IJSEM Papers in Press. Published April 17, 2014 as doi:10.1099/ijs.0.061747-0
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Loktanella maritima sp. nov. isolated from shallow sediments of the Sea of Japan.
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Naoto Tanaka, 1 Lyudmila A. Romanenko, 2 Valeriya V. Kurilenko, 2 Vassilii I. Svetashev, 3
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Natalia I. Kalinovskaya 2, and Valery V. Mikhailov 2.
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Setagaya-ku, Tokyo 156-8502, Japan
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of Sciences, Prospect 100 Let Vladivostoku, 159, Vladivostok 690022, Russia
NODAI Culture Collection Center, Tokyo University of Agriculture, 1-1-1 Sakuragaoka,
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy
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690041, Russia
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Author for correspondence: Naoto Tanaka. Tel: +81 3 5477 2549. Fax: +81 3 5477 2537.
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E-mail: [email protected]
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Subjective category: Proteobacteria
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Running title: Description of Loktanella maritima sp. nov., marine shallow sediments.
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The DDBJ/GenBank/EMBL accession number of the 16S rRNA gene sequence of strain KMM
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9530T is AB894236.
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Abbreviation: Bchl a, bacteriochlorophyll a.
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Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok
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An aerobic, Gram-negative, non-motile bacterium KMM 9530T was isolated from a sediment
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sample collected from the Sea of Japan seashore. Comparative 16S rRNA gene sequence
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analysis positioned novel strain KMM 9530T to the genus Loktanella as a separate line
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adjacent to Loktanella sediminilitoris KCTC 32383T, Loktanella tamlensis JCM 14020T and
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Loktanella maricola JCM 14564T with 98.5-98.2% sequence similarity. Strain KMM 9530T
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was characterized by the weak hydrolytic capacity and the inability to assimilate most
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organic substrates. The major isoprenoid quinone was Q-10, polar lipids consisted of
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phosphatidylcholine,
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phospholipid, an unknown aminolipid, and unknown lipids, and major fatty acid was
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C18:1ω7c. On the basis of phylogenetic analysis, DNA-DNA hybridization and phenotypic
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characterization, it can be concluded that novel strain KMM 9530T represents a novel species
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in the genus Loktanella, for which the name Loktanella maritima sp. nov. is proposed. The
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type strain of the species is strain KMM 9530T (=NRIC 0919T = JCM 19807T).
phosphatidylglycerol,
diphosphatidylglycerol,
an
unknown
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The genus Loktanella was initially proposed by Van Trappen et al. (2004) to accommodate three
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species, Loktanella salsilacus, as the type species of the genus, Loktanella vestfoldensis and
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Loktanella fryxellensis, and subsequently emended by Moon et al. (2010), Lee (2012), and
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Tsubouchi et al. (2013). The genus Loktanella was expanded to twelve more species, including L.
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hongkongensis (Lau et al., 2004), L. agnita, L.rosea (Ivanova et al., 2005), L. koreensis (Weon et
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al., 2006), L. atrilutea (Hosoya & Yokota, 2007), L. maricola (Yoon et al., 2007), L.
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pyoseonensis (Moon et al., 2010), L. tamlensis (Lee, 2012), L. cinnabarina (Tsubouchi et al.,
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2013), L. litorea (Yoon et al., 2013), L. sediminilitoris (Park et al., 2013a) and L. soesokkakensis
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(Park et al., 2013b). Here we report the polyphasic characterization of a Gram-negative, aerobic,
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beige-pigmented, non-motile bacterium, designated KMM 9530T, which was isolated from a
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shallow sediment sample. Phylogenetic analysis based on 16S rRNA gene sequence showed that
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strain KMM 9530T belongs to the genus Loktanella and may represent a novel species of this
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genus. Differential phenotypic properties, together with the phylogenetic distinctiveness and
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DNA relatedness demonstrated that strain KMM 9530T differed from related Loktanella species.
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On the basis of phenotypic and molecular data obtained, a novel species, Loktanella maritima, is
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described.
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Strain KMM 9530T was isolated from a shallow sediment sample, collected from Peter the Great
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Bay, the Sea of Japan, Russia, as described previously (Romanenko et al., 2004). Strain KMM
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9530T was grown aerobically on/in marine 2216 agar (MA) or marine broth (MB, BD Difco),
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and stored at –80 °C in MB supplemented with 30% (v/v) glycerol. The type strains, Loktanella
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sediminilitoris KCTC 32383T, Loktanella maricola JCM 14564T, Loktanella tamlensis JCM
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14020T, and Loktanella rosea KMM 6003T were kindly provided by the respective Culture
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Collections and used in the phenotypic and lipids analyses. Gram-staining, oxidase and catalase
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reactions, and motility (the hanging drop method) were tested as described by Gerhardt et al.
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(1994). The morphology of cells negatively stained with a 1% phosphotungstic acid was
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examined by electronic transmission microscopy [Libra 200 FE (Carl Zeiss), provided by the
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Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences] using carbon-
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coated 200-mesh copper grids. Nitrate reduction was determined for strains grown in nitrate
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broth supplemented with artificial sea water (ASW) (sulfanilic acid/α-naphthylamine test).
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Formation of H2S from thiosulfate was tested on the ASW-based medium using a lead acetate
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paper strip. Hydrolysis of L-tyrosine, chitin, xanthine and hypoxanthine was investigated by
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observing transparent zones on MA supplemented with a compound at a concentration of 1%
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each. Hydrolysis of DNA was examined using DNase Test Agar (BD BBLTM) prepared with
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ASW. Citrate utilization was tested on Simmons citrate agar (HiMedia laboratories Pvt. Ltd)
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supplemented with ASW. Artificial sea water (ASW) was prepared according to formula of
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Bruns et al. (2001) excepting 30 g l-1 NaCl. Degradation of starch (0.2%, w/v) and Tween 80
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(1%, w/v) was tested with ASW-based basal medium, containing 5 g l-1 Bactopeptone, 1 g l-1
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yeast extract, 0.1 g l-1 K2HPO4, 15 g l-1 agar. Hydrolysis of gelatin (0.4%, w/v) and casein (10%
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skim milk, w/v, BD Difco TM) was examined on ASW-based agar medium. Requirement for and
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tolerance of sodium chloride was tested on ASW-based medium using various concentrations of
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NaCl in the range 0-20%, supplemented with (per litre): 10.0 g Bacto peptone, 2.0 g yeast extract,
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0.028 g FeSO4, and 15.0 g agar. Biochemical tests were carried out using API 20NE, API 20E,
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API ID32 GN and API ZYM test kits (bioMérieux, France) as described by the manufacturer
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except that the cultures were suspended in ASW. Growth at different temperatures of 4, 7, 15, 28,
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30, and from 32 to 40 °C in increments of 1 °C. Antibiotic susceptibility was examined using
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commercial paper discs impregnated with the following antibiotics (μg per disc, unless otherwise
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indicated): ampicillin (10), benzylpenicillin (10 U), vancomycin (30), gentamicin (10),
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kanamycin (30), carbenicillin (100), chloramphenicol (30), neomycin (30), oxacillin (10),
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oleandomycin (15), ofloxacin (5), rifampicin (5), polymyxin (300 U), streptomycin (30),
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cephazolin (30), cephalexin (30), erythromycin (15), nalidixic acid (30), tetracycline (30) and
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doxocycline (10g). The pH range for growth was determined in MB that was adjusted to pH 5.0-
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11.5 (in increments of 0.5 pH unit) by the addition of 1 M HCl or 1 M NaOH. For polar lipid and
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fatty acid analyses, strains KMM 9530T, L. sediminilitoris KCTC 32383T, L. maricola JCM
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14564T, L. tamlensis JCM 14020T, and L. rosea KMM 6003T were grown on MA at 28 °C for
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three days. Lipids were extracted using the method of Folch et al. (1957). Two-dimensional thin
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layer chromatography of polar lipids was carried out on Silica gel 60 F254 (10 x 10 cm, Merck,
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Germany) using chloroform-methanol-water (65:25:4, v/v) for the first direction, and
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chloroform-methanol-acetic acid-water (80:12:15:4, v/v) for the second one (Collins & Shah,
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1984). Nonspecific detection of lipids on the two-dimensional TLC was conducted with 10%
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H2SO4 in methanol at 120 ° C. Amino-containing lipids were determined with ninhydrin,
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phospholipids with molybdate reagent, glycolipids with alpha-naphtol, and choline lipids with
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Dragendorff’s reagent. Respiratory lipoquinones were analyzed by reversed-phase high 4
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performance thin-layer chromatography as described by Mitchell & Fallon (1990). Fatty acid
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methyl esters (FAMEs) were prepared according to the procedure of the Microbial Identification
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System (MIDI) (Sasser, 1990). The analysis of FAMEs was performed as described previously
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(Romanenko et al., 2011a). Production of Bchl α was spectrophotometrically tested in
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methanolic extracts of cells grown on MA and MB in the dark as described by Lafay et al.
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(1995). The 16S rRNA gene sequence (1447 nt) of strain KMM 9530T was determined as
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described by Shida et al. (1997). The sequence obtained was compared with 16S rRNA gene
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sequences retrieved from the EMBL/GenBank/DDBJ databases by using the FASTA program
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(Pearson & Lipman, 1988). Phylogenetic analysis of 16S rRNA gene sequences was performed
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using the software package MEGA 5 (Tamura et al., 2011) after multiple alignment of data by
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CLUSTALW (version 1.83; Thompson et al., 2002). Phylogenetic trees were constructed by the
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neighbor-joining, the maximum-likelihood and maximum-parsimony methods and the distances
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were calculated according to the Kimura two-parameter model. The robustness of phylogenetic
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trees was estimated by the bootstrap analysis of 1000 replicates. The DNA-DNA hybridization
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experiments were performed by the photobiotin-labelled DNA probe microplate method of Ezaki
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et al. (1989). The DNA-DNA hybridization values are given as average means of at least two
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determination experiments.
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Comparative 16S rRNA gene sequence analysis established that strain KMM 9530T belonged to
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the genus Loktanella displaying the highest sequence similarity values of 98.5%, 98.4% and
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98.2% to its closely related Loktanella species, L. sediminilitoris KCTC 32383T, L. tamlensis
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JCM 14020T and L. maricola JCM 14564T, respectively, and similarity values below 97.7% to
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the remaining Loktanella species (Fig. 1). The 16S rRNA gene sequence similarities obtained for
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strain KMM 9530T and most Loktanella species were lower than the threshold similarity value of
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97% proposed by Stackebrandt & Goebel (1994) and re-evaluated to 98.7% by Stackebrandt &
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Ebers (2006), indicating that novel strain could be assigned to the genus Loktanella as an
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individual species. The DNA-DNA hybridization experiments were carried out between strain
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KMM 9530T and type strains, L. sediminilitoris KCTC 32383T, L. tamlensis JCM 14020T and L.
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maricola JCM 14564T and DNA relatedness values were measured to be 22%, 45% and 36%,
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respectively. The DNA-DNA hybridization values confirmed an assignment of the novel isolate
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KMM 9530T to the separate species according to the value of 70 % proposed by Wayne et al.
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(1987) for the bacterial species delineation. Cells of strain KMM 9530T were aerobic, Gram
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negative, non-motile ovoid or short rod-shaped (Supplementary figure S1). Cultural,
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physiological and metabolic properties of strain KMM 9530T and related members of the genus
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Loktanella are listed in Table 1 and in the species description. Fatty acid profiles were similar
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with a large proportion of C18:1ω7c and the presence of 11-methyl C18:1ω7c found in all strains
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tested (Table 2). These results are in accordance with the data reported for L. rosea KMM 6003T
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(Ivanova et al., 2005) and L. maricola JCM 14564T (Yoon et al., 2007), but disagreed with the
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results of Lee (2012) and Park et al. (2013) who did not find 11-methyl C18:1ω7c in L. tamlensis
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JCM 14020T and L. sediminilitoris KCTC 32383T. Strains KMM 9530T and L. tamlensis JCM
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14020T differed by a small proportion of 11-methyl C18:1ω7c, and only L. sediminilitoris KCTC
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32383T contained C12:1 compared with other related Loktanella strains (Table 2). The major
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isoprenoid quinone of strain KMM 9530T was ubiquinone Q-10. The polar lipid composition of
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strain KMM 9530T and related L. tamlensis JCM 14020T, L. rosea KMM 6003T, L. maricola
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JCM 14564T, L. sediminilitoris KCTC 32383T was found to be similar and included
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phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unknown aminolipid,
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unknown phospholipids, and unknown lipids. All strains tested contained minor amounts of DPG
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(Supplementary figure S2). The polar profile of L. sediminilitoris KCTC 32383T differed from
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those of four strains studied in that phosphatidylethanolamine was detected. This finding is
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congruent with the original description of Park et al. (2013) who reported the presence of
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phosphatidylcholine,
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aminolipid as dominant and diphosphatidylglycerol, an unknown phospholipid and an unknown
phosphatidylglycerol,
phosphatidylethanolamine
and
an
unknown
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lipid as minor components in L. sediminilitoris KCTC 32383T. In addition, unknown
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phospholipids PL1-PL3 were found in present study. The presence of phosphatidylethanolamine
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in L. sediminilitoris KCTC 32383T and its absence in KMM 9530T and three related Loktanella
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strains was confirmed by two-dimensional TCLs spraying with ninhydrin followed by staining
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with molybdate reagent as exemplified by strains KMM 9530T, L. maricola JCM 14564T and L.
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sediminilitoris KCTC 32383T. (Supplementary figure S2). Unlike our results with regard to the
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lack of PE in three above type strains, the occurrence of PE has been reported by Lee (2012),
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Yoon et al. (2007), and Ivanova et al. (2005) in L. tamlensis JCM 14020T, L. maricola JCM
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14564T, and L. rosea KMM 6003T, with minor and trace amounts for two latter. The genus
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Loktanella comprises both bacteria having PE together with PC and PG, L. litorea (Yoon et al.,
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2013), L. agnita (Ivanova et al., 2005), L. sediminilitoris KCTC 32383T (Park et al., 2013a), or L.
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cinnabarina (Tsubouchi et al., 2013), and bacteria not containing PE in their polar lipid profiles,
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L. pyoseonensis (Moon et al., 2010), L. hongkongensis (Tsubouchi et al., 2013), or L.
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soesokkakensis (Park et al., 2013b). At the same time Yoon et al. (2013) reported the presence of
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PE in L. salcilacus whereas PE was not found in this bacterium in the study of Tsubouchi et al.
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(2013). As reported by Tsubouchi et al. (2013) and Park et al. (2013b) that the same L.
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cinnabarina cluster includes bacteria containing PE (L. cinnabarina itself) and bacteria L.
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pyoseonensis, L. hongkongensis and L. soesokkakensis without PE. Chemotaxonomic properties
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(ubiquinone Q-10, the predominance of C18:1ω7c, and the presence of phosphatidylcholine and
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phosphatidylglycerol) obtained for the strain KMM 9530T supported its assignment to the genus
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Loktanella. It is evident from the results obtained that the isolate can be assigned to the genus
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Loktanella on the basis of its physiological, biochemical and chemotaxonomic characteristics. It
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is interesting to note that Loktanella type strains revealed similar antibiotic resistance profiles,
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being susceptible to 19 or 20 of 21 antibiotics applied, excepting strains KMM 9530T and L.
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sediminilitoris KCTC 32383T. Recently we have reported marine alphaproteobacteria showing a
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high sensitivity to antibiotics (Romanenko et al., 2011a, b). Strain KMM 9530T could be
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distinguished from related Loktanella type strains in colony pigmentation (excepting L. tamlensis
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JCM 14020T), in being able to grow in 7-8% NaCl (excepting L. rosea KMM 6003T), in being
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able to utilize citrate, as well as in enzyme activities in API ZYM tests and antibiotic sensitivity
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profile. It should be noted that recently described L. sediminilitoris KCTC 32383T revealed a
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number of distinctive traits compared with related Loktanella type strains tested including
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hydrolysis of Tween-80 and DNA and API ZYM enzyme activity profile. Differential
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phenotypic characteristics are listed in Table 1. Based on the results obtained it is proposed to
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assign a strain KMM 9530T to the genus Loktanella as representing novel species, Loktanella
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maritima sp. nov.
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Description of Loktanella maritima sp. nov.
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Loktanella maritima (ma.ri’ti.ma. L. fem. adj. maritima maritime, marine).
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Gram-negative, aerobic, oxidase-positive, catalase-positive, ovoid or short rod-shaped non-
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motile cells, 0.6-0.8 μm in diameter and 1.6-2.0 μm in length. Grew on MA 2216 and MB.
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Produced light beige-pigmented, shiny smooth colonies with the regular edges of 2-3 mm in
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diameter. No growth observed on commercial media tryptic soy agar or broth, nutrient agar and
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R2A agar. Bchl a is not produced. Required NaCl for growth; growth occurred between 2 and
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8% (w/v) NaCl with an optimum of 3-4 % NaCl; growth is weak with 2 and 8% NaCl. The
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temperature range for growth was 4-36 °C with an optimum of 28-30 °C; growth is weak at
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36 °C. The pH range for growth was 5.5-9.0 (optimal 6.5-7.5). Negative for hydrolysis of casein,
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DNA, chitin, starch, Tween 80, L-tyrosine and xanthine, and production of H2S in conventional
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tests. Negative for gelatin hydrolysis and nitrate reduction in routine and API 20NE tests.
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Positive for aesculin hydrolysis (API 20NE), PNPG test (β-galactosidase in API 20NE and API
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20E tests) and citrate utilization (API 20E); and negative for the remaining tests, that are
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included to the API 20NE and API 20E panels. According to the ID32 GN, strain could not
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assimilate any of substrates included to the ID32 GN gallery. Positive API ZYM test results are
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obtained for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid
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phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucosidase; weakly-positive for valine
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arylamidase and α-glucosidase; and negative for lipase (C14), cystine arylamidase, trypsin, α-
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chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-
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mannosidase and α-fucosidase. The major isoprenoid quinone is ubiquinone Q-10. The polar
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lipids included phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unknown
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aminolipid, an unknown phospholipid, and four unknown lipids. Fatty acid C18:1ω7c was
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predominant. Susceptible to ampicillin, benzylpenicillin, vancomycin, gentamicin, kanamycin,
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carbenicillin, chloramphenicol, neomycin, oxacillin, oleandomycin, ofloxacin, rifampicin,
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streptomycin, cephazolin, cephalexin and erythromycin; and resistant to lincomycin, polymyxin,
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nalidixic acid, tetracycline, and doxocycline.
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The type strain of the species is strain KMM 9530T (=NRIC 0919T =JCM 19807T) isolated from
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a shallow sediment sample, collected from Peter the Great Bay, the Sea of Japan, Russia,
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Van Trappen, S., Mergaert, J. & Swings, J. (2004). Loktanella salsilacus gen. nov., sp. nov.,
308
Loktanella fryxellensis sp. nov. and Loktanella vestfoldensis sp. nov., new members of the
309
Rhodobacter group, isolated from microbial mats in Antarctic lakes. Int J Syst Evol Microbiol 54,
310
1263-1269.
311
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky,
312
M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E., Stackebrandt, E., Starr, M. P. &
12
313
Trüper, H. G. (1987). Report of the ad hoc committee on reconciliation of approaches to
314
bacterial systematics. Int J Syst Bacteriol 37, 463-464.
315
Weon, H.Y., Kim, B.Y., Yoo, S.H., Kim, J.S., Kwon, S.W., Go, S.J. & Stackebrandt, E.
316
(2006). Loktanella koreensis sp. nov., isolated from sea sand in Korea. Int J Syst Evol Microbiol
317
56, 2199-2202.
318
Yoon, J.H., Kang, S.J., Lee, S.Y. & Oh, T.K. (2007). Loktanella maricola sp. nov., isolated
319
from seawater of the East Sea in Korea. Int J Syst Evol Microbiol 57, 1799-1802.
320
Yoon, J.H., Jung, Y.T. & Lee, J.S. (2013). Loktanella litorea sp. nov., isolated from seawater.
321
Int J Syst Evol Microbiol 63, 175-180.
322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338
13
339
Legend of Figures
340
Fig.1. Neighbor-joining tree based on 16S rRNA gene sequences showing relationships of the
341
isolate KMM 9530T and related taxa. Filled circles, generic branches that are present in
342
phylogenetic trees generated by the neighbor-joining, the maximum-likelihood and the
343
maximum-parsimony methods; filled triangles, generic branches that are present in trees
344
generated by the neighbor-joining and the maximum parsimony methods; filled squares, generic
345
branches that are present in trees generated by the neighbor-joining and the maximum likelihood
346
methods. Numbers indicate bootstrap values as percentage greater than 60 (neighbor-joining
347
probability/maximum-parsimony probability/maximum-likelihood probability). These values are
348
based on 1000 replicates. Bar, 0.01 substitutions per nucleotide position.
349 350
Supplementary Figure 1. Transmission electron micrograph of strain KMM 9530T grown in MA
351
for 24 h. Bar, 500 nm.
352 353
Supplementary Figure S2. Two-dimensional thin-layer chromatograms of polar lipids of
354
strains: (a, b, c) Loktanella maritima sp. nov. KMM 9530T; (d, e, f) Loktanella maricola JCM
355
14564T; (g, h, i) Loktanella sediminilitoris KCTC 32383T; (j) Loktanella tamlensis JCM 14020T;
356
(k) Loktanella rosea KMM 6003T. (a, d, g, j, k) non-specific detection of lipids prepared with
357
10% H2SO4 in methanol; (b, e, h) stained with ninhydrin; (c, f, i) stained with molybdate reagent.
358
Abbreviations:
359
diphosphatidylglycerol; PE, phosphatidylethanolamine; PL, PL1, PL2, PL3, unknown
360
phospholipids; AL, an unknown aminolipid; L1, L2, L3, L4, L5, unknown lipids.
PC,
phosphatidylcholine;
PG,
phosphatidylglycerol;
DPG,
361 362 363
14
364
Table 1. Differential phenotypic characteristics of strain KMM 9530T and type strains of related
365
Loktanella species.
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381
Strains: 1, Loktanella maritima sp. nov. KMM 9530T; 2, Loktanella tamlensis JCM 14020T; 3, Loktanella maricola JCM 14564T; 4, Loktanella rosea KMM 6003T; 5, Loktanella sediminilitoris KCTC 32383T. All data were obtained from the present study. +, Positive; -, negative; (+), weak reaction. All strains are positive for oxidase, catalase, sodium ions requirement for growth, aesculin hydrolysis and PNPG test (β-galactosidase) (in API 20NE), production of esterase C4, esterase lipase C8, and negative for gelatin hydrolysis and nitrate reduction (in routine and API 20NE tests), hydrolysis of casein, starch*, chitin, xanthine, production of lipase C14, cystine arylamidase, α-chymotrypsin, α-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, α-mannosidase, α-fucosidase (in API ZYM), arginine dihydrolase, urease, glucose fermentation, indol production, assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, D-gluconate, caprate, adipate, L-malate, citrate and phenylacetate (in API 20NE), and are susceptible to ampicillin, benzylpenicillin, vancomycin, gentamicin, kanamycin, carbenicillin, chloramphenicol, streptomycin, oleandomycin, ofloxacin, neomycin, oxacillin, rifampicin, cephazolin, cephalexin, erythromycin, and resistant to polymyxin§. Characteristic
1
2
3
4
5
Colony pigmentation
Beige
Beige
Dark-pink†
Pink
Greyish-yellow
Motility
-
+
-
+
-
Temperature range for
4-36
4-34*
4-34
4-39#
4-35§
2-8
2-4*
3-6†
2-8#
2-5§
Tyrosine
-
-
-
+
-
Hypoxanthine
(+)
+
+
+
-
Tween-80
-
-
-
-
+
DNA
-
-
-
-
+
Citrate utilization
+
-
-
-
-
-
-
-
-
+
Alkaline phosphatase
+
+
+
(+)
+
Leucine arylamidase
+
+
+
+
-
Valine arylamidase
(+)
-
-
-
-
Trypsin
-
-
-
-
+
Acid phosphatase
+
+
+
(+)
-
Naphthol-AS-BI-
+
+
+
+
(+)§
growth (ºC) NaCl range for growth (%) Hydrolysis of:
(Simmons citrate agar) H2S formation API ZYM tests:
15
phosphohydrolase α-glucosidase
(+)
-
-
-
-
β-glucosidase
+
-
-
-
-
Lincomycin (15)
-
+
+
-
-
Nalidixic acid (30)
-
+
+
+
-
Tetracycline (30)
-
+
+
+
+
Doxocycline (10)
-
+
+
+
+
Sensitivity to antibiotics (μg/disc):
382 383
*
Results differ from reported by Lee (2012).
384
†
Data not consistent with those reported by Yoon et al. (2007). Colony pigmentation for L.
385
maricola JCM 14564T was described by Yoon et al. (2007) as light orange.
386
#
Results differ from reported by Ivanova et al. (2005).
387
§
Results differ from reported by Park et al. (2013).
388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 16
407 408
Table 2. Fatty acid (%) profiles of strain KMM 9530T and related members of the genus
409
Loktanella.
410
Strains: 1, Loktanella maritima sp. nov. KMM 9530T; 2, Loktanella tamlensis JCM 14020T; 3,
411
Loktanella maricola JCM 14564T; 4, Loktanella rosea KMM 6003T; 5, Loktanella
412
sediminilitoris KCTC 32383T. The results were obtained from present study. All strains were
413
grown on MA at 28 °C for three days. -, Not detected; tr, - trace amount.
414 Fatty acid
1
2
3
4
5
C10:03-ОН
3.09
3.29
3.43
1.09
1.48
C12:13-ОН
2.45
1.95
4.37
3.53
3.37
-
-
-
tr
5.29
C16:1ω7c
0.49
1.92
0.39
1.09
1.12
C16:0
5.76
6.98
5.34
6.32
12.45
C17:1ω8c
1.07
-
-
0.26
-
C17:0
2.28
0.55
0.50
0.89
0.39
C18:2
3.40
1.65
1.45
3.69
4.69
C18:1ω7c
76.98
75.21
68.62
68.02
61.96
11-Methyl C18:1ω7c
2.62
4.56
9.70
11.47
6.78
C18:0
1.40
1.25
2.20
1.96
3.95
C12:1
415 416 417 418 419 420 421 17
422
Figure 1.
423
424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 18
441
Supplementary Figure S1.
442
443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 19
465
Supplementary Figure S2.
466
467 468
469 470
471 472
473 474 475 476 477 478
20
1
Loktanella maritima sp. nov. isolated from shallow sediments of the Sea of Japan.
2 3
Naoto Tanaka, 1 Lyudmila A. Romanenko, 2 Valeriya V. Kurilenko, 2 Vassilii I. Svetashev, 3
4
Natalia I. Kalinovskaya 2, and Valery V. Mikhailov 2.
5 6
1
7
Setagaya-ku, Tokyo 156-8502, Japan
8
2
9
of Sciences, Prospect 100 Let Vladivostoku, 159, Vladivostok 690022, Russia
NODAI Culture Collection Center, Tokyo University of Agriculture, 1-1-1 Sakuragaoka,
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy
10
3
11
690041, Russia
12
Author for correspondence: Naoto Tanaka. Tel: +81 3 5477 2549. Fax: +81 3 5477 2537.
13
E-mail: [email protected]
14
Subjective category: Proteobacteria
15
Running title: Description of Loktanella maritima sp. nov., marine shallow sediments.
16
The DDBJ/GenBank/EMBL accession number of the 16S rRNA gene sequence of strain KMM
17
9530T is AB894236.
18
Abbreviation: Bchl a, bacteriochlorophyll a.
19 20 21 22 23 24 25 26
Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok
27
An aerobic, Gram-negative, non-motile bacterium KMM 9530T was isolated from a sediment
28
sample collected from the Sea of Japan seashore. Comparative 16S rRNA gene sequence
29
analysis positioned novel strain KMM 9530T to the genus Loktanella as a separate line
30
adjacent to Loktanella sediminilitoris KCTC 32383T, Loktanella tamlensis JCM 14020T and
31
Loktanella maricola JCM 14564T with 98.5-98.2% sequence similarity. Strain KMM 9530T
32
was characterized by the weak hydrolytic capacity and the inability to assimilate most
33
organic substrates. The major isoprenoid quinone was Q-10, polar lipids consisted of
34
phosphatidylcholine,
35
phospholipid, an unknown aminolipid, and unknown lipids, and major fatty acid was
36
C18:1ω7c. On the basis of phylogenetic analysis, DNA-DNA hybridization and phenotypic
37
characterization, it can be concluded that novel strain KMM 9530T represents a novel species
38
in the genus Loktanella, for which the name Loktanella maritima sp. nov. is proposed. The
39
type strain of the species is strain KMM 9530T (=NRIC 0919T = JCM 19807T).
phosphatidylglycerol,
diphosphatidylglycerol,
an
unknown
40 41 42
The genus Loktanella was initially proposed by Van Trappen et al. (2004) to accommodate three
43
species, Loktanella salsilacus, as the type species of the genus, Loktanella vestfoldensis and
44
Loktanella fryxellensis, and subsequently emended by Moon et al. (2010), Lee (2012), and
45
Tsubouchi et al. (2013). The genus Loktanella was expanded to twelve more species, including L.
46
hongkongensis (Lau et al., 2004), L. agnita, L.rosea (Ivanova et al., 2005), L. koreensis (Weon et
47
al., 2006), L. atrilutea (Hosoya & Yokota, 2007), L. maricola (Yoon et al., 2007), L.
48
pyoseonensis (Moon et al., 2010), L. tamlensis (Lee, 2012), L. cinnabarina (Tsubouchi et al.,
49
2013), L. litorea (Yoon et al., 2013), L. sediminilitoris (Park et al., 2013a) and L. soesokkakensis
50
(Park et al., 2013b). Here we report the polyphasic characterization of a Gram-negative, aerobic,
51
beige-pigmented, non-motile bacterium, designated KMM 9530T, which was isolated from a
52
shallow sediment sample. Phylogenetic analysis based on 16S rRNA gene sequence showed that
2
53
strain KMM 9530T belongs to the genus Loktanella and may represent a novel species of this
54
genus. Differential phenotypic properties, together with the phylogenetic distinctiveness and
55
DNA relatedness demonstrated that strain KMM 9530T differed from related Loktanella species.
56
On the basis of phenotypic and molecular data obtained, a novel species, Loktanella maritima, is
57
described.
58 59
Strain KMM 9530T was isolated from a shallow sediment sample, collected from Peter the Great
60
Bay, the Sea of Japan, Russia, as described previously (Romanenko et al., 2004). Strain KMM
61
9530T was grown aerobically on/in marine 2216 agar (MA) or marine broth (MB, BD Difco),
62
and stored at –80 °C in MB supplemented with 30% (v/v) glycerol. The type strains, Loktanella
63
sediminilitoris KCTC 32383T, Loktanella maricola JCM 14564T, Loktanella tamlensis JCM
64
14020T, and Loktanella rosea KMM 6003T were kindly provided by the respective Culture
65
Collections and used in the phenotypic and lipids analyses. Gram-staining, oxidase and catalase
66
reactions, and motility (the hanging drop method) were tested as described by Gerhardt et al.
67
(1994). The morphology of cells negatively stained with a 1% phosphotungstic acid was
68
examined by electronic transmission microscopy [Libra 200 FE (Carl Zeiss), provided by the
69
Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences] using carbon-
70
coated 200-mesh copper grids. Nitrate reduction was determined for strains grown in nitrate
71
broth supplemented with artificial sea water (ASW) (sulfanilic acid/α-naphthylamine test).
72
Formation of H2S from thiosulfate was tested on the ASW-based medium using a lead acetate
73
paper strip. Hydrolysis of L-tyrosine, chitin, xanthine and hypoxanthine was investigated by
74
observing transparent zones on MA supplemented with a compound at a concentration of 1%
75
each. Hydrolysis of DNA was examined using DNase Test Agar (BD BBLTM) prepared with
76
ASW. Citrate utilization was tested on Simmons citrate agar (HiMedia laboratories Pvt. Ltd)
77
supplemented with ASW. Artificial sea water (ASW) was prepared according to formula of
78
Bruns et al. (2001) excepting 30 g l-1 NaCl. Degradation of starch (0.2%, w/v) and Tween 80
3
79
(1%, w/v) was tested with ASW-based basal medium, containing 5 g l-1 Bactopeptone, 1 g l-1
80
yeast extract, 0.1 g l-1 K2HPO4, 15 g l-1 agar. Hydrolysis of gelatin (0.4%, w/v) and casein (10%
81
skim milk, w/v, BD Difco TM) was examined on ASW-based agar medium. Requirement for and
82
tolerance of sodium chloride was tested on ASW-based medium using various concentrations of
83
NaCl in the range 0-20%, supplemented with (per litre): 10.0 g Bacto peptone, 2.0 g yeast extract,
84
0.028 g FeSO4, and 15.0 g agar. Biochemical tests were carried out using API 20NE, API 20E,
85
API ID32 GN and API ZYM test kits (bioMérieux, France) as described by the manufacturer
86
except that the cultures were suspended in ASW. Growth at different temperatures of 4, 7, 15, 28,
87
30, and from 32 to 40 °C in increments of 1 °C. Antibiotic susceptibility was examined using
88
commercial paper discs impregnated with the following antibiotics (μg per disc, unless otherwise
89
indicated): ampicillin (10), benzylpenicillin (10 U), vancomycin (30), gentamicin (10),
90
kanamycin (30), carbenicillin (100), chloramphenicol (30), neomycin (30), oxacillin (10),
91
oleandomycin (15), ofloxacin (5), rifampicin (5), polymyxin (300 U), streptomycin (30),
92
cephazolin (30), cephalexin (30), erythromycin (15), nalidixic acid (30), tetracycline (30) and
93
doxocycline (10g). The pH range for growth was determined in MB that was adjusted to pH 5.0-
94
11.5 (in increments of 0.5 pH unit) by the addition of 1 M HCl or 1 M NaOH. For polar lipid and
95
fatty acid analyses, strains KMM 9530T, L. sediminilitoris KCTC 32383T, L. maricola JCM
96
14564T, L. tamlensis JCM 14020T, and L. rosea KMM 6003T were grown on MA at 28 °C for
97
three days. Lipids were extracted using the method of Folch et al. (1957). Two-dimensional thin
98
layer chromatography of polar lipids was carried out on Silica gel 60 F254 (10 x 10 cm, Merck,
99
Germany) using chloroform-methanol-water (65:25:4, v/v) for the first direction, and
100
chloroform-methanol-acetic acid-water (80:12:15:4, v/v) for the second one (Collins & Shah,
101
1984). Nonspecific detection of lipids on the two-dimensional TLC was conducted with 10%
102
H2SO4 in methanol at 120 ° C. Amino-containing lipids were determined with ninhydrin,
103
phospholipids with molybdate reagent, glycolipids with alpha-naphtol, and choline lipids with
104
Dragendorff’s reagent. Respiratory lipoquinones were analyzed by reversed-phase high 4
105
performance thin-layer chromatography as described by Mitchell & Fallon (1990). Fatty acid
106
methyl esters (FAMEs) were prepared according to the procedure of the Microbial Identification
107
System (MIDI) (Sasser, 1990). The analysis of FAMEs was performed as described previously
108
(Romanenko et al., 2011a). Production of Bchl α was spectrophotometrically tested in
109
methanolic extracts of cells grown on MA and MB in the dark as described by Lafay et al.
110
(1995). The 16S rRNA gene sequence (1447 nt) of strain KMM 9530T was determined as
111
described by Shida et al. (1997). The sequence obtained was compared with 16S rRNA gene
112
sequences retrieved from the EMBL/GenBank/DDBJ databases by using the FASTA program
113
(Pearson & Lipman, 1988). Phylogenetic analysis of 16S rRNA gene sequences was performed
114
using the software package MEGA 5 (Tamura et al., 2011) after multiple alignment of data by
115
CLUSTALW (version 1.83; Thompson et al., 2002). Phylogenetic trees were constructed by the
116
neighbor-joining, the maximum-likelihood and maximum-parsimony methods and the distances
117
were calculated according to the Kimura two-parameter model. The robustness of phylogenetic
118
trees was estimated by the bootstrap analysis of 1000 replicates. The DNA-DNA hybridization
119
experiments were performed by the photobiotin-labelled DNA probe microplate method of Ezaki
120
et al. (1989). The DNA-DNA hybridization values are given as average means of at least two
121
determination experiments.
122 123
Comparative 16S rRNA gene sequence analysis established that strain KMM 9530T belonged to
124
the genus Loktanella displaying the highest sequence similarity values of 98.5%, 98.4% and
125
98.2% to its closely related Loktanella species, L. sediminilitoris KCTC 32383T, L. tamlensis
126
JCM 14020T and L. maricola JCM 14564T, respectively, and similarity values below 97.7% to
127
the remaining Loktanella species (Fig. 1). The 16S rRNA gene sequence similarities obtained for
128
strain KMM 9530T and most Loktanella species were lower than the threshold similarity value of
129
97% proposed by Stackebrandt & Goebel (1994) and re-evaluated to 98.7% by Stackebrandt &
130
Ebers (2006), indicating that novel strain could be assigned to the genus Loktanella as an
5
131
individual species. The DNA-DNA hybridization experiments were carried out between strain
132
KMM 9530T and type strains, L. sediminilitoris KCTC 32383T, L. tamlensis JCM 14020T and L.
133
maricola JCM 14564T and DNA relatedness values were measured to be 22%, 45% and 36%,
134
respectively. The DNA-DNA hybridization values confirmed an assignment of the novel isolate
135
KMM 9530T to the separate species according to the value of 70 % proposed by Wayne et al.
136
(1987) for the bacterial species delineation. Cells of strain KMM 9530T were aerobic, Gram
137
negative, non-motile ovoid or short rod-shaped (Supplementary figure S1). Cultural,
138
physiological and metabolic properties of strain KMM 9530T and related members of the genus
139
Loktanella are listed in Table 1 and in the species description. Fatty acid profiles were similar
140
with a large proportion of C18:1ω7c and the presence of 11-methyl C18:1ω7c found in all strains
141
tested (Table 2). These results are in accordance with the data reported for L. rosea KMM 6003T
142
(Ivanova et al., 2005) and L. maricola JCM 14564T (Yoon et al., 2007), but disagreed with the
143
results of Lee (2012) and Park et al. (2013) who did not find 11-methyl C18:1ω7c in L. tamlensis
144
JCM 14020T and L. sediminilitoris KCTC 32383T. Strains KMM 9530T and L. tamlensis JCM
145
14020T differed by a small proportion of 11-methyl C18:1ω7c, and only L. sediminilitoris KCTC
146
32383T contained C12:1 compared with other related Loktanella strains (Table 2). The major
147
isoprenoid quinone of strain KMM 9530T was ubiquinone Q-10. The polar lipid composition of
148
strain KMM 9530T and related L. tamlensis JCM 14020T, L. rosea KMM 6003T, L. maricola
149
JCM 14564T, L. sediminilitoris KCTC 32383T was found to be similar and included
150
phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unknown aminolipid,
151
unknown phospholipids, and unknown lipids. All strains tested contained minor amounts of DPG
152
(Supplementary figure S2). The polar profile of L. sediminilitoris KCTC 32383T differed from
153
those of four strains studied in that phosphatidylethanolamine was detected. This finding is
154
congruent with the original description of Park et al. (2013) who reported the presence of
155
phosphatidylcholine,
156
aminolipid as dominant and diphosphatidylglycerol, an unknown phospholipid and an unknown
phosphatidylglycerol,
phosphatidylethanolamine
and
an
unknown
6
157
lipid as minor components in L. sediminilitoris KCTC 32383T. In addition, unknown
158
phospholipids PL1-PL3 were found in present study. The presence of phosphatidylethanolamine
159
in L. sediminilitoris KCTC 32383T and its absence in KMM 9530T and three related Loktanella
160
strains was confirmed by two-dimensional TCLs spraying with ninhydrin followed by staining
161
with molybdate reagent as exemplified by strains KMM 9530T, L. maricola JCM 14564T and L.
162
sediminilitoris KCTC 32383T. (Supplementary figure S2). Unlike our results with regard to the
163
lack of PE in three above type strains, the occurrence of PE has been reported by Lee (2012),
164
Yoon et al. (2007), and Ivanova et al. (2005) in L. tamlensis JCM 14020T, L. maricola JCM
165
14564T, and L. rosea KMM 6003T, with minor and trace amounts for two latter. The genus
166
Loktanella comprises both bacteria having PE together with PC and PG, L. litorea (Yoon et al.,
167
2013), L. agnita (Ivanova et al., 2005), L. sediminilitoris KCTC 32383T (Park et al., 2013a), or L.
168
cinnabarina (Tsubouchi et al., 2013), and bacteria not containing PE in their polar lipid profiles,
169
L. pyoseonensis (Moon et al., 2010), L. hongkongensis (Tsubouchi et al., 2013), or L.
170
soesokkakensis (Park et al., 2013b). At the same time Yoon et al. (2013) reported the presence of
171
PE in L. salcilacus whereas PE was not found in this bacterium in the study of Tsubouchi et al.
172
(2013). As reported by Tsubouchi et al. (2013) and Park et al. (2013b) that the same L.
173
cinnabarina cluster includes bacteria containing PE (L. cinnabarina itself) and bacteria L.
174
pyoseonensis, L. hongkongensis and L. soesokkakensis without PE. Chemotaxonomic properties
175
(ubiquinone Q-10, the predominance of C18:1ω7c, and the presence of phosphatidylcholine and
176
phosphatidylglycerol) obtained for the strain KMM 9530T supported its assignment to the genus
177
Loktanella. It is evident from the results obtained that the isolate can be assigned to the genus
178
Loktanella on the basis of its physiological, biochemical and chemotaxonomic characteristics. It
179
is interesting to note that Loktanella type strains revealed similar antibiotic resistance profiles,
180
being susceptible to 19 or 20 of 21 antibiotics applied, excepting strains KMM 9530T and L.
181
sediminilitoris KCTC 32383T. Recently we have reported marine alphaproteobacteria showing a
182
high sensitivity to antibiotics (Romanenko et al., 2011a, b). Strain KMM 9530T could be
7
183
distinguished from related Loktanella type strains in colony pigmentation (excepting L. tamlensis
184
JCM 14020T), in being able to grow in 7-8% NaCl (excepting L. rosea KMM 6003T), in being
185
able to utilize citrate, as well as in enzyme activities in API ZYM tests and antibiotic sensitivity
186
profile. It should be noted that recently described L. sediminilitoris KCTC 32383T revealed a
187
number of distinctive traits compared with related Loktanella type strains tested including
188
hydrolysis of Tween-80 and DNA and API ZYM enzyme activity profile. Differential
189
phenotypic characteristics are listed in Table 1. Based on the results obtained it is proposed to
190
assign a strain KMM 9530T to the genus Loktanella as representing novel species, Loktanella
191
maritima sp. nov.
192 193
Description of Loktanella maritima sp. nov.
194
Loktanella maritima (ma.ri’ti.ma. L. fem. adj. maritima maritime, marine).
195 196
Gram-negative, aerobic, oxidase-positive, catalase-positive, ovoid or short rod-shaped non-
197
motile cells, 0.6-0.8 μm in diameter and 1.6-2.0 μm in length. Grew on MA 2216 and MB.
198
Produced light beige-pigmented, shiny smooth colonies with the regular edges of 2-3 mm in
199
diameter. No growth observed on commercial media tryptic soy agar or broth, nutrient agar and
200
R2A agar. Bchl a is not produced. Required NaCl for growth; growth occurred between 2 and
201
8% (w/v) NaCl with an optimum of 3-4 % NaCl; growth is weak with 2 and 8% NaCl. The
202
temperature range for growth was 4-36 °C with an optimum of 28-30 °C; growth is weak at
203
36 °C. The pH range for growth was 5.5-9.0 (optimal 6.5-7.5). Negative for hydrolysis of casein,
204
DNA, chitin, starch, Tween 80, L-tyrosine and xanthine, and production of H2S in conventional
205
tests. Negative for gelatin hydrolysis and nitrate reduction in routine and API 20NE tests.
206
Positive for aesculin hydrolysis (API 20NE), PNPG test (β-galactosidase in API 20NE and API
207
20E tests) and citrate utilization (API 20E); and negative for the remaining tests, that are
208
included to the API 20NE and API 20E panels. According to the ID32 GN, strain could not
8
209
assimilate any of substrates included to the ID32 GN gallery. Positive API ZYM test results are
210
obtained for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid
211
phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucosidase; weakly-positive for valine
212
arylamidase and α-glucosidase; and negative for lipase (C14), cystine arylamidase, trypsin, α-
213
chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-
214
mannosidase and α-fucosidase. The major isoprenoid quinone is ubiquinone Q-10. The polar
215
lipids included phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unknown
216
aminolipid, an unknown phospholipid, and four unknown lipids. Fatty acid C18:1ω7c was
217
predominant. Susceptible to ampicillin, benzylpenicillin, vancomycin, gentamicin, kanamycin,
218
carbenicillin, chloramphenicol, neomycin, oxacillin, oleandomycin, ofloxacin, rifampicin,
219
streptomycin, cephazolin, cephalexin and erythromycin; and resistant to lincomycin, polymyxin,
220
nalidixic acid, tetracycline, and doxocycline.
221
The type strain of the species is strain KMM 9530T (=NRIC 0919T =JCM 19807T) isolated from
222
a shallow sediment sample, collected from Peter the Great Bay, the Sea of Japan, Russia,
223 224 225 226 227 228 229 230 231 232 233 234
9
235
References
236
Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda ruestringensis gen. nov., sp. nov.,
237
a facultatively anaerobic, appendaged bacterium from German North sea intertidal sediment. Int
238
J Syst Evol Microbiol 51, 1997-2006.
239
Collins, M. D. & Shah, H. N. (1984). Fatty acid, menaquinone and polar lipid composition of
240
Rothia dentosacariosa. Arch Microbiol 137, 247-249.
241
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid–
242
deoxyribonucleic acid hybridization in micro-dilution wells as an alternative to membrane filter
243
hybridization in which radioisotopes are used to determine genetic relatedness among bacterial
244
strains. Int J Syst Bacteriol 39, 224–229.
245
Folch, J., Lees, M. & Sloane Stanley, G. H. (1957). A simple method for the isolation and
246
purification of total lipides from animal tissues. J Biol Chem 226, 497-509.
247
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for
248
General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
249
Lafay, B., Ruimy, R., Rausch de Traubenberg, C., Breittmayer, V., Gauthier, M. J. &
250
Christen, R. (1995). Roseobacter algicola sp. nov., a new marine bacterium isolated from
251
the phycosphere of the toxin-producing dinoflagellate Prorocentrum lima. Int J Syst Bacteriol 45,
252
290-296.
253
Hosoya, S. & Yokota, A. (2007). Loktanella atrilutea sp. nov., isolated from seawater in Japan.
254
Int J Syst Evol Microbiol 57, 1966-1969.
255
Ivanova, E.P., Zhukova, N.V., Lysenko, A.M., Gorshkova, N.M., Sergeev, A.F., Mikhailov,
256
V.V. & Bowman, J.P. (2005). Loktanella agnita sp. nov. and Loktanella rosea sp. nov., from
257
the north-west Pacific Ocean. Int J Syst Evol Microbiol 55, 2203-2207.
258
Lau, S.C.K., Tsoi, M.M.Y., Li, X., Plakhotnikova, I., Wu, M., Wong, P.K. & Qian, P.Y.
259
(2004). Loktanella hongkongensis sp. nov., a novel member of the α-Proteobacteria originating
260
from marine biofilms in Hong Kong waters. Int J Syst Evol Microbiol 54, 2281-2284.
10
261
Lee, S.D. (2012). Loktanella tamlensis sp. nov., isolated from seawater. Int J Syst Evol
262
Microbiol 62, 586-590.
263
Mitchell, K. & Fallon, R. J. (1990). The determination of ubiquinone profiles by reversed-
264
phase high performance thin-layer chromatography as an aid to the speciation of Legionellaceae.
265
J Gen Microbiol 136, 2035-2041.
266
Moon, Y.G., Seo, S.H., Lee, S.D. & Heo, M.S. (2010). Loktanella pyoseonensis sp. nov.,
267
isolated from beach sand, and emended description of the genus Loktanella. Int J Syst Evol
268
Microbiol 60, 785-789.
269
Park, S., Jung, Y.-T. & Yoon, J.-H. (2013). Loktanella sediminilitoris sp. nov., isolated from
270
tidal flat sediment. Int J Syst Evol Microbiol 63, 4118-4123.
271
Park, S., Lee, J.-S., Lee, K.-C. & Yoon, J.-H. (2013). Loktanella soesokkakensis sp. nov.,
272
isolated from the junction between the North Pacific Ocean and a freshwater spring. Antonie van
273
Leeuwenhoek 104, 397-404.
274
Pearson, W. & Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc
275
Natl Acad Sci USA 85, 2444-2448.
276
Romanenko, L. A., Schumann, P., Rohde, M., Mikhailov, V. V. & Stackebrandt, E. (2004).
277
Reinekea marinisedimentorum gen. nov., sp. nov., a novel gammaproteobacterium from marine
278
coastal sediments. Int J Syst Evol Microbiol 54, 669-673.
279
Romanenko, L. A., Tanaka, N., Svetashev, V. I. & Kalinovskaya, N. I. (2011a).
280
Pacificibacter maritimus gen. nov., sp. nov., isolated from shallow marine sediment. Int J Syst
281
Evol Microbiol 61, 1375-1381.
282
Romanenko, L. A., Tanaka, N., Svetashev, V. I. & Mikhailov, V. V. (2011b).
283
Primorskyibacter sedentarius gen. nov., sp. nov., a novel member of the class
284
Alphaproteobacteria from shallow marine sediments. Int J Syst Evol Microbiol 61, 1572-1578.
285
Sasser, M. (1990). Microbial identification by gas chromatographic analysis of fatty acid methyl
286
esters (GC-FAME).Technical Note 101. Newark, DE: MIDI.
11
287
Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K. (1997). Transfer of
288
Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus,
289
Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended
290
description of the genus Paenibacillus. Int J Syst Bacteriol 47, 289-298.
291
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA
292
reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology.
293
Int J Syst Bacteriol 44, 846-849.
294
Stackebrandt, E. & Ebers, J. (2006). Taxonomic parameters revisited: tarnished gold standards.
295
Microb Today 45, 153-155.
296
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5:
297
molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and
298
maximum parsimony methods. Mol Biol Evol 28, 2731-2739.
299
Thompson, J. D., Gibson, T. J., & Higgins, D. G. (2002). Multiple sequence alignment using
300
ClustalW and ClustalX. Chapter 2. In Curr Protoc Bioinformatics, Unit 2. 3. 1-2. 3. 22. Edited by
301
Baxevanis, A. D., Stein, L. D., Stormo, G. D. & Yates III, J. R. Hoboken, NJ: John Wiley & Sons,
302
Inc.
303
Tsubouchi, T., Shimane, Y., Mori, K., Miyazaki, M., Tame, A., Uematsu, K., Maruyama, T.
304
& Hatada, Y. (2013). Loktanella cinnabarina sp. nov., isolated from a deep subseafloor
305
sediment, and emended description of the genus Loktanella. Int J Syst Evol Microbiol 63, 1390-
306
1395.
307
Van Trappen, S., Mergaert, J. & Swings, J. (2004). Loktanella salsilacus gen. nov., sp. nov.,
308
Loktanella fryxellensis sp. nov. and Loktanella vestfoldensis sp. nov., new members of the
309
Rhodobacter group, isolated from microbial mats in Antarctic lakes. Int J Syst Evol Microbiol 54,
310
1263-1269.
311
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky,
312
M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E., Stackebrandt, E., Starr, M. P. &
12
313
Trüper, H. G. (1987). Report of the ad hoc committee on reconciliation of approaches to
314
bacterial systematics. Int J Syst Bacteriol 37, 463-464.
315
Weon, H.Y., Kim, B.Y., Yoo, S.H., Kim, J.S., Kwon, S.W., Go, S.J. & Stackebrandt, E.
316
(2006). Loktanella koreensis sp. nov., isolated from sea sand in Korea. Int J Syst Evol Microbiol
317
56, 2199-2202.
318
Yoon, J.H., Kang, S.J., Lee, S.Y. & Oh, T.K. (2007). Loktanella maricola sp. nov., isolated
319
from seawater of the East Sea in Korea. Int J Syst Evol Microbiol 57, 1799-1802.
320
Yoon, J.H., Jung, Y.T. & Lee, J.S. (2013). Loktanella litorea sp. nov., isolated from seawater.
321
Int J Syst Evol Microbiol 63, 175-180.
322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338
13
339
Legend of Figures
340
Fig.1. Neighbor-joining tree based on 16S rRNA gene sequences showing relationships of the
341
isolate KMM 9530T and related taxa. Filled circles, generic branches that are present in
342
phylogenetic trees generated by the neighbor-joining, the maximum-likelihood and the
343
maximum-parsimony methods; filled triangles, generic branches that are present in trees
344
generated by the neighbor-joining and the maximum parsimony methods; filled squares, generic
345
branches that are present in trees generated by the neighbor-joining and the maximum likelihood
346
methods. Numbers indicate bootstrap values as percentage greater than 60 (neighbor-joining
347
probability/maximum-parsimony probability/maximum-likelihood probability). These values are
348
based on 1000 replicates. Bar, 0.01 substitutions per nucleotide position.
349 350
Supplementary Figure 1. Transmission electron micrograph of strain KMM 9530T grown in MA
351
for 24 h. Bar, 500 nm.
352 353
Supplementary Figure S2. Two-dimensional thin-layer chromatograms of polar lipids of
354
strains: (a, b, c) Loktanella maritima sp. nov. KMM 9530T; (d, e, f) Loktanella maricola JCM
355
14564T; (g, h, i) Loktanella sediminilitoris KCTC 32383T; (j) Loktanella tamlensis JCM 14020T;
356
(k) Loktanella rosea KMM 6003T. (a, d, g, j, k) non-specific detection of lipids prepared with
357
10% H2SO4 in methanol; (b, e, h) stained with ninhydrin; (c, f, i) stained with molybdate reagent.
358
Abbreviations:
359
diphosphatidylglycerol; PE, phosphatidylethanolamine; PL, PL1, PL2, PL3, unknown
360
phospholipids; AL, an unknown aminolipid; L1, L2, L3, L4, L5, unknown lipids.
PC,
phosphatidylcholine;
PG,
phosphatidylglycerol;
DPG,
361 362 363
14
364
Table 1. Differential phenotypic characteristics of strain KMM 9530T and type strains of related
365
Loktanella species.
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381
Strains: 1, Loktanella maritima sp. nov. KMM 9530T; 2, Loktanella tamlensis JCM 14020T; 3, Loktanella maricola JCM 14564T; 4, Loktanella rosea KMM 6003T; 5, Loktanella sediminilitoris KCTC 32383T. All data were obtained from the present study. +, Positive; -, negative; (+), weak reaction. All strains are positive for oxidase, catalase, sodium ions requirement for growth, aesculin hydrolysis and PNPG test (β-galactosidase) (in API 20NE), production of esterase C4, esterase lipase C8, and negative for gelatin hydrolysis and nitrate reduction (in routine and API 20NE tests), hydrolysis of casein, starch*, chitin, xanthine, production of lipase C14, cystine arylamidase, α-chymotrypsin, α-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, α-mannosidase, α-fucosidase (in API ZYM), arginine dihydrolase, urease, glucose fermentation, indol production, assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, D-gluconate, caprate, adipate, L-malate, citrate and phenylacetate (in API 20NE), and are susceptible to ampicillin, benzylpenicillin, vancomycin, gentamicin, kanamycin, carbenicillin, chloramphenicol, streptomycin, oleandomycin, ofloxacin, neomycin, oxacillin, rifampicin, cephazolin, cephalexin, erythromycin, and resistant to polymyxin§. Characteristic
1
2
3
4
5
Colony pigmentation
Beige
Beige
Dark-pink†
Pink
Greyish-yellow
Motility
-
+
-
+
-
Temperature range for
4-36
4-34*
4-34
4-39#
4-35§
2-8
2-4*
3-6†
2-8#
2-5§
Tyrosine
-
-
-
+
-
Hypoxanthine
(+)
+
+
+
-
Tween-80
-
-
-
-
+
DNA
-
-
-
-
+
Citrate utilization
+
-
-
-
-
-
-
-
-
+
Alkaline phosphatase
+
+
+
(+)
+
Leucine arylamidase
+
+
+
+
-
Valine arylamidase
(+)
-
-
-
-
Trypsin
-
-
-
-
+
Acid phosphatase
+
+
+
(+)
-
Naphthol-AS-BI-
+
+
+
+
(+)§
growth (ºC) NaCl range for growth (%) Hydrolysis of:
(Simmons citrate agar) H2S formation API ZYM tests:
15
phosphohydrolase α-glucosidase
(+)
-
-
-
-
β-glucosidase
+
-
-
-
-
Lincomycin (15)
-
+
+
-
-
Nalidixic acid (30)
-
+
+
+
-
Tetracycline (30)
-
+
+
+
+
Doxocycline (10)
-
+
+
+
+
Sensitivity to antibiotics (μg/disc):
382 383
*
Results differ from reported by Lee (2012).
384
†
Data not consistent with those reported by Yoon et al. (2007). Colony pigmentation for L.
385
maricola JCM 14564T was described by Yoon et al. (2007) as light orange.
386
#
Results differ from reported by Ivanova et al. (2005).
387
§
Results differ from reported by Park et al. (2013).
388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 16
407 408
Table 2. Fatty acid (%) profiles of strain KMM 9530T and related members of the genus
409
Loktanella.
410
Strains: 1, Loktanella maritima sp. nov. KMM 9530T; 2, Loktanella tamlensis JCM 14020T; 3,
411
Loktanella maricola JCM 14564T; 4, Loktanella rosea KMM 6003T; 5, Loktanella
412
sediminilitoris KCTC 32383T. The results were obtained from present study. All strains were
413
grown on MA at 28 °C for three days. -, Not detected; tr, - trace amount.
414 Fatty acid
1
2
3
4
5
C10:03-ОН
3.09
3.29
3.43
1.09
1.48
C12:13-ОН
2.45
1.95
4.37
3.53
3.37
-
-
-
tr
5.29
C16:1ω7c
0.49
1.92
0.39
1.09
1.12
C16:0
5.76
6.98
5.34
6.32
12.45
C17:1ω8c
1.07
-
-
0.26
-
C17:0
2.28
0.55
0.50
0.89
0.39
C18:2
3.40
1.65
1.45
3.69
4.69
C18:1ω7c
76.98
75.21
68.62
68.02
61.96
11-Methyl C18:1ω7c
2.62
4.56
9.70
11.47
6.78
C18:0
1.40
1.25
2.20
1.96
3.95
C12:1
415 416 417 418 419 420 421 17
422
Figure 1.
423
424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 18
441
Supplementary Figure S1.
442
443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 19
465
Supplementary Figure S2.
466
467 468
469 470
471 472
473 474 475 476 477 478
20
