IJSEM Papers in Press. Published February 20, 2015 as doi:10.1099/ijs.0.000125
International Journal of Systematic and Evolutionary Microbiology Paracoccus panacisoli sp. nov., isolated from a forest soil cultivated with Vietnamese ginseng --Manuscript Draft-Manuscript Number:
IJS-D-15-00142
Full Title:
Paracoccus panacisoli sp. nov., isolated from a forest soil cultivated with Vietnamese ginseng
Short Title:
Paracoccus panacisoli sp. nov.
Article Type:
Standard
Section/Category:
New taxa - Proteobacteria
Keywords:
Paracoccus panacisoli, Vietnamese ginseng soil, polyamine, IAA production
Corresponding Author:
Deok-Chun Yang Kyung Hee University Yongin, Gyeonggi-do KOREA, REPUBLIC OF
First Author:
Ngoc-Lan Nguyen, Master
Order of Authors:
Ngoc-Lan Nguyen, Master Yeon-Ju Kim Van-An Hoang, PhD Bao-Tram Tran, Ms Huong-Son Pham, Dr Deok-Chun Yang
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A novel bacterial strain, designated DCY94T, was isolated from forest soil cultivated with ginseng in Vietnam, was Gram-reaction-negative, facultative anaerobic, nonmotile, rod-shaped, catalase- and oxidase-positive. The 16S rRNA gene sequence analysis demonstrated that strain DCY94T was closely related to Paracoccus sphaerophysae Zy-3T (97.5% 16S rRNA gene sequence similarity), and Paracoccus caeni MJ17T (96.9%). The fatty acid profile of strain DCY94T contained predominant amount of summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) (88.4%) and moderate to small quantities of C8:0 3-OH (1.0%), C10:0 3-OH (2.8%) and C18:0 (5.2%). Phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and one unidentified glycolipid were major polar lipids; one unidentified aminolipid, one unidentified aminophospholipid, one unidentified phospholipid, and four unidentified polar lipids were minor. The polyamine pattern comprised major compounds putrescine and spermidine, and minor amounts of sym-homospermidine and spermine. The ubiquinone of this strain was Q-10 and the G+C content of its genomic DNA was 68.3 mol%. All these results support the placement of strain DCY94T within the genus Paracoccus. Levels of DNA-DNA relatedness between strain DCY94T and Paracoccus sphaerophysae HAMBI 3106T, Paracoccus caeni KCTC 22480T were 52 and 50%, respectively. The phylogenetic analysis, phenotypic tests, chemotaxonomic characteristics, and DNA-DNA relatedness distinguished strain DCY94T from the closest recognized species of the genus Paracoccus suggest that this strain represents a novel species, for which the name Paracoccus panacisoli sp. nov. is proposed. The type strain is DCY94T (=KCTC 42086T=JCM 30337T).
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1
Paracoccus panacisoli sp. nov., isolated from a forest soil cultivated with Vietnamese ginseng
2 3
Ngoc-Lan Nguyen1, Yeon-Ju Kim1*, Van-An Hoang1, Bao-Tram Tran2, Huong-Son Pham2 and Deok-
4
Chun Yang1,3
5 6
1
Department of Oriental Medicinal Biotechnology, Kyung-Hee University, Seocheon-dong, Giheung-
7 8
gu, Yongin-si, Gyeonggi-do, 446-701, Republic of Korea. 2
Center for Experimental Biology, National Center for Technological Progress, Ministry of Science
9 10 11
and Technology, C6 Thanh Xuan Bac, Hanoi, Vietnam. 3
Graduate School of Biotechnology and Ginseng Bank, Kyung-Hee University, Seocheon-dong, Giheung-gu, Yongin-si, Gyeonggi-do, 446-701, Republic of Korea.
12 13
Subject category: New taxa; Subsection: Proteobacteria
14
Running title: Paracoccus panacisoli sp. nov.
15 16
* Corresponding author
17
Email: [email protected]
18
Tel: +82-31-201-2100, Fax: +82-31-202-2688
19 20
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DCY94T is
21
KJ653224.
22 23
One supplementary table and one supplementary figure are available with the online version of this
24
paper.
1
25
Abstract
26
A novel bacterial strain, designated DCY94T, was isolated from forest soil cultivated with
27
ginseng in Vietnam, was Gram-reaction-negative, facultative anaerobic, non-motile, rod-shaped,
28
catalase- and oxidase-positive. The 16S rRNA gene sequence analysis demonstrated that strain
29
DCY94T was closely related to Paracoccus sphaerophysae Zy-3T (97.5% 16S rRNA gene sequence
30
similarity), and Paracoccus caeni MJ17T (96.9%). The fatty acid profile of strain DCY94T contained
31
predominant amount of summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) (88.4%) and moderate to small
32
quantities of C8:0 3-OH (1.0%), C10:0 3-OH (2.8%) and C18:0 (5.2%). Phosphatidylethanolamine,
33
phosphatidylglycerol, phosphatidylcholine and one unidentified glycolipid were major polar lipids; one
34
unidentified aminolipid, one unidentified aminophospholipid, one unidentified phospholipid, and four
35
unidentified polar lipids were minor. The polyamine pattern comprised major compounds putrescine
36
and spermidine, and minor amounts of sym-homospermidine and spermine. The ubiquinone of this
37
strain was Q-10 and the G+C content of its genomic DNA was 68.3 mol%. All these results support the
38
placement of strain DCY94T within the genus Paracoccus. Levels of DNA-DNA relatedness between
39
strain DCY94T and Paracoccus sphaerophysae HAMBI 3106T, Paracoccus caeni KCTC 22480T were
40
52 and 50%, respectively. The phylogenetic analysis, phenotypic tests, chemotaxonomic
41
characteristics, and DNA-DNA relatedness distinguished strain DCY94T from the closest recognized
42
species of the genus Paracoccus suggest that this strain represents a novel species, for which the name
43
Paracoccus panacisoli sp. nov. is proposed. The type strain is DCY94 T (=KCTC 42086T=JCM
44
30337T).
45 46
Keywords: Paracoccus panacisoli, Vietnamese ginseng soil, polyamine, IAA production
2
47
The genus Paracoccus was proposed by Davis et al. (1969) and emended by Ludwig et al. (1993),
48
Katayama et al. (1995), and Liu et al. (2008). At the time of writing, the genus Paracoccus contains 40
49
species with validly published names (http://www.bacterio.net/paracoccus.html). Species of this genus
50
has been found in a variety of different habitats such as sediment (Liu et al., 2008; Roh et al., 2009; Li
51
et al., 2009), biofilter (Lipski et al., 1998), rhizosphere (Ghosh et al., 2006; Kämpfer et al., 2012),
52
sludge (La et al., 2005; Lee et al., 2011; Sun et al., 2013 ), fish (Kim et al., 2010), soil (Tsubokura et
53
al., 1999; Dastager et al., 2012), sea sand (Kim et al., 2006), sea water (Khan et al., 2008), and human
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infection (Daneshvar et al., 2003). Phylogenetic analyses have indicated that the genus Paracoccus
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belongs to the α-3 subclass of the phylum Proteobacteria with the closest relative being the genus
56
Rhodobacter (Tsubokura et al., 1999). Members of the genus Paracoccus consist of Gram-reaction-
57
negative coccid or short rods that shows substantial metabolic versatility. All species contain
58
ubiquinone-10 as the predominant respiratory, and C18:1 as major fatty acids. The G+C mol% content of
59
the genomic DNA is ranged 63–71 mol% (Kelly et al., 2006).
60
Though, many studies are focused to extract compounds from Vietnamese ginseng and their
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applications. Nonetheless, no study of resident bacterial populations in Vietnamese ginseng cultivated
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soil in Vietnam has been reported. Therefore, we used selective isolation and cultivation based on
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traditional culture-dependent methods to investigate microorganisms inhabiting in this soil. During the
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course of this study, we discovered strain DCY94T as a candidate for novel species of the genus
65
Paracoccus. Here, we used polyphasic approach to identify and classify this strain.
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The soil sample was collected from Nam Tra My district, Quang Nam province, Vietnam (15°01'54"N,
67
107°58'45"E) where Vietnamese ginseng is originally detected. The soil was thoroughly suspended
68
with sterilized 0.85% (w/v) of NaCl, following serial dilution steps of 10-5–10-6, was then spread onto a
69
modified Reasoner´s 2A (R2A, MB cell) agar medium (1/5-strength R2A). After three days culturing at
70
30 °C, isolates were picked up and purified. For long-term storage, isolates were mixed with 1/5-
71
strength R2A broth containing 30% (v/v) glycerol and maintained at -70 °C. Strain DCY94T was found
72
to form smooth, circular and creamy-coloured colony. Nutrient Broth (NB, MB cell) and Nutrient Agar
73
(NA) were used to maintain the isolate.
74
The 16S rRNA gene was amplified and sequenced from the chromosomal DNA of strain DCY94T
75
using the universal bacterial primer sets, 27F, 518F, 800R, and 1492R (Weisburg et al., 1991; Lane,
76
1991). These processes were performed by GenoTech Corp., Daejeon, Republic of Korea (Kim et al.,
3
77
2005). The partial sequences of the 16S rRNA gene were compiled with SeqMan software,
78
subsequently, the nearly complete sequence 1406 nucleotides was obtained and uploaded on the
79
EzTaxon-e server (Kim et al., 2012) to determine the pairwise similarity of the nearly complete 16S
80
rRNA gene sequences. The alignment of 16S rRNA gene sequences of strain DCY94 T and related type
81
strains were automatically performed by using the software CLUSTALX 2.0.10 program (Larkin et al.,
82
2007). Manually trimming of gaps was performed using the BioEdit program (Hall, 1999).
83
Evolutionary distances were calculated using the Tamura-Nei model (Tamura & Nei, 1993). The
84
phylogenetic affiliation of the retrieved sequences was inferred with the neighbour-joining (Saitou &
85
Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods
86
using the MEGA 6.0 programme (Tamura et al., 2013). Bootstrap analysis with 1,000 replicates was
87
also conducted in order to estimate the confidence levels of the tree topologies (Felsenstein, 1985).
88
Based on 16S rRNA gene sequence similarity, strain DCY94 T belongs to the genus Paracoccus and
89
was closely related to Paracoccus sphaerophysae Zy-3T (97.5% 16S rRNA gene sequence similarity),
90
and Paracoccus caeni MJ17T (96.9%). Paracoccus sphaerophysae HAMBI 3106T was obtained from
91
University of Helsinki, Faculty of Agriculture and Forestry, Division of Microbiology and
92
Biotechnology (HAMBI collection), and Paracoccus caeni KCTC 22480T was obtained from Korean
93
collection for type cultures (KCTC) for comparative purposes. Maximum-likelihood phylogenetic tree
94
clearly showed that strain DCY94T could be assigned into the genus Paracoccus (Fig. 1). Strain
95
DCY94T clustered with Paracoccus sphaerophysae Zy-3T and Paracoccus caeni MJ17T to form one
96
clade distinct from other Paracoccus species. These topologies were also supported in neighbour-
97
joining and maximum-parsimony phylogenetic trees (Fig. 1). Strain DCY94T shared high bootstrap
98
value to Paracoccus sphaerophysae Zy-3T, and low bootstrap value to Paracoccus caeni MJ17T.
99
The following tests were carried out on strain DCY94 T only. Colony morphology was observed after 2
100
days of incubation in NA agar at 30 °C. For transmission electron microscopy imaging, 20h-old
101
suspended cells were placed on formvar-carbon coated nickel grids for 30 s. The grids were floated on
102
drops of 0.1% (w/v) aqueous uranyl acetate for 1 min and then examined with transmission electron
103
microscopy (Carl Zeiss LEO912AB) at 80 kV under standard operating conditions. Gram type of the
104
cell was checked using a Gram staining kit (Sigma-Aldrich) according to the instructions of the
105
manufacturer. Anaerobic growth was tested by incubating cultures on NA plates in a BD GasPak™ EZ
106
anaerobe pouch system with indicator (Becton, Dickinson and Company) and AnaeroPack Rectangular
4
107
Jar (Mitsubishi gas chemical co., Inc.) that contained an Anaero pack-anaero (Mitsubishi gas chemical
108
co., Inc.) for 14 days at 30 ℃. Oxidase activity was detected by adding oxidase reagent (bioMérieux).
109
Catalase activity was assessed by observing bubble production after adding 3% (v/v) H 2O2 solution.
110
Motility test was done using the hanging drop method (Prescott & Harley, 2001). The temperature
111
range for growth was assayed at 4, 10, 15, 20, 25, 28, 30, 37 and 40 °C in NB broth for 5 days. Growth
112
in various media was assessed using R2A, NA, Trypticase Soya Agar (TSA, MB cell), Luria-Bertani
113
(LB, MB cell), and MacConkey agar (Difco) at 30 °C for 5 days. The pH range for growth was
114
examined between 4 and 10 at intervals of 0.5 units in R2A broth and incubation for 5 days at 30 °C;
115
pH values were adjusted by using the following buffers: citric acid/sodium citrate (pH 4.0-6.0),
116
Na2HPO4/NaH2PO4 (pH 6.0-8.0), Tris-HCl (pH 8.5-9.0) and glycine/NaOH (pH 9.5-10.0) (Gomori,
117
1955). The ability to grow in the presence of NaCl was performed at 30 °C in Difco nutrient broth
118
without additional NaCl and with up to 7% (w/v) NaCl in increments of 1%.
119
The following tests were carried out on strain DCY94T and two reference type strains Paracoccus
120
sphaerophysae HAMBI 3106T and Paracoccus caeni KCTC 22480T. Hydrolyses of starch, Tween 20,
121
Tween 80, DNA, L-tyrosine, casein, and gelatin were performed as described by Barrow & Feltham
122
(1993). Activity of phenylalanine deaminase, methyl red and Voges-Proskauber tests were examined
123
according to Lányí (1988). Thiosulfate iron H2S test was aslo checked (Levine et al., 1934).
124
Biochemical characteristics were further determined using API 20NE, ID 32GN, API ZYM, and API
125
50CH strips (bioMérieux). API tests were performed according to the instructions of the manufacturer.
126
API 20NE and ID 32GN strips were read after 24 and 48 h. API ZYM strips were checked after 12 h,
127
and API 50CH strips were observed after 2 and 5 days. Antibiotic susceptibility tests were checked by
128
disk diffusion method according to Chen et al., (2012), and Nokhal & Schlegel (1983). To test this,
129
inocula were prepared using the 12-h-old culture in NB broth at optical density adjusted 0.2 at 600 nm.
130
Then, the bacterial inocula were spread on Mueller-Hinton agar (Difco) plates. The following
131
antibiotics (Oxoid) were tested: carbenicillin, cefazolin, ceftazidine, erythromycin, lincomycin,
132
neomycin, novobiocin, oleandomycin, penicilin G, rifampicin, tetracycline, and vancomycin. The
133
susceptibility of cells to antibiotics was evaluated after 2 days incubation at 30 °C under aerobic
134
condition. The zones of inhibition were measured from the edges of antibiotics disc to the edges of
135
clear zones. Strain was considered to be resistant (inhibition zone 5 mm). Strain DCY94T and the reference type
5
137
strains were also screened for their plant growth promoting characteristics such as phosphate
138
solubilizing, siderophore and indole-3-acetic acid (IAA) producing. Phosphate solubilizing ability was
139
checked by streaking cells onto Pikovskaya agar medium (HiMedia) as described by Pikovskaya
140
(1948). After 7 days of incubation at 30 ℃, strains that induced clear zone around the growth were
141
considered as positive for phosphate solubilization. Siderophore producing capacity was tested by
142
streaking cells on Pseudomonas agar F (Difco) medium supplemented with a chrome azurol S complex
143
[CAS/iron(III)/hexadeciltrimethyl ammonium bromide], as described by Schwyn & Neilands (1987).
144
Inoculates were spotted on the plates and incubated at 30 ℃ for 5 days. Appearance of yellow-orange
145
halo around the growth proved strain is positive for siderosphore production. Production of IAA was
146
evaluated using King B (Glickmann & Dessaux, 1995) with the modifications as following ingredients
147
(per l): casein (10 g), peptone no.3 (10 g), dipotassium phosphate (1.5 g), magnesium sulfate (1.5 g),
148
glycerol (15 ml), and L-tryptophan (1 g). Optical density was measured at 540 nm.
149
For fatty acids analysis, cell biomass of strain DCY94T and the reference type strains was collected
150
from cells grown in NA at 30 °C for 18 to 24 h. Fatty acids were extracted, methylated using the
151
standard protocol of MIDI (Sherlock Microbial Identification System) as described by Sasser (1990)
152
and then separated by gas chromatography (Agilent GC 6890). Identification of the methyl esters was
153
conducted by using the TSBA library (version 6.1). Polar lipids of strain DCY94T were extracted and
154
analyzed by two dimensional TLC (Minnikin et al., 1984). Polar lipids extracts were spotted onto the
155
lower left-hand corner of a thin layer plates (silica gel 60 pre-prepared plates of Merck Art.No.5553,
156
10x10 cm). The plates were developed with chloroform/methanol/water (65:25:4, by vol.) in the first
157
direction and chloroform/acetic acid/methanol/water (80:15:12:4, by vol.) in the second direction. The
158
total polar lipids, aminolipids, phospholipids, glycolipids and phosphatidylcholine were detected by
159
staining with 5% of molybdophosphoric acid in ethanol, 0.2% of ninhydrin in saturated-butanol,
160
molybdenum blue reagent (1.3% molybdenum oxide in 4.2 M sulfuric acid, Sigma-Aldrich), 0.5% of
161
1-naphthol in methanol/water (1:1, v/v) and the sulfuric acid/ethanol (1:1, v/v), and Dragendorff’s
162
reagent (Merck), respectively. The respiratory quinones were extracted from 100 mg freeze-dried cells
163
and analyzed using HPLC (Hiraishi et al., 1996). The extracted ubiquinone was detected in Agilent
164
1260 infinity HPLC system (Agilent Technologies) with an Agilent Poroshell 120 EC-C18 column
165
(30x50 mm, 2.7 ㎛) at 270 nm. The mobiles phase was mixture of acetonitrile/isopropanol (65:35, v/v)
166
at the flow rate was 0.5 ml min-1. Polyamine was extracted and analyzed as described by Busse &
6
167
Auling (1988), and Taibi et al. (2000). The polyamine standards including spermine, spermidine, and
168
putrescine were purchased from Sigma-Aldrich. The internal standard was 1, 8-diaminooctane (Sigma-
169
Aldrich). Amines extract was applied to Agilent 1260 infinity HPLC system using an Agilent Poroshell
170
120 EC-C18 column (30x50 mm, 2.7 µm). The 60% methanol was used as mobile phase with the flow
171
rate of 0.3 ml min-1. The analysis was monitored by UV/VIS detector set at 234 nm.
172
To determine G+C mol% content, the genomic DNA was degraded into nucleosides by P1 nuclease
173
and alkaline phosphatase enzymes as described by Mesbah et al. (1989). Then nucleosides were
174
detected by HPLC (NS-4000, Futecs co. Ltd.) using an YMC-Triart C18 (250 x 4.6 mm, 5 µm).
175
Elution was mixture of 25 mM (NH4)H2PO4/acetonitrile (20:1, v/v) at the flow rate of 1.2 ml min-1.
176
Detection was performed by UV absorption at the wavelength of 270 nm. The genomic DNA of
177
Escherichia coli strain B (D4889, Sigma-Aldrich) was used as a standard. To confirm difference of
178
strain DCY94T from the type strains of closely related species, DNA-DNA hybridization was carried
179
out. Hybridization experiments were conducted fluorometrically using photobiotin-labeled DNA
180
probes and microdilution wells as reported by Ezaki et al., (1989). The hybridization temperature was
181
51 °C. The experiment was performed with five replications for each sample. The highest and lowest
182
values obtained from each sample were excluded, and the mean of the remaining three values were
183
calculated for DNA-DNA relatedness.
184
All strains in this study were susceptible to carbenicillin (100 µg), cefazolin (30 µg), ceftazidime (30
185
µg), erythromycin (15 µg), neomycin (30 µg), novobiocin (30 µg), rifampicin (5 µg) and tetracycline
186
(30 µg), and vancomycin (30 µg). However, strain DCY94T and Paracoccus sphaerophysae HAMBI
187
3106T were resistant to lincomycin (15 µg), and susceptible to oleandomycin (15 µg) and penicillin G.
188
(10 UI); whereas Paracoccus caeni KCTC 22480T exhibited opposite results. Physiological and
189
biochemical characteristics of strain DCY94T are summarized in the species description and the
190
comparison of selective characteristics with related type strains is shown in Table 1. Strain DCY94T
191
produced significantly higher amount of IAA than reference strains. The obtained results revealed
192
significantly differences between strain DCY94T and Paracoccus caeni KCTC 22480T. Moreover,
193
many contrast characteristics between strain DCY94T and the closest type strain Paracoccus
194
sphaerophysae HAMBI 3106T were detected such as hydrolyses of Tween 20, tyrosine; acid
195
production from arbutin, D-maltose, sucrose, and D-raffinose; assimilation of suberic acid, L-alanine,
196
potassium 5-ketogluconate, glycogen, L-serine, D-mannose, and phenyl acetic acid.
7
197
The overall fatty acid compositions of strain DCY94T and the two reference type strains were similar
198
(Supplementary Table S1). Strain DCY94T contained predominant quantities of summed feature 8
199
(C18:1 ω7c and/or C18:1 ω6c) (88.4%) and moderate to small quantities of C8:0 3-OH (1.0%), C10:0 3-OH
200
(2.8%), C18:0 (5.2%), which is in good agreement with fatty acids composition of members of the genus
201
Paracoccus. The absence of C16:0, C19:0 cyclo ω5c, and summed feature 2 (ante-C18:0 and/or C18:2
202
ω6,9c) which distinguished strain DCY94T from the two reference type strains. The same polyamines
203
patterns were detected in strain DCY94T and the type strain Paracoccus sphaerophysae HAMBI
204
3106T. Putrescine and spermidine were found as major quantities, whereas sym-homospermidine and
205
spermine as trace amount. The presence of highest amount of putrescine as well as trace amount of
206
sym-homospermidine in strain DCY94T is agreed with previous study on polyamines profile of the type
207
strain Paracoccus rhizosphaerae CC-CCM15-8T (Kämpfer et al., 2012). The polar lipids profile of
208
strain DCY94T is shown in supplementary Fig. S1. Phosphatidylethanolamine (PE) was one of major
209
polar lipids of strain DCY94 T that is consistent with data previously published by Kämpfer et al.
210
(2012). Moreover, the major polar lipid profile of strain DCY94T is similar to Paracoccus alcaliphilus
211
JCM 7363T (Sheu et al., 2011), Paracoccus marinus NBRC 100637T and Paracoccus homiensis
212
KACC 11518T (Chen et al., 2011), which consisted of major amount of phosphatidylethanolamine
213
(PE), phosphatidylglycerol (PG), phosphatidylcholine (PC) and one unidentified glycolipid (GL1).
214
However, minor to trace amounts of one unidentified aminolipid (AL1), one unidentified
215
aminophospholipid (APL1), one unidentified phospholipid (PL1), four unidentified polar lipids (L1-4)
216
that distinguished strain DCY94T with recognized species of the genus Paracoccus. The ubiquinone of
217
strain DCY94T was Q-10. Overall, chemotaxonomic data clearly show that strain DCY94 T contains
218
typical characteristics of the genus Paracoccus as well as strain-specific characteristics.
219
The G+C content of strain DCY94T was 68.3 mol%, a value within the range reported for Paracoccus
220
species which is from 58.7 mol% (Paracoccus caeni MJ17T; Lee et al., 2011) to 70 mol% (Paracoccus
221
solventivorans L1T; Siller et al., 2000). Levels of DNA-DNA relatedness between strain DCY94T and
222
Paracoccus sphaerophysae HAMBI 3106T, Paracoccus caeni KCTC 22480T were 52 and 50%,
223
respectively. These values was enough to separate strain DCY94T as distinct species of the genus
224
Paracoccus (Wayne et al., 1987).
8
225
On the basis of phylogenetic inference, phenotypic, chemotaxonomic and genetic data, strain DCY94T
226
should be classified in the genus Paracoccus as the type strain of a novel species, for which the name
227
Paracoccus panacisoli sp. nov. is proposed.
228 229
Description of Paracoccus panacisoli sp. nov.
230
Paracoccus panacisoli (pa.na.ci.so′li. N.L. n. Panax -acis scientific name of ginseng; L. n. solum –i
231
soil; N.L. gen. n. panacisoli of soil of a ginseng cultivation, the source of isolation of the type strain).
232
Cells are Gram-reaction-negative, non-motile, catalase- and oxidase-positive, facultative anaerobic
233
rods approximately 0.57 μm in diameter, 1.35 μm in length. Colonies on NA agar are creamy-coloured,
234
circular, convex, and 0.5-1.0 mm in diameter after 2 days of incubation. Growth occurs at 15-37 ˚C
235
(optimum, 30 ˚C), and at pH 5.5–9.0 (optimum, pH 6.5–7.0). Cells are able to grow in Difco nutrient
236
broth without added NaCl or with NaCl up to 6% (w/v), the optimum additional amount is 2% (w/v).
237
Growth also occurs on NA, R2A, LB and TSA medium, but not on MacConkey agar. IAA and
238
siderophore are produced but phosphate is not able to solubilize. L-tyrosine is degraded, but DNA,
239
casein, Tween 20, Tween 80, gelatin, and starch are not. Production of H2S, activity of phenylalanine
240
deaminase, methyl red and Voges-Proskauber tests are negative. In the API 20NE and 32 GN strips,
241
the following reactions are positive; assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol,
242
D-maltose, potassium gluconate, malic acid, phenylacetic acid, L-rhamnose, D-ribose, itaconic acid,
243
suberic acid, sodium malonate, sodium acetate, lactic acid, L-alanine, potassium 5-ketogluconate, 3-
244
hydroxybenzoic acid, L-serine, salicin, D-sorbitol, propionic acid, valeric acid, L-histidine, 3-
245
hyroxybutyric acid, 4-hydroxybenzoic acid, and L-proline; the following reactions are negative:
246
reduction of nitrates, fermentation of glucose, activity of urease, arginine dihydrolase and β-
247
galactosidase activity, hydrolysis of esculine, and assimilation of N-acetyl-glucosamine, adipic acid,
248
capric acid, trisodium citrate, D-saccharose, D-melibiose, L-fucose, and potassium 2-ketogluconate. In
249
the API ZYM strip, activity of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine
250
arylamidase,
251
phosphohydrolase, are detected; but activity of lipase, trypsin, α–chymotrypsin, α-galactosidase, β-
252
galactosidase,
253
mannosidase and α-fucosidase are not. Acid is produced from glycerol, D-arabinose, L-arabinose, D-
254
ribose, D-xylose, L-xylose, D-adonitol, D-glucose, D-fructose, D-mannose, dulcitol, D-mannitol, D-
valine
arylamidase,
α-glucosidase,
cystine
arylamidase,
β-glucuronidase,
acid
β-glucosidase,
9
phosphatase,
naphthol-AS-BI-
N-acetyl-β-glucosaminidase,
α-
255
sorbitol, amygdalin, arbutin, salicin, D-cellobiose, D-lactose, D-trehalose, xylitol, gentiobiose, D-
256
lyxose, D-fucose, D-arabitol, and L-arabitol; but not from erythritol, methyl-β-D-xylopyranoside, D-
257
galactose, L-sorbose, L-rhamnose, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, N-
258
acetyl-glucosamine, esculine, D-maltose, D-melibiose, sucrose, inulin, D-melezitose, D-raffinose,
259
starch, glycogen, D-turanose, D-tagatose, potassium gluconate, and potassium 5-ketogluconate. The
260
major cellular fatty acid is summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c). The major polar
261
lipids are phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and one unidentified
262
glycolipid. The major polyamines are putrescine and spermidine. The predominant ubiquinone is Q10
263
and the genomic DNA G+C content is 68.3 mol%.
264 265
The type strain, DCY94T (=KCTC 42086T=JCM 30337T), was isolated from a soil sample cultivated with ginseng in Quang Nam province, Vietnam.
266 267
Acknowledgments
268
This research was supported by a grant from Korea Institute of Planning & Evaluation for
269
Technology in Food, Agriculture, Forestry & Fisheries (KIPET No: 313038-03-1-SB010), and also
270
supported by Business for Cooperative R&D between Industry, Academy, and Research Institute
271
funded Korea Small and Medium Business Administration (Grants No: C0214183).
272
10
273
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gram-negative,
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15
bacterium
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Paracoccus
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Table 1. Different characteristics of strain DCY94T and related Paracoccus type strains.
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Strains: 1, strain DCY94T; 2, P. sphaerophysae HAMBI 3106T; 3, P. caeni KCTC 22480T
414
All data were from this study, except of DNA G+C mol%. +, positive; -, negative.
415
416
Characteristics 1 Hydrolysis of Tween 20 Tyrosine + Enzyme activity α-chymotrypsin α-glucosidase Assimilation of N-acetyl-glucosamine Inositol D-saccharose D-maltose Itaconic acid + Suberic acid + Sodium malonate + Sodium acetate + Lactic acid + L-alanine + Potassium 5-ketogluconate + Glycogen 3-hydroxybenzoic acid + L-serine + Salicin + Potassium 2-ketogluconate D-mannose + Phenylacetic acid + Acid production from Erythritol D-galactose L-sorbose L-rhamnose Dulcitol + Amygdalin + Arbutin + Salicin + D-maltose Sucrose D-melezitose D-raffinose D-turanose D-arabitol + L-arabitol + IAA production (µg ml-1) 134.4±0.58 G+C content (mol %) 68.3 Data cited from: †, Deng et al. (2010); ‡, Lee et al. (2011).
16
2
3
+ -
+
-
+ +
+ + + + + + + + -
+ + + + + + + + + +
+ + + + + + + + 40.7±0.20 67.1†
+ + + + + + + + 11.4±0.10 58.7‡
417
Legend of figure
418
Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences, showing the
419
taxonomic position of strain DCY94T in the genus Paracoccus. Palleronia marisminoris B33T was
420
used as outgroup. Filled circles indicate that the corresponding nodes were also recovered in the trees
421
generated with the neighbour-joining and maximum-parsimony algorithm. Bootstrap values >50%
422
based on 1000 replications to provide confident estimates for phylogenetic tree topology are shown at
423
branching points. Bar, 0.01 substitutions per nucleotide position.
17
Fig 1 Click here to download Figure: Fig. 1.pdf
Paracoccus aminovorans JCM 7685T (D32240) Paracoccus huijuniae FLN-7T (EU725799) Paracoccus yeei G1212T (AY014173) Paracoccus thiocyanatus THI 011T (D32242) Paracoccus denitrificans ATCC 17741T (Y16927) Paracoccus aminophilus JCM 7686T (D32239) Paracoccus lutimaris HDM-25T (KJ451483) 82 Paracoccus zhejiangensis J6T (JN561152) Paracoccus marinus KKL-A5T (AB185957) Paracoccus isoporae SW-3T (FJ593906) Paracoccus limosus NB88T (HQ336256) 99
0.01
66
Paracoccus halophilus HN-182T (DQ423482) Paracoccus sulfuroxidans LW36T (DQ512861) 97 Paracoccus bengalensis LMG 22700T (AJ864469) 93 Paracoccus versutus ATCC 25363T (Y16932) 91 Paracoccus pantotrophus ATCC 35512T (Y16933) 85 Paracoccus methylutens DM12T (AF250334) Paracoccus kondratievae GBT (AF250332) Paracoccus caeni MJ17T (GQ250442) Paracoccus panacisoli DCY94T (KJ653224) 99 Paracoccus sphaerophysae Zy-3T (GU129567) Paracoccus alcaliphilus JCM 7364T (D32238) 54 Paracoccus siganidrum M26T (JX398976) Paracoccus sediminis CMB17T (JX126474) Paracoccus oceanense JLT1679T (HQ638977) 88 Paracoccus stylophorae KTW-16T (GQ281379) Paracoccus seriniphilus MBT-A4T (AJ428275) 73 T 100 Paracoccus carotinifaciens E-396 (AB006899)
64
Paracoccus haeundaesis BC74171T (AY189743) Paracoccus zeaxanthinifaciens ATCC 21588T (AF461158) 89 Paracoccus homiensis DD-R11T (DQ342239) Paracoccus fistulariae KCTC 22803T (GQ260189) Paracoccus saliphilus JCM 7364T (DQ923133) Paracoccus tibetensis Tibet-S9a3T (DQ108402) 54 97 Paracoccus rhizosphaerae CC-CCM15-8T (JN662389) 73 Paracoccus beibuensis JLT1284T (EU650196) Paracoccus aestuarii B7T (EF660757) Paracoccus solventivorans DSM 6637T (Y07705) 98 Paracoccus alkenifer DSM 11593T (Y13827) 100 Paracoccus chinensis KS-11T (EU660389) Paracoccus niistensis NII-0918T (FJ842690) Paracoccus koreensis Ch05T (AB187584) Paracoccus kocurii JCM 7684T (D32241) Palleronia marisminoris B33T (AY926462)
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary material.pdf
International Journal of Systematic and Evolutionary Microbiology Paracoccus panacisoli sp. nov., isolated from a forest soil cultivated with Vietnamese ginseng --Manuscript Draft-Manuscript Number:
IJS-D-15-00142
Full Title:
Paracoccus panacisoli sp. nov., isolated from a forest soil cultivated with Vietnamese ginseng
Short Title:
Paracoccus panacisoli sp. nov.
Article Type:
Standard
Section/Category:
New taxa - Proteobacteria
Keywords:
Paracoccus panacisoli, Vietnamese ginseng soil, polyamine, IAA production
Corresponding Author:
Deok-Chun Yang Kyung Hee University Yongin, Gyeonggi-do KOREA, REPUBLIC OF
First Author:
Ngoc-Lan Nguyen, Master
Order of Authors:
Ngoc-Lan Nguyen, Master Yeon-Ju Kim Van-An Hoang, PhD Bao-Tram Tran, Ms Huong-Son Pham, Dr Deok-Chun Yang
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A novel bacterial strain, designated DCY94T, was isolated from forest soil cultivated with ginseng in Vietnam, was Gram-reaction-negative, facultative anaerobic, nonmotile, rod-shaped, catalase- and oxidase-positive. The 16S rRNA gene sequence analysis demonstrated that strain DCY94T was closely related to Paracoccus sphaerophysae Zy-3T (97.5% 16S rRNA gene sequence similarity), and Paracoccus caeni MJ17T (96.9%). The fatty acid profile of strain DCY94T contained predominant amount of summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) (88.4%) and moderate to small quantities of C8:0 3-OH (1.0%), C10:0 3-OH (2.8%) and C18:0 (5.2%). Phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and one unidentified glycolipid were major polar lipids; one unidentified aminolipid, one unidentified aminophospholipid, one unidentified phospholipid, and four unidentified polar lipids were minor. The polyamine pattern comprised major compounds putrescine and spermidine, and minor amounts of sym-homospermidine and spermine. The ubiquinone of this strain was Q-10 and the G+C content of its genomic DNA was 68.3 mol%. All these results support the placement of strain DCY94T within the genus Paracoccus. Levels of DNA-DNA relatedness between strain DCY94T and Paracoccus sphaerophysae HAMBI 3106T, Paracoccus caeni KCTC 22480T were 52 and 50%, respectively. The phylogenetic analysis, phenotypic tests, chemotaxonomic characteristics, and DNA-DNA relatedness distinguished strain DCY94T from the closest recognized species of the genus Paracoccus suggest that this strain represents a novel species, for which the name Paracoccus panacisoli sp. nov. is proposed. The type strain is DCY94T (=KCTC 42086T=JCM 30337T).
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1
Paracoccus panacisoli sp. nov., isolated from a forest soil cultivated with Vietnamese ginseng
2 3
Ngoc-Lan Nguyen1, Yeon-Ju Kim1*, Van-An Hoang1, Bao-Tram Tran2, Huong-Son Pham2 and Deok-
4
Chun Yang1,3
5 6
1
Department of Oriental Medicinal Biotechnology, Kyung-Hee University, Seocheon-dong, Giheung-
7 8
gu, Yongin-si, Gyeonggi-do, 446-701, Republic of Korea. 2
Center for Experimental Biology, National Center for Technological Progress, Ministry of Science
9 10 11
and Technology, C6 Thanh Xuan Bac, Hanoi, Vietnam. 3
Graduate School of Biotechnology and Ginseng Bank, Kyung-Hee University, Seocheon-dong, Giheung-gu, Yongin-si, Gyeonggi-do, 446-701, Republic of Korea.
12 13
Subject category: New taxa; Subsection: Proteobacteria
14
Running title: Paracoccus panacisoli sp. nov.
15 16
* Corresponding author
17
Email: [email protected]
18
Tel: +82-31-201-2100, Fax: +82-31-202-2688
19 20
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DCY94T is
21
KJ653224.
22 23
One supplementary table and one supplementary figure are available with the online version of this
24
paper.
1
25
Abstract
26
A novel bacterial strain, designated DCY94T, was isolated from forest soil cultivated with
27
ginseng in Vietnam, was Gram-reaction-negative, facultative anaerobic, non-motile, rod-shaped,
28
catalase- and oxidase-positive. The 16S rRNA gene sequence analysis demonstrated that strain
29
DCY94T was closely related to Paracoccus sphaerophysae Zy-3T (97.5% 16S rRNA gene sequence
30
similarity), and Paracoccus caeni MJ17T (96.9%). The fatty acid profile of strain DCY94T contained
31
predominant amount of summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) (88.4%) and moderate to small
32
quantities of C8:0 3-OH (1.0%), C10:0 3-OH (2.8%) and C18:0 (5.2%). Phosphatidylethanolamine,
33
phosphatidylglycerol, phosphatidylcholine and one unidentified glycolipid were major polar lipids; one
34
unidentified aminolipid, one unidentified aminophospholipid, one unidentified phospholipid, and four
35
unidentified polar lipids were minor. The polyamine pattern comprised major compounds putrescine
36
and spermidine, and minor amounts of sym-homospermidine and spermine. The ubiquinone of this
37
strain was Q-10 and the G+C content of its genomic DNA was 68.3 mol%. All these results support the
38
placement of strain DCY94T within the genus Paracoccus. Levels of DNA-DNA relatedness between
39
strain DCY94T and Paracoccus sphaerophysae HAMBI 3106T, Paracoccus caeni KCTC 22480T were
40
52 and 50%, respectively. The phylogenetic analysis, phenotypic tests, chemotaxonomic
41
characteristics, and DNA-DNA relatedness distinguished strain DCY94T from the closest recognized
42
species of the genus Paracoccus suggest that this strain represents a novel species, for which the name
43
Paracoccus panacisoli sp. nov. is proposed. The type strain is DCY94 T (=KCTC 42086T=JCM
44
30337T).
45 46
Keywords: Paracoccus panacisoli, Vietnamese ginseng soil, polyamine, IAA production
2
47
The genus Paracoccus was proposed by Davis et al. (1969) and emended by Ludwig et al. (1993),
48
Katayama et al. (1995), and Liu et al. (2008). At the time of writing, the genus Paracoccus contains 40
49
species with validly published names (http://www.bacterio.net/paracoccus.html). Species of this genus
50
has been found in a variety of different habitats such as sediment (Liu et al., 2008; Roh et al., 2009; Li
51
et al., 2009), biofilter (Lipski et al., 1998), rhizosphere (Ghosh et al., 2006; Kämpfer et al., 2012),
52
sludge (La et al., 2005; Lee et al., 2011; Sun et al., 2013 ), fish (Kim et al., 2010), soil (Tsubokura et
53
al., 1999; Dastager et al., 2012), sea sand (Kim et al., 2006), sea water (Khan et al., 2008), and human
54
infection (Daneshvar et al., 2003). Phylogenetic analyses have indicated that the genus Paracoccus
55
belongs to the α-3 subclass of the phylum Proteobacteria with the closest relative being the genus
56
Rhodobacter (Tsubokura et al., 1999). Members of the genus Paracoccus consist of Gram-reaction-
57
negative coccid or short rods that shows substantial metabolic versatility. All species contain
58
ubiquinone-10 as the predominant respiratory, and C18:1 as major fatty acids. The G+C mol% content of
59
the genomic DNA is ranged 63–71 mol% (Kelly et al., 2006).
60
Though, many studies are focused to extract compounds from Vietnamese ginseng and their
61
applications. Nonetheless, no study of resident bacterial populations in Vietnamese ginseng cultivated
62
soil in Vietnam has been reported. Therefore, we used selective isolation and cultivation based on
63
traditional culture-dependent methods to investigate microorganisms inhabiting in this soil. During the
64
course of this study, we discovered strain DCY94T as a candidate for novel species of the genus
65
Paracoccus. Here, we used polyphasic approach to identify and classify this strain.
66
The soil sample was collected from Nam Tra My district, Quang Nam province, Vietnam (15°01'54"N,
67
107°58'45"E) where Vietnamese ginseng is originally detected. The soil was thoroughly suspended
68
with sterilized 0.85% (w/v) of NaCl, following serial dilution steps of 10-5–10-6, was then spread onto a
69
modified Reasoner´s 2A (R2A, MB cell) agar medium (1/5-strength R2A). After three days culturing at
70
30 °C, isolates were picked up and purified. For long-term storage, isolates were mixed with 1/5-
71
strength R2A broth containing 30% (v/v) glycerol and maintained at -70 °C. Strain DCY94T was found
72
to form smooth, circular and creamy-coloured colony. Nutrient Broth (NB, MB cell) and Nutrient Agar
73
(NA) were used to maintain the isolate.
74
The 16S rRNA gene was amplified and sequenced from the chromosomal DNA of strain DCY94T
75
using the universal bacterial primer sets, 27F, 518F, 800R, and 1492R (Weisburg et al., 1991; Lane,
76
1991). These processes were performed by GenoTech Corp., Daejeon, Republic of Korea (Kim et al.,
3
77
2005). The partial sequences of the 16S rRNA gene were compiled with SeqMan software,
78
subsequently, the nearly complete sequence 1406 nucleotides was obtained and uploaded on the
79
EzTaxon-e server (Kim et al., 2012) to determine the pairwise similarity of the nearly complete 16S
80
rRNA gene sequences. The alignment of 16S rRNA gene sequences of strain DCY94 T and related type
81
strains were automatically performed by using the software CLUSTALX 2.0.10 program (Larkin et al.,
82
2007). Manually trimming of gaps was performed using the BioEdit program (Hall, 1999).
83
Evolutionary distances were calculated using the Tamura-Nei model (Tamura & Nei, 1993). The
84
phylogenetic affiliation of the retrieved sequences was inferred with the neighbour-joining (Saitou &
85
Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods
86
using the MEGA 6.0 programme (Tamura et al., 2013). Bootstrap analysis with 1,000 replicates was
87
also conducted in order to estimate the confidence levels of the tree topologies (Felsenstein, 1985).
88
Based on 16S rRNA gene sequence similarity, strain DCY94 T belongs to the genus Paracoccus and
89
was closely related to Paracoccus sphaerophysae Zy-3T (97.5% 16S rRNA gene sequence similarity),
90
and Paracoccus caeni MJ17T (96.9%). Paracoccus sphaerophysae HAMBI 3106T was obtained from
91
University of Helsinki, Faculty of Agriculture and Forestry, Division of Microbiology and
92
Biotechnology (HAMBI collection), and Paracoccus caeni KCTC 22480T was obtained from Korean
93
collection for type cultures (KCTC) for comparative purposes. Maximum-likelihood phylogenetic tree
94
clearly showed that strain DCY94T could be assigned into the genus Paracoccus (Fig. 1). Strain
95
DCY94T clustered with Paracoccus sphaerophysae Zy-3T and Paracoccus caeni MJ17T to form one
96
clade distinct from other Paracoccus species. These topologies were also supported in neighbour-
97
joining and maximum-parsimony phylogenetic trees (Fig. 1). Strain DCY94T shared high bootstrap
98
value to Paracoccus sphaerophysae Zy-3T, and low bootstrap value to Paracoccus caeni MJ17T.
99
The following tests were carried out on strain DCY94 T only. Colony morphology was observed after 2
100
days of incubation in NA agar at 30 °C. For transmission electron microscopy imaging, 20h-old
101
suspended cells were placed on formvar-carbon coated nickel grids for 30 s. The grids were floated on
102
drops of 0.1% (w/v) aqueous uranyl acetate for 1 min and then examined with transmission electron
103
microscopy (Carl Zeiss LEO912AB) at 80 kV under standard operating conditions. Gram type of the
104
cell was checked using a Gram staining kit (Sigma-Aldrich) according to the instructions of the
105
manufacturer. Anaerobic growth was tested by incubating cultures on NA plates in a BD GasPak™ EZ
106
anaerobe pouch system with indicator (Becton, Dickinson and Company) and AnaeroPack Rectangular
4
107
Jar (Mitsubishi gas chemical co., Inc.) that contained an Anaero pack-anaero (Mitsubishi gas chemical
108
co., Inc.) for 14 days at 30 ℃. Oxidase activity was detected by adding oxidase reagent (bioMérieux).
109
Catalase activity was assessed by observing bubble production after adding 3% (v/v) H 2O2 solution.
110
Motility test was done using the hanging drop method (Prescott & Harley, 2001). The temperature
111
range for growth was assayed at 4, 10, 15, 20, 25, 28, 30, 37 and 40 °C in NB broth for 5 days. Growth
112
in various media was assessed using R2A, NA, Trypticase Soya Agar (TSA, MB cell), Luria-Bertani
113
(LB, MB cell), and MacConkey agar (Difco) at 30 °C for 5 days. The pH range for growth was
114
examined between 4 and 10 at intervals of 0.5 units in R2A broth and incubation for 5 days at 30 °C;
115
pH values were adjusted by using the following buffers: citric acid/sodium citrate (pH 4.0-6.0),
116
Na2HPO4/NaH2PO4 (pH 6.0-8.0), Tris-HCl (pH 8.5-9.0) and glycine/NaOH (pH 9.5-10.0) (Gomori,
117
1955). The ability to grow in the presence of NaCl was performed at 30 °C in Difco nutrient broth
118
without additional NaCl and with up to 7% (w/v) NaCl in increments of 1%.
119
The following tests were carried out on strain DCY94T and two reference type strains Paracoccus
120
sphaerophysae HAMBI 3106T and Paracoccus caeni KCTC 22480T. Hydrolyses of starch, Tween 20,
121
Tween 80, DNA, L-tyrosine, casein, and gelatin were performed as described by Barrow & Feltham
122
(1993). Activity of phenylalanine deaminase, methyl red and Voges-Proskauber tests were examined
123
according to Lányí (1988). Thiosulfate iron H2S test was aslo checked (Levine et al., 1934).
124
Biochemical characteristics were further determined using API 20NE, ID 32GN, API ZYM, and API
125
50CH strips (bioMérieux). API tests were performed according to the instructions of the manufacturer.
126
API 20NE and ID 32GN strips were read after 24 and 48 h. API ZYM strips were checked after 12 h,
127
and API 50CH strips were observed after 2 and 5 days. Antibiotic susceptibility tests were checked by
128
disk diffusion method according to Chen et al., (2012), and Nokhal & Schlegel (1983). To test this,
129
inocula were prepared using the 12-h-old culture in NB broth at optical density adjusted 0.2 at 600 nm.
130
Then, the bacterial inocula were spread on Mueller-Hinton agar (Difco) plates. The following
131
antibiotics (Oxoid) were tested: carbenicillin, cefazolin, ceftazidine, erythromycin, lincomycin,
132
neomycin, novobiocin, oleandomycin, penicilin G, rifampicin, tetracycline, and vancomycin. The
133
susceptibility of cells to antibiotics was evaluated after 2 days incubation at 30 °C under aerobic
134
condition. The zones of inhibition were measured from the edges of antibiotics disc to the edges of
135
clear zones. Strain was considered to be resistant (inhibition zone 5 mm). Strain DCY94T and the reference type
5
137
strains were also screened for their plant growth promoting characteristics such as phosphate
138
solubilizing, siderophore and indole-3-acetic acid (IAA) producing. Phosphate solubilizing ability was
139
checked by streaking cells onto Pikovskaya agar medium (HiMedia) as described by Pikovskaya
140
(1948). After 7 days of incubation at 30 ℃, strains that induced clear zone around the growth were
141
considered as positive for phosphate solubilization. Siderophore producing capacity was tested by
142
streaking cells on Pseudomonas agar F (Difco) medium supplemented with a chrome azurol S complex
143
[CAS/iron(III)/hexadeciltrimethyl ammonium bromide], as described by Schwyn & Neilands (1987).
144
Inoculates were spotted on the plates and incubated at 30 ℃ for 5 days. Appearance of yellow-orange
145
halo around the growth proved strain is positive for siderosphore production. Production of IAA was
146
evaluated using King B (Glickmann & Dessaux, 1995) with the modifications as following ingredients
147
(per l): casein (10 g), peptone no.3 (10 g), dipotassium phosphate (1.5 g), magnesium sulfate (1.5 g),
148
glycerol (15 ml), and L-tryptophan (1 g). Optical density was measured at 540 nm.
149
For fatty acids analysis, cell biomass of strain DCY94T and the reference type strains was collected
150
from cells grown in NA at 30 °C for 18 to 24 h. Fatty acids were extracted, methylated using the
151
standard protocol of MIDI (Sherlock Microbial Identification System) as described by Sasser (1990)
152
and then separated by gas chromatography (Agilent GC 6890). Identification of the methyl esters was
153
conducted by using the TSBA library (version 6.1). Polar lipids of strain DCY94T were extracted and
154
analyzed by two dimensional TLC (Minnikin et al., 1984). Polar lipids extracts were spotted onto the
155
lower left-hand corner of a thin layer plates (silica gel 60 pre-prepared plates of Merck Art.No.5553,
156
10x10 cm). The plates were developed with chloroform/methanol/water (65:25:4, by vol.) in the first
157
direction and chloroform/acetic acid/methanol/water (80:15:12:4, by vol.) in the second direction. The
158
total polar lipids, aminolipids, phospholipids, glycolipids and phosphatidylcholine were detected by
159
staining with 5% of molybdophosphoric acid in ethanol, 0.2% of ninhydrin in saturated-butanol,
160
molybdenum blue reagent (1.3% molybdenum oxide in 4.2 M sulfuric acid, Sigma-Aldrich), 0.5% of
161
1-naphthol in methanol/water (1:1, v/v) and the sulfuric acid/ethanol (1:1, v/v), and Dragendorff’s
162
reagent (Merck), respectively. The respiratory quinones were extracted from 100 mg freeze-dried cells
163
and analyzed using HPLC (Hiraishi et al., 1996). The extracted ubiquinone was detected in Agilent
164
1260 infinity HPLC system (Agilent Technologies) with an Agilent Poroshell 120 EC-C18 column
165
(30x50 mm, 2.7 ㎛) at 270 nm. The mobiles phase was mixture of acetonitrile/isopropanol (65:35, v/v)
166
at the flow rate was 0.5 ml min-1. Polyamine was extracted and analyzed as described by Busse &
6
167
Auling (1988), and Taibi et al. (2000). The polyamine standards including spermine, spermidine, and
168
putrescine were purchased from Sigma-Aldrich. The internal standard was 1, 8-diaminooctane (Sigma-
169
Aldrich). Amines extract was applied to Agilent 1260 infinity HPLC system using an Agilent Poroshell
170
120 EC-C18 column (30x50 mm, 2.7 µm). The 60% methanol was used as mobile phase with the flow
171
rate of 0.3 ml min-1. The analysis was monitored by UV/VIS detector set at 234 nm.
172
To determine G+C mol% content, the genomic DNA was degraded into nucleosides by P1 nuclease
173
and alkaline phosphatase enzymes as described by Mesbah et al. (1989). Then nucleosides were
174
detected by HPLC (NS-4000, Futecs co. Ltd.) using an YMC-Triart C18 (250 x 4.6 mm, 5 µm).
175
Elution was mixture of 25 mM (NH4)H2PO4/acetonitrile (20:1, v/v) at the flow rate of 1.2 ml min-1.
176
Detection was performed by UV absorption at the wavelength of 270 nm. The genomic DNA of
177
Escherichia coli strain B (D4889, Sigma-Aldrich) was used as a standard. To confirm difference of
178
strain DCY94T from the type strains of closely related species, DNA-DNA hybridization was carried
179
out. Hybridization experiments were conducted fluorometrically using photobiotin-labeled DNA
180
probes and microdilution wells as reported by Ezaki et al., (1989). The hybridization temperature was
181
51 °C. The experiment was performed with five replications for each sample. The highest and lowest
182
values obtained from each sample were excluded, and the mean of the remaining three values were
183
calculated for DNA-DNA relatedness.
184
All strains in this study were susceptible to carbenicillin (100 µg), cefazolin (30 µg), ceftazidime (30
185
µg), erythromycin (15 µg), neomycin (30 µg), novobiocin (30 µg), rifampicin (5 µg) and tetracycline
186
(30 µg), and vancomycin (30 µg). However, strain DCY94T and Paracoccus sphaerophysae HAMBI
187
3106T were resistant to lincomycin (15 µg), and susceptible to oleandomycin (15 µg) and penicillin G.
188
(10 UI); whereas Paracoccus caeni KCTC 22480T exhibited opposite results. Physiological and
189
biochemical characteristics of strain DCY94T are summarized in the species description and the
190
comparison of selective characteristics with related type strains is shown in Table 1. Strain DCY94T
191
produced significantly higher amount of IAA than reference strains. The obtained results revealed
192
significantly differences between strain DCY94T and Paracoccus caeni KCTC 22480T. Moreover,
193
many contrast characteristics between strain DCY94T and the closest type strain Paracoccus
194
sphaerophysae HAMBI 3106T were detected such as hydrolyses of Tween 20, tyrosine; acid
195
production from arbutin, D-maltose, sucrose, and D-raffinose; assimilation of suberic acid, L-alanine,
196
potassium 5-ketogluconate, glycogen, L-serine, D-mannose, and phenyl acetic acid.
7
197
The overall fatty acid compositions of strain DCY94T and the two reference type strains were similar
198
(Supplementary Table S1). Strain DCY94T contained predominant quantities of summed feature 8
199
(C18:1 ω7c and/or C18:1 ω6c) (88.4%) and moderate to small quantities of C8:0 3-OH (1.0%), C10:0 3-OH
200
(2.8%), C18:0 (5.2%), which is in good agreement with fatty acids composition of members of the genus
201
Paracoccus. The absence of C16:0, C19:0 cyclo ω5c, and summed feature 2 (ante-C18:0 and/or C18:2
202
ω6,9c) which distinguished strain DCY94T from the two reference type strains. The same polyamines
203
patterns were detected in strain DCY94T and the type strain Paracoccus sphaerophysae HAMBI
204
3106T. Putrescine and spermidine were found as major quantities, whereas sym-homospermidine and
205
spermine as trace amount. The presence of highest amount of putrescine as well as trace amount of
206
sym-homospermidine in strain DCY94T is agreed with previous study on polyamines profile of the type
207
strain Paracoccus rhizosphaerae CC-CCM15-8T (Kämpfer et al., 2012). The polar lipids profile of
208
strain DCY94T is shown in supplementary Fig. S1. Phosphatidylethanolamine (PE) was one of major
209
polar lipids of strain DCY94 T that is consistent with data previously published by Kämpfer et al.
210
(2012). Moreover, the major polar lipid profile of strain DCY94T is similar to Paracoccus alcaliphilus
211
JCM 7363T (Sheu et al., 2011), Paracoccus marinus NBRC 100637T and Paracoccus homiensis
212
KACC 11518T (Chen et al., 2011), which consisted of major amount of phosphatidylethanolamine
213
(PE), phosphatidylglycerol (PG), phosphatidylcholine (PC) and one unidentified glycolipid (GL1).
214
However, minor to trace amounts of one unidentified aminolipid (AL1), one unidentified
215
aminophospholipid (APL1), one unidentified phospholipid (PL1), four unidentified polar lipids (L1-4)
216
that distinguished strain DCY94T with recognized species of the genus Paracoccus. The ubiquinone of
217
strain DCY94T was Q-10. Overall, chemotaxonomic data clearly show that strain DCY94 T contains
218
typical characteristics of the genus Paracoccus as well as strain-specific characteristics.
219
The G+C content of strain DCY94T was 68.3 mol%, a value within the range reported for Paracoccus
220
species which is from 58.7 mol% (Paracoccus caeni MJ17T; Lee et al., 2011) to 70 mol% (Paracoccus
221
solventivorans L1T; Siller et al., 2000). Levels of DNA-DNA relatedness between strain DCY94T and
222
Paracoccus sphaerophysae HAMBI 3106T, Paracoccus caeni KCTC 22480T were 52 and 50%,
223
respectively. These values was enough to separate strain DCY94T as distinct species of the genus
224
Paracoccus (Wayne et al., 1987).
8
225
On the basis of phylogenetic inference, phenotypic, chemotaxonomic and genetic data, strain DCY94T
226
should be classified in the genus Paracoccus as the type strain of a novel species, for which the name
227
Paracoccus panacisoli sp. nov. is proposed.
228 229
Description of Paracoccus panacisoli sp. nov.
230
Paracoccus panacisoli (pa.na.ci.so′li. N.L. n. Panax -acis scientific name of ginseng; L. n. solum –i
231
soil; N.L. gen. n. panacisoli of soil of a ginseng cultivation, the source of isolation of the type strain).
232
Cells are Gram-reaction-negative, non-motile, catalase- and oxidase-positive, facultative anaerobic
233
rods approximately 0.57 μm in diameter, 1.35 μm in length. Colonies on NA agar are creamy-coloured,
234
circular, convex, and 0.5-1.0 mm in diameter after 2 days of incubation. Growth occurs at 15-37 ˚C
235
(optimum, 30 ˚C), and at pH 5.5–9.0 (optimum, pH 6.5–7.0). Cells are able to grow in Difco nutrient
236
broth without added NaCl or with NaCl up to 6% (w/v), the optimum additional amount is 2% (w/v).
237
Growth also occurs on NA, R2A, LB and TSA medium, but not on MacConkey agar. IAA and
238
siderophore are produced but phosphate is not able to solubilize. L-tyrosine is degraded, but DNA,
239
casein, Tween 20, Tween 80, gelatin, and starch are not. Production of H2S, activity of phenylalanine
240
deaminase, methyl red and Voges-Proskauber tests are negative. In the API 20NE and 32 GN strips,
241
the following reactions are positive; assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol,
242
D-maltose, potassium gluconate, malic acid, phenylacetic acid, L-rhamnose, D-ribose, itaconic acid,
243
suberic acid, sodium malonate, sodium acetate, lactic acid, L-alanine, potassium 5-ketogluconate, 3-
244
hydroxybenzoic acid, L-serine, salicin, D-sorbitol, propionic acid, valeric acid, L-histidine, 3-
245
hyroxybutyric acid, 4-hydroxybenzoic acid, and L-proline; the following reactions are negative:
246
reduction of nitrates, fermentation of glucose, activity of urease, arginine dihydrolase and β-
247
galactosidase activity, hydrolysis of esculine, and assimilation of N-acetyl-glucosamine, adipic acid,
248
capric acid, trisodium citrate, D-saccharose, D-melibiose, L-fucose, and potassium 2-ketogluconate. In
249
the API ZYM strip, activity of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine
250
arylamidase,
251
phosphohydrolase, are detected; but activity of lipase, trypsin, α–chymotrypsin, α-galactosidase, β-
252
galactosidase,
253
mannosidase and α-fucosidase are not. Acid is produced from glycerol, D-arabinose, L-arabinose, D-
254
ribose, D-xylose, L-xylose, D-adonitol, D-glucose, D-fructose, D-mannose, dulcitol, D-mannitol, D-
valine
arylamidase,
α-glucosidase,
cystine
arylamidase,
β-glucuronidase,
acid
β-glucosidase,
9
phosphatase,
naphthol-AS-BI-
N-acetyl-β-glucosaminidase,
α-
255
sorbitol, amygdalin, arbutin, salicin, D-cellobiose, D-lactose, D-trehalose, xylitol, gentiobiose, D-
256
lyxose, D-fucose, D-arabitol, and L-arabitol; but not from erythritol, methyl-β-D-xylopyranoside, D-
257
galactose, L-sorbose, L-rhamnose, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, N-
258
acetyl-glucosamine, esculine, D-maltose, D-melibiose, sucrose, inulin, D-melezitose, D-raffinose,
259
starch, glycogen, D-turanose, D-tagatose, potassium gluconate, and potassium 5-ketogluconate. The
260
major cellular fatty acid is summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c). The major polar
261
lipids are phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and one unidentified
262
glycolipid. The major polyamines are putrescine and spermidine. The predominant ubiquinone is Q10
263
and the genomic DNA G+C content is 68.3 mol%.
264 265
The type strain, DCY94T (=KCTC 42086T=JCM 30337T), was isolated from a soil sample cultivated with ginseng in Quang Nam province, Vietnam.
266 267
Acknowledgments
268
This research was supported by a grant from Korea Institute of Planning & Evaluation for
269
Technology in Food, Agriculture, Forestry & Fisheries (KIPET No: 313038-03-1-SB010), and also
270
supported by Business for Cooperative R&D between Industry, Academy, and Research Institute
271
funded Korea Small and Medium Business Administration (Grants No: C0214183).
272
10
273
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15
bacterium
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Table 1. Different characteristics of strain DCY94T and related Paracoccus type strains.
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Strains: 1, strain DCY94T; 2, P. sphaerophysae HAMBI 3106T; 3, P. caeni KCTC 22480T
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All data were from this study, except of DNA G+C mol%. +, positive; -, negative.
415
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Characteristics 1 Hydrolysis of Tween 20 Tyrosine + Enzyme activity α-chymotrypsin α-glucosidase Assimilation of N-acetyl-glucosamine Inositol D-saccharose D-maltose Itaconic acid + Suberic acid + Sodium malonate + Sodium acetate + Lactic acid + L-alanine + Potassium 5-ketogluconate + Glycogen 3-hydroxybenzoic acid + L-serine + Salicin + Potassium 2-ketogluconate D-mannose + Phenylacetic acid + Acid production from Erythritol D-galactose L-sorbose L-rhamnose Dulcitol + Amygdalin + Arbutin + Salicin + D-maltose Sucrose D-melezitose D-raffinose D-turanose D-arabitol + L-arabitol + IAA production (µg ml-1) 134.4±0.58 G+C content (mol %) 68.3 Data cited from: †, Deng et al. (2010); ‡, Lee et al. (2011).
16
2
3
+ -
+
-
+ +
+ + + + + + + + -
+ + + + + + + + + +
+ + + + + + + + 40.7±0.20 67.1†
+ + + + + + + + 11.4±0.10 58.7‡
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Legend of figure
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Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences, showing the
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taxonomic position of strain DCY94T in the genus Paracoccus. Palleronia marisminoris B33T was
420
used as outgroup. Filled circles indicate that the corresponding nodes were also recovered in the trees
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generated with the neighbour-joining and maximum-parsimony algorithm. Bootstrap values >50%
422
based on 1000 replications to provide confident estimates for phylogenetic tree topology are shown at
423
branching points. Bar, 0.01 substitutions per nucleotide position.
17
Fig 1 Click here to download Figure: Fig. 1.pdf
Paracoccus aminovorans JCM 7685T (D32240) Paracoccus huijuniae FLN-7T (EU725799) Paracoccus yeei G1212T (AY014173) Paracoccus thiocyanatus THI 011T (D32242) Paracoccus denitrificans ATCC 17741T (Y16927) Paracoccus aminophilus JCM 7686T (D32239) Paracoccus lutimaris HDM-25T (KJ451483) 82 Paracoccus zhejiangensis J6T (JN561152) Paracoccus marinus KKL-A5T (AB185957) Paracoccus isoporae SW-3T (FJ593906) Paracoccus limosus NB88T (HQ336256) 99
0.01
66
Paracoccus halophilus HN-182T (DQ423482) Paracoccus sulfuroxidans LW36T (DQ512861) 97 Paracoccus bengalensis LMG 22700T (AJ864469) 93 Paracoccus versutus ATCC 25363T (Y16932) 91 Paracoccus pantotrophus ATCC 35512T (Y16933) 85 Paracoccus methylutens DM12T (AF250334) Paracoccus kondratievae GBT (AF250332) Paracoccus caeni MJ17T (GQ250442) Paracoccus panacisoli DCY94T (KJ653224) 99 Paracoccus sphaerophysae Zy-3T (GU129567) Paracoccus alcaliphilus JCM 7364T (D32238) 54 Paracoccus siganidrum M26T (JX398976) Paracoccus sediminis CMB17T (JX126474) Paracoccus oceanense JLT1679T (HQ638977) 88 Paracoccus stylophorae KTW-16T (GQ281379) Paracoccus seriniphilus MBT-A4T (AJ428275) 73 T 100 Paracoccus carotinifaciens E-396 (AB006899)
64
Paracoccus haeundaesis BC74171T (AY189743) Paracoccus zeaxanthinifaciens ATCC 21588T (AF461158) 89 Paracoccus homiensis DD-R11T (DQ342239) Paracoccus fistulariae KCTC 22803T (GQ260189) Paracoccus saliphilus JCM 7364T (DQ923133) Paracoccus tibetensis Tibet-S9a3T (DQ108402) 54 97 Paracoccus rhizosphaerae CC-CCM15-8T (JN662389) 73 Paracoccus beibuensis JLT1284T (EU650196) Paracoccus aestuarii B7T (EF660757) Paracoccus solventivorans DSM 6637T (Y07705) 98 Paracoccus alkenifer DSM 11593T (Y13827) 100 Paracoccus chinensis KS-11T (EU660389) Paracoccus niistensis NII-0918T (FJ842690) Paracoccus koreensis Ch05T (AB187584) Paracoccus kocurii JCM 7684T (D32241) Palleronia marisminoris B33T (AY926462)
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary material.pdf
