IJSEM Papers in Press. Published April 9, 2015 as doi:10.1099/ijs.0.000244
International Journal of Systematic and Evolutionary Microbiology Alcanivorax gelatiniphagus sp. nov., a marine bacterium isolated from tidal flat sediments enriched with crude oil --Manuscript Draft-Manuscript Number:
IJS-D-14-00445R1
Full Title:
Alcanivorax gelatiniphagus sp. nov., a marine bacterium isolated from tidal flat sediments enriched with crude oil
Short Title:
Alcanivorax gelatiniphagus sp. nov.
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Kae Kyoung Kwon, Ph.D. Korea Institute of Ocean Science & Technology Ansan, KOREA, REPUBLIC OF
First Author:
Kae Kyoung Kwon, Ph.D.
Order of Authors:
Kae Kyoung Kwon, Ph.D. Ji Hye Oh Sung-Hyun Yang Hyun-Seok Seo Jung-Hyun Lee
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A Gram-reaction-negative rod-shaped marine bacterium, designated as MEBiC08158T was isolated from sediments collected at the Taean County, Korea near the Hebei Spirit tanker oil spill accident. The 16S rRNA gene sequence analysis revealed that strain MEBiC08158T was closely related with Alcanivorax marinus R8-12T (99.5% similar) but distinguishable from other members of the genus Alcanivorax (93.7~97.1 %). The DNA-DNA hybridization value between strains MEBiC08158T and A. marinus R8-12T was 58.4%. Growth of strain MEBiC08158T was observed at 15-43°C (optimum 37~40°C), at pH 6.0-9.5 (optimum pH 7.0~8.0) and with 0.5-16 % (optimum 1.5~3.0%) NaCl. The dominant fatty acids were C16:0 (27.1%), C19:0 cyclo ω8c (19.1%), C12:0 (13.6%), C18:1 ω7c (12.4%), C12:0 3OH (8.4%), and summed feature 3 (comprising C15:0 2-OH and/or C16:1 ω7c; 6.1%). Several phenotypic characteristics differentiate strain MEBiC08158T from phylogenetically close members of the genus Alcanivorax. Therefore, strain MEBiC08158T should be classified as a novel species in the genus Alcanivorax, and it is proposed as A. gelatiniphagus sp. nov. The type strain is MEBiC08158T (=KCCM 42990 T =JCM 18425T).
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1
Alcanivorax gelatiniphagus sp. nov., a marine bacterium isolated from tidal flat sediments
2
enriched with crude oil
3 4 5
Kae Kyoung Kwon*, Ji Hye Oh, Sung-Hyun Yang, Hyun-Seok Seo, and Jung-Hyun Lee
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Marine Biotechnology Research Center, Korea Institute of Ocean Science & Technology, PO Box 29, Ansan
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425-600, Republic of Korea
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Running title: Alcanivorax gelatiniphagus sp. nov.
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Subject category; New Taxa (Proteobacteria)
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The GenBank accession number for the 16S rRNA gene sequence of strain MEBiC08158T is JQ937289.
17 18
*
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Phone : +82 31 400 6242.
Corresponding author Fax: +82 31 400 6232.
e-mail: [email protected]
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1
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A Gram-reaction-negative rod-shaped marine bacterium, designated as MEBiC08158T was isolated
22
from sediments collected at the Taean County, Korea near the Hebei Spirit tanker oil spill accident.
23
The 16S rRNA gene sequence analysis revealed that strain MEBiC08158T was closely related with
24
Alcanivorax marinus R8-12T (99.5% similar) but distinguishable from other members of the genus
25
Alcanivorax (93.7~97.1 %). The DNA-DNA hybridization value between strains MEBiC08158T and A.
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marinus R8-12T was 58.4%. Growth of strain MEBiC08158T was observed at 15-43°C (optimum
27
37~40°C), at pH 6.0-9.5 (optimum pH 7.0~8.0) and with 0.5-16 % (optimum 1.5~3.0%) NaCl. The
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dominant fatty acids were C16:0 (27.1%), C19:0 cyclo 8c (19.1%), C12:0 (13.6%), C18:1 7c (12.4%), C12:0
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3OH (8.4%), and summed feature 3 (comprising C15:0 2-OH and/or C16:1 7c; 6.1%). Several
30
phenotypic characteristics differentiate strain MEBiC08158T from phylogenetically close members of
31
the genus Alcanivorax. Therefore, strain MEBiC08158T should be classified as a novel species in the
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genus Alcanivorax, and it is proposed as A. gelatiniphagus sp. nov. The type strain is MEBiC08158T
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(=KCCM 42990 T =JCM 18425T).
34 35 36
Microorganisms highly specialized in hydrocarbon utilization has been discovered recently (Yakimov et al.,
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2007) and designated as 'obligate hydrocarbonoclastic bacteria' (OHCB). One of the OHCB genus is
38
Alcanivorax. Tthe genus Alcanivorax include OHCB and are distributed in low numbers in marine
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environments but dramatically increase in numbers in oil-polluted open and coastal seawaters, potentially
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comprising 80–90% of the oil-degrading microbial community (Harayama et al. 1999; Yakimov et al., 2005
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& 2007). Alcanivorax strains utilize a narrow range of substrates; predominantly alkanes and some organic
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acids (Golyshin et al., 2005). Recently, some members in this genus were revealed to utilize several amino
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acids and organic acids as well as glucose though weakly (Rivas et al., 2007; Lai et al, 2011). However, no
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detailed information on utilization patterns of organic acids was available. In the present study, results of the
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identification of a novel Alcanivoax microorganism isolated from sediments exposed to oil-pollution is
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reported.
2
47 48
Strain MEBiC08158T was isolated from a crude oil-enrichment culture of tidal flat sediments collected at
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Taean County, Korea where the Hebei Spirit oil spill accident occurred in 2007. Approx. 0.1 cm3 of
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sediments was inoculated into 20 ml MM2 inorganic medium (Sohn et al., 2004) supplemented with 0.3%
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Iranian Heavy oil and incubated for 2 weeks at 25 OC. Then, one ml was transferred into fresh media and
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cultivated for another two weeks. After cultivation appropriately diluted culture broth was spread onto
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marine agar 2216 medium (MA; BD). Inoculated plates were incubated at 25 OC for 3 days and individual
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colonies were isolated from MA depend on morphological differences. After primary isolation and
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purification, strain MEBiC08158T was cultivated at 25 OC on the same medium for biochemical and
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physiological characterization and stored at -80 OC in marine broth 2216 (MB; BD) supplemented with 20%
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(v/v) glycerol. For phenotypic comparisons, Alcanivorax venustensis ISO1T (= DSM 13974T, Fernández-
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Martínez et al., 2003), A. balearicus MACL04T (= LMG 22508T, Rivas et al., 2007), and A. dieselolei B-5T
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(= DSM 16502T, Liu & Shao, 2005) were purchased from DSMZ (Deutsche sammlung von
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Microorganismen und Zellkulturen GmbH) or BCCM/LMG (Belgian Co-ordinated Collections of
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Microorganisms) and A. marinus R8-12T (= MCCC 1A00382T, Lai et al., 2013) was provided from MCCC
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(Marine Culture Collection of China) and cultivated on MA at 25 OC. After checking the growth on volatile
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organic acids (Supplementary Fig. S4), strains were maintained with mineral salt medium (Yeast
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1.0 g, 1M phosphate buffer 1 ml, NH4Cl 1 g, NaCl 35 g in 1 liter DW) supplemented with 0.3% sodium-
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acetate (Designated it as Acetate-medium and abbreviated AA for agar medium and AB for liquid medium).
Extract
66 67
Extraction of genomic DNA was conducted by using a commercial DNA extraction kit (GeneAll). The 16S
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rRNA gene was amplified by using 27F and 1518R bacterial primer set (Giovannoni, 1991), the details of
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procedure and conditions were described in Lee et al. (2013). The amplified 16S rRNA gene was sequenced
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using an ABI 3730xl automatic DNA sequencer (ABI) according to the manufacturer’s instructions. Obtained
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sequences were assembled by using Vector NTI ver. 9.1 (Life Technologies) and compared by BLAST pair-
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wise alignment with sequences in EzTaxon-e database (Kim et al., 2012). Strain MEBiC08158T showed 99.5%
3
73
16S rRNA gene sequence similarity with the type strain of Alcanivorax marinus, however, similarity with
74
other validly reported members of the genus Alcanivorax was lower than 97.1%. Therefore, DNA-DNA
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hybridization with A. marinus was conducted according to the method described by Kaneko et al. (1978).
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The DNA-DNA relatedness value between strains MEBiC08158T and A. marinus MCCC 1A00382T was
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58.4% and was well below the threshold accepted for species delineation (Wayne et al., 1987).
78
Phylogenetic analysis based on almost complete 16S rRNA gene sequences (1,465 bps) of isolated strain
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with members of the family Alcanivoraceae was conducted using MEGA ver. 5.2 (Tamura et al., 2011). The
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neighbour-joining (Saitou & Nei, 1987) tree (Fig. 1) revealed that strain MEBiC08158T formed a coherent
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clade with the A. marinus and A. venustensis and the relationship was robustly supported by a bootstrap value
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of 100 %. The branch was also recovered in maximum-likelihood (Felsenstein, 1981) and maximum-
83
parsimony (Fitch, 1971) trees. Phylogenetic analysis performed using gyrB gene also conducted and
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obtained similar topology (Supplementary Fig. S2).
85 86
Unless otherwise stated, the physiological and morphological characterization was conducted according to
87
the methods described in Kwon et al. (2005) and Yang et al. (2006). Transmission electron micrographs were
88
taken using a LIBRA120 (Carl-Zeiss) electron microscope after negative staining of fixed cells using 2%
89
Phosphotungstic acid reagent at pH 7.0. The observations revealed rod-shaped cells with a long polar
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flagellum and numbers of inclusion bodies (Supplementary Fig. S1, available in IJSEM Online). The
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inclusion bodies stained with Sudan Black B dye and this result implied that major components of these
92
inclusion bodies are fatty storage matters such as poly-β-hydroxybutyrate. The growth temperature was
93
tested in AB at 12 different temperatures from 10 to 50 OC (10, 15, 21, 24.5, 28, 31, 34, 36.8, 39.8, 43.1, 46.6
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and 50 OC) in a temperature gradient incubator (TVS126MA; Adaventec) for up to 3 days. The tolerance
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range for NaCl was tested in AB supplemented with NaCl (Sigma; 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10,
96
12, 13, 14, 15, 16, 17, and 20%, w/v). The tolerance range for pH was determined (pH 4, 5, 6, 6.5, 7, 7.5, 8,
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8.5, 9, and 10) in AB with the pH adjusted using 10 mM MES (pH 4-6), HEPES (pH 6-8) or AMPSO (pH 8-
98
10) as biological buffers. To determine tolerance range of pH and NaCl range cells were cultivated at 37 OC
4
99
for 4 days. The bacterial suspension used to inoculate API 20E, 20NE, API ZYM, API 50CH kit
100
(bioMe´rieux) and a Microlog GN2 system (Biolog) was prepared in a 2% sea salt (Sigma) solution. The
101
biochemical characterization for strain MEBiC08108T was performed at 37 OC but reference strains were
102
cultivated at 28 OC for 2 days. Detailed results of the biochemical, morphological and physiological tests are
103
given in the species description and Table 1.
104 105
The cellular fatty acids profile was determined commercially by using the MIDI/Hewlett Packard Microbial
106
Identification System (Sasser, 1990) with Sherlock ver. 6.2 and RTSBA6 database was used for analysis
107
according to the manufacturer’s instruction on cells grown on AB for 1~2 days at 37 OC or 28 OC considering
108
the growth rates. The dominant fatty acids were determined as C16:0 (27.1%), C19:0 cyclo 8c (19.1%), C12:0
109
(13.6%), C18:1 7c (12.4%), C12:0 3OH (8.4%), and summed feature 3 (comprising C15:0 2-OH and/or C16:1
110
7c; 6.1%). The fatty acid composition was similar to that of the members of genus Alcanivorax, though the
111
proportion of some components was markedly different (Table 2). This result implied that novel isolate
112
belonging to the genus Alcanivorax but could be distinguished from existing type strains.
113
Polar lipids were extracted using a chloroform/methanol system and separated by two-dimensional TLC
114
using silica gel 60 F254 aluminum-backed thin-layer plates (Merck) (Minnkin, 1984). The details of
115
procedure and reagents were described in Yang et al. (2013). The predominant polar lipids were
116
diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminolipid, and
117
four unidentified aminophospholipids (Supplementary Fig. S3). The respiratory quinone could not
118
determined by HPLC analysis according to Collins (1985). The DNA G+C content was 65.2 mol%, as
119
determined from genome sequence analysis (data not published) and is within the reported range of that of
120
the genus Alcanivorax (Lai et al., 2013).
121 122
The results of the phylogentic anaylsis based on 16S rRNA gene sequences and gyrase B subunit sequences
123
indicates that strain MEBiC08158T is affiliated with the genus Alcanivorax (Fig. 1 & Fig. S2) and shares
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common features of this genus including Gram-staining properties, halophilic nature, major fatty acids
5
125
constituents, utilization of relatively short chain aliphatic hydrocarbons (data not shown) and some short
126
chain organic acids (Supplementary Fig. S4) as sole or principal carbon sources, and positive for oxidase and
127
catalase activities but could not utilize almost all other kinds of organic substrates (Table 1). But the isolate
128
could be distinguished from closely related members of the genus Alcanivorax (A. marinus, A. venustensis, A.
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dieselolei, and A. balearicus) by hydrolysis of gelatin, trypsin activity, and utilization of dextrin. Strain
130
MEBiC08158T could be further distinguished from A. marinus by the utilization pattern of hydroxy butyric
131
acids, from A. venustensis in assimilation of phenyl-acetate and D-mannose, utilization pattern of hydroxy
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butyric acids, and Naphtol-AS-BI phosphohydrolase activity, from A. dieselolei in assimilation of gluconate,
133
phenyl-acetate, acetate, cis-aconitic acid, phenyl ethyl- amine, α-keto glutaric acids, p-hydroxy phenylacetic
134
acid, bromo succinic acid and utilization of β- & γ-butyric acid, and from A. balearicus in assimilation of
135
citrate, phenyl-acetate, citrate, cis-aconitic acid, α-keto glutaric acids, formic acid, phenyl ethyl amine, and
136
utilization of β- and γ-butyric acid and activities of cystine arylamidase and N-acetyl-β-glucosaminidase
137
(Table 1). On the basis of this polyphasic taxonomical evidence, we propose that strain MEBiC08158T should
138
be classified as a novel species in the genus Alcanivorax with the name Alcanivorax gelatiniphagus sp. nov.
139 140
Description of Alcanivorax gelatiniphagus sp. nov.
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Alcanivorax gelatiniphagus (gel.la.ti.ni.pha'gus. N. L. neut. n. gelatininum gelatin; Gr. masc. n. phagos,
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glutton; N.L. masc. n. gelatiniphagus gelatin eater).
143
Cells are Gram-reaction--negative, rod-shaped, 0.5-0.6 m wide 1.2-2.4 m long, and forms beige colored
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colonies. Motile by means of a long polar flagellum. Produces extracellular matrix, bleb-like structures on
145
membrane, and fatty intracellular granules. Growth was observed at 15-43OC (optimum 37~40OC), at pH
146
6.0-9.5 (optimum 7.0~8.0) and with 0.5-16 % (optimum 1.5~3.0%) NaCl. Could not reduce nitrate. The
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strain could actively degrade relatively short-chain linear hydrocarbons (C10~C18) and growth was
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accelerated with 0.3% pyruvate, acetate or propionate. When assayed with API strips and Microlog GN2
149
plate, degrade dextrin, tweens 40 & 80, and gelatin. Enzyme activities of acid-& alkaline phosphatase,
150
esterase, esterase-lipase, lipase, trypsin, cystein-arylamidase, leucine-arylamidase & valine-arylamidase, and
6
151
phosphohydrolase are present. Produces acetoin and assimilates caprate, adipate, formate, phenyl acetate,
152
methyl pyruvate, mono-methyl succinate, acetate, butyrate, α-hydroxy butyrate, α-keto butyrate D,L-lactate,
153
propionate, sebacinate, succinate, and succinamate. However, almost no carbohydrates and amino acids were
154
utilized in API test strips or GN2 Microplate. The dominant fatty acids were C16:0, C19:0 cyclo 8c, C12:0, C18:1
155
7c,C12:0 3OH, and summed feature 3 (C15:0 2-OH and/or C16:1 7c). The type strain is MEBiC08158T
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(=KCCM 42990 T =JCM 18425T) isolated from tidal flat sediments of Taean County, Korea near the Hebei
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Spirit tanker oil spill accident.
158 159
Acknowledgements
160
This work was supported by the project titled "Oil spill environmental impact assessment and environmental
161
restoration" of the Ministry of Ocean & Fisheries, Korea & KIOST in-house program (PE99314).
162 163
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Kwon, K. K., Lee, H-S., Yang, S. H. & Kim, S-J. (2005). Kordiimonas gwangyangensis gen. nov., sp. nov.,
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a marine bacterium isolated from marine sediments that forms a distinct phyletic lineage (Kordiimonadales
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ord. nov.) in the ‘Alpha-Proteobacteria’. Int J Syst Evol Microbiol 55, 2033-2037.
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Lai, Q., Wang, L., Liu, Y., Fu, Y., Zhong, H., Wang, B., Chen, L., Wang, J., Sun, F. & Shao, Z. (2011).
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Alcanivorax pacificus sp. nov., isolated from a deep-sea pyrene-degrading consortium. Int J Syst Evol
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Alcanivorax balearicus sp. nov., isolated from Lake Martel. Int J Syst Evol Microbiol 57, 1331–1335.
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230
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9
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Table 1. Differential characteristics between MEBiC08158T and closely related species in the genus
233
Alcanivorax.
234
Strains: 1. MEBiC08158T (data were obtained in the present study); 2. A. marinus R8-12T (data from this study & from
235
Lai et al., 2013); 3. A. venustensis ISO1T (data from this study & from Fernández-Martínez et al., 2003); 4. A.
236
balearicus MACL04T (data from this study & from Rivas et al., 2007); 5. A. dieselolei B-5T (data from this study &
237
from Liu & Shao, 2005). +, Positive reaction; -, negative reaction; nd, no data. Positive for all strains; Gram negative
238
and motile by flagella. Oxidase, Catalase, Acid-& Alkaline phosphatase, Esterase, Esterase-lipase, Lipase, and Leu-
239
&Val-arylamidase activities on API ZYM strip. Acetoin production and assimilation of caprate and adipate on API 20E
240
& 20NE strips. Utilization of tweens 40 & 80, methyl pyruvate, mono-methyl succinate, acetate, D,L-lactate, propionate,
241
sebaccinate, and succinate on Microlog GN2. All other enzymatic activities and substrates utilization were negative for
242
all strains except described in the table. Characteristics Growth range of§: Temperature† pH† NaCl† Degradation of: Gelatin, Dextrin
1 15-43 (37-40) 6-9.5 (7-8) 0.5-16 (1.5-3)
2
3
4
5
10-42 (28) 4-40(23-25) 4-37 (28) 15-45(28) 6-10 (7-8) nd 5.5-9 (7) nd 0.5-15 (3) 1-15(3-10) 0-15 (28) 1-15(3-7.5)
+
-
-
-
-
+ + +
+ +
+ -
+ +
+ +
-
-
-
+
+ +
+ + + + +
+ + + + -
+ + -
+ + +
+ + + + +
65.2
66.1
66.4
62.8
62.1
Enzyme activities of ; Cystine arylamidase Trypsin N-acetyl-β-glucosaminidase Naphtol-AS-BI phosphohydrolase Assimilation of; Gluconate, Malate, Citrate, cis-Aaconitate, phenyl-ethyl amine, αketo glutaric acid Phenyl-acetate, Formate Mannose β-&γ-OH butyric acids Succinate, Mono-methyl succinate phenyl-acetate, formate Bromo succinic acid, p-OH phenylacetic acid α-keto butyric acid α-OH butyric acid G+C ratio§ (mol%) †
243 244
§
Values in parentheses are the optimum range.
Data for reference strains were cited from original papers.
245
10
246
Table 2. Fatty acid compositions(%) of strain MEBiC06500T and the type strains of closely related
247
Alcanivorax species
248
Strains: 1. MEBiC08158T (data from this study); 2. A. marinus R8-12T (data from this study); 3. A. venustensis ISO1T
249
(data from this study); 4. A. balearicus MACL04T (data from Lai et al., 2013); 5. A. dieselolei B-5T (data from Lai et al.,
250
2013). Strains analyzed in this study were cultivated with 0.3% sodium-acetate as carbon source at 37 OC or 28 OC
251
depending on optimal growth temperature. The proportions lower than 1% in all strains were not recorded. Characters
252
are scored as: -, not detected: tr, trace amount (
International Journal of Systematic and Evolutionary Microbiology Alcanivorax gelatiniphagus sp. nov., a marine bacterium isolated from tidal flat sediments enriched with crude oil --Manuscript Draft-Manuscript Number:
IJS-D-14-00445R1
Full Title:
Alcanivorax gelatiniphagus sp. nov., a marine bacterium isolated from tidal flat sediments enriched with crude oil
Short Title:
Alcanivorax gelatiniphagus sp. nov.
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Kae Kyoung Kwon, Ph.D. Korea Institute of Ocean Science & Technology Ansan, KOREA, REPUBLIC OF
First Author:
Kae Kyoung Kwon, Ph.D.
Order of Authors:
Kae Kyoung Kwon, Ph.D. Ji Hye Oh Sung-Hyun Yang Hyun-Seok Seo Jung-Hyun Lee
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A Gram-reaction-negative rod-shaped marine bacterium, designated as MEBiC08158T was isolated from sediments collected at the Taean County, Korea near the Hebei Spirit tanker oil spill accident. The 16S rRNA gene sequence analysis revealed that strain MEBiC08158T was closely related with Alcanivorax marinus R8-12T (99.5% similar) but distinguishable from other members of the genus Alcanivorax (93.7~97.1 %). The DNA-DNA hybridization value between strains MEBiC08158T and A. marinus R8-12T was 58.4%. Growth of strain MEBiC08158T was observed at 15-43°C (optimum 37~40°C), at pH 6.0-9.5 (optimum pH 7.0~8.0) and with 0.5-16 % (optimum 1.5~3.0%) NaCl. The dominant fatty acids were C16:0 (27.1%), C19:0 cyclo ω8c (19.1%), C12:0 (13.6%), C18:1 ω7c (12.4%), C12:0 3OH (8.4%), and summed feature 3 (comprising C15:0 2-OH and/or C16:1 ω7c; 6.1%). Several phenotypic characteristics differentiate strain MEBiC08158T from phylogenetically close members of the genus Alcanivorax. Therefore, strain MEBiC08158T should be classified as a novel species in the genus Alcanivorax, and it is proposed as A. gelatiniphagus sp. nov. The type strain is MEBiC08158T (=KCCM 42990 T =JCM 18425T).
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1
Alcanivorax gelatiniphagus sp. nov., a marine bacterium isolated from tidal flat sediments
2
enriched with crude oil
3 4 5
Kae Kyoung Kwon*, Ji Hye Oh, Sung-Hyun Yang, Hyun-Seok Seo, and Jung-Hyun Lee
6 7
Marine Biotechnology Research Center, Korea Institute of Ocean Science & Technology, PO Box 29, Ansan
8
425-600, Republic of Korea
9 10 11
Running title: Alcanivorax gelatiniphagus sp. nov.
12 13
Subject category; New Taxa (Proteobacteria)
14 15 16
The GenBank accession number for the 16S rRNA gene sequence of strain MEBiC08158T is JQ937289.
17 18
*
19
Phone : +82 31 400 6242.
Corresponding author Fax: +82 31 400 6232.
e-mail: [email protected]
20
1
21
A Gram-reaction-negative rod-shaped marine bacterium, designated as MEBiC08158T was isolated
22
from sediments collected at the Taean County, Korea near the Hebei Spirit tanker oil spill accident.
23
The 16S rRNA gene sequence analysis revealed that strain MEBiC08158T was closely related with
24
Alcanivorax marinus R8-12T (99.5% similar) but distinguishable from other members of the genus
25
Alcanivorax (93.7~97.1 %). The DNA-DNA hybridization value between strains MEBiC08158T and A.
26
marinus R8-12T was 58.4%. Growth of strain MEBiC08158T was observed at 15-43°C (optimum
27
37~40°C), at pH 6.0-9.5 (optimum pH 7.0~8.0) and with 0.5-16 % (optimum 1.5~3.0%) NaCl. The
28
dominant fatty acids were C16:0 (27.1%), C19:0 cyclo 8c (19.1%), C12:0 (13.6%), C18:1 7c (12.4%), C12:0
29
3OH (8.4%), and summed feature 3 (comprising C15:0 2-OH and/or C16:1 7c; 6.1%). Several
30
phenotypic characteristics differentiate strain MEBiC08158T from phylogenetically close members of
31
the genus Alcanivorax. Therefore, strain MEBiC08158T should be classified as a novel species in the
32
genus Alcanivorax, and it is proposed as A. gelatiniphagus sp. nov. The type strain is MEBiC08158T
33
(=KCCM 42990 T =JCM 18425T).
34 35 36
Microorganisms highly specialized in hydrocarbon utilization has been discovered recently (Yakimov et al.,
37
2007) and designated as 'obligate hydrocarbonoclastic bacteria' (OHCB). One of the OHCB genus is
38
Alcanivorax. Tthe genus Alcanivorax include OHCB and are distributed in low numbers in marine
39
environments but dramatically increase in numbers in oil-polluted open and coastal seawaters, potentially
40
comprising 80–90% of the oil-degrading microbial community (Harayama et al. 1999; Yakimov et al., 2005
41
& 2007). Alcanivorax strains utilize a narrow range of substrates; predominantly alkanes and some organic
42
acids (Golyshin et al., 2005). Recently, some members in this genus were revealed to utilize several amino
43
acids and organic acids as well as glucose though weakly (Rivas et al., 2007; Lai et al, 2011). However, no
44
detailed information on utilization patterns of organic acids was available. In the present study, results of the
45
identification of a novel Alcanivoax microorganism isolated from sediments exposed to oil-pollution is
46
reported.
2
47 48
Strain MEBiC08158T was isolated from a crude oil-enrichment culture of tidal flat sediments collected at
49
Taean County, Korea where the Hebei Spirit oil spill accident occurred in 2007. Approx. 0.1 cm3 of
50
sediments was inoculated into 20 ml MM2 inorganic medium (Sohn et al., 2004) supplemented with 0.3%
51
Iranian Heavy oil and incubated for 2 weeks at 25 OC. Then, one ml was transferred into fresh media and
52
cultivated for another two weeks. After cultivation appropriately diluted culture broth was spread onto
53
marine agar 2216 medium (MA; BD). Inoculated plates were incubated at 25 OC for 3 days and individual
54
colonies were isolated from MA depend on morphological differences. After primary isolation and
55
purification, strain MEBiC08158T was cultivated at 25 OC on the same medium for biochemical and
56
physiological characterization and stored at -80 OC in marine broth 2216 (MB; BD) supplemented with 20%
57
(v/v) glycerol. For phenotypic comparisons, Alcanivorax venustensis ISO1T (= DSM 13974T, Fernández-
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Martínez et al., 2003), A. balearicus MACL04T (= LMG 22508T, Rivas et al., 2007), and A. dieselolei B-5T
59
(= DSM 16502T, Liu & Shao, 2005) were purchased from DSMZ (Deutsche sammlung von
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Microorganismen und Zellkulturen GmbH) or BCCM/LMG (Belgian Co-ordinated Collections of
61
Microorganisms) and A. marinus R8-12T (= MCCC 1A00382T, Lai et al., 2013) was provided from MCCC
62
(Marine Culture Collection of China) and cultivated on MA at 25 OC. After checking the growth on volatile
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organic acids (Supplementary Fig. S4), strains were maintained with mineral salt medium (Yeast
64
1.0 g, 1M phosphate buffer 1 ml, NH4Cl 1 g, NaCl 35 g in 1 liter DW) supplemented with 0.3% sodium-
65
acetate (Designated it as Acetate-medium and abbreviated AA for agar medium and AB for liquid medium).
Extract
66 67
Extraction of genomic DNA was conducted by using a commercial DNA extraction kit (GeneAll). The 16S
68
rRNA gene was amplified by using 27F and 1518R bacterial primer set (Giovannoni, 1991), the details of
69
procedure and conditions were described in Lee et al. (2013). The amplified 16S rRNA gene was sequenced
70
using an ABI 3730xl automatic DNA sequencer (ABI) according to the manufacturer’s instructions. Obtained
71
sequences were assembled by using Vector NTI ver. 9.1 (Life Technologies) and compared by BLAST pair-
72
wise alignment with sequences in EzTaxon-e database (Kim et al., 2012). Strain MEBiC08158T showed 99.5%
3
73
16S rRNA gene sequence similarity with the type strain of Alcanivorax marinus, however, similarity with
74
other validly reported members of the genus Alcanivorax was lower than 97.1%. Therefore, DNA-DNA
75
hybridization with A. marinus was conducted according to the method described by Kaneko et al. (1978).
76
The DNA-DNA relatedness value between strains MEBiC08158T and A. marinus MCCC 1A00382T was
77
58.4% and was well below the threshold accepted for species delineation (Wayne et al., 1987).
78
Phylogenetic analysis based on almost complete 16S rRNA gene sequences (1,465 bps) of isolated strain
79
with members of the family Alcanivoraceae was conducted using MEGA ver. 5.2 (Tamura et al., 2011). The
80
neighbour-joining (Saitou & Nei, 1987) tree (Fig. 1) revealed that strain MEBiC08158T formed a coherent
81
clade with the A. marinus and A. venustensis and the relationship was robustly supported by a bootstrap value
82
of 100 %. The branch was also recovered in maximum-likelihood (Felsenstein, 1981) and maximum-
83
parsimony (Fitch, 1971) trees. Phylogenetic analysis performed using gyrB gene also conducted and
84
obtained similar topology (Supplementary Fig. S2).
85 86
Unless otherwise stated, the physiological and morphological characterization was conducted according to
87
the methods described in Kwon et al. (2005) and Yang et al. (2006). Transmission electron micrographs were
88
taken using a LIBRA120 (Carl-Zeiss) electron microscope after negative staining of fixed cells using 2%
89
Phosphotungstic acid reagent at pH 7.0. The observations revealed rod-shaped cells with a long polar
90
flagellum and numbers of inclusion bodies (Supplementary Fig. S1, available in IJSEM Online). The
91
inclusion bodies stained with Sudan Black B dye and this result implied that major components of these
92
inclusion bodies are fatty storage matters such as poly-β-hydroxybutyrate. The growth temperature was
93
tested in AB at 12 different temperatures from 10 to 50 OC (10, 15, 21, 24.5, 28, 31, 34, 36.8, 39.8, 43.1, 46.6
94
and 50 OC) in a temperature gradient incubator (TVS126MA; Adaventec) for up to 3 days. The tolerance
95
range for NaCl was tested in AB supplemented with NaCl (Sigma; 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10,
96
12, 13, 14, 15, 16, 17, and 20%, w/v). The tolerance range for pH was determined (pH 4, 5, 6, 6.5, 7, 7.5, 8,
97
8.5, 9, and 10) in AB with the pH adjusted using 10 mM MES (pH 4-6), HEPES (pH 6-8) or AMPSO (pH 8-
98
10) as biological buffers. To determine tolerance range of pH and NaCl range cells were cultivated at 37 OC
4
99
for 4 days. The bacterial suspension used to inoculate API 20E, 20NE, API ZYM, API 50CH kit
100
(bioMe´rieux) and a Microlog GN2 system (Biolog) was prepared in a 2% sea salt (Sigma) solution. The
101
biochemical characterization for strain MEBiC08108T was performed at 37 OC but reference strains were
102
cultivated at 28 OC for 2 days. Detailed results of the biochemical, morphological and physiological tests are
103
given in the species description and Table 1.
104 105
The cellular fatty acids profile was determined commercially by using the MIDI/Hewlett Packard Microbial
106
Identification System (Sasser, 1990) with Sherlock ver. 6.2 and RTSBA6 database was used for analysis
107
according to the manufacturer’s instruction on cells grown on AB for 1~2 days at 37 OC or 28 OC considering
108
the growth rates. The dominant fatty acids were determined as C16:0 (27.1%), C19:0 cyclo 8c (19.1%), C12:0
109
(13.6%), C18:1 7c (12.4%), C12:0 3OH (8.4%), and summed feature 3 (comprising C15:0 2-OH and/or C16:1
110
7c; 6.1%). The fatty acid composition was similar to that of the members of genus Alcanivorax, though the
111
proportion of some components was markedly different (Table 2). This result implied that novel isolate
112
belonging to the genus Alcanivorax but could be distinguished from existing type strains.
113
Polar lipids were extracted using a chloroform/methanol system and separated by two-dimensional TLC
114
using silica gel 60 F254 aluminum-backed thin-layer plates (Merck) (Minnkin, 1984). The details of
115
procedure and reagents were described in Yang et al. (2013). The predominant polar lipids were
116
diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminolipid, and
117
four unidentified aminophospholipids (Supplementary Fig. S3). The respiratory quinone could not
118
determined by HPLC analysis according to Collins (1985). The DNA G+C content was 65.2 mol%, as
119
determined from genome sequence analysis (data not published) and is within the reported range of that of
120
the genus Alcanivorax (Lai et al., 2013).
121 122
The results of the phylogentic anaylsis based on 16S rRNA gene sequences and gyrase B subunit sequences
123
indicates that strain MEBiC08158T is affiliated with the genus Alcanivorax (Fig. 1 & Fig. S2) and shares
124
common features of this genus including Gram-staining properties, halophilic nature, major fatty acids
5
125
constituents, utilization of relatively short chain aliphatic hydrocarbons (data not shown) and some short
126
chain organic acids (Supplementary Fig. S4) as sole or principal carbon sources, and positive for oxidase and
127
catalase activities but could not utilize almost all other kinds of organic substrates (Table 1). But the isolate
128
could be distinguished from closely related members of the genus Alcanivorax (A. marinus, A. venustensis, A.
129
dieselolei, and A. balearicus) by hydrolysis of gelatin, trypsin activity, and utilization of dextrin. Strain
130
MEBiC08158T could be further distinguished from A. marinus by the utilization pattern of hydroxy butyric
131
acids, from A. venustensis in assimilation of phenyl-acetate and D-mannose, utilization pattern of hydroxy
132
butyric acids, and Naphtol-AS-BI phosphohydrolase activity, from A. dieselolei in assimilation of gluconate,
133
phenyl-acetate, acetate, cis-aconitic acid, phenyl ethyl- amine, α-keto glutaric acids, p-hydroxy phenylacetic
134
acid, bromo succinic acid and utilization of β- & γ-butyric acid, and from A. balearicus in assimilation of
135
citrate, phenyl-acetate, citrate, cis-aconitic acid, α-keto glutaric acids, formic acid, phenyl ethyl amine, and
136
utilization of β- and γ-butyric acid and activities of cystine arylamidase and N-acetyl-β-glucosaminidase
137
(Table 1). On the basis of this polyphasic taxonomical evidence, we propose that strain MEBiC08158T should
138
be classified as a novel species in the genus Alcanivorax with the name Alcanivorax gelatiniphagus sp. nov.
139 140
Description of Alcanivorax gelatiniphagus sp. nov.
141
Alcanivorax gelatiniphagus (gel.la.ti.ni.pha'gus. N. L. neut. n. gelatininum gelatin; Gr. masc. n. phagos,
142
glutton; N.L. masc. n. gelatiniphagus gelatin eater).
143
Cells are Gram-reaction--negative, rod-shaped, 0.5-0.6 m wide 1.2-2.4 m long, and forms beige colored
144
colonies. Motile by means of a long polar flagellum. Produces extracellular matrix, bleb-like structures on
145
membrane, and fatty intracellular granules. Growth was observed at 15-43OC (optimum 37~40OC), at pH
146
6.0-9.5 (optimum 7.0~8.0) and with 0.5-16 % (optimum 1.5~3.0%) NaCl. Could not reduce nitrate. The
147
strain could actively degrade relatively short-chain linear hydrocarbons (C10~C18) and growth was
148
accelerated with 0.3% pyruvate, acetate or propionate. When assayed with API strips and Microlog GN2
149
plate, degrade dextrin, tweens 40 & 80, and gelatin. Enzyme activities of acid-& alkaline phosphatase,
150
esterase, esterase-lipase, lipase, trypsin, cystein-arylamidase, leucine-arylamidase & valine-arylamidase, and
6
151
phosphohydrolase are present. Produces acetoin and assimilates caprate, adipate, formate, phenyl acetate,
152
methyl pyruvate, mono-methyl succinate, acetate, butyrate, α-hydroxy butyrate, α-keto butyrate D,L-lactate,
153
propionate, sebacinate, succinate, and succinamate. However, almost no carbohydrates and amino acids were
154
utilized in API test strips or GN2 Microplate. The dominant fatty acids were C16:0, C19:0 cyclo 8c, C12:0, C18:1
155
7c,C12:0 3OH, and summed feature 3 (C15:0 2-OH and/or C16:1 7c). The type strain is MEBiC08158T
156
(=KCCM 42990 T =JCM 18425T) isolated from tidal flat sediments of Taean County, Korea near the Hebei
157
Spirit tanker oil spill accident.
158 159
Acknowledgements
160
This work was supported by the project titled "Oil spill environmental impact assessment and environmental
161
restoration" of the Ministry of Ocean & Fisheries, Korea & KIOST in-house program (PE99314).
162 163
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emended genus Alcanivorax. Int J Syst Evol Microbiol 53, 331–338.
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Kwon, K. K., Lee, H-S., Yang, S. H. & Kim, S-J. (2005). Kordiimonas gwangyangensis gen. nov., sp. nov.,
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a marine bacterium isolated from marine sediments that forms a distinct phyletic lineage (Kordiimonadales
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ord. nov.) in the ‘Alpha-Proteobacteria’. Int J Syst Evol Microbiol 55, 2033-2037.
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Lai, Q., Wang, L., Liu, Y., Fu, Y., Zhong, H., Wang, B., Chen, L., Wang, J., Sun, F. & Shao, Z. (2011).
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Alcanivorax pacificus sp. nov., isolated from a deep-sea pyrene-degrading consortium. Int J Syst Evol
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Alcanivorax balearicus sp. nov., isolated from Lake Martel. Int J Syst Evol Microbiol 57, 1331–1335.
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Yang S-H, Seo H-S., Oh H-M., Kim S-J., Lee J-H. & Kwon K. K. (2013). Brumimicrobium mesophilum
229
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230
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Int J Syst Evol Microbiol 63, 1105-1110
231
9
232
Table 1. Differential characteristics between MEBiC08158T and closely related species in the genus
233
Alcanivorax.
234
Strains: 1. MEBiC08158T (data were obtained in the present study); 2. A. marinus R8-12T (data from this study & from
235
Lai et al., 2013); 3. A. venustensis ISO1T (data from this study & from Fernández-Martínez et al., 2003); 4. A.
236
balearicus MACL04T (data from this study & from Rivas et al., 2007); 5. A. dieselolei B-5T (data from this study &
237
from Liu & Shao, 2005). +, Positive reaction; -, negative reaction; nd, no data. Positive for all strains; Gram negative
238
and motile by flagella. Oxidase, Catalase, Acid-& Alkaline phosphatase, Esterase, Esterase-lipase, Lipase, and Leu-
239
&Val-arylamidase activities on API ZYM strip. Acetoin production and assimilation of caprate and adipate on API 20E
240
& 20NE strips. Utilization of tweens 40 & 80, methyl pyruvate, mono-methyl succinate, acetate, D,L-lactate, propionate,
241
sebaccinate, and succinate on Microlog GN2. All other enzymatic activities and substrates utilization were negative for
242
all strains except described in the table. Characteristics Growth range of§: Temperature† pH† NaCl† Degradation of: Gelatin, Dextrin
1 15-43 (37-40) 6-9.5 (7-8) 0.5-16 (1.5-3)
2
3
4
5
10-42 (28) 4-40(23-25) 4-37 (28) 15-45(28) 6-10 (7-8) nd 5.5-9 (7) nd 0.5-15 (3) 1-15(3-10) 0-15 (28) 1-15(3-7.5)
+
-
-
-
-
+ + +
+ +
+ -
+ +
+ +
-
-
-
+
+ +
+ + + + +
+ + + + -
+ + -
+ + +
+ + + + +
65.2
66.1
66.4
62.8
62.1
Enzyme activities of ; Cystine arylamidase Trypsin N-acetyl-β-glucosaminidase Naphtol-AS-BI phosphohydrolase Assimilation of; Gluconate, Malate, Citrate, cis-Aaconitate, phenyl-ethyl amine, αketo glutaric acid Phenyl-acetate, Formate Mannose β-&γ-OH butyric acids Succinate, Mono-methyl succinate phenyl-acetate, formate Bromo succinic acid, p-OH phenylacetic acid α-keto butyric acid α-OH butyric acid G+C ratio§ (mol%) †
243 244
§
Values in parentheses are the optimum range.
Data for reference strains were cited from original papers.
245
10
246
Table 2. Fatty acid compositions(%) of strain MEBiC06500T and the type strains of closely related
247
Alcanivorax species
248
Strains: 1. MEBiC08158T (data from this study); 2. A. marinus R8-12T (data from this study); 3. A. venustensis ISO1T
249
(data from this study); 4. A. balearicus MACL04T (data from Lai et al., 2013); 5. A. dieselolei B-5T (data from Lai et al.,
250
2013). Strains analyzed in this study were cultivated with 0.3% sodium-acetate as carbon source at 37 OC or 28 OC
251
depending on optimal growth temperature. The proportions lower than 1% in all strains were not recorded. Characters
252
are scored as: -, not detected: tr, trace amount (
