IJSEM Papers in Press. Published March 4, 2015 as doi:10.1099/ijs.0.000168
International Journal of Systematic and Evolutionary Microbiology Algibacter psychrophilus sp. nov., a psychrophilic bacterium isolated from Antarctic marine sediment --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00344R1
Full Title:
Algibacter psychrophilus sp. nov., a psychrophilic bacterium isolated from Antarctic marine sediment
Short Title:
Algibacter psychrophilus sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
HONG KUM LEE KOREA POLAR RSEARCH INSTITUTE Incheon, KOREA, REPUBLIC OF
First Author:
You-Jung Jung
Order of Authors:
You-Jung Jung YUNG MI LEE Kiwoon Baek Chung Yeon Hwang Yirang Cho Soon Gyu Hong Ji Hee Kim HONG KUM LEE
Manuscript Region of Origin:
ANTARCTICA
Abstract:
A Gram-staining-negative, aerobic, yellow-pigmented, flexirubin-negative, rod-shaped, non-motile and psychrophilic bacterial strain, PAMC 27237T, was isolated from the marine sediment of the Ross Sea, Antarctica. Strain PAMC 27237T grew at 0-20 C (optimally at 17 C), at pH 5.0-9.5 (optimally at 7.0) and in the presence of 0-3.5% NaCl (w/v) (optimally at 1.5-2.5%). The major fatty acids (≥ 5%) were iso-C17:0 3-OH, C17:0 2-OH, anteiso-C15:0, summed feature 3 (16:1 ω6c/16:1 ω7c), iso-C15:0 3-OH, anteiso-C17:1 ω9c, anteiso-C15:1 A, iso-C16:0 3-OH, and iso-C15:1 G. The major polar lipids consisted of phosphatidylethanolamine, two unidentified aminolipids, four unidentified lipids, and a glycolipid. The major respiratory quinone was identified as MK-6. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain PAMC 27237T belongs to the genus Algibacter showing the high similarities with A. agarivorans (97.2 %), A. agarilyticus (97.0 %), and A. mikhailovii (96.4 %). Average nucleotide identity values between strain PAMC 27237T and the type strains of A. agarivorans and A. agarilyticus were 83.1 and 84.2 %, respectively and the genome-togenome distance were 22.4-24.2 % on average, indicating PAMC 27237T is clearly distinguished from the most closely related Algibacter species. The genomic DNA G+C content calculated from genome sequences was 33.5 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, strain PAMC 27237T is considered to represent a novel species of the genus Algibacter, for which the name Algibacter psychrophilus sp. nov. is proposed with the strain PAMC 27237T (= KCTC 42130T = JCM 30370T).
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1
Algibacter psychrophilus sp. nov., a psychrophilic bacterium isolated from Antarctic marine
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sediment
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You-Jung Jung1†, Yung Mi Lee1,2†, Kiwoon Baek1, Chung Yeon Hwang1, Yirang Cho1, Soon Gyu Hong1, Ji Hee
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Kim1, and Hong Kum Lee1*
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1
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840, Republic of Korea
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2
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Division of Polar Life Sciences, Korea Polar Research Institute, 26 Songdomirae-ro, Yeonsu-gu, Incheon 406-
School of Biological Sciences, College of Natural Science, Seoul National University, 599 Gwanak-ro,
Gwanak-gu, Seoul 151-747, Republic of Korea
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Running title: Algibacter psychrophilus sp. nov.
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Subject category: New taxa - Bacteroidetes
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Keywords: Algibacter psychrophilus sp. nov. ; Polyphasic taxonomy; Bacteroidetes
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The Genbank accession number for the 16S rRNA gene sequence of strain PAMC 27237 T is KJ475138.
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†
Equally contributed to this work
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* Corresponding author
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Name: Hong Kum Lee
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Address: Division of Polar Life Sciences, Korea Polar Research Institute, 26 Songdomirae-ro, Yeonsu-gu,
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Incheon 406-840, Republic of Korea
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Phone: +82-32-760-5569
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Fax: +82-32-760-5509
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E-mail: [email protected]
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Abstract
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A Gram-staining-negative, aerobic, yellow-pigmented, flexirubin-negative, rod-shaped, non-motile and
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psychrophilic bacterial strain, PAMC 27237T, was isolated from the marine sediment of the Ross Sea, Antarctica.
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Strain PAMC 27237T grew at 0-20 C (optimally at 17 C), at pH 5.0-9.5 (optimally at 7.0) and in the presence
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of 0-3.5% (w/v) (optimally at 1.5–2.5%) of NaCl. The major fatty acids (≥ 5%) were iso-C17:0 3-OH, C17:0 2-
34
OH, anteiso-C15:0, summed feature 3 (16:1 ω6c/16:1 ω7c), iso-C15:0 3-OH, anteiso-C17:1 ω9c, anteiso-C15:1
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A, iso-C16:0 3-OH, and iso-C15:1 G. The major polar lipids consisted of phosphatidylethanolamine, two
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unidentified aminolipids, four unidentified lipids, and a glycolipid. The major respiratory quinone was identified
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as MK-6. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain PAMC 27237T
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belongs to the genus Algibacter showing the high similarities with A. agarivorans (97.2 %), A. agarilyticus
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(97.0 %), and A. mikhailovii (96.4 %). Average nucleotide identity values between strain PAMC 27237T and the
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type strains of A. agarivorans and A. agarilyticus were 83.1 and 84.2 %, respectively and the genome-to-
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genome distance were 22.4-24.2 % on average, indicating PAMC 27237T is clearly distinguished from the most
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closely related Algibacter species. The genomic DNA G+C content calculated from genome sequences was 33.5
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mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, strain PAMC 27237T is
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considered to represent a novel species of the genus Algibacter, for which the name Algibacter psychrophilus sp.
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nov. is proposed with the strain PAMC 27237T (= KCTC 42130T = JCM 30370T).
46 47 48
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The genus Algibacter, a member of the family Flavobacteriaceae was first described with the species Algibacter
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lectus (Bernardet et al. 2002; Nedashkovskaya et al. 2004; Bernardet et al. 2006). At the time of writing, the
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genus Algibacter encompasses ten validly published species in the List of Prokaryotic Names with Standing in
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Nomenclature (Parte 2014). All type strains of the genus Aligibacter were originated from marine environments
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such as seawater, algae, urchin or marine sediments. The type species A. aestuarii, A. agarilyticus, A.
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agarivorans, A. aquimarinus, and A. pectinivorans were isolated from seawater (Yi et al. 2011; Park et al.
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2013a; Park et al. 2013b; Park et al. 2013c). Other type species A. letus, A. mikhailovii, A. miyuki, A. undariae,
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and A. wandonensis were isolated from green algae, sea urchin, brown algae, or sediments from brown algae
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reservoir (Nedashkovskaya et al. 2004; Nedashkovskaya et al. 2007; Park et al. 2013d; Park et al. 2013e; Yoon
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et al. 2013). The genus Aligibacter is characterized as being Gram-staining-negative, aerobic, rod-shape, non-
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motile, and yellow- or orange-pigmented and includes members such as A. agarivorans, A. agarilyticus, A. letus,
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and A. mikhailovii with agarolytic activity (Nedashkovskaya et al. 2004; Nedashkovskaya et al. 2007; Park et al.
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2013c). In this study, a bacterial strain PAMC 27237T was isolated from Antarctic marine sediment and
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subjected to polyphasic taxonomic analysis. The strain, PAMC 27237T, is considered to represent a novel
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species of the genus Algibacter, for which the name Algibacter psychrophilus sp. nov. is proposed here.
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Sediment samples were collected at a depth of 156 m in the Ross Sea in the Southern Ocean (74° 38' 46.20" S,
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164° 13' 24.60" E) on February 6, 2011 by using a box corer. The sediment samples were suspended in 20%
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glycerol and preserved at -80C until the use. For cultivation, a serially diluted aliquot (100 µl) of the sample
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suspension was spread on marine agar (MA, BD Difco) plates and incubated at 10C for 15 days. Total 12
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colonies belonged to the genus Algibacter, Loktanella, Octadecabacter, Psychroserpens, Sulfitobacter, and
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Winogradskyella were isolated (Lee et al. 2014) and all strains were preserved as glycerol suspensions (20% in
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distilled water, v/v) at -80 C. In this study, one of these strains, PAMC 27237T, was routinely cultured on MA
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at 15C after the optimal temperature for growth was determined on solid medium. A. agarivorans KCTC
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23855T and A. agarilyticus KCTC 23857T were purchased from Korean Collection of Type Cultures (KCTC)
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and used as reference strains following cultivation under comparable conditions as PAMC 27237 T.
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The 16S rRNA gene sequence (1510 nt) retrieved from whole genome sequencing was compared with those of
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all type strains in EzTaxon-e database (Kim et al. 2012) and aligned with those of type strains showing high
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similarities using the PHYDIT (ver. 3.2) program (http://plaza.snu.ac.kr/~jchun/phydit/). Phylogenetic trees
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were reconstructed using three tree-making algorithms, neighbour-joining (Saitou and Nei 1987) and maximum-
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parsimony (Fitch 1971) using PAUP (ver 4.0) program (Swofford 2002) and maximum-likelihood (Felsenstein
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1981) methods using the PhyML (ver 3.0) program (Guindon and Gascuel 2003). The robustness of the tree
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topologies was assessed by bootstrap analyses based on 1000 replications of the sequences. Comparison of 16S
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rRNA gene sequences showed that strain PAMC 27237T was closely related to A. agarivorans (97.2 % sequence
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similarity), A. agarilyticus (97.0 % sequence similarity), A. mikhailovii (96.4 % sequence similarity), A.
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aestuarii (96.0 %), A. wandonensis (96.0 %), A. miyuki (96.0 %), A. undariae (96.0 %), A. lectus (95.7%), A.
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pectinivorans (95.5%), and A. aquimarinus (94.8 %). In phylogenetic trees inferred three tree-making
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algorithms, PAMC 27237T formed a clade with strains A. agarivorans and A. agarilyticus and was separated
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from other genera in the family Flavobacteriaceae, indicating strain PAMC 27237T is a member of
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genus Algibacter (Fig.1).
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Genome relatedness was investigated by whole genome sequencing of strain PAMC 27237T and the most related
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Algibacter species, A. agarivorans KCTC 23855T and A. agarilyticus KCTC 23857T. Genomic DNAs were
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extracted using DNeasy Blood & Tissue kit (Qiagen) and genome sequencing was performed using the MiSeq
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sequencer system (Illumina) at Chun Lab (Seoul, Korea). The degree of pairwise genome-based relatedness was
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estimated by both an average nucleotide identity (ANI) value following the BLAST-based ANI calculation
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method described by Goris et al. (2007), and the genome-to-genome distance calculation (GGDC) method
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described by Auch et al. (2010). The genomic DNA G+C content was directly calculated from its genome
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sequence. The details of draft genomes for the three strains, PAMC 27237T and 2 reference strains, are
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summarized in Table S1. The ANI values calculated for the estimation of the degree of pairwise genome-based
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relatedness between strain PAMC 27237T and the type strains of A. agarivorans and A. agarilyticus were 83.1
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and 84.2 %, respectively (Table S2) and this level is well below the ANI cut-off values (95-96 %) that proposed
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for delineating bacterial species (Goris et al. 2007; Richter and Rosselló-Mora 2009). DNA-DNA hybridization
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values between strain PAMC 27237T and the other type strains estimated by GGDC were 22.4-24.2 % on
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average (Table S2), indicating that strain PAMC 27237T is distinguishable from other Algibacter species
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(Rosselló-Mora and Amann 2001). The genomic DNA G+C content of strain PAMC 27237 T was determined to
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be 33.5 mol% from the draft genome sequence (Table 1), a value within the range for the genus Algibacter (31-
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41.7 mol%) determined by the HPLC analysis.
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The temperature range and optimum for growth were determined by culturing strain PAMC 27237T and the 2
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reference strains in marine broth (BD Difco) at different temperatures (0, 4, 10, 15, 17, 20, 22, 25, 30, and 37 C)
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for 14 days. Two reference strains, A. agarivorans KCTC 23855T and A. agarilyticus KCTC 23857T grew at 0-
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25 C and PAMC 27237T grew at 0-20C. The pH range and optimum for growth were determined in artificial
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seawater (ASW) medium (Choo et al. 2007) supplemented with 0.5% peptone and 0.1% yeast extract at pH 5.0-
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10.0 (at intervals of 0.5 pH unit). The pH was adjusted using the following buffering systems; MES pH 5.0-6.0,
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MOPS pH 6.5-7.0, HEPES pH 7.5-8.0, Tris pH 8.5-9.0, and CHES pH 9.5-10.0. Salt tolerance test was carried
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on ASW medium supplemented with 0-3% (at intervals of 0.5%), 4, 5, 7.5, 10, and 15% of NaCl (w/v). The
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growth of each culture was assessed by measuring optical density at 600 nm (EnVision plate reader;
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PerkinElmer) everyday for up to 15 days. The presence of flexirubin-type pigments was determined by the
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bathochromatic shift test with 20 % KOH solution. Catalase activity was tested with 3% H2O2 and oxidase
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activity was determined using tetramethyl-p-phenylenediamine as the methods described in Kovacs (1956).
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Hydrolysis was tested on MA supplemented with Tweens 20, 40, 60, and 80, starch, carboxymethylcellulose,
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chitin, dextran, and xylan (each of 1% (w/v). Agarolytic activity was detected as the hollow zone around
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colonies after staining with 5% iodine solution and the depression on agar medium. Other biochemical activities
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of PAMC 27237T were determined by using API 20NE, API ZYM, and API 50CH kit (bioMérieux) according to
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manufacturer's instructions except that bacterial strains were suspended in ASW medium. Susceptibility to
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antibiotics, ampicillin (10 µg /disc), chloramphenicol (30 µg /disc), erythromycin (15 µg /disc), gentamicin (10
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µg /disc), kanamycin (30 µg /disc), nalidixic acid (30 µg /disc), rifampicin (30 µg /disc), tetracycline (30 µg
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/disc), and vancomycin (30 µg /disc) was analysed by the disc diffusion method. The morphological,
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physiological, and biochemical characteristics of strain PAMC 27237T are described in Supplementary Fig. S1,
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Table 1 and the species description. Among the number of physiological characteristics, especially temperature
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range, optimal temperature, and salt requirement for growth of PAMC 27237T was different. The temperature
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range for growth of PAMC 27237T was 0-20 C while A. agarivorans KCTC 23855T and A. agarilyticus KCTC
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23857T can grow at up to 25 C and 30 C, respectively. Salt tolerance for growth of PAMC 27237T was narrow
130
compared to that of A. agarivorans KCTC 23855T and A. agarilyticus KCTC 23857T. In addition, acid
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production from a variety of substrates, enzyme activities, susceptibility to antibiotics, and genomic data
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differentiated strain PAMC 27237T from closely related type strains of the genus Algibacter, A. agarivorans
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KCTC 23855T and A. agarilyticus KCTC 23857T.
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For cellular fatty acids analysis, strain PAMC 27237T and two reference strains were grown on MA at 15°C for
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5 days. Cellular fatty acids were extracted as described by Sasser (1990) and were analysed according to the
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method described by the Sherlock Microbial Identification System version 6.1 (MIDI) using the TSBA6.1
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database. The isoprenoid quinones extracted according to the method described in Minnikin et al. (1984) were
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separated by TLC and analysed by using high-performance liquid chromatography (HPLC) (Collins 1985).
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Polar lipids of strain PAMC 27237T were extracted from lyophilized bacterial cells, separated using two-
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dimensional thin layer chromatography (TLC), and detected by spraying the reagents molybdatophosphoric acid,
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ninhydrin, molybdenum blue, α-naphthol, Dragendorff’s solution, and schiff’s solution (Minnikin et al. 1984).
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The major fatty acids (≥ 5%) of strain PAMC 27237T were iso-C17:0 3-OH (10.8 %), C17:0 2-OH (10.0 %),
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anteiso-C15:0 (9.4 %), summed feature 3 (16:1 ω6c/16:1 ω7c) (6.7 %), iso-C15:0 3-OH (6.3 %), anteiso-C17:1
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ω9c (6.2 %), anteiso-C15:1 A (5.8 %), iso-C16:0 3-OH (5.6 %), and iso-C15:1 G (5.0 %) (Table 2). Overall
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profiles of fatty acids of 3 strains were similar each other while the high composition of anteiso-C17:1 ω9c, 6.2 %
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compared to that of A. agarivorans KCTC 23855T (1.2 %) and A. agarilyticus 23857T (0.7 %) was
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distinguishable. MK-6 was the only menaquinone present in strain PAMC 27237T. The polar lipids of strain
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PAMC 27237T were found to consist of phosphatidylethanolamine (PE), two unidentified aminolipids (AL1-2),
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four unidentified lipids (L1-4), and a glycolipid (GL) (Supplementary Fig. S2). This composition was similar to
150
that closely related type strains of Algibacter, A. agarivorans KCTC 23855T and A. agarilyticus KCTC 23857T
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with respect to the presence of PE, unidentified aminolipids, and unidentified lipids (Park et al. 2013). However,
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the presence of a GL in PAMC 27237T distinguished it from the reference strains.
153 154
Overall, strain PAMC 27237T formed a phylogenetic clade with the two type strains of the genus Algibacter
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(Fig. 1). However, genomic data, physiological characteristics such as temperature range and optimal
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temperature, salt tolerance, acid production from a variety of substrates, enzyme activities, and susceptibility to
157
antibiotics differentiated strain PAMC 27237T from closely related type strains A. agarivorans and A.
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agarilyticus. Therefore, strain PAMC 27237T represents a novel species of the genus Algibacter for which the
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name Algibacter psychrophilus sp. nov. is proposed.
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Description of Algibacter psychrophilus sp. nov.
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Algibacter psychrophilus (psy.chro′phi.lus. Gr. adj. psychros cold; Gr. adj. philos liking, loving; N.L. masc.
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adj.psychrophilus cold-loving).
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Cells are Gram-staining-negative, aerobic, yellow-pigmented, oxidase- and catalase- positive, non-motile, non-
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gliding, flexirubin-negative, and rod-shaped. Colonies are circular, convex, and glittering on MA plates after 7
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days incubation at 15C. Growth occurs at 0-20C (optimum, 17C), pH 5.0-9.5 (optimum, pH 7.0), and in the
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presence of 0-3.5 % (w/v) NaCl (optimum, 1.5–2.5 %). Starch is hydrolysed, but carboxymethylcellulose, chitin,
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dextran, and xylan are not. In the API 20NE system, nitrate is reduced to nitrite, aesculin is hydrolysed, and D-
170
glucose was assimilated. In the API ZYM system, alkaline phosphatase, esterase (C4), leucine arylamidase,
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valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and α-mannosidase are positive. In
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the API 50CH system, acid is produced from D-xylose, D-fructose, esculin ferric citrate, D-cellobiose, amidon,
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and glycogen. Susceptible to chloramphenicol, erythromycin, and rifampin. The major cellular fatty acids iso-
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C17:0 3-OH, C17:0 2-OH, anteiso-C15:0, summed feature 3 (16:1 ω6c/16:1 ω7c), iso-C15:0 3-OH, anteiso-
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C17:1 ω9c, anteiso-C15:1 A, iso-C16:0 3-OH, and iso-C15:1 G. The major isoprenoid quinone is MK-6. The
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polar lipids are phosphatidylethanolamine, two unidentifiedaminolipids, four unidentified lipids, and a
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glycolipid.
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The type strain PAMC 27237T (= KCTC 42130T = JCM 30370T) was isolated from a sediment sample collected
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from the Ross Sea, Antarctica. The GenBank accession number for the 16S rRNA gene sequence of strain
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PAMC 27237T is KJ475138.
181 182
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Acknowledgments
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We thank the crew of the R/V ARAON for their support at sea. and Prof. Jang-Cheon Cho (Inha University) for
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his help on the fatty acid analysis. This work was supported by Korea Polar Research Institute (Grant PE14080).
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253-260.
242 243 244 245 246
Park, S., Lee, J.S., Lee, K. C. & Yoon, J. H. (2013e). Algibacter undariae sp. nov., isolated from a brown algae reservoir. Int J Syst Evol Microbiol 63, 3704–3709. Parte, A. C. (2014). LPSN—list of prokaryotic names with standing in nomenclature. Nucleic Acids Res 42, 613-616. Richter, M. & Rosselló-Mora, R. (2009). Shifting the genomic gold standard for the prokaryotic species
247
definition. Proc Natl Acad Sci USA 106, 19126-19131.
248
Rosselló-Mora, R. & Amann, R. (2001). The species concept for prokaryotes. FEMS Microbiol Rev 25, 39–67.
249
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees.
250 251 252 253 254 255 256 257 258
Mol Biol Evol 4, 406–425. Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. USFCC News Lett 20,16. Swofford D.L. (2002). PAUP*. Phylogenetic analysis using parsimony (* and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Yi, H., Cho, J.-C. & Chun, J. (2011). Pontirhabdus pectinivorans gen. nov., sp. nov., isolated from seawater. Int J Syst Evol Microbiol 61, 2475-2481. Yoon, J. H. & Park, S. (2013). Algibacter wandonensis sp. nov., isolated from sediment around a brown algae (Undaria pinnatifida) reservoir. Int J Syst Evol Microbiol 63, 4771-4776.
259
Table 1. Major characteristics that differentiate strain PAMC 27237T from closely related type strains
260
of Algibacter species
261
Strains: 1, Algibacter psychrophilus PAMC 27237T; 2, A. agarivorans KCTC 23855T; 3, A.
262
agarilyticus KCTC 23857T. All data were obtained in this study. All strains were non-motile, rod-
263
shaped, yellow-pigmented, flexirubin-negative, aerobic, and have MK-6 as a major isoprenoid
264
quinone. All strains were positive for the following characteristics: reduction of nitrate to nitrite,
265
hydrolysis of aesculin and starch and presence of oxidase, catalase, alkaline phosphatase, esterase
266
(C4), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase,
267
and α-mannosidase; acid production from esculin ferric citrate and glycogen. All strains were negative
268
for the following characteristics: hydrolysis of chitin, carboxymethylcellulose, chitin, dextran, and
269
xylan; assimilation of L-tryptophan, L-arginine, urea, L-arabinose, D-mannose, D-mannitol, N-
270
acetylglucosamine, potassium gluconate, caprate, adipate, malate, citrate, and phenyl acetate, and
271
hydrolysis of gelatin in the API 20NE system; the presence of esterase lipase (C8), lipase (C14),
272
trypsin, α-chymotrypsin, α-galactosidase, ß-glucosidase, N-acetyl-ß-glucosaminidase, and α-
273
fucosidase activities: acid production from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, L-
274
xylose, D-adonitol, methyl-β-D-xylopyranoside, L-sorbose, dulcitol, inositol, D-mannitol, D-sorbitol,
275
methyl-α-D-mannopyranoside,
276
arbutin, salicin, D-maltose, D-melibiose, D-trehalose, inulin, D-melezitose, D-raffinose, xylitol, D-
277
lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, and potassium 2-
278
ketogluconate in the API 50CH system.
Characteristics Growth temperature range (°C) † ‡ pH range (optimal pH) NaCl range (%) (optimal %) G+C mol% (genome sequencing) Assimilation of D-Glucose 4-nitrophenyl-ß-Dgalactopyranoside Enzyme activities of
methyl-α-D-glucopyranoside,
N-acetylglucosamine,
1
2
3
0-20 (17) 5-9.5 (7) 0-3.5 (1.5-2.5) 33.5
0-25 (17) 5-9.5 (7) 0-6 (1-4) 33.3
0-30 (22) 5-9.5 (7-7.5) 0-6 (0.5-3.5) 33.0
+
-
-
-
+
+
amygdalin,
Cystine arylamidase ß-Glucuronidase ß-Glucosidase α-Glucosidase ß-Galactosidase α-Mannosidase N-acetyl-ß-glucosaminidase Acid production from D-xylose D-galactose D-glucose D-fructose D-mannose L-rhamnose D-celiobiose D-lactose D-saccharose Amidon Gentiobiose D-turanose Potassium 5-ketogluconate G+C contents (mol%)
-
+* +*
+*
+ -
-* +* + +* -*
+* +* +* -
+ + -
+ + + +
+ + -
+ -
+
+ -
+ 33.5
+ 33.3
+ -
+ + 33.0
279 280
†
281
agarilyticus KCTC 23857T according to the results from Park et al. 2013c.
282
‡
283
agarilyticus KCTC 23857T according to the results from Park et al. 2013c.
284
*
285 286 287
Growth temperature range was 10-28 °C for A. agarivorans KCTC 23855T and 10-30 °C for A.
pH range (optimal pH) was 6-11 (7-8) for A. agarivorans KCTC 23855T and 6-11 (7-8) for A.
means different results with that from Park et al. 2013c.
288
Table 2. Cellular fatty acid composition of strain PAMC 27237 T and other type strains of the genus
289
Algibacter
290
Strains: 1, Algibacter psychrophilus PAMC 27237T; 2, A. agarivorans KCTC 23855T; 3, A.
291
agarilyticus KCTC 23857T. Fatty acids
1
2
3
C14:0
-
0.9
-
C15:0
3.7
6.2
5.5
C16:0
4.4
1.9
1.9
C18:0
2.4
-
1.2
C15:0 2-OH
3.6
1.8
3.3
C16:0 3-OH
-
2.0
1.7
C17:0 2-OH
10.0
3.4
6.3
iso-C15:0
4.7
9.1
11.1
iso-C16:0
2.1
4.2
4.4
iso-C15:0 3-OH
6.3
7.9
6.8
iso-C16:0 3-OH
5.6
6.7
3.8
iso-C17:0 3-OH
10.8
11.2
13.3
anteiso-C15:1 A
5.8
3.4
4.1
anteiso-C15:0
9.4
8.7
7.6
anteiso-C17:1ω9c
6.2
1.2
0.7
iso-C15:1 G
5.0
8.6
5.1
iso-C16:1 G
-
1.8
-
iso-C16:1 H
2.6
-
-
C15:1ω6c
-
1.5
1.2
C16:1ω5c
-
2.2
1.9
C17:1ω6c
2.6
3.7
5.5
C18:1ω5c
3.6
-
-
3 (C16:1 ω6c and/or C16:1 ω7c)
6.7
11.4
12.1
9 (iso-C17:1 ω9c and/or C16:0 10-methly)
4.5
2.2
2.5
Straight-chain saturated
Branched saturated
Monounsaturated
Summed feature*
292 293 294 295 296
-,
not detected.
297
Figure legends
298
Fig. 1 Neighbor-joining tree based on 16S rRNA gene sequences showing the relationships of Algibacter
299
psychrophilus PAMC 27237T with species of the genus Algibacter and other closely related members of the
300
family Flavobacteriaceae. Filled circles indicate that the corresponding nodes were recovered by all treeing
301
methods. Percent bootstrap supports (>50%) are given at each node (NJ/MP/ML). Flavobacterium aquidurense
302
WB 1.1-56T was used as an outgroup.
303
Figure Click here to download Figure: PAMC_27237_figure_revised.pptx Flavobacterium aquidurense WB 1.1-56 (NR042470) Algibacter psychrophilus PAMC 27237 (KJ475138) Algibacter agarilyticus KYW563 (JN864027)
63/ 52/ *
Algibacter agarivorans KYW560 (HQ405792) Algibacter aestuarii KYW371 (HQ405792) Algibacter aquimarinus KYW589 (JQ259859) 76/ 88/ 81
Arenitalea lutea P7-3-5 (JQ807811)
88/ 73/ 78
Algibacter pectinivorans JC2675 (HM475134) Algibacter mikhailovii LMG 23988 (AM491809)
100/ */ 74 92/ 87/ 91
Algibacter lectus KMM 3902 (AY187689) Algibacter wandonensis WS-MY22 (KC987358)
Algibacter undariae WS-MY9 (KC261664))
90/ 54 / 55
Algibacter miyuki WS-MY6 (KC662118) Flaviramulus basaltis H35 (DQ361033)
*/ 63/ *
Flaviramulus ichthyoenteri Th78 (JX412958) 67/62/ 75 79/ */ *
Gaetbulibacter lutimaris D1-y4 (JF739861) Gaetbulibacter marinus IMCC1914 (EF108219) Tamlana agarivorans JW-26 (EU221275)
64/ 70/ *
Tamlana sedimentorum KMM 9545 (AB894238) 75/ 67/ 80 100/ 96/ 97
Olleya aquimaris L-4 (FJ886713) Olleya marilimosa CAM030 (EF660466) Olleya namhaensis WT-MY15 (JQ327134)
90/ 72/ 71
100/ 100/ 100
Mesoflavibacter sabulilitoris GJMS-9 (KJ816860) Mesoflavibacter zeaxanthinifaciens DSM 18436 TD-ZX30 (AB265181)
99/ 99/ 98 92/ 87/ 88
Formosa arctica IMCC9485 (KF148059) Formosa undariae WS-MY3 (KC832835) Formosa spongicola A2 (FJ348469)
100/ 98/ 82
Bizionia hallyeonensis T-y7 (JN885199)
Bizionia echini KMM 6177 (FJ716799) 0.01
Fig. 1
Supplementary Material Files Click here to download Supplementary Material Files: PAMC_27237_supplementary.pdf
International Journal of Systematic and Evolutionary Microbiology Algibacter psychrophilus sp. nov., a psychrophilic bacterium isolated from Antarctic marine sediment --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00344R1
Full Title:
Algibacter psychrophilus sp. nov., a psychrophilic bacterium isolated from Antarctic marine sediment
Short Title:
Algibacter psychrophilus sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
HONG KUM LEE KOREA POLAR RSEARCH INSTITUTE Incheon, KOREA, REPUBLIC OF
First Author:
You-Jung Jung
Order of Authors:
You-Jung Jung YUNG MI LEE Kiwoon Baek Chung Yeon Hwang Yirang Cho Soon Gyu Hong Ji Hee Kim HONG KUM LEE
Manuscript Region of Origin:
ANTARCTICA
Abstract:
A Gram-staining-negative, aerobic, yellow-pigmented, flexirubin-negative, rod-shaped, non-motile and psychrophilic bacterial strain, PAMC 27237T, was isolated from the marine sediment of the Ross Sea, Antarctica. Strain PAMC 27237T grew at 0-20 C (optimally at 17 C), at pH 5.0-9.5 (optimally at 7.0) and in the presence of 0-3.5% NaCl (w/v) (optimally at 1.5-2.5%). The major fatty acids (≥ 5%) were iso-C17:0 3-OH, C17:0 2-OH, anteiso-C15:0, summed feature 3 (16:1 ω6c/16:1 ω7c), iso-C15:0 3-OH, anteiso-C17:1 ω9c, anteiso-C15:1 A, iso-C16:0 3-OH, and iso-C15:1 G. The major polar lipids consisted of phosphatidylethanolamine, two unidentified aminolipids, four unidentified lipids, and a glycolipid. The major respiratory quinone was identified as MK-6. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain PAMC 27237T belongs to the genus Algibacter showing the high similarities with A. agarivorans (97.2 %), A. agarilyticus (97.0 %), and A. mikhailovii (96.4 %). Average nucleotide identity values between strain PAMC 27237T and the type strains of A. agarivorans and A. agarilyticus were 83.1 and 84.2 %, respectively and the genome-togenome distance were 22.4-24.2 % on average, indicating PAMC 27237T is clearly distinguished from the most closely related Algibacter species. The genomic DNA G+C content calculated from genome sequences was 33.5 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, strain PAMC 27237T is considered to represent a novel species of the genus Algibacter, for which the name Algibacter psychrophilus sp. nov. is proposed with the strain PAMC 27237T (= KCTC 42130T = JCM 30370T).
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1
Algibacter psychrophilus sp. nov., a psychrophilic bacterium isolated from Antarctic marine
2
sediment
3 4
You-Jung Jung1†, Yung Mi Lee1,2†, Kiwoon Baek1, Chung Yeon Hwang1, Yirang Cho1, Soon Gyu Hong1, Ji Hee
5
Kim1, and Hong Kum Lee1*
6 7
1
8
840, Republic of Korea
9
2
10
Division of Polar Life Sciences, Korea Polar Research Institute, 26 Songdomirae-ro, Yeonsu-gu, Incheon 406-
School of Biological Sciences, College of Natural Science, Seoul National University, 599 Gwanak-ro,
Gwanak-gu, Seoul 151-747, Republic of Korea
11 12
Running title: Algibacter psychrophilus sp. nov.
13
Subject category: New taxa - Bacteroidetes
14
Keywords: Algibacter psychrophilus sp. nov. ; Polyphasic taxonomy; Bacteroidetes
15 16
The Genbank accession number for the 16S rRNA gene sequence of strain PAMC 27237 T is KJ475138.
17 18 19 20
†
Equally contributed to this work
21 22
* Corresponding author
23
Name: Hong Kum Lee
24
Address: Division of Polar Life Sciences, Korea Polar Research Institute, 26 Songdomirae-ro, Yeonsu-gu,
25
Incheon 406-840, Republic of Korea
26
Phone: +82-32-760-5569
27
Fax: +82-32-760-5509
28
E-mail: [email protected]
29
Abstract
30
A Gram-staining-negative, aerobic, yellow-pigmented, flexirubin-negative, rod-shaped, non-motile and
31
psychrophilic bacterial strain, PAMC 27237T, was isolated from the marine sediment of the Ross Sea, Antarctica.
32
Strain PAMC 27237T grew at 0-20 C (optimally at 17 C), at pH 5.0-9.5 (optimally at 7.0) and in the presence
33
of 0-3.5% (w/v) (optimally at 1.5–2.5%) of NaCl. The major fatty acids (≥ 5%) were iso-C17:0 3-OH, C17:0 2-
34
OH, anteiso-C15:0, summed feature 3 (16:1 ω6c/16:1 ω7c), iso-C15:0 3-OH, anteiso-C17:1 ω9c, anteiso-C15:1
35
A, iso-C16:0 3-OH, and iso-C15:1 G. The major polar lipids consisted of phosphatidylethanolamine, two
36
unidentified aminolipids, four unidentified lipids, and a glycolipid. The major respiratory quinone was identified
37
as MK-6. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain PAMC 27237T
38
belongs to the genus Algibacter showing the high similarities with A. agarivorans (97.2 %), A. agarilyticus
39
(97.0 %), and A. mikhailovii (96.4 %). Average nucleotide identity values between strain PAMC 27237T and the
40
type strains of A. agarivorans and A. agarilyticus were 83.1 and 84.2 %, respectively and the genome-to-
41
genome distance were 22.4-24.2 % on average, indicating PAMC 27237T is clearly distinguished from the most
42
closely related Algibacter species. The genomic DNA G+C content calculated from genome sequences was 33.5
43
mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, strain PAMC 27237T is
44
considered to represent a novel species of the genus Algibacter, for which the name Algibacter psychrophilus sp.
45
nov. is proposed with the strain PAMC 27237T (= KCTC 42130T = JCM 30370T).
46 47 48
49
The genus Algibacter, a member of the family Flavobacteriaceae was first described with the species Algibacter
50
lectus (Bernardet et al. 2002; Nedashkovskaya et al. 2004; Bernardet et al. 2006). At the time of writing, the
51
genus Algibacter encompasses ten validly published species in the List of Prokaryotic Names with Standing in
52
Nomenclature (Parte 2014). All type strains of the genus Aligibacter were originated from marine environments
53
such as seawater, algae, urchin or marine sediments. The type species A. aestuarii, A. agarilyticus, A.
54
agarivorans, A. aquimarinus, and A. pectinivorans were isolated from seawater (Yi et al. 2011; Park et al.
55
2013a; Park et al. 2013b; Park et al. 2013c). Other type species A. letus, A. mikhailovii, A. miyuki, A. undariae,
56
and A. wandonensis were isolated from green algae, sea urchin, brown algae, or sediments from brown algae
57
reservoir (Nedashkovskaya et al. 2004; Nedashkovskaya et al. 2007; Park et al. 2013d; Park et al. 2013e; Yoon
58
et al. 2013). The genus Aligibacter is characterized as being Gram-staining-negative, aerobic, rod-shape, non-
59
motile, and yellow- or orange-pigmented and includes members such as A. agarivorans, A. agarilyticus, A. letus,
60
and A. mikhailovii with agarolytic activity (Nedashkovskaya et al. 2004; Nedashkovskaya et al. 2007; Park et al.
61
2013c). In this study, a bacterial strain PAMC 27237T was isolated from Antarctic marine sediment and
62
subjected to polyphasic taxonomic analysis. The strain, PAMC 27237T, is considered to represent a novel
63
species of the genus Algibacter, for which the name Algibacter psychrophilus sp. nov. is proposed here.
64
Sediment samples were collected at a depth of 156 m in the Ross Sea in the Southern Ocean (74° 38' 46.20" S,
65
164° 13' 24.60" E) on February 6, 2011 by using a box corer. The sediment samples were suspended in 20%
66
glycerol and preserved at -80C until the use. For cultivation, a serially diluted aliquot (100 µl) of the sample
67
suspension was spread on marine agar (MA, BD Difco) plates and incubated at 10C for 15 days. Total 12
68
colonies belonged to the genus Algibacter, Loktanella, Octadecabacter, Psychroserpens, Sulfitobacter, and
69
Winogradskyella were isolated (Lee et al. 2014) and all strains were preserved as glycerol suspensions (20% in
70
distilled water, v/v) at -80 C. In this study, one of these strains, PAMC 27237T, was routinely cultured on MA
71
at 15C after the optimal temperature for growth was determined on solid medium. A. agarivorans KCTC
72
23855T and A. agarilyticus KCTC 23857T were purchased from Korean Collection of Type Cultures (KCTC)
73
and used as reference strains following cultivation under comparable conditions as PAMC 27237 T.
74
The 16S rRNA gene sequence (1510 nt) retrieved from whole genome sequencing was compared with those of
75
all type strains in EzTaxon-e database (Kim et al. 2012) and aligned with those of type strains showing high
76
similarities using the PHYDIT (ver. 3.2) program (http://plaza.snu.ac.kr/~jchun/phydit/). Phylogenetic trees
77
were reconstructed using three tree-making algorithms, neighbour-joining (Saitou and Nei 1987) and maximum-
78
parsimony (Fitch 1971) using PAUP (ver 4.0) program (Swofford 2002) and maximum-likelihood (Felsenstein
79
1981) methods using the PhyML (ver 3.0) program (Guindon and Gascuel 2003). The robustness of the tree
80
topologies was assessed by bootstrap analyses based on 1000 replications of the sequences. Comparison of 16S
81
rRNA gene sequences showed that strain PAMC 27237T was closely related to A. agarivorans (97.2 % sequence
82
similarity), A. agarilyticus (97.0 % sequence similarity), A. mikhailovii (96.4 % sequence similarity), A.
83
aestuarii (96.0 %), A. wandonensis (96.0 %), A. miyuki (96.0 %), A. undariae (96.0 %), A. lectus (95.7%), A.
84
pectinivorans (95.5%), and A. aquimarinus (94.8 %). In phylogenetic trees inferred three tree-making
85
algorithms, PAMC 27237T formed a clade with strains A. agarivorans and A. agarilyticus and was separated
86
from other genera in the family Flavobacteriaceae, indicating strain PAMC 27237T is a member of
87
genus Algibacter (Fig.1).
88
Genome relatedness was investigated by whole genome sequencing of strain PAMC 27237T and the most related
89
Algibacter species, A. agarivorans KCTC 23855T and A. agarilyticus KCTC 23857T. Genomic DNAs were
90
extracted using DNeasy Blood & Tissue kit (Qiagen) and genome sequencing was performed using the MiSeq
91
sequencer system (Illumina) at Chun Lab (Seoul, Korea). The degree of pairwise genome-based relatedness was
92
estimated by both an average nucleotide identity (ANI) value following the BLAST-based ANI calculation
93
method described by Goris et al. (2007), and the genome-to-genome distance calculation (GGDC) method
94
described by Auch et al. (2010). The genomic DNA G+C content was directly calculated from its genome
95
sequence. The details of draft genomes for the three strains, PAMC 27237T and 2 reference strains, are
96
summarized in Table S1. The ANI values calculated for the estimation of the degree of pairwise genome-based
97
relatedness between strain PAMC 27237T and the type strains of A. agarivorans and A. agarilyticus were 83.1
98
and 84.2 %, respectively (Table S2) and this level is well below the ANI cut-off values (95-96 %) that proposed
99
for delineating bacterial species (Goris et al. 2007; Richter and Rosselló-Mora 2009). DNA-DNA hybridization
100
values between strain PAMC 27237T and the other type strains estimated by GGDC were 22.4-24.2 % on
101
average (Table S2), indicating that strain PAMC 27237T is distinguishable from other Algibacter species
102
(Rosselló-Mora and Amann 2001). The genomic DNA G+C content of strain PAMC 27237 T was determined to
103
be 33.5 mol% from the draft genome sequence (Table 1), a value within the range for the genus Algibacter (31-
104
41.7 mol%) determined by the HPLC analysis.
105
The temperature range and optimum for growth were determined by culturing strain PAMC 27237T and the 2
106
reference strains in marine broth (BD Difco) at different temperatures (0, 4, 10, 15, 17, 20, 22, 25, 30, and 37 C)
107
for 14 days. Two reference strains, A. agarivorans KCTC 23855T and A. agarilyticus KCTC 23857T grew at 0-
108
25 C and PAMC 27237T grew at 0-20C. The pH range and optimum for growth were determined in artificial
109
seawater (ASW) medium (Choo et al. 2007) supplemented with 0.5% peptone and 0.1% yeast extract at pH 5.0-
110
10.0 (at intervals of 0.5 pH unit). The pH was adjusted using the following buffering systems; MES pH 5.0-6.0,
111
MOPS pH 6.5-7.0, HEPES pH 7.5-8.0, Tris pH 8.5-9.0, and CHES pH 9.5-10.0. Salt tolerance test was carried
112
on ASW medium supplemented with 0-3% (at intervals of 0.5%), 4, 5, 7.5, 10, and 15% of NaCl (w/v). The
113
growth of each culture was assessed by measuring optical density at 600 nm (EnVision plate reader;
114
PerkinElmer) everyday for up to 15 days. The presence of flexirubin-type pigments was determined by the
115
bathochromatic shift test with 20 % KOH solution. Catalase activity was tested with 3% H2O2 and oxidase
116
activity was determined using tetramethyl-p-phenylenediamine as the methods described in Kovacs (1956).
117
Hydrolysis was tested on MA supplemented with Tweens 20, 40, 60, and 80, starch, carboxymethylcellulose,
118
chitin, dextran, and xylan (each of 1% (w/v). Agarolytic activity was detected as the hollow zone around
119
colonies after staining with 5% iodine solution and the depression on agar medium. Other biochemical activities
120
of PAMC 27237T were determined by using API 20NE, API ZYM, and API 50CH kit (bioMérieux) according to
121
manufacturer's instructions except that bacterial strains were suspended in ASW medium. Susceptibility to
122
antibiotics, ampicillin (10 µg /disc), chloramphenicol (30 µg /disc), erythromycin (15 µg /disc), gentamicin (10
123
µg /disc), kanamycin (30 µg /disc), nalidixic acid (30 µg /disc), rifampicin (30 µg /disc), tetracycline (30 µg
124
/disc), and vancomycin (30 µg /disc) was analysed by the disc diffusion method. The morphological,
125
physiological, and biochemical characteristics of strain PAMC 27237T are described in Supplementary Fig. S1,
126
Table 1 and the species description. Among the number of physiological characteristics, especially temperature
127
range, optimal temperature, and salt requirement for growth of PAMC 27237T was different. The temperature
128
range for growth of PAMC 27237T was 0-20 C while A. agarivorans KCTC 23855T and A. agarilyticus KCTC
129
23857T can grow at up to 25 C and 30 C, respectively. Salt tolerance for growth of PAMC 27237T was narrow
130
compared to that of A. agarivorans KCTC 23855T and A. agarilyticus KCTC 23857T. In addition, acid
131
production from a variety of substrates, enzyme activities, susceptibility to antibiotics, and genomic data
132
differentiated strain PAMC 27237T from closely related type strains of the genus Algibacter, A. agarivorans
133
KCTC 23855T and A. agarilyticus KCTC 23857T.
134
For cellular fatty acids analysis, strain PAMC 27237T and two reference strains were grown on MA at 15°C for
135
5 days. Cellular fatty acids were extracted as described by Sasser (1990) and were analysed according to the
136
method described by the Sherlock Microbial Identification System version 6.1 (MIDI) using the TSBA6.1
137
database. The isoprenoid quinones extracted according to the method described in Minnikin et al. (1984) were
138
separated by TLC and analysed by using high-performance liquid chromatography (HPLC) (Collins 1985).
139
Polar lipids of strain PAMC 27237T were extracted from lyophilized bacterial cells, separated using two-
140
dimensional thin layer chromatography (TLC), and detected by spraying the reagents molybdatophosphoric acid,
141
ninhydrin, molybdenum blue, α-naphthol, Dragendorff’s solution, and schiff’s solution (Minnikin et al. 1984).
142
The major fatty acids (≥ 5%) of strain PAMC 27237T were iso-C17:0 3-OH (10.8 %), C17:0 2-OH (10.0 %),
143
anteiso-C15:0 (9.4 %), summed feature 3 (16:1 ω6c/16:1 ω7c) (6.7 %), iso-C15:0 3-OH (6.3 %), anteiso-C17:1
144
ω9c (6.2 %), anteiso-C15:1 A (5.8 %), iso-C16:0 3-OH (5.6 %), and iso-C15:1 G (5.0 %) (Table 2). Overall
145
profiles of fatty acids of 3 strains were similar each other while the high composition of anteiso-C17:1 ω9c, 6.2 %
146
compared to that of A. agarivorans KCTC 23855T (1.2 %) and A. agarilyticus 23857T (0.7 %) was
147
distinguishable. MK-6 was the only menaquinone present in strain PAMC 27237T. The polar lipids of strain
148
PAMC 27237T were found to consist of phosphatidylethanolamine (PE), two unidentified aminolipids (AL1-2),
149
four unidentified lipids (L1-4), and a glycolipid (GL) (Supplementary Fig. S2). This composition was similar to
150
that closely related type strains of Algibacter, A. agarivorans KCTC 23855T and A. agarilyticus KCTC 23857T
151
with respect to the presence of PE, unidentified aminolipids, and unidentified lipids (Park et al. 2013). However,
152
the presence of a GL in PAMC 27237T distinguished it from the reference strains.
153 154
Overall, strain PAMC 27237T formed a phylogenetic clade with the two type strains of the genus Algibacter
155
(Fig. 1). However, genomic data, physiological characteristics such as temperature range and optimal
156
temperature, salt tolerance, acid production from a variety of substrates, enzyme activities, and susceptibility to
157
antibiotics differentiated strain PAMC 27237T from closely related type strains A. agarivorans and A.
158
agarilyticus. Therefore, strain PAMC 27237T represents a novel species of the genus Algibacter for which the
159
name Algibacter psychrophilus sp. nov. is proposed.
160 161
Description of Algibacter psychrophilus sp. nov.
162
Algibacter psychrophilus (psy.chro′phi.lus. Gr. adj. psychros cold; Gr. adj. philos liking, loving; N.L. masc.
163
adj.psychrophilus cold-loving).
164 165
Cells are Gram-staining-negative, aerobic, yellow-pigmented, oxidase- and catalase- positive, non-motile, non-
166
gliding, flexirubin-negative, and rod-shaped. Colonies are circular, convex, and glittering on MA plates after 7
167
days incubation at 15C. Growth occurs at 0-20C (optimum, 17C), pH 5.0-9.5 (optimum, pH 7.0), and in the
168
presence of 0-3.5 % (w/v) NaCl (optimum, 1.5–2.5 %). Starch is hydrolysed, but carboxymethylcellulose, chitin,
169
dextran, and xylan are not. In the API 20NE system, nitrate is reduced to nitrite, aesculin is hydrolysed, and D-
170
glucose was assimilated. In the API ZYM system, alkaline phosphatase, esterase (C4), leucine arylamidase,
171
valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and α-mannosidase are positive. In
172
the API 50CH system, acid is produced from D-xylose, D-fructose, esculin ferric citrate, D-cellobiose, amidon,
173
and glycogen. Susceptible to chloramphenicol, erythromycin, and rifampin. The major cellular fatty acids iso-
174
C17:0 3-OH, C17:0 2-OH, anteiso-C15:0, summed feature 3 (16:1 ω6c/16:1 ω7c), iso-C15:0 3-OH, anteiso-
175
C17:1 ω9c, anteiso-C15:1 A, iso-C16:0 3-OH, and iso-C15:1 G. The major isoprenoid quinone is MK-6. The
176
polar lipids are phosphatidylethanolamine, two unidentifiedaminolipids, four unidentified lipids, and a
177
glycolipid.
178
The type strain PAMC 27237T (= KCTC 42130T = JCM 30370T) was isolated from a sediment sample collected
179
from the Ross Sea, Antarctica. The GenBank accession number for the 16S rRNA gene sequence of strain
180
PAMC 27237T is KJ475138.
181 182
183
Acknowledgments
184
We thank the crew of the R/V ARAON for their support at sea. and Prof. Jang-Cheon Cho (Inha University) for
185
his help on the fatty acid analysis. This work was supported by Korea Polar Research Institute (Grant PE14080).
186
187
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Kovacs, N. (1956). Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature 178, 703.
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Lee, Y. M., Jung, Y.-J., Hong, S. G., Kim, J. H. & Lee, H. K. (2014). Diversity and physiological
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characteristics of culturable bacteria from marine sediments of Ross Sea, Antarctica. Kor J Microbiol 50,
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Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H.
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(1984). Anintegrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J
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Microbiol Meth 2, 233–241.
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Nedashkovskaya, O. I., Kim, S. B., Han, S. K., Rhee, M. S., Lysenko, A. M., Rohde, M., Zhukova, N. V.,
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Frolova, G. M., Mikhailov, V. V. & Bae, K. S. (2004). Algibacter lectus gen. nov., sp. nov., a novel
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member of the family Flavobacteriaceae isolated from green algae. Int J Syst Evol Microbiol 54, 1257–
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Nedashkovskaya, O. I., Vancanneyt, M., Kim, S. B., Hoste, B. & Bae, K. S. (2007). Algibacter mikhailovii
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sp. nov., a novel marine bacterium of the family Flavobacteriaceae, and emended description of the
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genus Algibacter. Int J Syst Evol Microbiol 57, 2147–2150.
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Park, S. C., Choe, H. N., Hwang, Y. M., Baik, K. S., Kim, S. N., Lee, Y. S., Jung, J. S. & Seong, C. N.
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(2013a). Marinivirga aestuarii gen. nov., sp. nov., a member of the family Flavobacteriaceae, isolated
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from marine environments, and emended descriptions of the genera Hyunsoonleella, Jejuia and
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Pontirhabdus and the species Hyunsoonleella jejuensis, Jejuia pallidilutea and Pontirhabdus
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pectinivorans. Int J Syst Evol Microbiol 63, 1524–1531.
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Park, S. C., Hwang, Y. M., Choe, H. N., Baik, K. S., Kim, H. & Seong, C. N. (2013b). Algibacter
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aquimarinus sp. nov., isolated from a marine environment, and reclassification of Pontirhabdus
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pectinivorans as Algibacter pectinivorans comb. nov. Int J Syst Evol Microbiol 63, 2038–2042.
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Park, S. C., Hwang, Y. M., Lee, J. H., Baik, K. S. & Seong, C. N. (2013c). Algibacter agarivorans sp. nov.
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and Algibacter agarilyticus sp. nov., isolated from seawater, reclassification of Marinivirga
237
aestuarii as Algibacter aestuarii comb. nov. and emended description of the genus Algibacter. Int J Syst
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Evol Microbiol 63, 3494–3500.
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Park, S., Jung, Y. T. & Yoon, J. H. (2013d). Algibacter miyuki sp. nov., a member of the family
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Flavobacteriaceae isolated from leachate of a brown algae reservoir. Antonie Van Leeuwenhoek 104,
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Park, S., Lee, J.S., Lee, K. C. & Yoon, J. H. (2013e). Algibacter undariae sp. nov., isolated from a brown algae reservoir. Int J Syst Evol Microbiol 63, 3704–3709. Parte, A. C. (2014). LPSN—list of prokaryotic names with standing in nomenclature. Nucleic Acids Res 42, 613-616. Richter, M. & Rosselló-Mora, R. (2009). Shifting the genomic gold standard for the prokaryotic species
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definition. Proc Natl Acad Sci USA 106, 19126-19131.
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Rosselló-Mora, R. & Amann, R. (2001). The species concept for prokaryotes. FEMS Microbiol Rev 25, 39–67.
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Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees.
250 251 252 253 254 255 256 257 258
Mol Biol Evol 4, 406–425. Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. USFCC News Lett 20,16. Swofford D.L. (2002). PAUP*. Phylogenetic analysis using parsimony (* and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Yi, H., Cho, J.-C. & Chun, J. (2011). Pontirhabdus pectinivorans gen. nov., sp. nov., isolated from seawater. Int J Syst Evol Microbiol 61, 2475-2481. Yoon, J. H. & Park, S. (2013). Algibacter wandonensis sp. nov., isolated from sediment around a brown algae (Undaria pinnatifida) reservoir. Int J Syst Evol Microbiol 63, 4771-4776.
259
Table 1. Major characteristics that differentiate strain PAMC 27237T from closely related type strains
260
of Algibacter species
261
Strains: 1, Algibacter psychrophilus PAMC 27237T; 2, A. agarivorans KCTC 23855T; 3, A.
262
agarilyticus KCTC 23857T. All data were obtained in this study. All strains were non-motile, rod-
263
shaped, yellow-pigmented, flexirubin-negative, aerobic, and have MK-6 as a major isoprenoid
264
quinone. All strains were positive for the following characteristics: reduction of nitrate to nitrite,
265
hydrolysis of aesculin and starch and presence of oxidase, catalase, alkaline phosphatase, esterase
266
(C4), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase,
267
and α-mannosidase; acid production from esculin ferric citrate and glycogen. All strains were negative
268
for the following characteristics: hydrolysis of chitin, carboxymethylcellulose, chitin, dextran, and
269
xylan; assimilation of L-tryptophan, L-arginine, urea, L-arabinose, D-mannose, D-mannitol, N-
270
acetylglucosamine, potassium gluconate, caprate, adipate, malate, citrate, and phenyl acetate, and
271
hydrolysis of gelatin in the API 20NE system; the presence of esterase lipase (C8), lipase (C14),
272
trypsin, α-chymotrypsin, α-galactosidase, ß-glucosidase, N-acetyl-ß-glucosaminidase, and α-
273
fucosidase activities: acid production from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, L-
274
xylose, D-adonitol, methyl-β-D-xylopyranoside, L-sorbose, dulcitol, inositol, D-mannitol, D-sorbitol,
275
methyl-α-D-mannopyranoside,
276
arbutin, salicin, D-maltose, D-melibiose, D-trehalose, inulin, D-melezitose, D-raffinose, xylitol, D-
277
lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, and potassium 2-
278
ketogluconate in the API 50CH system.
Characteristics Growth temperature range (°C) † ‡ pH range (optimal pH) NaCl range (%) (optimal %) G+C mol% (genome sequencing) Assimilation of D-Glucose 4-nitrophenyl-ß-Dgalactopyranoside Enzyme activities of
methyl-α-D-glucopyranoside,
N-acetylglucosamine,
1
2
3
0-20 (17) 5-9.5 (7) 0-3.5 (1.5-2.5) 33.5
0-25 (17) 5-9.5 (7) 0-6 (1-4) 33.3
0-30 (22) 5-9.5 (7-7.5) 0-6 (0.5-3.5) 33.0
+
-
-
-
+
+
amygdalin,
Cystine arylamidase ß-Glucuronidase ß-Glucosidase α-Glucosidase ß-Galactosidase α-Mannosidase N-acetyl-ß-glucosaminidase Acid production from D-xylose D-galactose D-glucose D-fructose D-mannose L-rhamnose D-celiobiose D-lactose D-saccharose Amidon Gentiobiose D-turanose Potassium 5-ketogluconate G+C contents (mol%)
-
+* +*
+*
+ -
-* +* + +* -*
+* +* +* -
+ + -
+ + + +
+ + -
+ -
+
+ -
+ 33.5
+ 33.3
+ -
+ + 33.0
279 280
†
281
agarilyticus KCTC 23857T according to the results from Park et al. 2013c.
282
‡
283
agarilyticus KCTC 23857T according to the results from Park et al. 2013c.
284
*
285 286 287
Growth temperature range was 10-28 °C for A. agarivorans KCTC 23855T and 10-30 °C for A.
pH range (optimal pH) was 6-11 (7-8) for A. agarivorans KCTC 23855T and 6-11 (7-8) for A.
means different results with that from Park et al. 2013c.
288
Table 2. Cellular fatty acid composition of strain PAMC 27237 T and other type strains of the genus
289
Algibacter
290
Strains: 1, Algibacter psychrophilus PAMC 27237T; 2, A. agarivorans KCTC 23855T; 3, A.
291
agarilyticus KCTC 23857T. Fatty acids
1
2
3
C14:0
-
0.9
-
C15:0
3.7
6.2
5.5
C16:0
4.4
1.9
1.9
C18:0
2.4
-
1.2
C15:0 2-OH
3.6
1.8
3.3
C16:0 3-OH
-
2.0
1.7
C17:0 2-OH
10.0
3.4
6.3
iso-C15:0
4.7
9.1
11.1
iso-C16:0
2.1
4.2
4.4
iso-C15:0 3-OH
6.3
7.9
6.8
iso-C16:0 3-OH
5.6
6.7
3.8
iso-C17:0 3-OH
10.8
11.2
13.3
anteiso-C15:1 A
5.8
3.4
4.1
anteiso-C15:0
9.4
8.7
7.6
anteiso-C17:1ω9c
6.2
1.2
0.7
iso-C15:1 G
5.0
8.6
5.1
iso-C16:1 G
-
1.8
-
iso-C16:1 H
2.6
-
-
C15:1ω6c
-
1.5
1.2
C16:1ω5c
-
2.2
1.9
C17:1ω6c
2.6
3.7
5.5
C18:1ω5c
3.6
-
-
3 (C16:1 ω6c and/or C16:1 ω7c)
6.7
11.4
12.1
9 (iso-C17:1 ω9c and/or C16:0 10-methly)
4.5
2.2
2.5
Straight-chain saturated
Branched saturated
Monounsaturated
Summed feature*
292 293 294 295 296
-,
not detected.
297
Figure legends
298
Fig. 1 Neighbor-joining tree based on 16S rRNA gene sequences showing the relationships of Algibacter
299
psychrophilus PAMC 27237T with species of the genus Algibacter and other closely related members of the
300
family Flavobacteriaceae. Filled circles indicate that the corresponding nodes were recovered by all treeing
301
methods. Percent bootstrap supports (>50%) are given at each node (NJ/MP/ML). Flavobacterium aquidurense
302
WB 1.1-56T was used as an outgroup.
303
Figure Click here to download Figure: PAMC_27237_figure_revised.pptx Flavobacterium aquidurense WB 1.1-56 (NR042470) Algibacter psychrophilus PAMC 27237 (KJ475138) Algibacter agarilyticus KYW563 (JN864027)
63/ 52/ *
Algibacter agarivorans KYW560 (HQ405792) Algibacter aestuarii KYW371 (HQ405792) Algibacter aquimarinus KYW589 (JQ259859) 76/ 88/ 81
Arenitalea lutea P7-3-5 (JQ807811)
88/ 73/ 78
Algibacter pectinivorans JC2675 (HM475134) Algibacter mikhailovii LMG 23988 (AM491809)
100/ */ 74 92/ 87/ 91
Algibacter lectus KMM 3902 (AY187689) Algibacter wandonensis WS-MY22 (KC987358)
Algibacter undariae WS-MY9 (KC261664))
90/ 54 / 55
Algibacter miyuki WS-MY6 (KC662118) Flaviramulus basaltis H35 (DQ361033)
*/ 63/ *
Flaviramulus ichthyoenteri Th78 (JX412958) 67/62/ 75 79/ */ *
Gaetbulibacter lutimaris D1-y4 (JF739861) Gaetbulibacter marinus IMCC1914 (EF108219) Tamlana agarivorans JW-26 (EU221275)
64/ 70/ *
Tamlana sedimentorum KMM 9545 (AB894238) 75/ 67/ 80 100/ 96/ 97
Olleya aquimaris L-4 (FJ886713) Olleya marilimosa CAM030 (EF660466) Olleya namhaensis WT-MY15 (JQ327134)
90/ 72/ 71
100/ 100/ 100
Mesoflavibacter sabulilitoris GJMS-9 (KJ816860) Mesoflavibacter zeaxanthinifaciens DSM 18436 TD-ZX30 (AB265181)
99/ 99/ 98 92/ 87/ 88
Formosa arctica IMCC9485 (KF148059) Formosa undariae WS-MY3 (KC832835) Formosa spongicola A2 (FJ348469)
100/ 98/ 82
Bizionia hallyeonensis T-y7 (JN885199)
Bizionia echini KMM 6177 (FJ716799) 0.01
Fig. 1
Supplementary Material Files Click here to download Supplementary Material Files: PAMC_27237_supplementary.pdf
