IJSEM Papers in Press. Published May 26, 2015 as doi:10.1099/ijs.0.000352

International Journal of Systematic and Evolutionary Microbiology Pontivivens insulae gen. nov., sp. nov., isolated from seawater --Manuscript Draft-Manuscript Number:

IJS-D-15-00096R1

Full Title:

Pontivivens insulae gen. nov., sp. nov., isolated from seawater

Short Title:

Pontivivens insulae gen. nov., sp. nov.

Article Type:

Note

Section/Category:

New taxa - Proteobacteria

Corresponding Author:

Jung-Hoon Yoon Sungkyunkwan University Suwon, KOREA, REPUBLIC OF

First Author:

Sooyeon Park

Order of Authors:

Sooyeon Park Sung-Min Won Ji-MIn Park Yong-Taek Jung Jung-Hoon Yoon

Manuscript Region of Origin:

KOREA, REPUBLIC OF

Abstract:

A Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated GYSW-23T, was isolated from seawater off Geoje island on the South Sea, South Korea. Strain GYSW-23T grew optimally at 25 C, at pH 7.0-8.0 and in the presence of approximately 2.0-3.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain GYSW-23T forms a distinct evolutionary lineage independent of other taxa of the family Rhodobacteraceae. It exhibited 16S rRNA gene sequence similarity values of 94.0, 93.6, 93.5, 93.4 and 93.4 % to the type strains of Roseovarius aestuarii, 'Actinobacterium atlanticum', Ruegeria marina, Roseovarius pacificus and Oceanicola litoreus, respectively. Strain GYSW-23T contained Q-10 as the predominant ubiquinone and C18:1 ω7c as the major fatty acid. The major polar lipids of strain GYSW-23T were phosphatidylcholine, phosphatidylglycerol and one unidentified aminolipid. The fatty acid and polar lipid profiles of strain GYSW-23T were distinguished from those of the phylogenetically related taxa. The DNA G+C content of strain GYSW-23T was 60.6 mol%. On the basis of the phylogenetic, chemotaxonomic and other phenotypic properties, strain GYSW23T is considered to represent a new genus and species within the family Rhodobacteraceae, for which the name Pontivivens insulae gen. nov., sp. nov. is proposed. The type strain of Pontivivens insulae is GYSW-23T (= KCTC 42458T = CECT 8812T).

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1

Pontivivens insulae gen. nov., sp. nov., isolated from seawater

2 3

Sooyeon Park,1† Sung-Min Won,1† Ji-Min Park,1 Yong-Taek Jung1,2 and Jung-Hoon Yoon1

4 5

1

6

Suwon, South Korea

7

2

8

Korea

Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu,

University of Science and Technology (UST), 113 Gwahak-ro, Yuseong-gu, Daejeon, South

9 10 11

Running title: Pontivivens insulae gen. nov., sp. nov.

12 13

Subject category: New taxa - Proteobacteria

14 15 16 17 18 19 20

Author for correspondence: Prof. Jung-Hoon Yoon Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, South Korea Tel : +82-31-290-7800 Fax : +82-31-290-7882 e-mail : [email protected]

21 22



These authors contributed equally to this work.

23 24 25 26

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GYSW-23T is KP662553. Four supplementary figures are available with the online version of this paper.

1

27

(Abstract)

28

A Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain,

29

designated GYSW-23T, was isolated from seawater off Geoje island on the South Sea, South

30

Korea. Strain GYSW-23T grew optimally at 25 C, at pH 7.0-8.0 and in the presence of

31

approximately 2.0-3.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences

32

revealed that strain GYSW-23T forms a distinct evolutionary lineage independent of other taxa

33

of the family Rhodobacteraceae. It exhibited 16S rRNA gene sequence similarity values of 94.0,

34

93.6, 93.5, 93.4 and 93.4 % to the type strains of Roseovarius aestuarii, ‘Actinobacterium

35

atlanticum’, Ruegeria marina, Roseovarius pacificus and Oceanicola litoreus, respectively.

36

Strain GYSW-23T contained Q-10 as the predominant ubiquinone and C18:1 ω7c as the major

37

fatty acid. The major polar lipids of strain GYSW-23T were phosphatidylcholine,

38

phosphatidylglycerol and one unidentified aminolipid. The fatty acid and polar lipid profiles of

39

strain GYSW-23T were distinguished from those of the phylogenetically related taxa. The DNA

40

G+C content of strain GYSW-23T was 60.6 mol%. On the basis of the phylogenetic,

41

chemotaxonomic and other phenotypic properties, strain GYSW-23T is considered to represent a

42

new genus and species within the family Rhodobacteraceae, for which the name Pontivivens

43

insulae gen. nov., sp. nov. is proposed. The type strain of Pontivivens insulae is GYSW-23T (=

44

KCTC 42458T = CECT 8812T).

45 2

46

The family Rhodobacteraceae, belonging to the order Rhodobacterales of the class

47

Alphaproteobacteria (Garrity et al., 2005, 2006), has been known to be one of the most

48

abundant groups in global marine environments (Rappé et al., 2000; Buchan et al., 2005;

49

Brinkhoff et al., 2008). From our studies to screen novel bacteria from a variety of seawaters off

50

Geoje island on the South Sea of the Korean Peninsula, many novel bacterial strains belonging

51

to the Roseobacter clade have recently been isolated and characterized taxonomically (Park et

52

al., 2014a, d). One of these isolates, designated GYSW-23T, is described in this study.

53

Comparative 16S rRNA gene sequence analysis showed that this novel strain is

54

phylogenetically affiliated to members of the family Rhodobacteraceae but its exact taxonomic

55

status is not clear due to equidistant 16S rRNA gene sequence similarities with representatives

56

of several genera. The aim of the present work was to determine the exact taxonomic position of

57

strain GYSW-23T by using a polyphasic characterization which included the determination of

58

chemotaxonomic and other phenotypic properties and detailed phylogenetic investigations

59

based on 16S rRNA gene sequences.

60 61 62

A seawater sample was collected from Geoje island on the South Sea, South Korea, and used as

63

the source for the isolation of bacterial strains. Strain GYSW-23T was isolated by the standard

64

dilution plating technique at 25 °C on marine agar 2216 (MA; Becton, Dickinson and Company)

3

65

and cultivated routinely under the same culture conditions. Ruegeria marina JCM 16262T,

66

Roseovarius pacificus LMG 24575T and ‘Actibacterium atlanticum’ LMG 27158T, and

67

Roseovarius aestuarii SMK-122T and Oceanicola litoreus M-M22T, which were used as

68

reference strains for phenotypic characterization and the analyses of fatty acids and polar lipids,

69

were obtained from the Japan Collection of Microorganisms (JCM; Japan), the Laboratorium

70

voor Microbiologie Universiteit Gent (LMG; Belgium) and our previous studies (Yoon et al.,

71

2008; Park et al., 2013), respectively. The cell morphology, Gram reaction, pH range for growth

72

and anaerobic growth were determined as described by Park et al. (2014b). The presence of

73

poly- -hydroxybutyrate granules was investigated by epifluorescence microscopy (BX51;

74

Olympus) after staining with Nile blue A as described by Ostle & Holt (1982). Growth at 4, 10,

75

20, 25, 28, 30, 35, 37 and 40 °C was measured on MA to determine the optimal temperature and

76

temperature range for growth. Growth at various concentrations of NaCl (0, 0.5 and 1.0-10.0 %,

77

at increments of 1.0 %) was investigated by supplementing with appropriate concentrations of

78

NaCl in marine broth 2216 (MB) prepared according to the formula of the Becton, Dickinson

79

and Company medium except that NaCl was excluded. The requirement for Mg2+ ions was

80

investigated by using MB, prepared according to the formula of the Becton, Dickinson and

81

Company medium, that comprised all of the constituents except MgCl2 and MgSO4. Catalase

82

and oxidase activities were determined as described by Lányí (1987). Hydrolysis of aesculin,

83

casein, starch, hypoxanthine, Tween 80, L-tyrosine and xanthine was tested on MA using the

4

84

substrate concentrations described by Barrow & Feltham (1993). Nitrate reduction was

85

investigated as described previously (Lányí, 1987) with the modification that artificial seawater

86

was used for the preparation of media. Hydrolysis of gelatin and urea were investigated by

87

using nutrient gelatin and urea agar base media (Becton, Dickinson and Company), respectively,

88

with the modification that artificial seawater was used for the preparation of media. The

89

artificial seawater contained (l-1 distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2·6H2O,

90

5.94 g MgSO4·7H2O and 1.3 g CaCl2·2H2O (Bruns et al., 2001). Utilization of various

91

substrates for growth was tested according to Baumann & Baumann (1981), using

92

supplementation with 1 % (v/v) vitamin solution (Staley, 1968) and 2 % (v/v) Hutner’s mineral

93

salts (Cohen-Bazire et al., 1957). Susceptibility to antibiotics was tested on MA plates using

94

antibiotic discs (Advantec) containing the following (g per disc unless otherwise stated):

95

ampicillin (10), carbenicillin (100), cephalothin (30), chloramphenicol (100), gentamicin (30),

96

kanamycin (30), lincomycin (15), neomycin (30), novobiocin (5), oleandomycin (15), penicillin

97

G (20 IU), polymyxin B (100 IU), streptomycin (50) and tetracycline (30). Enzyme activities

98

were determined, after incubation for 8 h at 25 ºC, by using the API ZYM system (bioMérieux);

99

the strip was inoculated with cells suspended in artificial seawater from which CaCl 2 was

100

excluded to avoid the formation of precipitates. For spectral analysis of in vivo pigment

101

absorption, strain GYSW-22T was cultivated aerobically in the dark at 25 °C in MB. The culture

102

was washed twice using centrifugation with a MOPS buffer (MOPS/NaOH, 0.01 M; KCl, 0.1

5

103

M; MgCl2, 0.001 M; pH 7.5) and disrupted by means of sonication (VC505; Sonics & Materials,

104

Inc.). After removal of cell debris by centrifugation, the absorption spectrum of the supernatant

105

was examined on the Eon Microplate spectrophotometer (Biotek).

106 107

Cell biomass of strain GYSW-23T for DNA extraction and for the analyses of isoprenoid

108

quinones and polar lipids was obtained from cultures grown for 3 days in MB at 25 °C, and cell

109

biomass of R. aestuarii SMK-122T, R. pacificus LMG 24575T, ‘A. atlanticum’ LMG 27158T and

110

R.marina JCM 16262T for polar lipid analysis was obtained from the same culture conditions.

111

Chromosomal DNA was extracted and purified according to Yoon et al. (1996), with the

112

modification that RNase T1 was used in combination with RNase A to minimize contamination

113

of RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al.,

114

1998)

115

ACGGTTACCTTGTTACGACTT-3’. Sequencing of the amplified 16S rRNA gene and

116

phylogenetic analysis were performed as described by Yoon et al. (2003).

using

two

universal

primers,

5’-GAGTTTGATCCTGGCTCAG-3’

and

5’-

117 118

Isoprenoid quinones were extracted and analysed as described by Komagata & Suzuki (1987),

119

using reversed-phase HPLC and a YMC ODS-A (250×4.6 mm) column. The isoprenoid

120

quinones were eluted by a mixture of methanol/isopropanol (2:1, v/v) using a flow rate of 1 ml

121

min-1 at room temperature and detected by UV absorbance at 275 nm. For cellular fatty acid

6

122

analysis, cell mass of strain GYSW-23T was harvested from MA plates after cultivation for 3, 5

123

and 7 days at 25 °C, and cell mass of R. aestuarii SMK-122T, R. pacificus LMG 24575T, ‘A.

124

atlanticum’ LMG 27158T, R. marina JCM 16262T and O. litoreus M-M22T was harvested from

125

MA plates after cultivation for 5 days at 25 °C. Fatty acids were saponified, methylated and

126

extracted using the standard MIDI protocol (Sherlock Microbial Identification System, version

127

6.2B). The fatty acids were analysed by GC (Hewlett Packard 6890) and identified using the

128

TSBA6 database of the Microbial Identification System (Sasser, 1990). Polar lipids were

129

extracted according to the procedures described by Minnikin et al. (1984), and separated by

130

two-dimensional TLC using chloroform/methanol/water (65:25:3.8, by vol.) for the first

131

dimension and chloroform/methanol/acetic acid/water (40:7.5:6:1.8, by vol.) for the second

132

dimension as described by Embley & Wait (1994). Individual polar lipids were identified by

133

spraying the plates with 10 % ethanolic molybdophosphoric acid, molybdenum blue, ninhydrin

134

and -naphthol reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987) and with

135

Dragendorff’s reagent (Sigma). The DNA G+C content was determined by the method of

136

Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant

137

nucleotides were analysed by reversed-phase HPLC equipped with a YMC ODS-A (2504.6

138

mm) column. The nucleotides were eluted by a mixture of 0.55 M NH4H2PO4 (pH 4.0) and

139

acetonitrile (40:1, v/v), using a flow rate of 1 ml min-1 at room temperature and detected by UV

140

absorbance at 270 nm.

7

141 142 143

Morphological, cultural, physiological and biochemical characteristics of strain GYSW-23T are

144

given in the genus and species descriptions (see below) or in Table 1 or Fig. S1 (available in the

145

online Supplementary Material). The almost-complete 16S rRNA gene sequence of strain

146

GYSW-23T determined in this study comprised 1385 nucleotides, representing approximately

147

95 % of the E. coli 16S rRNA gene sequence. Strain GYSW-23T exhibited the highest 16S

148

rRNA gene sequence similarity value (94.0 %) to the type strain of Roseovarius aestuarii. It

149

exhibited 16S rRNA gene sequence similarity values of 93.6, 93.5, 93.4 and 93.4 % to

150

‘Actibacterium atlanticum’ 22II-S11-z10T, Ruegeria marina ZH17T, Roseovarius pacificus 81-

151

2T and Oceanicola litoreus M-M22T, respectively. In the neighbour-joining, maximum-

152

likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences,

153

strain GYSW-23T was found to form a distinct evolutionary lineage independent of other taxa of

154

the family Rhodobacteraceae (Fig. 1; Fig. S2 & S3).

155 156

The predominant isoprenoid quinone detected in strain GYSW-23T was ubiquinone-10 (Q-10),

157

which is typical of the vast majority of the class Alphaproteobacteria. In Table 2, the cellular

158

fatty acid profile of strain GYSW-23T is compared with those of the type strains of some

159

phylogenetically related species (Table 2). The major fatty acid (> 10 % of total fatty acids)

8

160

detected in strain GYSW-23T was C18:1 ω7c. The fatty acid profile of strain GYSW-23T was

161

similar to those of the type strains of R. aestuarii, R. pacificus, ‘A. atlanticum’, R. marina and O.

162

litoreus in that C18:1 ω7c is major fatty acid, but distinguishable from the reference strains by

163

differences in proportions of some fatty acids, particularly C16:0, C18:0 and 11-methyl C18:1 ω7c

164

(Table 2). The major polar lipids detected in strain GYSW-23T were phosphatidylcholine,

165

phosphatidylglycerol and one unidentified aminolipid; minor amounts of one unidentified lipid

166

and one unidentified phospholipid were also present (Fig. S4). The polar lipid profile of strain

167

GYSW-23T was distinguished from those of the type strains of R. aestuarii, R. pacificus, ‘A.

168

atlanticum’, R. marina and O. litoreus by the absence of phosphatidylethanolamine,

169

sulfoquinovosyldiacylglycerol and other unidentified lipid(s) as major components (Fig. S4;

170

Park et al., 2013). The polar lipid profile of strain GYSW-23T was also distinguished from those

171

of other members of the genera Roseovarius, Actibacterium, Ruegeria and Oceanicola by the

172

absence of phosphatidylethanolamine and/or unidentified lipid(s) as major components (Kim et

173

al., 2012a, b; Lucena et al., 2012; Choi et al., 2013; Kämpfer et al., 2013; Park et al., 2013,

174

2014c; Rajasabapathy et al., 2014). The DNA G+C content of strain GYSW-23T was 60.6

175

mol%, a value in the range reported for members of the phylogenetically related taxa (Table 1).

176 177

From the results of the phylogenetic analyses, it does not seem appropriate to assign strain

178

GYSW-23T to any of the recognized genera of the family Rhodobacteraceae (Fig. 1; Fig. S2 &

9

179

S3). The differences in fatty acid and polar lipid profiles make it reasonable to differentiate

180

strain GYSW-23T from the phylogenetically related taxa (Table 2; Fig. S4). Strain GYSW-23T

181

could be distinguished from the type strains of some phylogenetically related species by

182

differences in several phenotypic characteristics, including motility, growth at 40 C, nitrate

183

reduction, hydrolysis and utilization of some substrates, activity of some enzymes and

184

susceptibility to some antibiotics (Table 1). Accordingly, on the basis of the phylogenetic and

185

chemotaxonomic distinctiveness and other differential phenotypic properties, strain GYSW-23T

186

is considered to represent a novel genus and species within the family Rhodobacteraceae, for

187

which the name Pontivivens insulae gen. nov., sp. nov., is proposed.

188 189 190

Description of Pontivivens gen. nov.

191 192

Pontivivens (Pon.ti.vi’vens. L. n. pontus the sea; L. part. adj. vivens living; N.L. part. adj.

193

Pontivivens living in the sea).

194 195

Cells are Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped. Catalase-

196

and oxidase-positive. Nitrate reduction is positive. The predominant ubiquinone is Q-10. The

197

major fatty acid is C18:1 ω7c. The major polar lipids are phosphatidylcholine,

10

198

phosphatidylglycerol and one unidentified aminolipid. The DNA G+C content is 60.6 mol%.

199

The type species is Pontivivens insulae. A member of the family Rhodobacteraceae, the class

200

Alphaproteobacteria, according to 16S rRNA gene sequence analysis.

201 202 203

Description of Pontivivens insulae sp. nov.

204 205

Pontivivens insulae (in’su.lae. L. gen. n. insulae of an island, referring to the source of

206

isolation of the type strain).

207 208

Cells are Gram-stain-negative, non-flagellated and coccoid, ovoid or rod-shaped, approximately

209

0.2-0.7 μm in diameter and 0.3-6.0 μm in length. Colonies on MA are circular, slightly convex,

210

smooth, glistening, greyish yellow in colour and 0.5-1.0 mm in diameter after incubation for 5

211

days at 25 °C. Optimal temperature for growth is 25 °C; growth occurs at 15 and 35 °C, but not

212

at 10 and 37 °C. Optimal pH for growth is 7.0-8.0; growth occurs at pH 6.0, but not at pH 5.5.

213

Optimal growth occurs in the presence of approximately 2.0-3.0 % (w/v) NaCl; growth occurs

214

in the presence of 1.0-8.0 % (w/v) NaCl. Mg2+ ions are required for growth. Growth does not

215

occur under anaerobic conditions on MA and on MA supplemented with nitrate.

216

Bacterichlorophyll a is not produced. Catalase- and oxidase-positive. Nitrate is reduced to

11

217

nitrite. Aesculin and L-tyrosine are hydrolysed, but casein, gelatin, hypoxanthine, starch, Tween

218

80, urea and xanthine are not. D-Cellobiose, D-galactose, D-glucose, maltose, sucrose, D-xylose,

219

L-malate, pyruvate, succinate and salicin are utilized as sole carbon and energy sources, but L-

220

arabinose, D-fructose, D-mannose, D-trehalose, acetate, benzoate, citrate, formate and L-

221

glutamate are not. Susceptible to chloramphenicol, kanamycin, neomycin, novobiocin,

222

oleandomycin, polymyxin B, streptomycin and tetracycline, but not to ampicillin, carbenicillin,

223

cephalothin, gentamicin, lincomycin and penicillin G. Activity of alkaline phosphatase, esterase

224

(C 4) and esterase lipase (C 8) is present and activity of leucine arylamidase is weakly present,

225

but activity of lipase (C 14), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin,

226

acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-

227

glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and

228

α-fucosidase is absent. The predominant ubiquinone is Q-10. The major fatty acid (> 10 % of

229

total fatty acids) is C18:1 ω7c. The major polar lipids are phosphatidylcholine,

230

phosphatidylglycerol and one unidentified aminolipid. The DNA G+C content of the type strain

231

is 60.6 mol%.

232

The type strain, GYSW-23T (= KCTC 42458T = CECT 8812T), was isolated from seawater off

233

Geoje island on the South Sea, South Korea.

234 235 12

236

Acknowledgements

237 238

This work was supported by the project on survey of indigenous species of Korea of the

239

National Institute of Biological Resources (NIBR) under the Ministry of Environment (MOE)

240

and the Programs for Collection, Management and Utilization of Biological Resources (grant

241

NRF-2013M3A9A5075953) from the Ministry of Science, ICT & Future Planning (MSIP) of

242

the Republic of Korea.

243 244 245

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Park, S., Lee, M.-H. & Yoon, J.-H. (2013). Oceanicola litoreus sp. nov., an

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aquaeponti sp. nov., an alphaproteobacterium isolated from seawater. Antonie van Leeuwenhoek

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Park, S., Park, D.-S., Bae, K. S. & Yoon, J.-H. (2014b). Phaeobacter aquaemixtae sp. nov.,

17

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333 334

Park, S., Park, J.-M., Kang, C.-H. & Yoon, J.-H. (2014c). Confluentimicrobium lipolyticum

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Park, S., Park, J.-M., Park, D.-S. & Yoon, J.-H. (2014d). Litoreibacter ponti sp. nov.,

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isolated from seawater. Int J Syst Evol Microbiol 64, 3810-3815.

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Rajasabapathy, R., Mohandass, C., Dastager, S. G., Liu, Q., Khieu, T.-N., Son, C. K., Li,

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W.-J. & Colaco, A. (2014). Roseovarius azorensis sp. nov., isolated from seawater at

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Espalamaca, Azores. Antonie van Leeuwenhoek 105, 571-578.

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Microbiol Ecol 33, 219-232.

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19

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375 376 377 378 379 380 381 382 383 384 385 386 387 20

388 389 390 391 392 393 394 395 396 397 398 399 400 401 402

Table 1. Differential characteristics of strain GYSW-23T and the type strains of some phylogenetically related species. Strain: 1, GYSW-23T; 2, Roseovarius aestuarii SMK-122T, data from Yoon et al. (2008); 3, Roseovarius pacificus LMG 24575T, data from Jung et al. (2012) and Kim et al. (2012) unless indicated otherwise; 4, ‘Actibacterium atlanticum’ LMG 27158T, data from this study unless indicated otherwise; 5, Ruegeria marina JCM 16262T, data from this study unless indicated otherwise; 6, Oceanicola litoreus M-M22T, data from Park et al. (2013). +, positive reaction; , negative reaction; w, weakly positive reaction; ND, not determined. All strains are positive for activity of catalase and oxidase; utilization of L-malate (weak for Ruegeria marina JCM 16262T) and succinate (weak for Ruegeria marina JCM 16262T and Oceanicola litoreus M-M22T); activity of alkaline phosphatase (weak for Roseovarius aestuarii SMK-122T), esterase (C 4) and esterase lipase (C 8); and susceptibility to chloramphenicol. All strains are negative for Gram-staining; hydrolysis of starch; utilization of D-trehalose, formate and L-glutamate; activity of lipase (C 14), cystine arylamidase, trypsin, -chymotrypsin, -galactosidase, -galactosidase, glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase; and susceptibility to lincomycin.

Characteristic Motility Growth at 4 C 40 C Nitrate reduction to nitrite Hydrolysis of Aesculin Casein Gelatin Tween 80 Utilization of L-Arabinose D-Cellobiose D-Fructose D-Galactose, sucrose, salicin D-Glucose Maltose D-Mannose D-Xylose Acetate Benzoate Citrate Pyruvate Enzyme activity (by API ZYM) Leucine arylamidase Valine arylamidase Acid phosphatase Naphthol-AS-BIphosphohydrolase Susceptibility to antibiotics Ampicillin Carbenicillin, cephalothin Gentamicin Kanamycin Neomycin Novobiocin Oleandomycin Penicillin G

1 

2 

3 *

4 *

5 *

6 

  

  

W* ND *

* * *

* * *

  

   

   

   

* * * *

* * * *

   

           

           

           

     w      

           w

           

w  

  

  

  

  

  













       

       

  w  w   

       

       

      w 

21

Polymyxin B Streptomycin Tetracycline DNA G+C content (mol%)

403 404

   60.6

   58.6

   62.3*

   59.0*

   63.5*

  w 67.6

*Data of R. pacificus LMG 24575T, A. atlanticum LMG 27158T and R. marina JCM 16262T taken from Wang et al. (2009), Li et al. (2014) and Huo et al. (2011), respectively.

405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 22

422 423 424 425 426 427 428

Table 2. Cellular fatty acid compositions (%) of strain GYSW-23T and the type strains of some phylogenetically related species. Strains: 1, GYSW-23T (3 days); 2, GYSW-23T (5 days); 3, GYSW-23T (7 days); 4, Roseovarius aestuarii SMK-122T; 5, Roseovarius pacificus LMG 24575T; 6, ‘Actibacterium atlanticum’ LMG 27158T; 7, Ruegeria marina JCM 16262T; 8, Oceanicola litoreus M-M22T. Fatty acids that represented < 0.5 % in all columns were omitted. TR, Traces (< 0.5 %); –, Not detected; ECL, Equivalent chain length.

Fatty acid Straight-chain C10:0 C12:0 C16:0 C17:0 C18:0 Branched iso-C15:1 F anteiso-C15:1 A Unsaturated C17:1 ω8c C18:1 ω7c anteiso-C17:1 ω9c Hydroxy C10:0 3-OH C12:0 3-OH C12:1 3-OH C16:0 2-OH C18:1 2-OH iso-C13:0 3-OH 11-methyl C18:1 ω7c Cyclo C19:0 ω8c Summed features* 1 3 7

429 430 431

1

2

3

4

5

6

7

8

TR – 1.3 1.3 3.2

– – 1.1 1.7 3.3

– – 1.2 1.0 3.2

0.6 4.7 14.6 TR 1.2

0.6 3.9 3.4 TR 0.6

– – 1.2 – 4.0

2.0 1.7 5.8 TR 1.1

TR – 4.7 1.3 8.3

– –

TR –

2.1 0.9

– –

– –

– –

TR –

– –

1.0 84.1 –

1.3 81.4 –

1.4 73.4 1.2

– 69.1 –

– 66.5 –

– 75.6 –

– 72.8 –

– 79.3 –

0.8 – – – – – 2.4 –

1.1 – – – – TR 2.1 –

1.1 – – – 0.8 2.4 3.1 –

TR 5.4 – – – – 0.8 1.3

TR 2.9 2.2 2.8 – – 10.0 5.1

5.1 1.8 – – – – 10.3 1.4

TR 4.5 TR 2.3 TR TR 6.9 –

3.3 – – – – – TR 0.8

– 0.8 4.4

– 1.2 5.3

0.9 1.3 4.3

– TR –

– 0.7 –

– – –

TR TR –

– 1.5 –

*Summed feature 1 contains iso-C15:1 H and/or C13:0 3-OH; summed feature 3 contains C16:1 ω7c and/or C16:1 ω6c; summed feature 7 contains unknown fatty acid 18.846(ECL), C19:1 ω6c and/or cyclo C19:0 ω10c.

432 433 434 435 23

436 437

Legend to Figure

438 439

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

440

positions of Pontivivens insulae GYSW-23T and representatives of some other related taxa.

441

Only bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are

442

shown at branching points. Filled circles indicate that the corresponding nodes were also

443

recovered in the trees generated with the maximum-likelihood and maximum-parsimony

444

algorithms. Stappia stellulata IAM 12621T (GenBank accession number, D88525) was used as

445

an outgroup. Scale bar, 0.01 substitutions per nucleotide position.

24

Figure 1 Click here to download Figure: Fig. 1.ppt

Sulfitobacter dubius KMM 3554T (AY180102) Sulfitobacter pontiacus ChLG 10T (Y13155) Roseobacter litoralis ATCC 49566T (X78312) Aestuariihabitans beolgyonensis BB-MW15T (KC577450) Pelagicola litoralis CL-ES2T (EF192392) Pacificibacter maritimus KMM 9031T (AB558927) 99.6 Leisingera aquimarina LMG 24366T (AM900415) 55.9 Leisingera methylohalidivorans MB2T (AY005463) 66.5 Phaeobacter caeruleus LMG 24369T (AM943630) Phaeobacter gallaeciensis BS107T (Y13244) Marinovum algicola ATCC 51440T (X78315) 98.4 Ruegeria lacuscaerulensis ITI-1157T (U77644) 58.0 Ruegeria atlantica IAM 14463T (D88526) Ruegeria pomeroyi DSS-3T (AF098491) Ruegeria marina ZH17T (FJ872535) Ruegeria scottomollicae LMG 24367T (AM905330) 100 Marivita litorea CL-JM1T (EU512918) 84.2 Marivita cryptomonadis CL-SK44T (EU512919) Thalassobius maritimus GSW-M6T (HM748766) 50.6 Thalassococcus lentus YCS-24T (JX090308) Thalassococcus halodurans UST050418-052T (DQ397336) 95.1 69.4 Roseovarius pacificus 81-2T (DQ120726) 100 Roseovarius halotolerans HJ50T (EU431217) Roseovarius litoreus GSW-M15T (JQ390520) Donghicola eburneus SW-277T (DQ667965) 51.9 Citreimonas salinaria CL-SP20T (AY962295) Donghicola xiamenensis Y-2T (DQ120728) 56.2 Cribrihabitans marinus CZ-M5T (JX306766) 88.3 Roseovarius aestuarii SMK-122T (EU156066) Roseovarius nubinhibens ISMT (AF098495) 89.1 Roseovarius tolerans EL-172T (Y11551) Pontivivens insulae GYSW-23T (KP662553) 87.5 Rhodovulum kholense JA297T (AM748927) 99.7 Rhodovulum sulfidophilum DSM 1374T (D16423) Rhodovulum marinum JA128T (AJ891122) Rhodobacter capsulatus ATCC 11166T (D16428) Rhodobacter sphaeroides 2.4.1T (X53853) 99.7 ‘Confluentimicrobium lipolyticum’ SSK1-4T (KJ889015) 95.3 Paracoccus aminophilus ATCC 49673T (AY014176) Paracoccus denitrificans ATCC 17741T (Y16927) 100 Paracoccus saliphilus YIM 90738T (DQ923133) ‘Actibacterium atlanticum’ 22II-S11-z10T (KJ159064) Actibacterium mucosum R46T (HE590855) 99.7 69.1 Pseudoruegeria aquimaris SW-255T (DQ675021) Pseudoruegeria lutimaris HD-43T (FJ374173) Celeribacter neptunius H 14T (FJ535354) Celeribacter baekdonensis L-6T (HM997022) 99.1 99.6 Tropicimonas sediminicola M97T (JF748735) Tropicimonas isoalkanivorans B51T (AB302379) Oceanicola litoreus M-M22T (JX291104) Oceanicola granulosus HTCC2516T (AY424896) Roseisalinus antarcticus EL-88T (AJ605747) 90.1 Wenxinia marina HY34T (DQ640643) Rubellimicrobium thermophilum C-lvk-R2A-2T (AJ844281) Stappia stellulata IAM 12621T (D88525) 71.5 99.2

70.9

0.01

Fig. 1

Supplementary Materials (PDF) Click here to download Supplementary Material Files: Supplementary materials.pdf

Pontivivens insulae gen. nov., sp. nov., isolated from seawater.

A Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated GYSW-23(T), was isolated from seawater off Ge...
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