IJSEM Papers in Press. Published May 11, 2015 as doi:10.1099/ijs.0.000321

International Journal of Systematic and Evolutionary Microbiology Phaeodactylibacter luteus sp. nov., isolated from the oleaginous microalga Picochlorum sp. --Manuscript Draft-Manuscript Number:

IJSEM-D-14-00410R2

Full Title:

Phaeodactylibacter luteus sp. nov., isolated from the oleaginous microalga Picochlorum sp.

Short Title:

Phaeodactylibacter luteus sp. nov.

Article Type:

Note

Section/Category:

New taxa - Bacteroidetes

Corresponding Author:

Tianling Zheng Xiamen University CHINA

First Author:

Xueqian Lei

Order of Authors:

Xueqian Lei Yi Li Guanghua Wang Yao Chen Qiliang Lai Zhangran Chen Jingyan Zhang Pingping Liao Hong Zhu Wei Zheng Tianling Zheng

Manuscript Region of Origin:

CHINA

Abstract:

A Gram-staining-negative, orange-pigmented, non-motile, aerobic bacterial strain, designated GYP20T, was isolated from a culture of the alga Picochlorum sp., a promising feedstock for biodiesel production, which was isolated from the India Ocean. Growth was observed at temperatures from 20 to 37 °C, at salinities from 0 to 3% and at pH from 5 to 9. Mg2+ and Ca2+ ions are required for growth. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain was a member of the genus Phaeodactylibacter, which belongs to the family Saprospiraceae. Strain GYP20T was most closely related to Phaeodactylibacter xiamenensis KD52T (95.5% sequence similarity). The dominant fatty acids were iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH and Summed Feature 3. The predominant respiratory quinone was menaquinone-7 (MK-7). The polar lipids of strain GYP20T were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified glycolipid, two unidentified phospholipid and three unidentified aminolipid. According to its morphology, physiology, fatty acid composition and 16S rRNA sequence data, the novel strain most appropriately belongs to the genus Phaeodactylibacter, but can readily be distinguished from known Phaeodactylibacter xiamenensis GYP20T. The name Phaeodactylibacter luteus sp. nov. is proposed (type strain GYP20T =MCCC 1F01222T =KCTC 42180T).

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Phaeodactylibacter luteus sp. nov., isolated from the oleaginous microalga Picochlorum sp.

4

Xueqian Lei1†, Yi Li1†, Guanghua Wang2, Yao Chen1, Qiliang Lai3, Zhangran Chen1,

5

Jingyan Zhang1, Pingping Liao1, Hong Zhu1, Wei Zheng1, Tianling Zheng1*

6

1

7

Xiamen University, Xiamen 361005, China

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2

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Guangdong Key Laboratory of Marine Materia Medica (LMMM-GD), South China

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Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301,

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China

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3

13

State Oceanic Administration, People’ s Republic of China

Key Laboratory of MOE for Coast and Wetland Ecosystems, School of Life Sciences,

CAS Key Laboratory of Tropical Marine Bio-resources and Ecology (LMB-CAS),

Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography,

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Co-first author: These authors contributed equally to this work.

16

*

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+86-592-2184528; E-mail: [email protected];

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Subjective category: Bacteroidetes

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Running title: Phaeodactylibacter luteus sp. nov.

20

Abbreviations: KCTC, Korean Collection For Type Cultures; MCCC, Marine

21

Culture Collection of China.

22

The GenBank [/EMBL/DDBJ] accession number for the 16S rRNA gene sequence of

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strain GYP20T is KM235292.

24

Four supplementary figures are available with the online Supplementary Material.

Corresponding

author:

Tianling

Zheng.

Tel:

+86-592-2183217.

Fax:

25

Abstract:

26

A Gram-staining-negative, orange-pigmented, non-motile, aerobic bacterial strain,

27

designated GYP20T, was isolated from a culture of the alga Picochlorum sp., a

28

promising feedstock for biodiesel production, which was isolated from the India

29

Ocean. Growth was observed at temperatures from 20 to 37 °C, at salinities from 0 to

30

3% and at pH from 5 to 9. Mg2+ and Ca2+ ions were required for growth. Phylogenetic

31

analysis based on 16S rRNA gene sequencing revealed that the strain was a member

32

of the genus Phaeodactylibacter, which belongs to the family Saprospiraceae. Strain

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GYP20T was most closely related to Phaeodactylibacter xiamenensis KD52T (95.5%

34

sequence similarity). The major fatty acids were iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH

35

and Summed Feature 3. The predominant respiratory quinone was menaquinone-7

36

(MK-7). The polar lipids of strain GYP20T were found to consist of

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diphosphatidylglycerol,

38

unidentified glycolipid, two unidentified phospholipid and three unidentified

39

aminolipid. According to its morphology, physiology, fatty acid composition and 16S

40

rRNA sequence data, the novel strain most appropriately belongs to the genus

41

Phaeodactylibacter, but can readily be distinguished from known Phaeodactylibacter

42

xiamenensis GYP20T. The name Phaeodactylibacter luteus sp. nov. is proposed for

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strain GYP20T (=MCCC 1F01222T =KCTC 42180T).

phosphatidylethanolamine,

phosphatidylglycerol,

four

44 45

Main text:

46

Members of the family Saprospiraceae, are considered to be important members of

47

the bacterial community involved in ecophysiological activities in a variety of natural

48

environments (Yoon et al., 2011). The family Saprospiraceae consists of the genera

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Saprospira (Reichenbach et al., 1989), Haliscomenobacter (Van Veen et al., 1973),

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Lewinella (Sly et al., 1998), ‘Portibacter’ (Yoon et al., 2011), Aureispira (Hosoya, et

51

al., 2006), ‘Rubidimonas’ (Yoon et al., 2012) and Phaeodactylibacter (Chen et al.,

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2014), and they are found in various habitats. Comparative 16S rRNA gene sequence

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analysis indicated that strain GYP20T formed a clade within the genus

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Phaeodactylibacter. The genus Phaeodactylibacter was first proposed by Chen et al.

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(2014). At present, this genus includes one species with validly published names:

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Phaeodactylibacter xiamenensis KD52T. Accordingly, the aim of the present work

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was to determine the exact taxonomic position of strain GYP20T by using polyphasic

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characterization including the determination of phenotypic properties and a detailed

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phylogenetic analysis based on 16S rRNA gene sequences.

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Strain GYP20T was isolated from a culture of the alga Picochlorum sp., a promising

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feedstock for biodiesel production, which was isolated from the India Ocean (Yang et

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al., 2014). The samples were serially diluted (10-fold dilution) by sterile seawater and

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0.1 mL aliquots of each dilution were spread onto marine agar 2216 (MA; Difco)

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followed by incubation for 7 days at 28 °C. Individual colonies of distinct

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morphology were further purified three times and stored at -80 °C in marine broth

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2216 (MB; Difco) supplemented with 10% (v/v) glycerol.

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Genomic DNA was extracted according to the method of Ausubel et al. (1995). The

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16S rRNA gene sequence was amplified by PCR using primers 27F and 1492R

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(DeLong, 1992). Purification of the PCR product was carried out according to the

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protocol of the TIANquick midi purification kit (TIANGEN, China). The purified

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DNA was cloned into vector pMD19-T and sequenced. Sequences of related taxa

72

were

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(http://eztaxon-e.ezbiocloud.net/) (Kim et al., 2012). Phylogenetic analysis was

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performed using MEGA version 5.0 (Tamura et al., 2011) on the basis of

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neighbour-joining (Saitou & Nei, 1987), minimum evolution (Rzhetsky & Nei, 1993)

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and maximum likelihood (Felsenstein 1981) algorithms, with bootstrap resampling of

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1,000 replications.

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A nearly full-length 16S rRNA gene sequence (1484 bp) of strain GYP20T was

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determined. Phylogenetic analysis of the strain GYP20T based on the 16S rRNA gene

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sequence indicated that this strain belonged to the family Saprospiraceae, forming a

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robust clade within the genus Phaeodactylibacter (Fig. 1, Fig. S2 and Fig. S3,

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available in the online Supplementary Material). The closest related species was P.

83

xiamenensis KD52T (95.5%). Strain GYP20T formed a separate phylogenetic clade

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with P. xiamenensis KD52T.

85

Cell morphology and motility were observed by using transmission electron

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microscopy (model JEM-2100HC; JEOL) and phase-contrast light microscopy (model

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50i; Nikon), with cells from the early exponential phase grown on MA at 28 °C.

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Colony morphology was examined from cultures grown on MA for 2 days. The

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presence of flexirubin-type pigments was assessed using the bathochromic shift test

obtained

from

the

GenBank

database

and

EzTaxon-e

server

90

with 20% KOH, as described by Bernardet et al. (2002). The Gram reaction was

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determined by using the bioMérieux Gram stain kit according to the manufacturer’s

92

instructions. Gliding motility was investigated as described by Bowman (2000).

93

Anaerobic growth was examined according to the protocol of Li et al. (2013).

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Triplicate cultures were grown in 50 ml anaerobic serum bottles sealed with thick

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butyl rubber stoppers and aluminum caps, and incubated statically in the dark at 30 °C

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for 21 days. The optimal growth temperature was determined over the range 0–45 °C

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(0, 4, 15, 20, 25, 28, 30, 32, 35, 37, 38, 39, 40, 42 and 45°C) in MB with triplicate and

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measured by spectrophotometer method with OD600 every 12 h in total 3 days. The

99

specific ion requirements and NaCl tolerance were performed as described by Sohn et

100

al. (2004). Specific ion requirements and NaCl tolerance were tested by using

101

NaCl-free 2216 medium (Tryptone 5.0 g/L, yeast extract 1.0 g/L, 1 L distilled water,

102

pH 7.6-7.8) that was supplemented with NaCl or combinatorial artificial sea salts. The

103

strain showed requirement for cations, namely Na+, Mg2+ and Ca2+, as no growth was

104

observed in medium that had not been supplemented with Na+, Mg2+ and Ca2+ ions.

105

The pH range for growth was determined in MB that was adjusted to pH 3.0-12.0 (at

106

1 pH unit intervals), as previously described (Lei et al., 2014). Verification of the pH

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values after autoclaving revealed only minor changes (Su et al., 2013). The catalase

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activity was performed by addition of 3% (v/v) hydrogen peroxide to

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exponential-phase colonies, and oxidase reaction was tested by using oxidase reagent

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(bioMérieux). Hydrolysis of starch, chitin, tyrosine, casein, gelatin, urea and Tweens

111

20, 40, 60 and 80 was tested using MA supplemented with 0.5% (w/v) of starch and 1%

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(w/v) of the other substrates. Results were examined twice after growth on agar plates

113

for 3 and 5 days. Further biochemical test of strain GYP20T and P. xiamenensis

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KD52T were conducted using the API 20NE and API 20E test kits (bioMérieux) at

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30 °C for 3 days and the API ZYM test kit (bioMérieux) at 30 °C for 24 h according

116

to the manufacturer’s instructions. All the commercial kits were inoculated with

117

bacterial suspensions prepared in 3% (w/v) NaCl. The physiological and biochemical

118

characteristics of strain GYP20T are shown in the species description and in Table 1.

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Fatty acids of strain GYP20T and P. xiamenensis KD52T in whole cells grown on MA

120

at 28 °C for 48 h were extracted, saponified and esterified using the standard protocol

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of MIDI (Sherlock Microbial Identification System, version 6.0B). The two strains

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had similar growth rates and had same physiological age after growth on MA plates at

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28 °C for 48 h. The fatty acids were analysed by GC (Agilent Technologies 6850) and

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identified by using the TSBA 6.0 database of the Microbial Identification System

125

(Sasser, 1990). P. xiamenensis KD52T was tested under the same condition in this

126

study with strain GYP20T. As showed in Table 2, the major fatty acids of GYP20T

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were Summed Feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c, 23.5%), iso-C15:0

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(13.3%), iso-C17:0 3-OH (13.1%), iso-C15:1 G (12.0%), which accounted for 61.9%.

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Compared with P. xiamenensis KD52T, strain GYP20T possessed a lower content of

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iso-C15:0 and a higher content of Summed Feature 3 (comprising C16:1 ω7c and/or C16:1

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ω6c, 23.5%).

132

The G+C content of the chromosomal DNA was determined according to the methods

133

(Mesbah & Whitman, 1989) using a reverse-phase HPLC. The DNA G+C contents of

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the new isolate GYP20T was 53 mol%, which was similar to reference strain P.

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xiamenensis KD52T.

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The major respiratory quinone of the strain GYP20T was determined to be

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menaquinone-7 (MK-7), which was carried out by the Identification Service of the

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DSMZ, Braunschweig, Germany. This trait was in accordance with the properties of

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the genus Phaeodactylibacter.

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Polar lipids of strain GYP20T were extracted using a chloroform / methanol system

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and analysed using one- and two-dimensional TLC, as described previously (Kates,

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1986). Merck silica gel 60 F254 aluminium-backed thin-layer plates were used in

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TLC analysis. The plate dotted with sample was subjected to two-dimensional

144

development, with the first solvent of chloroform-methanol-water (65:25:4, by vol.)

145

followed by second solvent of chloroform-methanol-acetic acid-water (85:12:15:4, by

146

vol.). The TLC plates were sprayed with sulfuric acid/ethanol (1:  2, v/v) followed by

147

heating at 150 °C for 3 min to detect phospholipids and glycolipids. The TLC plates

148

were also visualized by treating the plates with 10 % (w/v) molybdatophosphoric acid

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followed by heating at 150 °C for 5 min. The polar lipids of strain GYP20T were

150

found

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phosphatidylglycerol, four unidentified glycolipid, two unidentified phospholipid and

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three

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phosphatidylethanolamine, phosphatidylglycerol, unidentified glycolipid were found

154

in strain GYP20T. (Fig. S4, available in the online Supplementary Material).

to

consist

unidentified

of

diphosphatidylglycerol,

aminolipid.

Compared

with

phosphatidylethanolamine,

the

strain

KD52T,

155 156

On the basis of morphological, physiological and chemotaxonomic characteristics,

157

together with data from 16S rRNA gene sequences comparison described above,

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strain GYP20T should be assigned to a novel species within the genus

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Phaeodactylibacter, for which a name Phaeodactylibacter luteus sp. nov. is proposed.

160 161

Description of Phaeodactylibacter luteus sp. nov.

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Phaeodactylibacter luteus (lu'te.us. L.masc. adj. luteus, orange-colored, referring to

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the color of the colony).

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Cells are Gram-staining-negative, rod-shaped, non-motile, non-gliding rods, 5.0-11.2

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μm in length and 0.5-0.6 μm in diameter, as show in Fig. S1 (available in the online

166

Supplementary Material). Growth is visible after 48 h of incubation on MA at 28 °C.

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Colonies on MA are orange and circular with regular, smooth edges that are 1-2 mm

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in diameter after 48 h incubation at 28 °C, opaque and raised in the centre. Growth

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occurs at 20-37 ºC (optimum 28 ºC). Growth occurs at pH 5–9 (optimum pH 7).

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Growth occurs at NaCl concentrations of 0-3% (w/v), with optimal growth at 2%

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(w/v). No growth was observed in medium that has not been supplemented with Na+,

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Mg2+ and Ca2+ ions. Growth does not occur under anaerobic conditions on MB.

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Flexirubin-type pigments are not produced. Positive for catalase, oxidase, reduction of

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nitrate and hydrolysis of Tweens 20, 40 and 60 and tyrosine. Negative for hydrolysis

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of casein, chitin, starch, Tween 80, urea and gelatin. In API 20NE strips positive for

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reduction of nitrate to nitrite, denitrification, β-glucosidase (aesculin hydrolysis),

177

β-galactosidase; negative for indole production, arginine dihydrolase, urease activity,

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gelatin hydrolysis, D-glucose fermentation and utilization of D-glucose, L-arabinose,

179

D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate,

180

capric acid, adipic acid, malic acid and trisodium citrate phenylacetic acid. According

181

to API 20E strips, positive for ß-galactosidase, tryptophan deaminase and acetoin

182

production; negative for arginine dihydrolase, lysine decarboxylase, ornithine

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decarboxylase, citrate utilization, H2S production, urease activity, indole production,

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gelatinase and acid production from glucose, mannitol, inositol, sorbitol, rhamnose,

185

saccharose, melibiose, amygdalin and arabinose. In the test of API ZYM, positive for

186

alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine aminopeptidase,

187

valine aminopeptidase, cystine aminopeptidase, trypsin, α-chymotrypsin, acid

188

phosphatase, naphtol-AS-Bl-phosphoamidase and β-glucosidase; weakly positive for

189

lipase (C14), β-galactosidase and α-glucosidase; negative for α-galactosidase,

190

β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase or α-fucosidase. The

191

major fatty acids are iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH and Summed Feature 3. The

192

DNA G+C contents of the type strain is 53 mol%. The predominant respiratory

193

quinone is menaquinone-7 (MK-7). The major polar lipids are diphosphatidylglycerol,

194

phosphatidylethanolamine, phosphatidylglycerol, four unidentified glycolipid, two

195

unidentified phospholipid and three unidentified aminolipid.

196

The type strain, GYP20T (= MCCC 1F01222T =KCTC 42180T) was isolated from a

197

culture of Picochlorum sp. collected from the India Ocean.

198 199

Acknowledgements

200

This work was supported by the National Nature Science Foundation of China

201

(41376119, 40930847), Public Science and Technology Research Funds Projects of

202

Ocean (201305016). We also thank Prof. I. J. Hodgkiss of The University of Hong

203

Kong for help with English.

204 205

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280

281

282

283

284

285

286 287

Figure Legends

288 289

Fig. 1. Neighbour-joining tree showing the phylogenetic positions of strain GYP20T

290

and representatives of some other related taxa, based on 16S rRNA gene sequences.

291

Filled circles indicate nodes that were also recovered in maximum-likelihood, and

292

minimum evolution trees based on the same sequences. Bootstrap values (expressed

293

as percentages of 1000 replications) are shown at branch points. Only bootstrap

294

values >70% are showed. Taibaiella smilacinae KCTC 32316T (KC571459) was used

295

as an outgroup. Bar, 0.02 nucleotide substitution rate (Knuc) units.

296

297

298

299

300

301

302

303

304 305 306

Table 1. Differential characteristics between strain GYP20T and P. xiamenensis

307

KD52T.

308

Strains: 1, GYP20T; 2, P. xiamenensis KD52T. Data of catalase, oxidase, API 20NE, API 20E and

309

API ZYM for two strains were done at the same time in this study. Two strains are positive for

310

oxidase activities, Tweens 20, 40 and 60; negative for Tween 80, starch, chitin, casein, gelatin,

311

urea, flexirubin-type pigments. In the API 20NE strip, two strains are positive for reduction of

312

nitrate, β-glucosidase (aesculin hydrolysis) and β-galactosidase; negative for indole production,

313

arginine dihydrolase, urease activity, gelatin hydrolysis and utilization of D-glucose, L-arabinose,

314

D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid,

315

adipic acid, malic acid, trisodium citrate and phenylacetic acid. In the test of API 20E, two strains

316

are positive for β-galactosidase, acetoin production; negative for citrate utilization, H2S

317

production, indole production and acid production from glucose, mannitol, inositol, sorbitol,

318

rhamnose, saccharose, melibiose, amygdalin and arabinose. In the API ZYM strip, two strains are

319

positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine aminopeptidase,

320

valine aminopeptidase, cystine aminopeptidase, trypsin, α-chymotrypsin, acid phosphatase,

321

naphtol-AS-Bl-phosphoamidase and β-glucosidase; weakly positive for lipase (C14); negative for

322

α-mannosidase. +, Positive; w, weakly positive; -, negative. All data from this study except growth

323

temperature, salinity range, growth pH and the DNA G+C contents of P. xiamenensis KD52T,

324

which are from Chen et al. (2014).

Characteristic

1

2

Colony colour

Orange

Reddish orange

NaCl concentration (optimum) (%, w/v)

0-3 (2)

1-9 (2.5)

20-37(28)

20-37(28)

pH range (optimal)

5-9 (7)

5-8.5 (6)

Catalase activities

+

-

+

-

Denitrification

+

-

D-Glucose fermentation

-

w

Arginine dihydrolase

-

+

Lysine decarboxylase

-

+

Ornithine decarboxylase

-

+

Urease activity

-

+

Tryptophane deaminase

+

-

Gelatinase

-

+

α-Galactosidase

-

+

β-Galactosidase

w

+

β-Glucuronidase

-

+

Growth temperature (optimum) (°C)

Hydrolysis of: Tyrosine API 20NE

API 20E

API ZYM

α-Glucosidase

w

+

N-acetyl-β-glucosaminidase

-

+

α-Fucosidase

-

+

53

51

DNA G+C content (mol%) 325

Table 2. Cellular fatty acid content of strain GYP20T and P. xiamenensis KD52T.

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Strains: 1, GYP20T; 2, P. xiamenensis KD52T. All data were generated in this study. Values are

327

percentages of total fatty acids; tr, traces amount (

Phaeodactylibacter luteus sp. nov., isolated from the oleaginous microalga Picochlorum sp.

A Gram-staining-negative, orange-pigmented, non-motile, aerobic bacterial strain, designated GYP20T, was isolated from a culture of the alga Picochlor...
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