International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1841–1846
DOI 10.1099/ijs.0.060772-0
Aestuariispira insulae gen. nov., sp. nov., a lipolytic bacterium isolated from a tidal flat Sooyeon Park, Ji-Min Park, Chul-Hyung Kang and Jung-Hoon Yoon Correspondence Jung-Hoon Yoon
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea
[email protected]
A Gram-stain-negative, non-motile, aerobic, curved-to-spiral-rod-shaped bacterium, designated AH-MY2T, was isolated from a tidal flat on Aphae island in the sea to the south-west of South Korea, and its taxonomic position was investigated using a polyphasic taxonomic approach. Strain AH-MY2T grew optimally at 30 6C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain AH-MY2T clustered with the type strain of Terasakiella pusilla and that this cluster joined the clade comprising the type strains of species of the genus Thalassospira. Strain AH-MY2T exhibited 16S rRNA gene sequence similarity values of 90.6 % to the type strain of Terasakiella pusilla and of less than 91.0 % to the type strains of other species with validly published names. Strain AH-MY2T contained Q-10 as the predominant ubiquinone and C18 : 1v7c as the major fatty acid. The major polar lipids detected in strain AHMY2T were phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminolipids and one unidentified glycolipid. The DNA G+C content of strain AH-MY2T was 56.0 mol%. The phylogenetic data and differential chemotaxonomic and other phenotypic properties revealed that strain AH-MY2T represented a novel genus and species within the family Rhodospirillaceae of the class Alphaproteobacteria, for which the name Aestuariispira insulae gen. nov., sp. nov. is proposed. The type strain of Aestuariispira insulae is AH-MY2T (5KCTC 32577T5CECT 8488T).
From our studies to screen novel bacteria from a variety of tidal flats on the Yellow Sea and the South Sea coasts of the Korean peninsula, many novel bacterial taxa belonging to the class Alphaproteobacteria have been described (Yoon et al., 2009a, 2009b, 2010, 2013a, 2013b; Jung et al., 2010; and others). The majority of the novel bacterial taxa have been those belonging to the family Rhodobacteraceae (Garrity et al., 2005, 2006), which is known to be one of the most abundant groups in marine environments (Rappe´ et al. 2000; Buchan et al. 2005). In this study, we describe a lipolytic bacterial strain, designated AH-MY2T, isolated from a tidal flat on Aphae island, located in the sea southwest of the Korean peninsula. Comparative 16S rRNA gene sequence analysis indicated that the novel strain forms a deep branch within the family Rhodospirillaceae, showing 16S rRNA gene sequence similarities of less than 91.0 % to any known bacterial taxa. The aim of the present work was to determine the exact taxonomic position of strain AHMY2T by using a polyphasic characterization which included determination of the chemotaxonomic and other
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AH-MY2T is KF876014. A supplementary figure is is available with the online version of this paper.
060772 G 2014 IUMS
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phenotypic properties and detailed phylogenetic investigations based on 16S rRNA gene sequences. A tidal flat sediment was collected from Aphae island and used as a source for the isolation of bacterial strains. Strain AH-MY2T was isolated at 25 uC on tricaprylin (TCN)marine agar 2216 (MA; Becton Dickinson). The TCN-MA for screening of lipolytic bacterial strains was prepared as follows: a TCN emulsion was made by emulsifying 5 ml TCN in 45 ml gum arabic solution (200 mM NaCl, 10 mM CaCl2 and 5 %, w/v, gum arabic) for 2 min using a Waring blender, and the TCN emulsion (50 ml) was mixed with 450 ml MA. The morphological, physiological and biochemical characteristics of strain AH-MY2T were investigated using routine cultivation on MA at 30 uC. Terasakiella pusilla LMG 7372T was used as a reference strain for the analyses of fatty acids and polar lipids and for phenotypic characterization. Cell morphology was examined by light microscopy (BX51; Olympus) and transmission electron microscopy (JEM1010; JEOL). The latter technique was also used to assess the presence of flagella on cells from an exponentially growing MA culture. For this purpose, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. Motility was assessed under 61500 magnification by using the hanging drop technique with cells grown for 3–5 days at 30 uC. The Gram reaction was 1841
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determined by using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Growth under anaerobic conditions was determined after incubation for 10 days in an anaerobic jar (MGC) with AnaeroPack (MGC) on MA and on MA with potassium nitrate (0.1 %, w/v); the jar was kept overnight at 4 uC to create anoxic conditions before incubation at 30 uC. Growth at 4, 10, 15, 20, 25, 30, 37, 40 and 45 uC was measured on MA to measure the optimal temperature and temperature range for growth. The pH range for growth was determined in marine broth 2216 (MB; Becton Dickinson) adjusted to pH 4.5–9.5 (using increments of 0.5 pH units) by using sodium acetate/acetic acid and Na2CO3 buffers. The pH values were verified after autoclaving. Growth at various concentrations of NaCl (0, 0.5 and 1.0–10.0 %, at increments of 1.0 %) was investigated by adding appropriate concentrations of NaCl to MB prepared according to the formula of the BD medium except that NaCl was excluded. The requirement for Mg2+ ions was investigated by using MB, prepared according to the formula of the BD medium, which comprised all of the constituents except MgCl2 and MgSO4. Catalase and oxidase activities were determined as described by La´nyı´ (1987). Hydrolysis of casein, starch, hypoxanthine, Ltyrosine and xanthine was investigated on MA using the substrate concentrations described by Barrow & Feltham (1993). Hydrolysis of aesculin and Tweens 20, 40, 60 and 80 and nitrate reduction were investigated as described previously (La´nyı´, 1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea were investigated by using nutrient gelatin and urea agar base media (Becton Dickinson), respectively, with the modification that artificial seawater was used for the preparation of media. The artificial seawater contained (l21 distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g MgSO4 . 7H2O and 1.3 g CaCl2 . 2H2O (Bruns et al., 2001). Utilization of various substrates for growth was tested according to the protocols of Baumann & Baumann (1981), using supplementation with 1 % (v/v) vitamin solution (Staley, 1968) and 2 % (v/v) Hutner’s mineral salts (Cohen-Bazire et al., 1957). Susceptibility to antibiotics was tested on MA plates using antibiotic discs (Advantec) containing the following (mg per disc unless otherwise stated): ampicillin (10), carbenicillin (100), cephalothin (30), chloramphenicol (100), gentamicin (30), kanamycin (30), lincomycin (15), neomycin (30), novobiocin (5), oleandomycin (15), penicillin G (20 U), polymyxin B (100 U), streptomycin (50) and tetracycline (30). Enzyme activities were determined, after incubation for 8 h at 30 uC, by using the API ZYM system (bioMe´rieux).
purified by the procedure described by Hunter (1985), with the exception that RNase T1 was used in combination with RNase A to minimize the contamination with RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al., 1998) using two universal primers (59-GAGTTTGATCCTGGCTCAG-39 and 59-ACGGTTACCTTGTTACGACTT-39). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described previously (Yoon et al., 2003). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC in a chromatograph equipped with a YMC ODS-A (25064.6 mm) column. The isoprenoid quinones were eluted using a mixture of methanol and 2propanol (2 : 1, v/v) using a flow rate of 1 ml min21 at room temperature and detected by UV absorbance (A275). For cellular fatty acid analysis, cell masses of strain AHMY2T and Terasakiella pusilla LMG 7372T were harvested from MA plates after cultivation for 3 days at 30 uC. The physiological age of the cell masses was standardized by observing the growth development of colonies on agar Table 1. Differential characteristics of strain AH-MY2T and the type strain of Terasakiella pusilla Strains: 1, AH-MY2T; 2, Terasakiella pusilla LMG 7372T. Data obtained from this study unless indicated otherwise. +, Positive reaction; 2, negative reaction. Both strains are positive for catalase and oxidase activities; nitrate reduction; utilization of acetate, citrate, L-malate, pyruvate and succinate; susceptibility to chloramphenicol, kanamycin, oleandomycin and streptomycin; and activity of alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and valine arylamidase. Both strains are negative for anaerobic growth; Gram-staining; hydrolysis of aesculin, gelatin and starch; utilization of L-arabinose, cellobiose, D-fructose, D-galactose, maltose, D-mannose, sucrose, trehalose, D-xylose, formate, L-glutamate and salicin; susceptibility to ampicillin, carbenicillin, cephalothin, lincomycin, neomycin, novobiocin, penicillin G and polymyxin B; and activity of lipase (C 14), cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Characteristic Motility Growth at 10 and 40 uC NaCl range (%) for growth Utilization of: D-Glucose Benzoate Susceptibility to: Gentamicin Tetracycline DNA G+C content (mol%)
1
2
2 2 1.0–7.0
+* +* 0.5–8.0*
+ 2
2 +
+ + 56.0
2 2 48D
Cell biomass of strain AH-MY2T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 2 days in MB at 30 uC, and cell biomass of Terasakiella pusilla LMG 7372T for polar lipid analysis was obtained under the same culture conditions. Chromosomal DNA was extracted and
*Data taken from Terasaki (1979). DData taken from Sakane & Yokota (1994).
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Aestuariispira insulae gen. nov., sp. nov.
98.8 Thalassospira xianhensis P-4T (EU017546) Thalassospira xiamenensis M-5T (AY189753)
0.02
100 83.1
Thalassospira profundimaris WP0211T (AY186195)
100
Thalassospira tepidiphila 1-1BT (AB265822) Thalassospira lucentensis DSM 14000T (AM294944) Aestuariispira insulae AH-MY2T (KF876014)
51.9
72.6
Terasakiella pusilla IFO 13613T (AB006768) Roseospirillum parvum 930IT (AJ011919) Pelagibius litoralis CL-UU02T (DQ401091) Caenispirillum bisanense K92T (EF100694)
61.1
53.5 99.9
Insolitispirillum peregrinum LMG 4340T (EF612767) Novispirillum itersonii LMG 4337T (EF612765) Roseospira mediosalina BN 280T (AJ000989) Rhodospira trueperi ATCC 700224T (X99671)
98.0
Rhodospirillum rubrum ATCC 11170T (D30778) 100
Phaeospirillum fulvum NCIMB 11762T (D14433) Magnetospirillum gryphiswaldense MSR-1T (Y10109) Escherichia coli ATCC 11775T (X80725)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Aestuariispira insulae AH-MY2T and representatives of some other related taxa. Only bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branching points. Filled circles indicate that the corresponding nodes were also recovered in the trees reconstructed with the maximum-likelihood and maximum-parsimony algorithms. Escherichia coli ATCC 11775T (GenBank accession no. X80725) was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.
plates followed by harvesting them from the same quadrant on the agar plates according to the standard MIDI protocol (Sherlock Microbial Identification System, version 6.1). Fatty acids were saponified, methylated and extracted using the standard protocol of the MIDI (Sherlock Microbial Identification System, version 6.1). The fatty acids were analysed by GC (model 6890; Hewlett Packard) and identified by using the TSBA6 database of the Microbial Identification System (Sasser, 1990). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and separated by two-dimensional TLC using chloroform/methanol/water (65 : 25 : 3.8, by vol.) for the first dimension and chloroform/methanol/acetic acid/water (40 : 7.5 : 6 : 1.8, by vol.) for the second dimension as described by Minnikin et al. (1977). Individual polar lipids were identified by spraying with molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987) and with Dragendorff’s reagent (Sigma). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversedphase HPLC using a chromatograph equipped with a YMC ODS-A (25064.6 mm) column. The nucleotides were eluted by a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40 : 1, v/v), using flow rate of 1 ml min21 at room temperature and detected by UV absorbance (A270). Morphological, cultural, physiological and biochemical characteristics of strain AH-MY2T are given in the genus http://ijs.sgmjournals.org
and species descriptions (see below) or in Table 1 and Fig. S1 available with the online Supplementary Material. The almost-complete 16S rRNA gene sequence of strain AHMY2T determined in this study comprised 1417 nt, representing approximately 95 % of the Escherichia coli 16S rRNA gene sequence. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequence, strain AH-MY2T clustered with the type strain of Terasakiella pusilla with a bootstrap resampling value of 72.6 %, and this cluster joined the clade comprising the type strains of species of the genus Thalassospira with a bootstrap resampling value of 83.1 % (Fig. 1). These tree topologies were also found in the trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). The relationships among the type strains of the five species of the genus Thalassospira analysed, as shown in the neighbour-joining phylogenetic tree, were also found in the maximum-likelihood and maximum-parsimony trees (Fig. 1). Strain AH-MY2T exhibited 16S rRNA gene sequence similarity values of 90.6 and 90.5– 91.0 % to the type strain of Terasakiella pusilla and to those of the five species of the genus Thalassospira, respectively. It exhibited 16S rRNA gene sequence similarity values of less than 90.1 % to those of other species with validly published names. The predominant isoprenoid quinone detected in strain AH-MY2T was ubiquinone-10 (Q-10), which is the same as that of the genus Terasakiella (Satomi et al., 2002); minor amounts of Q-8 (approximately 3.6 %) and Q-9 1843
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Table 2. Cellular fatty acid compositions (percentages) of strain AH-MY2T and the type strain of Terasakiella pusilla
(a)
Strains: 1, AH-MY2T; 2, Terasakiella pusilla LMG 7372T. All data obtained from this study. Fatty acids that represented ,0.5 % in both strains were omitted. TR, trace (,0.5 %); 2, Not detected. Fatty acid Straight-chain C12 : 0 C14 : 0 C16 : 0 C18 : 0 C20 : 0 Unsaturated C16 : 1v5c C18 : 1v5c C18 : 1v7c C20 : 1v7c Branched iso-C18 : 1 H Hydroxy C16 : 0 3-OH C18 : 0 3-OH C18 : 1 2-OH Summed features* 2 3
1
GL PG L2
2 PL2
2.9 2.3 8.6 1.8 0.8
4.2 2.2 9.9 0.7 2
1.0 65.0 1.3
0.8 0.8 53.6 2
2
0.7
8.6 1.1 2
0.7 1.2 6.3
3.0 2.5
8.6 9.9
TR
*Summed feature 2 contained iso-C16 : 1 I and/or C14 : 0 3-OH. Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
(b) L1
PS
AL1
PL1 PE AL2
GL AL1 PG AL3 AL4 PL2
L3 APL PE
PS
Fig. 2. Thin layer chromatograms of the polar lipids of Aestuariispira insulae AH-MY2T (a) and Terasakiella pusilla LMG 7372T (b). Spots were revealed by spraying the plates with 10 % ethanolic molybdophosphoric acid. PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PS, phosphatidylserine; AL1–4, unidentified aminolipids; GL, unidentified glycolipid; L1–3, unidentified lipids; PL1–2, unidentified phospholipids; APL, unidentified aminophospholipid.
strain AH-MY2T is different from that (AL3) of Terasakiella pusilla LMG 7372T and in that several unidentified polar lipids are present or absent (Fig. 2). The DNA G+C content of strain AH-MY2T was 56.0 mol%, a value higher than that reported for of the type strain of Terasakiella pusilla (Table 1). The differences in several phenotypic properties also served to differentiate strain AH-MY2T from Terasakiella pusilla (Table 1). Strain AH-MY2T was differentiated from Terasakiella pusilla LMG 7372T by differences in phenotypic characteristics, including motility, growth at 10 and 40 uC, utilization of some substrates and susceptibility to some antibiotics (Table 1). The phylogenetic data and differential chemotaxonomic and other phenotypic properties indicate that strain AH-MY2T represents a novel genus and species within family Rhodospirillaceae, for which the name Aestuariispira insulae gen. nov., sp. nov. is proposed.
(approximately 4.6 %) were present. Among the species of the genus Thalassospira, Thalassospira xianhensis, whose predominant isoprenoid quinone has been reported, has been described as having Q-9 (Zhao et al. 2010). In Table 2, the cellular fatty acid profile of strain AH-MY2T is compared with that of the type strain of Terasakiella pusilla grown and analysed under identical conditions in this study. The major fatty acid (.10 % of the total fatty acids) detected in strain AH-MY2T was C18 : 1v7c (65.0 %). The fatty acid profiles of strain AH-MY2T and Terasakiella pusilla LMG 7372T were similar in that C18 : 1v7c was the predominant fatty acid, but their profiles were distinguishable by the differences in the proportions of some fatty acids, particularly C18 : 1v7c, C16 : 0 3-OH, C18 : 1 2-OH and summed features 2 and 3 (Table 2). The major polar lipids detected in strain AH-MY2T were phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminolipids and one unidentified glycolipid (Fig. 2). The polar lipid profile of strain AH-MY2T was similar to that of Terasakiella pusilla LMG 7372T in that phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified glycolipid were the major polar lipids. However, their polar lipid profiles were distinguishable from each other in that one major unidentified aminolipid (AL2) of
The type species is Aestuariispira insulae. A member of the family Rhodospirillaceae, the class Alphaproteobacteria, according to 16S rRNA gene sequence analysis. The DNA G+C content of the type strain is 56.0 mol%.
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Description of Aestuariispira gen. nov. Aestuariispira (Aes.tu.a.ri.i.spi9ra. L. neut. n. aestuarium -i tidal flat; L. fem. n. spira a spiral; N.L. fem. n. Aestuariispira a spiral from a tidal flat). Cells are Gram-stain-negative, aerobic, non-flagellated and curved-to-spiral rods. Catalase- and oxidase-positive. Nitrate reduction is positive. The predominant ubiquinone is Q-10. The major fatty acid (.10 % of the total fatty acids) is C18 : 1v7c. The major polar lipids are phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminolipids and one unidentified glycolipid.
Aestuariispira insulae gen. nov., sp. nov.
Description of Aestuariispira insulae sp. nov.
Baumann, P. & Baumann, L. (1981). The marine Gram-negative
Aestuariispira insulae (in9su.lae. L. fem. gen. n. insulae of an island, referring to the source of isolation of the type strain).
eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes. In The Prokaryotes, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Tru¨per, A. Balows & H. G. Schlegel. Berlin: Springer.
Cells are Gram-stain-negative, non-flagellated and curvedto-spiral rods, approximately 0.2–0.8 mm in width and 1.0– 8.0 mm in length. Colonies on MA are circular, slightly convex, smooth, glistening, light yellow and 1.0–1.5 mm in diameter after incubation for 3 days at 30 uC. Optimal temperature for growth is 30 uC; growth occurs at 15 and 37 uC, but not at 10 and 40 uC. Optimal pH for growth is pH 7.0–8.0; growth occurs at pH 6.0, but not at pH 5.5. Optimal growth occurs in the presence of approximately 2.0 % (w/v) NaCl; growth occurs in the presence of 1.0– 7.0 % (w/v) NaCl. Growth does not occur under anaerobic conditions on MA or on MA supplemented with nitrate. Catalase- and oxidase-positive. Nitrate is reduced to nitrite. H2S is not produced. Tricaprylin is degraded. Casein, hypoxanthine, Tweens 20, 40, 60 and 80, L-tyrosine and xanthines are hydrolysed, but aesculin, gelatin, starch and urea are not. D-Glucose, acetate, citrate, L-malate, pyruvate and succinate are utilized as sole carbon and energy sources, but L-arabinose, cellobiose, D-fructose, D-galactose, maltose, D-mannose, sucrose, trehalose, D-xylose, benzoate, formate, salicin and L-glutamate are not. Susceptible to chloramphenicol, gentamicin, kanamycin, oleandomycin, streptomycin and tetracycline, but not to ampicillin, carbenicillin, cephalothin, lincomycin, neomycin, novobiocin, penicillin G and polymyxin B. In assays with the API ZYM system, alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and valine arylamidase activities are present, but lipase (C 14), cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, bgalactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities are absent. The predominant ubiquinone is Q-10. The major fatty acid (.10 % of the total fatty acids) is C18 : 1v7c. The major polar lipids are phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminolipids and one unidentified glycolipid. The type strain, AH-MY2T (5KCTC 32577T5CECT 8488T), was isolated from a tidal flat on Aphae island, South Korea. The DNA G+C content of the type strain is 56.0 mol%.
ACKNOWLEDGEMENTS This work was supported by Mid-career Researcher Program through a National Research Foundation (NRF) grant from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea (2012R1A2A2A01047369).
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sp. nov., a polycyclic aromatic hydrocarbon-degrading marine bacterium. Int J Syst Evol Microbiol 60, 1125–1129.
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DOI 10.1099/ijs.0.060772-0
Aestuariispira insulae gen. nov., sp. nov., a lipolytic bacterium isolated from a tidal flat Sooyeon Park, Ji-Min Park, Chul-Hyung Kang and Jung-Hoon Yoon Correspondence Jung-Hoon Yoon
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea
[email protected]
A Gram-stain-negative, non-motile, aerobic, curved-to-spiral-rod-shaped bacterium, designated AH-MY2T, was isolated from a tidal flat on Aphae island in the sea to the south-west of South Korea, and its taxonomic position was investigated using a polyphasic taxonomic approach. Strain AH-MY2T grew optimally at 30 6C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain AH-MY2T clustered with the type strain of Terasakiella pusilla and that this cluster joined the clade comprising the type strains of species of the genus Thalassospira. Strain AH-MY2T exhibited 16S rRNA gene sequence similarity values of 90.6 % to the type strain of Terasakiella pusilla and of less than 91.0 % to the type strains of other species with validly published names. Strain AH-MY2T contained Q-10 as the predominant ubiquinone and C18 : 1v7c as the major fatty acid. The major polar lipids detected in strain AHMY2T were phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminolipids and one unidentified glycolipid. The DNA G+C content of strain AH-MY2T was 56.0 mol%. The phylogenetic data and differential chemotaxonomic and other phenotypic properties revealed that strain AH-MY2T represented a novel genus and species within the family Rhodospirillaceae of the class Alphaproteobacteria, for which the name Aestuariispira insulae gen. nov., sp. nov. is proposed. The type strain of Aestuariispira insulae is AH-MY2T (5KCTC 32577T5CECT 8488T).
From our studies to screen novel bacteria from a variety of tidal flats on the Yellow Sea and the South Sea coasts of the Korean peninsula, many novel bacterial taxa belonging to the class Alphaproteobacteria have been described (Yoon et al., 2009a, 2009b, 2010, 2013a, 2013b; Jung et al., 2010; and others). The majority of the novel bacterial taxa have been those belonging to the family Rhodobacteraceae (Garrity et al., 2005, 2006), which is known to be one of the most abundant groups in marine environments (Rappe´ et al. 2000; Buchan et al. 2005). In this study, we describe a lipolytic bacterial strain, designated AH-MY2T, isolated from a tidal flat on Aphae island, located in the sea southwest of the Korean peninsula. Comparative 16S rRNA gene sequence analysis indicated that the novel strain forms a deep branch within the family Rhodospirillaceae, showing 16S rRNA gene sequence similarities of less than 91.0 % to any known bacterial taxa. The aim of the present work was to determine the exact taxonomic position of strain AHMY2T by using a polyphasic characterization which included determination of the chemotaxonomic and other
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AH-MY2T is KF876014. A supplementary figure is is available with the online version of this paper.
060772 G 2014 IUMS
Printed in Great Britain
phenotypic properties and detailed phylogenetic investigations based on 16S rRNA gene sequences. A tidal flat sediment was collected from Aphae island and used as a source for the isolation of bacterial strains. Strain AH-MY2T was isolated at 25 uC on tricaprylin (TCN)marine agar 2216 (MA; Becton Dickinson). The TCN-MA for screening of lipolytic bacterial strains was prepared as follows: a TCN emulsion was made by emulsifying 5 ml TCN in 45 ml gum arabic solution (200 mM NaCl, 10 mM CaCl2 and 5 %, w/v, gum arabic) for 2 min using a Waring blender, and the TCN emulsion (50 ml) was mixed with 450 ml MA. The morphological, physiological and biochemical characteristics of strain AH-MY2T were investigated using routine cultivation on MA at 30 uC. Terasakiella pusilla LMG 7372T was used as a reference strain for the analyses of fatty acids and polar lipids and for phenotypic characterization. Cell morphology was examined by light microscopy (BX51; Olympus) and transmission electron microscopy (JEM1010; JEOL). The latter technique was also used to assess the presence of flagella on cells from an exponentially growing MA culture. For this purpose, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. Motility was assessed under 61500 magnification by using the hanging drop technique with cells grown for 3–5 days at 30 uC. The Gram reaction was 1841
S. Park and others
determined by using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Growth under anaerobic conditions was determined after incubation for 10 days in an anaerobic jar (MGC) with AnaeroPack (MGC) on MA and on MA with potassium nitrate (0.1 %, w/v); the jar was kept overnight at 4 uC to create anoxic conditions before incubation at 30 uC. Growth at 4, 10, 15, 20, 25, 30, 37, 40 and 45 uC was measured on MA to measure the optimal temperature and temperature range for growth. The pH range for growth was determined in marine broth 2216 (MB; Becton Dickinson) adjusted to pH 4.5–9.5 (using increments of 0.5 pH units) by using sodium acetate/acetic acid and Na2CO3 buffers. The pH values were verified after autoclaving. Growth at various concentrations of NaCl (0, 0.5 and 1.0–10.0 %, at increments of 1.0 %) was investigated by adding appropriate concentrations of NaCl to MB prepared according to the formula of the BD medium except that NaCl was excluded. The requirement for Mg2+ ions was investigated by using MB, prepared according to the formula of the BD medium, which comprised all of the constituents except MgCl2 and MgSO4. Catalase and oxidase activities were determined as described by La´nyı´ (1987). Hydrolysis of casein, starch, hypoxanthine, Ltyrosine and xanthine was investigated on MA using the substrate concentrations described by Barrow & Feltham (1993). Hydrolysis of aesculin and Tweens 20, 40, 60 and 80 and nitrate reduction were investigated as described previously (La´nyı´, 1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea were investigated by using nutrient gelatin and urea agar base media (Becton Dickinson), respectively, with the modification that artificial seawater was used for the preparation of media. The artificial seawater contained (l21 distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g MgSO4 . 7H2O and 1.3 g CaCl2 . 2H2O (Bruns et al., 2001). Utilization of various substrates for growth was tested according to the protocols of Baumann & Baumann (1981), using supplementation with 1 % (v/v) vitamin solution (Staley, 1968) and 2 % (v/v) Hutner’s mineral salts (Cohen-Bazire et al., 1957). Susceptibility to antibiotics was tested on MA plates using antibiotic discs (Advantec) containing the following (mg per disc unless otherwise stated): ampicillin (10), carbenicillin (100), cephalothin (30), chloramphenicol (100), gentamicin (30), kanamycin (30), lincomycin (15), neomycin (30), novobiocin (5), oleandomycin (15), penicillin G (20 U), polymyxin B (100 U), streptomycin (50) and tetracycline (30). Enzyme activities were determined, after incubation for 8 h at 30 uC, by using the API ZYM system (bioMe´rieux).
purified by the procedure described by Hunter (1985), with the exception that RNase T1 was used in combination with RNase A to minimize the contamination with RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al., 1998) using two universal primers (59-GAGTTTGATCCTGGCTCAG-39 and 59-ACGGTTACCTTGTTACGACTT-39). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described previously (Yoon et al., 2003). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC in a chromatograph equipped with a YMC ODS-A (25064.6 mm) column. The isoprenoid quinones were eluted using a mixture of methanol and 2propanol (2 : 1, v/v) using a flow rate of 1 ml min21 at room temperature and detected by UV absorbance (A275). For cellular fatty acid analysis, cell masses of strain AHMY2T and Terasakiella pusilla LMG 7372T were harvested from MA plates after cultivation for 3 days at 30 uC. The physiological age of the cell masses was standardized by observing the growth development of colonies on agar Table 1. Differential characteristics of strain AH-MY2T and the type strain of Terasakiella pusilla Strains: 1, AH-MY2T; 2, Terasakiella pusilla LMG 7372T. Data obtained from this study unless indicated otherwise. +, Positive reaction; 2, negative reaction. Both strains are positive for catalase and oxidase activities; nitrate reduction; utilization of acetate, citrate, L-malate, pyruvate and succinate; susceptibility to chloramphenicol, kanamycin, oleandomycin and streptomycin; and activity of alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and valine arylamidase. Both strains are negative for anaerobic growth; Gram-staining; hydrolysis of aesculin, gelatin and starch; utilization of L-arabinose, cellobiose, D-fructose, D-galactose, maltose, D-mannose, sucrose, trehalose, D-xylose, formate, L-glutamate and salicin; susceptibility to ampicillin, carbenicillin, cephalothin, lincomycin, neomycin, novobiocin, penicillin G and polymyxin B; and activity of lipase (C 14), cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Characteristic Motility Growth at 10 and 40 uC NaCl range (%) for growth Utilization of: D-Glucose Benzoate Susceptibility to: Gentamicin Tetracycline DNA G+C content (mol%)
1
2
2 2 1.0–7.0
+* +* 0.5–8.0*
+ 2
2 +
+ + 56.0
2 2 48D
Cell biomass of strain AH-MY2T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 2 days in MB at 30 uC, and cell biomass of Terasakiella pusilla LMG 7372T for polar lipid analysis was obtained under the same culture conditions. Chromosomal DNA was extracted and
*Data taken from Terasaki (1979). DData taken from Sakane & Yokota (1994).
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Aestuariispira insulae gen. nov., sp. nov.
98.8 Thalassospira xianhensis P-4T (EU017546) Thalassospira xiamenensis M-5T (AY189753)
0.02
100 83.1
Thalassospira profundimaris WP0211T (AY186195)
100
Thalassospira tepidiphila 1-1BT (AB265822) Thalassospira lucentensis DSM 14000T (AM294944) Aestuariispira insulae AH-MY2T (KF876014)
51.9
72.6
Terasakiella pusilla IFO 13613T (AB006768) Roseospirillum parvum 930IT (AJ011919) Pelagibius litoralis CL-UU02T (DQ401091) Caenispirillum bisanense K92T (EF100694)
61.1
53.5 99.9
Insolitispirillum peregrinum LMG 4340T (EF612767) Novispirillum itersonii LMG 4337T (EF612765) Roseospira mediosalina BN 280T (AJ000989) Rhodospira trueperi ATCC 700224T (X99671)
98.0
Rhodospirillum rubrum ATCC 11170T (D30778) 100
Phaeospirillum fulvum NCIMB 11762T (D14433) Magnetospirillum gryphiswaldense MSR-1T (Y10109) Escherichia coli ATCC 11775T (X80725)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Aestuariispira insulae AH-MY2T and representatives of some other related taxa. Only bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branching points. Filled circles indicate that the corresponding nodes were also recovered in the trees reconstructed with the maximum-likelihood and maximum-parsimony algorithms. Escherichia coli ATCC 11775T (GenBank accession no. X80725) was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.
plates followed by harvesting them from the same quadrant on the agar plates according to the standard MIDI protocol (Sherlock Microbial Identification System, version 6.1). Fatty acids were saponified, methylated and extracted using the standard protocol of the MIDI (Sherlock Microbial Identification System, version 6.1). The fatty acids were analysed by GC (model 6890; Hewlett Packard) and identified by using the TSBA6 database of the Microbial Identification System (Sasser, 1990). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and separated by two-dimensional TLC using chloroform/methanol/water (65 : 25 : 3.8, by vol.) for the first dimension and chloroform/methanol/acetic acid/water (40 : 7.5 : 6 : 1.8, by vol.) for the second dimension as described by Minnikin et al. (1977). Individual polar lipids were identified by spraying with molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987) and with Dragendorff’s reagent (Sigma). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversedphase HPLC using a chromatograph equipped with a YMC ODS-A (25064.6 mm) column. The nucleotides were eluted by a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40 : 1, v/v), using flow rate of 1 ml min21 at room temperature and detected by UV absorbance (A270). Morphological, cultural, physiological and biochemical characteristics of strain AH-MY2T are given in the genus http://ijs.sgmjournals.org
and species descriptions (see below) or in Table 1 and Fig. S1 available with the online Supplementary Material. The almost-complete 16S rRNA gene sequence of strain AHMY2T determined in this study comprised 1417 nt, representing approximately 95 % of the Escherichia coli 16S rRNA gene sequence. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequence, strain AH-MY2T clustered with the type strain of Terasakiella pusilla with a bootstrap resampling value of 72.6 %, and this cluster joined the clade comprising the type strains of species of the genus Thalassospira with a bootstrap resampling value of 83.1 % (Fig. 1). These tree topologies were also found in the trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). The relationships among the type strains of the five species of the genus Thalassospira analysed, as shown in the neighbour-joining phylogenetic tree, were also found in the maximum-likelihood and maximum-parsimony trees (Fig. 1). Strain AH-MY2T exhibited 16S rRNA gene sequence similarity values of 90.6 and 90.5– 91.0 % to the type strain of Terasakiella pusilla and to those of the five species of the genus Thalassospira, respectively. It exhibited 16S rRNA gene sequence similarity values of less than 90.1 % to those of other species with validly published names. The predominant isoprenoid quinone detected in strain AH-MY2T was ubiquinone-10 (Q-10), which is the same as that of the genus Terasakiella (Satomi et al., 2002); minor amounts of Q-8 (approximately 3.6 %) and Q-9 1843
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Table 2. Cellular fatty acid compositions (percentages) of strain AH-MY2T and the type strain of Terasakiella pusilla
(a)
Strains: 1, AH-MY2T; 2, Terasakiella pusilla LMG 7372T. All data obtained from this study. Fatty acids that represented ,0.5 % in both strains were omitted. TR, trace (,0.5 %); 2, Not detected. Fatty acid Straight-chain C12 : 0 C14 : 0 C16 : 0 C18 : 0 C20 : 0 Unsaturated C16 : 1v5c C18 : 1v5c C18 : 1v7c C20 : 1v7c Branched iso-C18 : 1 H Hydroxy C16 : 0 3-OH C18 : 0 3-OH C18 : 1 2-OH Summed features* 2 3
1
GL PG L2
2 PL2
2.9 2.3 8.6 1.8 0.8
4.2 2.2 9.9 0.7 2
1.0 65.0 1.3
0.8 0.8 53.6 2
2
0.7
8.6 1.1 2
0.7 1.2 6.3
3.0 2.5
8.6 9.9
TR
*Summed feature 2 contained iso-C16 : 1 I and/or C14 : 0 3-OH. Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
(b) L1
PS
AL1
PL1 PE AL2
GL AL1 PG AL3 AL4 PL2
L3 APL PE
PS
Fig. 2. Thin layer chromatograms of the polar lipids of Aestuariispira insulae AH-MY2T (a) and Terasakiella pusilla LMG 7372T (b). Spots were revealed by spraying the plates with 10 % ethanolic molybdophosphoric acid. PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PS, phosphatidylserine; AL1–4, unidentified aminolipids; GL, unidentified glycolipid; L1–3, unidentified lipids; PL1–2, unidentified phospholipids; APL, unidentified aminophospholipid.
strain AH-MY2T is different from that (AL3) of Terasakiella pusilla LMG 7372T and in that several unidentified polar lipids are present or absent (Fig. 2). The DNA G+C content of strain AH-MY2T was 56.0 mol%, a value higher than that reported for of the type strain of Terasakiella pusilla (Table 1). The differences in several phenotypic properties also served to differentiate strain AH-MY2T from Terasakiella pusilla (Table 1). Strain AH-MY2T was differentiated from Terasakiella pusilla LMG 7372T by differences in phenotypic characteristics, including motility, growth at 10 and 40 uC, utilization of some substrates and susceptibility to some antibiotics (Table 1). The phylogenetic data and differential chemotaxonomic and other phenotypic properties indicate that strain AH-MY2T represents a novel genus and species within family Rhodospirillaceae, for which the name Aestuariispira insulae gen. nov., sp. nov. is proposed.
(approximately 4.6 %) were present. Among the species of the genus Thalassospira, Thalassospira xianhensis, whose predominant isoprenoid quinone has been reported, has been described as having Q-9 (Zhao et al. 2010). In Table 2, the cellular fatty acid profile of strain AH-MY2T is compared with that of the type strain of Terasakiella pusilla grown and analysed under identical conditions in this study. The major fatty acid (.10 % of the total fatty acids) detected in strain AH-MY2T was C18 : 1v7c (65.0 %). The fatty acid profiles of strain AH-MY2T and Terasakiella pusilla LMG 7372T were similar in that C18 : 1v7c was the predominant fatty acid, but their profiles were distinguishable by the differences in the proportions of some fatty acids, particularly C18 : 1v7c, C16 : 0 3-OH, C18 : 1 2-OH and summed features 2 and 3 (Table 2). The major polar lipids detected in strain AH-MY2T were phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminolipids and one unidentified glycolipid (Fig. 2). The polar lipid profile of strain AH-MY2T was similar to that of Terasakiella pusilla LMG 7372T in that phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified glycolipid were the major polar lipids. However, their polar lipid profiles were distinguishable from each other in that one major unidentified aminolipid (AL2) of
The type species is Aestuariispira insulae. A member of the family Rhodospirillaceae, the class Alphaproteobacteria, according to 16S rRNA gene sequence analysis. The DNA G+C content of the type strain is 56.0 mol%.
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Description of Aestuariispira gen. nov. Aestuariispira (Aes.tu.a.ri.i.spi9ra. L. neut. n. aestuarium -i tidal flat; L. fem. n. spira a spiral; N.L. fem. n. Aestuariispira a spiral from a tidal flat). Cells are Gram-stain-negative, aerobic, non-flagellated and curved-to-spiral rods. Catalase- and oxidase-positive. Nitrate reduction is positive. The predominant ubiquinone is Q-10. The major fatty acid (.10 % of the total fatty acids) is C18 : 1v7c. The major polar lipids are phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminolipids and one unidentified glycolipid.
Aestuariispira insulae gen. nov., sp. nov.
Description of Aestuariispira insulae sp. nov.
Baumann, P. & Baumann, L. (1981). The marine Gram-negative
Aestuariispira insulae (in9su.lae. L. fem. gen. n. insulae of an island, referring to the source of isolation of the type strain).
eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes. In The Prokaryotes, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Tru¨per, A. Balows & H. G. Schlegel. Berlin: Springer.
Cells are Gram-stain-negative, non-flagellated and curvedto-spiral rods, approximately 0.2–0.8 mm in width and 1.0– 8.0 mm in length. Colonies on MA are circular, slightly convex, smooth, glistening, light yellow and 1.0–1.5 mm in diameter after incubation for 3 days at 30 uC. Optimal temperature for growth is 30 uC; growth occurs at 15 and 37 uC, but not at 10 and 40 uC. Optimal pH for growth is pH 7.0–8.0; growth occurs at pH 6.0, but not at pH 5.5. Optimal growth occurs in the presence of approximately 2.0 % (w/v) NaCl; growth occurs in the presence of 1.0– 7.0 % (w/v) NaCl. Growth does not occur under anaerobic conditions on MA or on MA supplemented with nitrate. Catalase- and oxidase-positive. Nitrate is reduced to nitrite. H2S is not produced. Tricaprylin is degraded. Casein, hypoxanthine, Tweens 20, 40, 60 and 80, L-tyrosine and xanthines are hydrolysed, but aesculin, gelatin, starch and urea are not. D-Glucose, acetate, citrate, L-malate, pyruvate and succinate are utilized as sole carbon and energy sources, but L-arabinose, cellobiose, D-fructose, D-galactose, maltose, D-mannose, sucrose, trehalose, D-xylose, benzoate, formate, salicin and L-glutamate are not. Susceptible to chloramphenicol, gentamicin, kanamycin, oleandomycin, streptomycin and tetracycline, but not to ampicillin, carbenicillin, cephalothin, lincomycin, neomycin, novobiocin, penicillin G and polymyxin B. In assays with the API ZYM system, alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and valine arylamidase activities are present, but lipase (C 14), cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, bgalactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities are absent. The predominant ubiquinone is Q-10. The major fatty acid (.10 % of the total fatty acids) is C18 : 1v7c. The major polar lipids are phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminolipids and one unidentified glycolipid. The type strain, AH-MY2T (5KCTC 32577T5CECT 8488T), was isolated from a tidal flat on Aphae island, South Korea. The DNA G+C content of the type strain is 56.0 mol%.
ACKNOWLEDGEMENTS This work was supported by Mid-career Researcher Program through a National Research Foundation (NRF) grant from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea (2012R1A2A2A01047369).
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saemankumensis gen. nov., sp. nov., isolated from a tidal flat of the Yellow Sea. Int J Syst Evol Microbiol 59, 48–52.
sp. nov., a polycyclic aromatic hydrocarbon-degrading marine bacterium. Int J Syst Evol Microbiol 60, 1125–1129.
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