IJSEM Papers in Press. Published November 26, 2014 as doi:10.1099/ijs.0.070680-0
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Loktanella sediminum sp. nov., isolated from sediment of the northern Okinawa
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Trough
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Jing Liang, Zenghu Zhang, Yan Liu, Min Wang, Xiao-Hua Zhang
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College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
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Running title: Loktanella sediminum sp. nov.
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Author for correspondence:
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Xiao-Hua Zhang, College of Marine Life Sciences, Ocean University of China, 5
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Yushan Road, Qingdao 266003, P. R. China, Tel/Fax: +86-532-82032767, Email:
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[email protected]
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The GenBank accession number for the 16S rRNA gene sequence of Loktanella
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sediminum S3B03T is KJ620985. The maximum-likelihood tree (Fig. S1) and
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maximum–parsimony tree (Fig. S2) based on 16S rRNA gene sequences, total polar
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lipids of strain S3B03T separated by two-dimensional TLC and detected with 10 %
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ethanolic molybdophosphoric acid (Fig. S3) are available as supplementary materials
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with the online version of this paper.
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Summary:
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A Gram-staining-negative, strictly aerobic and short rod-shaped bacterium, designated
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strain S3B03T, was isolated from the sediment of the northern Okinawa Trough.
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Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain
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S3B03T belonged to the genus Loktanella (family Rhodobacteraceae) and showed the
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highest sequence similarity with Loktanella litorea KCTC 23883T (96.16 %) and
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92.99–95.90 % to other members of the genus Loktanella. Optimal growth occurred in
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the presence of 2–5 % (w/v) NaCl, at pH 7.0–8.0 and at 28–32 ºC. Ubiquinone-10
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(Q-10) was the predominent respiratory quinone. The major fatty acids (>10 % of
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total fatty acids) were C18:1ω7c and/or C18:1ω6c. The major polar lipids were
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phosphatidylglycerol,
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phosphatidylethanolamine and two unidentified polar lipids. The DNA G+C content
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of strain S3B03T was 57.6 mol%. On the basis of polyphasic analysis, strain S3B03T
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is considered to represent a novel species of the genus Loktanella, for which the name
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Loktanella sediminum sp. nov. is proposed. The type strain is S3B03T (= JCM 30120T
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= DSM 28715T = MCCC 1K00257T).
diphosphatidylglycerol,
phosphatidylcholine,
38 39 40
The genus Loktanella, a member of the class Alphaproteobacteria, was proposed for
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novel bacteria isolated from Antarctic lakes (Van Trappen et al., 2004), with the
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description of three novel species, including Loktanella salsilacus as the type species
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of the genus, and subsequently emended by Moon et al. (2010), Lee (2012) and
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Tsubouchi et al. (2013). At the time of writing, the genus Loktanella comprised 16
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species with validly published names: Loktanella salsilacus, L. fryxellensis and L.
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vestfoldensis (Van Trappen et al., 2004), L. hongkongensis (Lau et al., 2004), L.
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agnita and L. rosea (Ivanova et al., 2005), L. koreensis (Weon et al., 2006), L.
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maricola (Yoon et al., 2007), L. atrilutea (Hosoya & Yokota, 2007), L. pyoseonensis
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(Moon et al., 2010), L. tamlensis (Lee, 2012), L. litorea (Yoon et al., 2013), L.
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cinnabarina (Tsubouchi et al., 2013), L. sediminilitoris (Park et al., 2013a), L.
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soesokkakensis (Park et al., 2013b) and L. maritime (Tanaka et al., 2014). These
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bacteria have been isolated from various sources including water, sediment, sand,
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marine biofilms, etc. In the course of study on bacterial diversity in marine sediments,
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we describe a Loktanella-like bacterial strain, designated S3B03T, which was isolated
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from surface sediment at water depth of 873.9 m in the northern Okinawa Trough at
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station 3B (30º14.6053
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No. 1 in August 2013. The aim of the present study was to determine the exact
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taxonomic position of strain S3B03T by using a polyphasic characterization that
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included the determination of chemotaxonomic and other phenotypic properties and
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detailed phylogenetic investigation based on 16S rRNA gene sequences.
N 128º26.8606
E) during the expedition on the R/V Kexue
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Strain S3B03T was isolated by the standard dilution plating technique on marine agar
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2216 (MA; Becton Dickinson) at 4 ºC for 3 months or 28 ºC for up to 2 weeks. After
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primary isolation and purification, strain S3B03T was routinely cultured on MA at 28
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ºC and preserved at -80 ºC as a suspension in sterile 0.85 % (w/v) saline supplemented
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with 15 % (w/v) glycerol. Loktanella salsilacus LMG 21507T and Loktanella litorea
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KCTC 23883T were chosen as the reference strains in phenotypic characterization and
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fatty acid analysis.
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Genomic DNA extraction, PCR amplification, cloning and sequencing of the 16S
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rRNA gene were performed according to Yu et al. (2012). The almost complete 16S
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rRNA gene sequence (1421 nt) was manually checked and submitted to GenBank.
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Pairwise similarity values between strain S3B03T and closely related type strains were
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calculated using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al.,
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2012). The 16S rRNA gene sequences of related strains were downloaded from the
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NCBI database (http://www.ncbi.nlm.nih.gov). Phylogenetic analysis was performed
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by the software package MEGA version 5.0 (Tamura et al. 2011) after multiple
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alignment of the sequence data with CLUSTAL_X (Thompson et al. 1997).
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Phylogenetic trees based on the neighbour-joining (NJ), maximum-likelihood (ML)
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and maximum-parsimony (MP) algorithms were constructed and the genetic distances
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were calculated by the Kimura two-parameter model (Kimura, 1980). The topology of
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the phylogenetic trees was evaluated by the bootstrap resampling method of
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Felsenstein (1981) with 1000 replicates.
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Strain S3B03T showed the highest 16S rRNA gene sequence similarity with
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Loktanella litorea KCTC 23883T (96.16 %) followed by Loktanella koreensis
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GA2-M3T (95.90 %), exhibiting sequence similarity values of 94.06 % to the type
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species, Loktanella salsilacus LMG 21507T, and 92.99–95.38 % to other members of
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the genus Loktanella. The NJ tree (Fig. 1) showed that S3B03T formed a tight
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phylogenetic cluster with the type strains of species in the genus Loktanella. The
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topology of trees generated with the ML (Fig. S1) and MP (Fig. S2) algorithms were
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essentially the same. However, the low levels of sequence similarity to the type strains
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of recognized species of the genus Loktanella implied that it represented a novel
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species in the genus Loktanella (Stackebrandt & Goebel, 1994).
95 96
Cell morphology of strain S3B03T was studied by transmission electron microscopy
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(JEOL; JEM-1200EX) after cells in exponential phase were collected from MA and
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negatively stained with 1 % (w/v) phosphotungstic acid. Gram-staining and flagellum
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staining were
performed
according to Beveridge
et al (2007). In
the
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poly-β-hydroxybutyrate (PHB) experiment, cells in exponential phase were collected
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from MA and a modified marine ammonium mineral salt medium (MAMS) (Raina et
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al., 2009; supplied with D-fructose, D-glucose, lactic acid and succinic acid as sole
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carbon source, respectively). PHB was tested in the wavelength of 460 nm by
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fluorescence microscopy CX; OLYMPUS
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(Ostle & Holt, 1982). Salinity and pH ranges supporting growth were investigated in
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96-well microplates by measuring the optical densities (wavelength 590 nm). In the
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salinity experiment, NaCl concentration was adjusted to 0–15.0 % (w/v, at intervals of
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1.0 %) in marine broth 2216 (MB; BD) (seawater was replaced by distilled water).
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Growth was evaluated at 0, 4, 10, 16, 25, 28, 32, 37, 42 and 46 ºC on MA and at pH
（
）according to the standard approaches
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2.0–11.0 in MB using the following buffer systems: Na2HPO4/citric acid (pH 2.0–7.0),
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Tris/HCl (pH 8.0–9.0) and Na2CO3/NaHCO3 (pH 10.0–11.0). To test for anaerobic
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growth, strain S3B03T was cultured at 28 ºC for one month on MA with resazurin
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(0.02 %, w/v) added as an indicator of anaerobic condition. Inoculated plates were
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incubated in an anaerobic jar filled with nitrogen and a bag of AneroPack-Anaero
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(Mitsibishi Gas Chemical Co., Inc). The presence of various phenotypic
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characterizations of strain S3B03T and two reference strains were tested according to
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the standard approaches (Tindall et al., 2007) with sterile seawater substituted for
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distilled water: activities of catalase, oxidase (method 2) and hydrolysis of starch,
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casein, gelatin, agar, and Tweens 20, 40 and 80 (method 2). DNase agar (Qingdao 96
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Hope Bio-technology Co., Ltd) prepared with sterile seawater was used to detect the
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DNase activity. Chitin (1 %, w/v) and sodium alginate (2 %, w/v) were added to MA
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plates to determine the degradation by the formation of clear zones around colonies
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directly or after flooding with appropriate solutions (Teather & Wood, 1982).
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Activities of constitutive enzymes, the fermentation/oxidation profile, acid production,
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substrate utilization as sole carbon and energy source were performed using API 20E,
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API 20NE, API 50CH, API ZYM strips (bioMérieux) and the GN2 MicroPlate kit
127
(Biolog) according to the manufacturers’ instructions except that sterile seawater was
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used to prepare the inocula.
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For PHB tests, growth of cells and existence of PHB granules occurred on all kinds of
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media mentioned above. The morphological, physiological and biochemical
132
characteristics of strain S3B03T are given in Table 1, Fig. 2 and the species
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description.
134 135
For cellular fatty acid analysis, strain S3B03T and the two reference strains were
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grown on MA at 28 ºC for 48 h when the bacterial communities reached the late
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exponential stage of growth. Fatty acid methyl esters were prepared and analyzed
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according to the standard protocol of MIDI (Sherlock Microbial Identification System,
139
version 6.10), and identified by the TSBA6.0 database of the Microbial Identification
140
System (Sasser, 1990). For analysis of respiratory quinones and polar lipids, cells
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were harvested from MB after incubation at 28 °C for 48 h and freeze-dried. Polar
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lipids were extracted according to Minnikin et al. (1984), separated by
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two-dimensional TLC on silica gel 60 F254 plates (Merck), and identified by spraying
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with the appropriate detection reagents (Komagata & Suzuki, 1987). The respiratory
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quinones of strain S3B03T were extracted with chloroform/methanol (2:1, v/v) and
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identified by HPLC as described by Xie & Yokota (2003). The genomic DNA of
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strain S3B03T was extracted according to Moore et al. (1999) and the G+C content
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was determined by reversed-phase HPLC according to Mesbah et al. (1989).
149 150
Similar to those of Loktanella salsilacus LMG 21507T and Loktanella litorea KCTC
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23883T, the predominant cellular fatty acid (>10 % of total fatty acids) of strain
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S3B03T was summed feature 8 (C18:1ω7c and/or C18:1ω6c). However, in strain S3B03T,
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the proportion of 11-methyl C18:1ω7c was also significant. Differences in fatty acid
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compositions and the proportion of some components were identified between
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S3B03T and two reference strains (Table 2). The predominant isoprenoid quinone
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detected in strain S3B03T was Q-10, in line with other species of the genus Loktanella
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(Weon et al., 2006; Yoon et al., 2007; Hosoya & Yokota, 2007; Moon et al., 2010; Lee,
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2012; Yoon et al., 2013; Tsubouchi et al., 2013; Park et al., 2013a; Park et al., 2013b;
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Tanaka et al., 2014). The major polar lipids detected in S3B03T were
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phosphatidylglycerol,
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phosphatidylethanolamine and two unidentified polar lipids (Fig. S3), and the polar
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lipid profile was similar to the reference strains (Yoon et al., 2013) except that
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diphosphatidylglycerol and two unidentified polar lipids were present in S3B03T. The
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G+C content of S3B03T is 57.6 mol%, which is within the range reported for
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members of the genus Loktanella (55.0–69.3 mol%) (Tsubouchi et al., 2013).
diphosphatidylglycerol,
phosphatidylcholine,
166 167
Strain S3B03T shared several common traits with Loktanella salsilacus LMG 21507T
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and Loktanella litorea KCTC 23883T in both phenotypic and chemical characteristics.
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However, S3B03T could be clearly differentiated from these two reference strains by
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NaCl and temperature range for growth, hydrolysis of various macromolecules and
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the existence of PHB granules in the cell. Based on the phenotypic and phylogenetic
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data, strain S3B03T represents a novel species in the genus Loktanella, for which the
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name Loktanella sediminum sp. nov. is proposed.
174 175
Description of Loktanella sediminum sp. nov.
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Loktanella sediminum (se.di.mi′num. L. gen. pl. n. sediminum of sediments,
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pertaining to source of isolation).
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Cells are Gram-staining-negative, strictly aerobic, non-motile short rods (0.4–0.5 μm
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in width, 0.8–1.2 μm in length). Can grow on MA ,and R2A medium. Colonies on MA
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are light beige (light pink after incubation for 5–7 days), regular, translucent, shiny
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and smooth with the regular margin of 0.5–1 mm in diameter after incubation for 3
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days at 28 ºC. Growth occurs at 10 to 46 °C with an optimum temperature at
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28–32
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for growth is 0–6 % (optimum 2–5 %). Strain is positive for oxidase and catalase
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activity and hydrolysis of DNA, gelatin, and Tween 20; negative for hydrolysis of
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starch, casein, agar, alginate, chitin, Tween 40 and Tween 80. In the API 20E and
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20NE strips, there are positive results for aesculin hydrolysis and β-galactosidase, and
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negative
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decarboxylase, citrate utilization, H2S production, urea hydrolysis, nitrate reduction to
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nitrite, deaminase, indole production and gelatinase. In the API ZYM strip, alkaline
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phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid
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phosphatase, naphthol-AS-BI-phosphohydrolase,
193
(weakly-positive) activities are present; lipase (C14), valine arylamidase, cystine
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arylamidase, trypsin, α-chymotrypsin, α-galactosidase, α-glucosidase, β-glucuronidase,
195
α-mannosidase, α-fucosidase
196
According to the results of API 50CH strip, acid production is positive for D-ribose,
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D-glucose, D-fructose, D-mannitol, D-cellobiose, esculin, melibiose, sucrose,
°C. The pH range for growth is 7.0–10.0 (optimum 7.0–8.0). The NaCl range
results
for
arginine
dihydrolase,
lysine
decarboxylase,
β-galactosidase
and
ornithine
β-glucosidase
and N-acetyl-β-glucosaminidase activities are absent.
198
trehalose; weakly-positive for L-arabinose, D-galactose, D-mannose, D-fucose,
199
amygdalin, salicin, maltose, lactose, turanose; and other substrates are negative.
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D-fructose, D-glucose, lactic acid, succinic acid can be weakly utilized in the Biolog
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GN2 MicroPlate system. The major respiratory quinone is Q-10. The predominant
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polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine,
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phosphatidylethanolamine and two unidentified polar lipids. The major fatty acid is
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C18:1ω7c and/or C18:1ω6c. The genome G+C content is 57.6 mol%.
205 206
The type strain, S3B03T (= JCM 30120T = DSM 28715T = MCCC 1K00257T), was
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isolated from sediment of the northern Okinawa Trough.
208 209
Acknowledgements
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We thank all of the scientists and crew members on the R/V Kexue No. 1 during the
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expedition for their great efforts and help in sample collection. This work was
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supported by grants from the Major State Basic Research Development Program of
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China (2013CB429700), the China Ocean Mineral Resources Research &
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Development Association (COMRA, No. DY125-15-R-03), and the National High
215
Technology Research & Development Program of China (863 Programs, No.
216
2012AA091605).
217 218
Reference
219
Beveridge, T., Lawrence, J. & Murray, R. (2007). Sampling and staining for light
220
microscopy. In Methods for General and Molecular Microbiology, 19–33. Edited by
221
C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. Marzluf, T. M. Schmidt & L. R.
222
Snyder (ed.) ASM Press, Washington DC.
223
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum
224
likelihood approach. J Mol Evol 17, 368–376.
225
Hosoya, S. & Yokota, A. (2007). Loktanella atrilutea sp. nov., isolated from seawater
226
in Japan. Int J Syst Evol Microbiol 57, 1966–1969.
227
Ivanova, E. P., Zhukova, N. V., Lysenko, A. M., Gorshkova, N. M., Sergeev, A. F.,
228
Mikhailov, V. V. & Bowman, J. P. (2005). Loktanella agnita sp. nov. and Loktanella
229
rosea sp. nov., from the north-west Pacific Ocean. Int J Syst Evol Microbiol 55,
230
2203–2207.
231
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y.
232
S., Lee, J. H. & Yi, H. (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene
233
sequence database with phylotypes that represent uncultured species. Int J Syst Evol
234
Microbiol 62, 716-721.
235
Kimura, M. (1980). A simple method for estimating evolutionary rates of base
236
substitutions through comparative studies of nucleotide sequences. J Mol Evol 16,
237
111–120.
238
Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial
239
systematics. Methods Microbiol 19, 1–207.
240
Lau, S. C. K., Tsoi, M. M. Y., Li, X., Plakhotnikova, I., Wu, M., Wong, P.-K. &
241
Qian, P.-Y. (2004). Loktanella hongkongensis sp. nov., a novel member of the
242
α-Proteobacteria originating from marine biofilms in Hong Kong waters. Int J Syst
243
Evol Microbiol 54, 2281–2284.
244
Lee, S. D. (2012). Loktanella tamlensis sp. nov., isolated from seawater. Int J Syst
245
Evol Microbiol 62, 586–590.
246
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement
247
of the G+C content of deoxyribonucleic acid by highperformance liquid
248
chromatography. Int J Syst Bacteriol 39, 159–167
249
Minnikin, D., O'Donnell, A., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A.
250
& Parlett, J. (1984). An integrated procedure for the extraction of bacterial
251
isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
252
Montero-Calasanz, M. del C., Göker, M., Rohde, M., Spröer, C., Schumann, P.,
253
Busse, H.-J., Schmid, M., Tindall, B. J., Klenk, H.-P. & Camacho, M. (2013).
254
Chryseobacterium hispalense sp. nov., a plant-growth-promoting bacterium isolated
255
from a rainwater pond in an olive plant nursery, and emended descriptions of
256
Chryseobacterium
257
wanjuense and Chryseobacterium gregarium. Int J Syst Evol Microbiol 63,
258
4386–4395.
259
Moon, Y. G., Seo, S. H., Lee, S. D. & Heo, M. S. (2010). Loktanella pyoseonensis sp.
260
nov., isolated from beach sand, and emended description of the genus Loktanella. Int J
261
Syst Evol Microbiol 60, 785–789.
262
Moore, E. R. B., Arnscheidt, A., Krüger, A., Strömpl, C. & Mau, M. (1999).
263
Simplified protocols for the preparation of genomic DNA from bacterial cultures. In
defluvii,
Chryseobacterium
indologenes,
Chryseobacterium
264
Molecular Microbial Ecology Manual 1.6.1, pp. 1–15. Edited by A. D. L. Akkermans,
265
J. D. van Elsas & F. J. de Bruijn. Dordrecht: Kluwer.
266
Ostle A.G., Holt J.G. (1982). Nile Blue A as a fluorescent stain for poly
267
β-hydroxybutirate. Appl Environ Microbiol 44, 238–241.
268
Park, S., Jung, Y.-T. & Yoon, J.-H. (2013). Loktanella sediminilitoris sp. nov.,
269
isolated from tidal flat sediment. Int J Syst Evol Microbiol 63, 4118–4123.
270
Park, S., Lee, J.-S., Lee, K.-C. & Yoon, J.-H. (2013). Loktanella soesokkakensis sp.
271
nov., isolated from the junction between the North Pacific Ocean and a freshwater
272
spring. Antonie van Leeuwenhoek 104, 397–404.
273
Raina J.-B., Tapiolas D., Willis B.L., Bourne D.G. (2009). Coral-associated bacteria
274
and their role in the biogeochemical cycling of sulfur. Applied and Environmental
275
Microbiology 75:3492-3501.
276
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty
277
acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
278
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA–DNA
279
reassociation and 16S rRNA sequence analysis in the present species definition in
280
bacteriology. 44, 846–849.
281
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011).
282
MEGA5: molecular evolutionary genetics analysis using maximum likelihood,
283
evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28,
284
2731–2739.
285
Tanaka, N., Romanenko, L. A., Kurilenko, V. V., Svetashev, V. I., Kalinovskaya,
286
N. I., and Mikhailov, V. V. (2014). Loktanella maritima sp. nov. isolated from
287
shallow sediments of the Sea of Japan. Int J Syst Evol Microbiol 64, 2370–2375.
288
Teather, R. M. & Wood, P. J. (1982). Use of Congo red-polysaccharide interactions
289
in enumeration and characterization of cellulolytic bacteria from the bovine rumen.
290
Appl Environ Microbiol 43, 777–780.
291
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.
292
(1997). The CLUSTAL_X windows interface: flexible strategies for multiple
293
sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882.
294
Tindall, B., Sikorski, J., Smibert, R. & Krieg, N. (2007). Phenotypic
295
characterization and the principles of comparative systematics. In Methods for
296
General and Molecular Microbiology 3, 330–393. Edited by C. A. Reddy, T. J.
297
Beveridge, J. A. Breznak, G. Marzluf, T. M. Schmidt, & L. R. Snyder (ed.) ASM Press,
298
Washington DC.
299
Tsubouchi, T., Shimane, Y., Mori, K., Miyazaki, M., Tame, A., Uematsu, K.,
300
Maruyama, T. & Hatada, Y. (2013). Loktanella cinnabarina sp. nov., isolated from a
301
deep subseafloor sediment, and emended description of the genus Loktanella. Int J
302
Syst Evol Microbiol 63, 1390–1395.
303
Van Trappen, S., Mergaert, J. & Swings, J. (2004). Loktanella salsilacus gen. nov.,
304
sp. nov., Loktanella fryxellensis sp. nov. and Loktanella vestfoldensis sp. nov., new
305
members of the Rhodobacter group, isolated from microbial mats in Antarctic lakes.
306
Int J Syst Evol Microbiol 54, 1263–1269.
307
Weon, H.-Y., Kim, B.-Y., Yoo, S.-H., Kim, J.-S., Kwon, S.-W., Go, S.-J. &
308
Stackebrandt, E. (2006). Loktanella koreensis sp. nov., isolated from sea sand in
309
Korea. Int J Syst Evol Microbiol 56, 2199–2202.
310
Xie, C.-H. & Yokota, A. (2003). Phylogenetic analyses of Lampropedia hyalina
311
based on the 16S rRNA gene sequence. J Gen Appl Microbiol 49, 345–349.
312
Yoon, J.-H., Jung, Y.-T. & Lee, J.-S. (2013). Loktanella litorea sp. nov., isolated
313
from seawater. Int J Syst Evol Microbiol 63, 175–180.
314
Yoon, J.-H., Kang, S.-J., Lee, S.-Y. & Oh, T.-K. (2007). Loktanella maricola sp.
315
nov., isolated from seawater of the East Sea in Korea. Int J Syst Evol Microbiol 57,
316
1799–1802.
317
Yu, T., Yin, Q.,Song, X., Zhao. R.,Shi, X. & Zhang, X.-H (2012). Aquimarina longa
318
sp. nov., isolated from the seawater of South Pacific Gyre. Int J Syst Evol Microbiol
319
63, 1235–1240.
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Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences
321
showing the phylogenetic position of strain S3B03T, the type strains of other species
322
of the genus Loktanella and representatives of some other related taxa. Bootstrap
323
percentages (>70 %) based on 1000 replicates are shown at branch points. Stappia
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stellulata DSM 5886T (AUIM01000013) was used as an outgroup. Bar, 0.01
325
substitution per nucleotide position.
326
Fig. 2. Transmission electron micrographs of negatively stained cells of Loktanella
327
sediminum S3B03T cultured on MA at 28 ºC for 24 h. Bar, 0.1 μm. a: a large cell; b: a
328
small cell.
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Table 1. Differential characteristics between strain S3B03T and the type strains of
330
phylogenetically related species of the genus Loktanella
331 332 333
Strains: 1. S3B03T 2. Loktanella litorea KCTC 23883T; 3. Loktanella salsilacus LMG 21507T. All data from this study unless otherwise indicated. +, positive reaction; -, negative reaction; w, weakly positive. 1 2 3* Characteristics Colony colour (on MA) light beige-pink light orange–yellow beige Growth at/in 10 % NaCl + 10 °C + + 40 °C + Hydrolysis of DNA + Gelatin + Alginate + -† Tween 20 + +† Tween 40 +† Tween 80 + Citrate utilization (API 20E) ‡ + Enzymes activity (API ZYM) ‡ Leucine arylamidase + + α-Galactosidase + β-galactosidase + α-Glucosidase w β-Glucosidase w w
（
Acid production from API 50CH) ‡
334 335 336
L-Arabinose w + D-Glucose + D-Mannitol + Melibiose + + DNA G+C content (mol%)‡ 57.6 57.6 T *All data for L. salsilacus LMG 21507 taken from Van Trappen et al.(2004). † Data not reported in Van Trappen et al.(2004). ‡ Data for reference strains were taken from Yoon et al.(2013).
60.4
337 338 339 340 341 342
343 344 345 346 347 348 349 350
Table 2. Cellular fatty acid contents (%) of strain S3B03T and the type strains of phylogenetically closely related species Strains: 1. S3B03T; 2. Loktanella litorea KCTC 23883T; 3. Loktanella salsilacus LMG 21507T. All data are from this study. Only fatty acids more than 1 % of the total fatty acids of at least one of the strains are shown. Tr, trace amount (< 1 % of total); -, not detected. Fatty acid 1 2 3 Straight chain C16:0 5.29 11 18.54 C17:0 1.27 C18:0 4.38 3.37 2.57 Branched anteiso-C15:0 1.54 anteiso-C16:0 Tr anteiso-C17:0 1.69 1.39 1.05 Hydroxy C10:0 3-OH 1.18 5.38 1.43 C12:1 3-OH 2.52 2.87 C16:0 2-OH 1.01 1.07 0.8 C18:1 2-OH 1.7 Unsaturated C20:1ω7c 1.81 11-methyl C18:1ω7c 2.26 Tr 7.34 C16:0 N alcohol 1.47 1.71 1.44 Summed feature 2† Tr Tr Summed feature 3† 1.23 2.8 Summed feature 7† 3.08 1.76 1.97 Summed feature 8† 64.2 65.35 63.76 † As indicated by Montero-Calasanz et al.(2013) summed features are groups of two or three fatty acids that are treated together for the purpose of evaluation in the MIDI system and include both peaks with discrete equivalent chain lengths (ECLs) as well as those where the ECLs are not reported separately. Summed feature 2 was listed as C12:0 aldehyde, an unknown fatty acid (ECL 10.928), C14:0 3-OH and/or iso-C16:1 I. Summed feature 3 was listed as C16:1ω7c and/or C16:1ω6c. Summed feature 7 was listed as an unknown fatty acid (ECL 18.846), C19:1ω6c and/or C19:0 ω10c cyclo. Summed feature 8 was listed as C18:1ω7c and/or C18:1ω6c.
1
Loktanella sediminum sp. nov., isolated from sediment of the northern Okinawa
2
Trough
3 4
Jing Liang, Zenghu Zhang, Yan Liu, Min Wang, Xiao-Hua Zhang
5
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
6 7
Running title: Loktanella sediminum sp. nov.
8 9
Author for correspondence:
10
Xiao-Hua Zhang, College of Marine Life Sciences, Ocean University of China, 5
11
Yushan Road, Qingdao 266003, P. R. China, Tel/Fax: +86-532-82032767, Email:
12
[email protected]
13 14 15
The GenBank accession number for the 16S rRNA gene sequence of Loktanella
16
sediminum S3B03T is KJ620985. The maximum-likelihood tree (Fig. S1) and
17
maximum–parsimony tree (Fig. S2) based on 16S rRNA gene sequences, total polar
18
lipids of strain S3B03T separated by two-dimensional TLC and detected with 10 %
19
ethanolic molybdophosphoric acid (Fig. S3) are available as supplementary materials
20
with the online version of this paper.
21
22
Summary:
23
A Gram-staining-negative, strictly aerobic and short rod-shaped bacterium, designated
24
strain S3B03T, was isolated from the sediment of the northern Okinawa Trough.
25
Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain
26
S3B03T belonged to the genus Loktanella (family Rhodobacteraceae) and showed the
27
highest sequence similarity with Loktanella litorea KCTC 23883T (96.16 %) and
28
92.99–95.90 % to other members of the genus Loktanella. Optimal growth occurred in
29
the presence of 2–5 % (w/v) NaCl, at pH 7.0–8.0 and at 28–32 ºC. Ubiquinone-10
30
(Q-10) was the predominent respiratory quinone. The major fatty acids (>10 % of
31
total fatty acids) were C18:1ω7c and/or C18:1ω6c. The major polar lipids were
32
phosphatidylglycerol,
33
phosphatidylethanolamine and two unidentified polar lipids. The DNA G+C content
34
of strain S3B03T was 57.6 mol%. On the basis of polyphasic analysis, strain S3B03T
35
is considered to represent a novel species of the genus Loktanella, for which the name
36
Loktanella sediminum sp. nov. is proposed. The type strain is S3B03T (= JCM 30120T
37
= DSM 28715T = MCCC 1K00257T).
diphosphatidylglycerol,
phosphatidylcholine,
38 39 40
The genus Loktanella, a member of the class Alphaproteobacteria, was proposed for
41
novel bacteria isolated from Antarctic lakes (Van Trappen et al., 2004), with the
42
description of three novel species, including Loktanella salsilacus as the type species
43
of the genus, and subsequently emended by Moon et al. (2010), Lee (2012) and
44
Tsubouchi et al. (2013). At the time of writing, the genus Loktanella comprised 16
45
species with validly published names: Loktanella salsilacus, L. fryxellensis and L.
46
vestfoldensis (Van Trappen et al., 2004), L. hongkongensis (Lau et al., 2004), L.
47
agnita and L. rosea (Ivanova et al., 2005), L. koreensis (Weon et al., 2006), L.
48
maricola (Yoon et al., 2007), L. atrilutea (Hosoya & Yokota, 2007), L. pyoseonensis
49
(Moon et al., 2010), L. tamlensis (Lee, 2012), L. litorea (Yoon et al., 2013), L.
50
cinnabarina (Tsubouchi et al., 2013), L. sediminilitoris (Park et al., 2013a), L.
51
soesokkakensis (Park et al., 2013b) and L. maritime (Tanaka et al., 2014). These
52
bacteria have been isolated from various sources including water, sediment, sand,
53
marine biofilms, etc. In the course of study on bacterial diversity in marine sediments,
54
we describe a Loktanella-like bacterial strain, designated S3B03T, which was isolated
55
from surface sediment at water depth of 873.9 m in the northern Okinawa Trough at
56
station 3B (30º14.6053
57
No. 1 in August 2013. The aim of the present study was to determine the exact
58
taxonomic position of strain S3B03T by using a polyphasic characterization that
59
included the determination of chemotaxonomic and other phenotypic properties and
60
detailed phylogenetic investigation based on 16S rRNA gene sequences.
N 128º26.8606
E) during the expedition on the R/V Kexue
61 62
Strain S3B03T was isolated by the standard dilution plating technique on marine agar
63
2216 (MA; Becton Dickinson) at 4 ºC for 3 months or 28 ºC for up to 2 weeks. After
64
primary isolation and purification, strain S3B03T was routinely cultured on MA at 28
65
ºC and preserved at -80 ºC as a suspension in sterile 0.85 % (w/v) saline supplemented
66
with 15 % (w/v) glycerol. Loktanella salsilacus LMG 21507T and Loktanella litorea
67
KCTC 23883T were chosen as the reference strains in phenotypic characterization and
68
fatty acid analysis.
69 70
Genomic DNA extraction, PCR amplification, cloning and sequencing of the 16S
71
rRNA gene were performed according to Yu et al. (2012). The almost complete 16S
72
rRNA gene sequence (1421 nt) was manually checked and submitted to GenBank.
73
Pairwise similarity values between strain S3B03T and closely related type strains were
74
calculated using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al.,
75
2012). The 16S rRNA gene sequences of related strains were downloaded from the
76
NCBI database (http://www.ncbi.nlm.nih.gov). Phylogenetic analysis was performed
77
by the software package MEGA version 5.0 (Tamura et al. 2011) after multiple
78
alignment of the sequence data with CLUSTAL_X (Thompson et al. 1997).
79
Phylogenetic trees based on the neighbour-joining (NJ), maximum-likelihood (ML)
80
and maximum-parsimony (MP) algorithms were constructed and the genetic distances
81
were calculated by the Kimura two-parameter model (Kimura, 1980). The topology of
82
the phylogenetic trees was evaluated by the bootstrap resampling method of
83
Felsenstein (1981) with 1000 replicates.
84 85
Strain S3B03T showed the highest 16S rRNA gene sequence similarity with
86
Loktanella litorea KCTC 23883T (96.16 %) followed by Loktanella koreensis
87
GA2-M3T (95.90 %), exhibiting sequence similarity values of 94.06 % to the type
88
species, Loktanella salsilacus LMG 21507T, and 92.99–95.38 % to other members of
89
the genus Loktanella. The NJ tree (Fig. 1) showed that S3B03T formed a tight
90
phylogenetic cluster with the type strains of species in the genus Loktanella. The
91
topology of trees generated with the ML (Fig. S1) and MP (Fig. S2) algorithms were
92
essentially the same. However, the low levels of sequence similarity to the type strains
93
of recognized species of the genus Loktanella implied that it represented a novel
94
species in the genus Loktanella (Stackebrandt & Goebel, 1994).
95 96
Cell morphology of strain S3B03T was studied by transmission electron microscopy
97
(JEOL; JEM-1200EX) after cells in exponential phase were collected from MA and
98
negatively stained with 1 % (w/v) phosphotungstic acid. Gram-staining and flagellum
99
staining were
performed
according to Beveridge
et al (2007). In
the
100
poly-β-hydroxybutyrate (PHB) experiment, cells in exponential phase were collected
101
from MA and a modified marine ammonium mineral salt medium (MAMS) (Raina et
102
al., 2009; supplied with D-fructose, D-glucose, lactic acid and succinic acid as sole
103
carbon source, respectively). PHB was tested in the wavelength of 460 nm by
104
fluorescence microscopy CX; OLYMPUS
105
(Ostle & Holt, 1982). Salinity and pH ranges supporting growth were investigated in
106
96-well microplates by measuring the optical densities (wavelength 590 nm). In the
107
salinity experiment, NaCl concentration was adjusted to 0–15.0 % (w/v, at intervals of
108
1.0 %) in marine broth 2216 (MB; BD) (seawater was replaced by distilled water).
109
Growth was evaluated at 0, 4, 10, 16, 25, 28, 32, 37, 42 and 46 ºC on MA and at pH
（
）according to the standard approaches
110
2.0–11.0 in MB using the following buffer systems: Na2HPO4/citric acid (pH 2.0–7.0),
111
Tris/HCl (pH 8.0–9.0) and Na2CO3/NaHCO3 (pH 10.0–11.0). To test for anaerobic
112
growth, strain S3B03T was cultured at 28 ºC for one month on MA with resazurin
113
(0.02 %, w/v) added as an indicator of anaerobic condition. Inoculated plates were
114
incubated in an anaerobic jar filled with nitrogen and a bag of AneroPack-Anaero
115
(Mitsibishi Gas Chemical Co., Inc). The presence of various phenotypic
116
characterizations of strain S3B03T and two reference strains were tested according to
117
the standard approaches (Tindall et al., 2007) with sterile seawater substituted for
118
distilled water: activities of catalase, oxidase (method 2) and hydrolysis of starch,
119
casein, gelatin, agar, and Tweens 20, 40 and 80 (method 2). DNase agar (Qingdao 96
120
Hope Bio-technology Co., Ltd) prepared with sterile seawater was used to detect the
121
DNase activity. Chitin (1 %, w/v) and sodium alginate (2 %, w/v) were added to MA
122
plates to determine the degradation by the formation of clear zones around colonies
123
directly or after flooding with appropriate solutions (Teather & Wood, 1982).
124
Activities of constitutive enzymes, the fermentation/oxidation profile, acid production,
125
substrate utilization as sole carbon and energy source were performed using API 20E,
126
API 20NE, API 50CH, API ZYM strips (bioMérieux) and the GN2 MicroPlate kit
127
(Biolog) according to the manufacturers’ instructions except that sterile seawater was
128
used to prepare the inocula.
129 130
For PHB tests, growth of cells and existence of PHB granules occurred on all kinds of
131
media mentioned above. The morphological, physiological and biochemical
132
characteristics of strain S3B03T are given in Table 1, Fig. 2 and the species
133
description.
134 135
For cellular fatty acid analysis, strain S3B03T and the two reference strains were
136
grown on MA at 28 ºC for 48 h when the bacterial communities reached the late
137
exponential stage of growth. Fatty acid methyl esters were prepared and analyzed
138
according to the standard protocol of MIDI (Sherlock Microbial Identification System,
139
version 6.10), and identified by the TSBA6.0 database of the Microbial Identification
140
System (Sasser, 1990). For analysis of respiratory quinones and polar lipids, cells
141
were harvested from MB after incubation at 28 °C for 48 h and freeze-dried. Polar
142
lipids were extracted according to Minnikin et al. (1984), separated by
143
two-dimensional TLC on silica gel 60 F254 plates (Merck), and identified by spraying
144
with the appropriate detection reagents (Komagata & Suzuki, 1987). The respiratory
145
quinones of strain S3B03T were extracted with chloroform/methanol (2:1, v/v) and
146
identified by HPLC as described by Xie & Yokota (2003). The genomic DNA of
147
strain S3B03T was extracted according to Moore et al. (1999) and the G+C content
148
was determined by reversed-phase HPLC according to Mesbah et al. (1989).
149 150
Similar to those of Loktanella salsilacus LMG 21507T and Loktanella litorea KCTC
151
23883T, the predominant cellular fatty acid (>10 % of total fatty acids) of strain
152
S3B03T was summed feature 8 (C18:1ω7c and/or C18:1ω6c). However, in strain S3B03T,
153
the proportion of 11-methyl C18:1ω7c was also significant. Differences in fatty acid
154
compositions and the proportion of some components were identified between
155
S3B03T and two reference strains (Table 2). The predominant isoprenoid quinone
156
detected in strain S3B03T was Q-10, in line with other species of the genus Loktanella
157
(Weon et al., 2006; Yoon et al., 2007; Hosoya & Yokota, 2007; Moon et al., 2010; Lee,
158
2012; Yoon et al., 2013; Tsubouchi et al., 2013; Park et al., 2013a; Park et al., 2013b;
159
Tanaka et al., 2014). The major polar lipids detected in S3B03T were
160
phosphatidylglycerol,
161
phosphatidylethanolamine and two unidentified polar lipids (Fig. S3), and the polar
162
lipid profile was similar to the reference strains (Yoon et al., 2013) except that
163
diphosphatidylglycerol and two unidentified polar lipids were present in S3B03T. The
164
G+C content of S3B03T is 57.6 mol%, which is within the range reported for
165
members of the genus Loktanella (55.0–69.3 mol%) (Tsubouchi et al., 2013).
diphosphatidylglycerol,
phosphatidylcholine,
166 167
Strain S3B03T shared several common traits with Loktanella salsilacus LMG 21507T
168
and Loktanella litorea KCTC 23883T in both phenotypic and chemical characteristics.
169
However, S3B03T could be clearly differentiated from these two reference strains by
170
NaCl and temperature range for growth, hydrolysis of various macromolecules and
171
the existence of PHB granules in the cell. Based on the phenotypic and phylogenetic
172
data, strain S3B03T represents a novel species in the genus Loktanella, for which the
173
name Loktanella sediminum sp. nov. is proposed.
174 175
Description of Loktanella sediminum sp. nov.
176
Loktanella sediminum (se.di.mi′num. L. gen. pl. n. sediminum of sediments,
177
pertaining to source of isolation).
178
Cells are Gram-staining-negative, strictly aerobic, non-motile short rods (0.4–0.5 μm
179
in width, 0.8–1.2 μm in length). Can grow on MA ,and R2A medium. Colonies on MA
180
are light beige (light pink after incubation for 5–7 days), regular, translucent, shiny
181
and smooth with the regular margin of 0.5–1 mm in diameter after incubation for 3
182
days at 28 ºC. Growth occurs at 10 to 46 °C with an optimum temperature at
183
28–32
184
for growth is 0–6 % (optimum 2–5 %). Strain is positive for oxidase and catalase
185
activity and hydrolysis of DNA, gelatin, and Tween 20; negative for hydrolysis of
186
starch, casein, agar, alginate, chitin, Tween 40 and Tween 80. In the API 20E and
187
20NE strips, there are positive results for aesculin hydrolysis and β-galactosidase, and
188
negative
189
decarboxylase, citrate utilization, H2S production, urea hydrolysis, nitrate reduction to
190
nitrite, deaminase, indole production and gelatinase. In the API ZYM strip, alkaline
191
phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid
192
phosphatase, naphthol-AS-BI-phosphohydrolase,
193
(weakly-positive) activities are present; lipase (C14), valine arylamidase, cystine
194
arylamidase, trypsin, α-chymotrypsin, α-galactosidase, α-glucosidase, β-glucuronidase,
195
α-mannosidase, α-fucosidase
196
According to the results of API 50CH strip, acid production is positive for D-ribose,
197
D-glucose, D-fructose, D-mannitol, D-cellobiose, esculin, melibiose, sucrose,
°C. The pH range for growth is 7.0–10.0 (optimum 7.0–8.0). The NaCl range
results
for
arginine
dihydrolase,
lysine
decarboxylase,
β-galactosidase
and
ornithine
β-glucosidase
and N-acetyl-β-glucosaminidase activities are absent.
198
trehalose; weakly-positive for L-arabinose, D-galactose, D-mannose, D-fucose,
199
amygdalin, salicin, maltose, lactose, turanose; and other substrates are negative.
200
D-fructose, D-glucose, lactic acid, succinic acid can be weakly utilized in the Biolog
201
GN2 MicroPlate system. The major respiratory quinone is Q-10. The predominant
202
polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine,
203
phosphatidylethanolamine and two unidentified polar lipids. The major fatty acid is
204
C18:1ω7c and/or C18:1ω6c. The genome G+C content is 57.6 mol%.
205 206
The type strain, S3B03T (= JCM 30120T = DSM 28715T = MCCC 1K00257T), was
207
isolated from sediment of the northern Okinawa Trough.
208 209
Acknowledgements
210
We thank all of the scientists and crew members on the R/V Kexue No. 1 during the
211
expedition for their great efforts and help in sample collection. This work was
212
supported by grants from the Major State Basic Research Development Program of
213
China (2013CB429700), the China Ocean Mineral Resources Research &
214
Development Association (COMRA, No. DY125-15-R-03), and the National High
215
Technology Research & Development Program of China (863 Programs, No.
216
2012AA091605).
217 218
Reference
219
Beveridge, T., Lawrence, J. & Murray, R. (2007). Sampling and staining for light
220
microscopy. In Methods for General and Molecular Microbiology, 19–33. Edited by
221
C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. Marzluf, T. M. Schmidt & L. R.
222
Snyder (ed.) ASM Press, Washington DC.
223
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum
224
likelihood approach. J Mol Evol 17, 368–376.
225
Hosoya, S. & Yokota, A. (2007). Loktanella atrilutea sp. nov., isolated from seawater
226
in Japan. Int J Syst Evol Microbiol 57, 1966–1969.
227
Ivanova, E. P., Zhukova, N. V., Lysenko, A. M., Gorshkova, N. M., Sergeev, A. F.,
228
Mikhailov, V. V. & Bowman, J. P. (2005). Loktanella agnita sp. nov. and Loktanella
229
rosea sp. nov., from the north-west Pacific Ocean. Int J Syst Evol Microbiol 55,
230
2203–2207.
231
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y.
232
S., Lee, J. H. & Yi, H. (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene
233
sequence database with phylotypes that represent uncultured species. Int J Syst Evol
234
Microbiol 62, 716-721.
235
Kimura, M. (1980). A simple method for estimating evolutionary rates of base
236
substitutions through comparative studies of nucleotide sequences. J Mol Evol 16,
237
111–120.
238
Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial
239
systematics. Methods Microbiol 19, 1–207.
240
Lau, S. C. K., Tsoi, M. M. Y., Li, X., Plakhotnikova, I., Wu, M., Wong, P.-K. &
241
Qian, P.-Y. (2004). Loktanella hongkongensis sp. nov., a novel member of the
242
α-Proteobacteria originating from marine biofilms in Hong Kong waters. Int J Syst
243
Evol Microbiol 54, 2281–2284.
244
Lee, S. D. (2012). Loktanella tamlensis sp. nov., isolated from seawater. Int J Syst
245
Evol Microbiol 62, 586–590.
246
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement
247
of the G+C content of deoxyribonucleic acid by highperformance liquid
248
chromatography. Int J Syst Bacteriol 39, 159–167
249
Minnikin, D., O'Donnell, A., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A.
250
& Parlett, J. (1984). An integrated procedure for the extraction of bacterial
251
isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
252
Montero-Calasanz, M. del C., Göker, M., Rohde, M., Spröer, C., Schumann, P.,
253
Busse, H.-J., Schmid, M., Tindall, B. J., Klenk, H.-P. & Camacho, M. (2013).
254
Chryseobacterium hispalense sp. nov., a plant-growth-promoting bacterium isolated
255
from a rainwater pond in an olive plant nursery, and emended descriptions of
256
Chryseobacterium
257
wanjuense and Chryseobacterium gregarium. Int J Syst Evol Microbiol 63,
258
4386–4395.
259
Moon, Y. G., Seo, S. H., Lee, S. D. & Heo, M. S. (2010). Loktanella pyoseonensis sp.
260
nov., isolated from beach sand, and emended description of the genus Loktanella. Int J
261
Syst Evol Microbiol 60, 785–789.
262
Moore, E. R. B., Arnscheidt, A., Krüger, A., Strömpl, C. & Mau, M. (1999).
263
Simplified protocols for the preparation of genomic DNA from bacterial cultures. In
defluvii,
Chryseobacterium
indologenes,
Chryseobacterium
264
Molecular Microbial Ecology Manual 1.6.1, pp. 1–15. Edited by A. D. L. Akkermans,
265
J. D. van Elsas & F. J. de Bruijn. Dordrecht: Kluwer.
266
Ostle A.G., Holt J.G. (1982). Nile Blue A as a fluorescent stain for poly
267
β-hydroxybutirate. Appl Environ Microbiol 44, 238–241.
268
Park, S., Jung, Y.-T. & Yoon, J.-H. (2013). Loktanella sediminilitoris sp. nov.,
269
isolated from tidal flat sediment. Int J Syst Evol Microbiol 63, 4118–4123.
270
Park, S., Lee, J.-S., Lee, K.-C. & Yoon, J.-H. (2013). Loktanella soesokkakensis sp.
271
nov., isolated from the junction between the North Pacific Ocean and a freshwater
272
spring. Antonie van Leeuwenhoek 104, 397–404.
273
Raina J.-B., Tapiolas D., Willis B.L., Bourne D.G. (2009). Coral-associated bacteria
274
and their role in the biogeochemical cycling of sulfur. Applied and Environmental
275
Microbiology 75:3492-3501.
276
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty
277
acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
278
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA–DNA
279
reassociation and 16S rRNA sequence analysis in the present species definition in
280
bacteriology. 44, 846–849.
281
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011).
282
MEGA5: molecular evolutionary genetics analysis using maximum likelihood,
283
evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28,
284
2731–2739.
285
Tanaka, N., Romanenko, L. A., Kurilenko, V. V., Svetashev, V. I., Kalinovskaya,
286
N. I., and Mikhailov, V. V. (2014). Loktanella maritima sp. nov. isolated from
287
shallow sediments of the Sea of Japan. Int J Syst Evol Microbiol 64, 2370–2375.
288
Teather, R. M. & Wood, P. J. (1982). Use of Congo red-polysaccharide interactions
289
in enumeration and characterization of cellulolytic bacteria from the bovine rumen.
290
Appl Environ Microbiol 43, 777–780.
291
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.
292
(1997). The CLUSTAL_X windows interface: flexible strategies for multiple
293
sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882.
294
Tindall, B., Sikorski, J., Smibert, R. & Krieg, N. (2007). Phenotypic
295
characterization and the principles of comparative systematics. In Methods for
296
General and Molecular Microbiology 3, 330–393. Edited by C. A. Reddy, T. J.
297
Beveridge, J. A. Breznak, G. Marzluf, T. M. Schmidt, & L. R. Snyder (ed.) ASM Press,
298
Washington DC.
299
Tsubouchi, T., Shimane, Y., Mori, K., Miyazaki, M., Tame, A., Uematsu, K.,
300
Maruyama, T. & Hatada, Y. (2013). Loktanella cinnabarina sp. nov., isolated from a
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deep subseafloor sediment, and emended description of the genus Loktanella. Int J
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Syst Evol Microbiol 63, 1390–1395.
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Van Trappen, S., Mergaert, J. & Swings, J. (2004). Loktanella salsilacus gen. nov.,
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sp. nov., Loktanella fryxellensis sp. nov. and Loktanella vestfoldensis sp. nov., new
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members of the Rhodobacter group, isolated from microbial mats in Antarctic lakes.
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Int J Syst Evol Microbiol 54, 1263–1269.
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Weon, H.-Y., Kim, B.-Y., Yoo, S.-H., Kim, J.-S., Kwon, S.-W., Go, S.-J. &
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Stackebrandt, E. (2006). Loktanella koreensis sp. nov., isolated from sea sand in
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Korea. Int J Syst Evol Microbiol 56, 2199–2202.
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Xie, C.-H. & Yokota, A. (2003). Phylogenetic analyses of Lampropedia hyalina
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based on the 16S rRNA gene sequence. J Gen Appl Microbiol 49, 345–349.
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Yoon, J.-H., Jung, Y.-T. & Lee, J.-S. (2013). Loktanella litorea sp. nov., isolated
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from seawater. Int J Syst Evol Microbiol 63, 175–180.
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Yoon, J.-H., Kang, S.-J., Lee, S.-Y. & Oh, T.-K. (2007). Loktanella maricola sp.
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nov., isolated from seawater of the East Sea in Korea. Int J Syst Evol Microbiol 57,
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1799–1802.
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Yu, T., Yin, Q.,Song, X., Zhao. R.,Shi, X. & Zhang, X.-H (2012). Aquimarina longa
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sp. nov., isolated from the seawater of South Pacific Gyre. Int J Syst Evol Microbiol
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63, 1235–1240.
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Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences
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showing the phylogenetic position of strain S3B03T, the type strains of other species
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of the genus Loktanella and representatives of some other related taxa. Bootstrap
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percentages (>70 %) based on 1000 replicates are shown at branch points. Stappia
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stellulata DSM 5886T (AUIM01000013) was used as an outgroup. Bar, 0.01
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substitution per nucleotide position.
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Fig. 2. Transmission electron micrographs of negatively stained cells of Loktanella
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sediminum S3B03T cultured on MA at 28 ºC for 24 h. Bar, 0.1 μm. a: a large cell; b: a
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small cell.
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Table 1. Differential characteristics between strain S3B03T and the type strains of
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phylogenetically related species of the genus Loktanella
331 332 333
Strains: 1. S3B03T 2. Loktanella litorea KCTC 23883T; 3. Loktanella salsilacus LMG 21507T. All data from this study unless otherwise indicated. +, positive reaction; -, negative reaction; w, weakly positive. 1 2 3* Characteristics Colony colour (on MA) light beige-pink light orange–yellow beige Growth at/in 10 % NaCl + 10 °C + + 40 °C + Hydrolysis of DNA + Gelatin + Alginate + -† Tween 20 + +† Tween 40 +† Tween 80 + Citrate utilization (API 20E) ‡ + Enzymes activity (API ZYM) ‡ Leucine arylamidase + + α-Galactosidase + β-galactosidase + α-Glucosidase w β-Glucosidase w w
（
Acid production from API 50CH) ‡
334 335 336
L-Arabinose w + D-Glucose + D-Mannitol + Melibiose + + DNA G+C content (mol%)‡ 57.6 57.6 T *All data for L. salsilacus LMG 21507 taken from Van Trappen et al.(2004). † Data not reported in Van Trappen et al.(2004). ‡ Data for reference strains were taken from Yoon et al.(2013).
60.4
337 338 339 340 341 342
343 344 345 346 347 348 349 350
Table 2. Cellular fatty acid contents (%) of strain S3B03T and the type strains of phylogenetically closely related species Strains: 1. S3B03T; 2. Loktanella litorea KCTC 23883T; 3. Loktanella salsilacus LMG 21507T. All data are from this study. Only fatty acids more than 1 % of the total fatty acids of at least one of the strains are shown. Tr, trace amount (< 1 % of total); -, not detected. Fatty acid 1 2 3 Straight chain C16:0 5.29 11 18.54 C17:0 1.27 C18:0 4.38 3.37 2.57 Branched anteiso-C15:0 1.54 anteiso-C16:0 Tr anteiso-C17:0 1.69 1.39 1.05 Hydroxy C10:0 3-OH 1.18 5.38 1.43 C12:1 3-OH 2.52 2.87 C16:0 2-OH 1.01 1.07 0.8 C18:1 2-OH 1.7 Unsaturated C20:1ω7c 1.81 11-methyl C18:1ω7c 2.26 Tr 7.34 C16:0 N alcohol 1.47 1.71 1.44 Summed feature 2† Tr Tr Summed feature 3† 1.23 2.8 Summed feature 7† 3.08 1.76 1.97 Summed feature 8† 64.2 65.35 63.76 † As indicated by Montero-Calasanz et al.(2013) summed features are groups of two or three fatty acids that are treated together for the purpose of evaluation in the MIDI system and include both peaks with discrete equivalent chain lengths (ECLs) as well as those where the ECLs are not reported separately. Summed feature 2 was listed as C12:0 aldehyde, an unknown fatty acid (ECL 10.928), C14:0 3-OH and/or iso-C16:1 I. Summed feature 3 was listed as C16:1ω7c and/or C16:1ω6c. Summed feature 7 was listed as an unknown fatty acid (ECL 18.846), C19:1ω6c and/or C19:0 ω10c cyclo. Summed feature 8 was listed as C18:1ω7c and/or C18:1ω6c.
