol/vol) as the first direction and

393 chloroform-acetic acid-methanol-water 80:15:12:4 (vol/vol) as the second direction. All

394 polar lipids were detected with 5 % ethanolic molybdophosphoric acid.

395 Phosphatidylglycerol (PG) was identified using a commercial standard (DOOSAN

396 Serdary Research Laboratories). PG, phosphatidylglycerol; GUDG,

397 glucuronopyranosyldiglyceride; MGDG, mono-glycosyldiglyceride; PL1-3, unidentified

398 phospholipid; GL1-2, unidentified glycolipid. 77 Hyphomonas hirschiana VP-5T (AF082794) 93 Hyphomonas rosenbergii VP-6 T (AF082795) 100 Hyphomonas neptunium ATCC 15444T (AF082798) Hyphomonas polymorpha DSM2665T (AJ227813) Hyphomonas adhaerens MHS-3T (AF082790) 100 100 Hyphomonas jannaschiana ATCC 33883T (AJ227814) Hyphomonas oceanitis SCH-89T (AF082797) 97 87 Hyphomonas johnsonii MHS-2T (AF082791) Ponticaulis koreensis GSW-23T (FM202497) T 68 87 Henriciella litoralis SD10 (FJ230835) T 100 Henriciella marina Iso4 (EF660760) 98 Henriciella aquimarina P38T (EU819081) 98 Hirschia baltica DSM 5838T (AJ421782) 100 Hirschia maritima GSW-2T (FM202386) Robiginitomaculum antarcticum IMCC3195T (EF495229) Hellea balneolensis 26III/A02/215T (AY576758) 100 Litorimonas taeanensis G5T (FJ230838) 85 Algimonas porphyrae 0C-2-2T (AB689189) 99 Algimonas porphyrae 0C-17 (AB689190) 100 Algimonas porphyrae LNM-3 (AB689191) 80 Oceanicaulis alexandrii C116-18T (AJ309862) Woodsholea maritima CM243T (AJ578476) 100 Maricaulis salignorans MCS 18T (AJ227806) 67 Maricaulis washingtonensis MCS 6T (AJ227804) 99 Maricaulis maris ATCC 15268T (AJ227802) T 81 Maricaulis parjimensis MCS 25 (AJ227808) 100 Maricaulis virginensis VC-5T (AJ301667) Escherichia coli ATCC 11775T (X80725)

0.02 Fig. 1. Fukui et al. (a) (b)

Fig. 2. Fukui et al. (a) PL1 MGDG (b)PL1 MGDG (c) PL1 MGDG PL2 PL2 PL2 GUDG GUDG GUDG PG PG PG

PL3 PL3 PL3 GL1 GL1 GL1

PL1 (d) MGDG (e) PL1 MGDG PL2 GUDG PL2 GUDG PG GL2 PG PL3

PL3 GL1 GL1

Fig. 3. Fukui et al. Table 1. DNA G+C contents and DNA-DNA hybridization values among novel strains and other related genera Strains DNA G+C content DNA-DNA reassociation value (%) with biotinylated DNA from: (mol%) A. porphyrae sp. nov. L. taeanensis H. balneolensis 0C-2-2T G5T DSM 19091T Algimonas porphyrae sp. nov.: 0C-2-2T 58.5 100.0 1.2 1.2 0C-17 59.0 101.6 1.5 1.9 LNM-3 60.2 99.0 2.0 2.4 Litorimonas taeanensis G5T 47.1 1.8 100.0 3.9 Hellea balneolensis DSM19091T 47.9 2.5 3.4 100.0 Table 2. Differential phenotypic characteristics among novel strains and other related species

Species/strains: 1, A. porphyrae sp. nov. 0C-2-2T; 2, A. porphyrae sp. nov. 0C-17; 3, A. porphyrae sp. nov. LNM- 3; 4, L. taeanensis G5T; 5, H. balneolensis DSM 19091T. +, Positive; -, negative; S, susceptible; R, resistant.

Characteristic 1 2 3 4 5 Mode of division Binary fission Binary fission Binary fission Budding Budding Pigmentation Orange Orange Orange Pale-orange Orange Growth at 5 °C---+- 10 °C+--+- 35 °C---++ Growth in pH 6.0, pH 9.0 + + + + - 0.5 % NaCl - - - + - 1 % NaCl + + + + - 5 % NaCl + - - + - 6-7 % NaCl - - - + - Oxidase activity - - - + - Hydrolysis of DNA - - - - + Tween 40, 80 + + + - + API 20NE Nitrate reduction + + + - - Hydrolysis of gelatin - - - + + Enzyme activity (API ZYM) Lipase - - - + + Cystine arylamidase - - - + + Acid phosphatase + - - + + Carbon utilization (API 50CH) Erythritol - - - - + Ribose - - - + - D-xylose - - - + - Adonitol - - - - + Galactose + - - - - Glucose + + + + - Mannose + - + - - Rhamnose - - - - + Dulcitol - - - - + Mannitol - - - - + Arbutin - - - - + Cellobiose - - - + - Maltose + + + - + Sucrose - - - + + Inulin - - - - + Starch + + + - - Glycogen - - - + + D-tagatose - - - - + L-arabitol - - - - + 2-keto-gluconate - - - + + Susceptibility to Carbenicillin (100 μg) S S R R S Kanamycin (30 μg) S S S R S Polymyxin B (300 U) R S R R R Streptomycin (10 μg) S S S R R Vancomycin (30 μg) S R S R S Table 3. Cellular fatty acid contents (%) of Algimonas porphyrae sp. nov. and other related genera Species/strains: 1, A. porphyrae sp. nov. 0C-2-2T; 2, A. porphyrae sp. nov. 0C-17; 3, A. porphyrae sp. nov. LNM-3; 4, L. taeanensis G5T; 5, H. balneolensis DSM 19091T. tr, trace amount (less than 0.5 %); ND, not detected. Fatty acid (%) 1 2 3 4 5

C17:0 0.6 6.1 0.5 4.4 3.5

C18:0 4.9 2.1 4.9 1.6 1.4

C19:0 1.4 0.5 1.2 ND ND

C16:1ω7c tr tr tr 1.1 ND

C17:1ω8c ND tr ND tr 1.3

C17:1ω7c tr 1.1 tr 5.1 0.9

C17:1ω6c tr 2.9 tr 6.2 2.8

C18:1ω7c 71.4 34.9 71.6 42.6 27.4

C18:2ω7c, 13c ND ND ND 1.4 2.6

C19:1ω9c tr 1.7 tr 0.7 1.3

C19:1ω8c 2.4 26.9 2.2 13.9 24.8

C19:1ω7c tr 3.8 tr 1.6 5.2

C19:2ω7c, 14c ND ND ND ND 5.7

C17:1 2-OH tr 1.3 tr 2.2 1.3

C18:1 2-OH 11.1 7.7 11.1 13.0 1.3

C19:1 2-OH tr 6.6 tr 1.1 1.3

iso-C18:1 2-OH tr tr ND tr 1.6

iso-C19:1 2-OH tr ND tr ND 8.8

iso-C20:1 2-OH tr tr ND ND 3.9

Unknown ECL 26.141 1.3 0.7 1.5 1.3 ND

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