International Journal of Systematic and Evolutionary Microbiology (2015), 65, 141–146
DOI 10.1099/ijs.0.069500-0
Litorivivens lipolytica 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, flagellated, aerobic and rod-shaped or ovoid bacterial strain, designated HJTF-7T, was isolated from a tidal flat on the South Sea of South Korea, and its taxonomic position was investigated using a polyphasic approach. Strain HJTF-7T grew optimally at 25 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 HJTF-7T joined the cluster comprising the type strains of species of the genera Spongiibacter and Zhongshania. Strain HJTF-7T exhibited 16S rRNA gene sequence similarities of 90.4–92.5 % to the type strains of species of the genera Spongiibacter and Zhongshania and of less than 91.5 % to the type strains of other recognized species. Strain HJTF-7T contained Q-8 as the predominant ubiquinone. The major fatty acids were iso-C17 : 1v9c, iso-C15 : 0, iso-C17 : 0, iso-C11 : 0 3-OH and C17 : 1v8c and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The fatty acid and polar lipid profiles of strain HJTF-7T were distinct from those of members of the genera Spongiibacter and Zhongshania. The DNA G+C content of strain HJTF-7T was 55.9 mol%. The phylogenetic data and differential chemotaxonomic and other phenotypic properties revealed that strain HJTF-7T represents a novel genus and species within the class Gammaproteobacteria, for which the name Litorivivens lipolytica gen. nov., sp. nov. is proposed. The type strain of Litorivivens lipolytica is HJTF-7T (5KCTC 42157T5CECT 8654T).
From our studies aimed at discovering novel bacteria from marine environments of the South Sea of the Korean peninsula, many new bacterial taxa belonging to the class Proteobacteria have been described (Lee et al., 2013; Park et al., 2013; Park & Yoon, 2013; Yoon et al., 2013a, b). The majority of these novel taxa belong to the family Rhodobacteraceae within the class Alphaproteobacteria (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 HJTF-7T, isolated from a tidal flat at Haenam, which is located at the southernmost point of the Korean peninsula. Comparative 16S rRNA gene sequence analysis indicated that the novel strain forms a deep branch within the class Gammaproteobacteria, showing 16S rRNA gene sequence similarities of less than 92.5 % to any known bacterial taxa. The aim of the present work was to determine the exact taxonomic position of strain HJTF-7T by using polyphasic characterization, which included the determination of chemotaxonomic and other phenotypic properties as well as detailed phylogenetic investigations based on 16S rRNA gene sequences. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain HJTF-7T is KM017973. Two supplementary figures are available with the online Supplementary Material.
069500 G 2015 IUMS
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Tidal flat sediment was collected from Haenam, South Korea, and used as the source sample for the isolation of bacterial strains. Strain HJTF-7T was isolated by the standard dilution plating technique at 25 uC on marine agar 2216 (MA; Becton Dickinson and cultivated routinely under the same culture conditions. Spongiibacter marinus DSM 17750T, Spongiibacter tropicus KCCM 90065T and Zhongshania borealis KCCM 90094T, which were used as reference strains for the analyses of fatty acids and polar lipids and other phenotypic characterization, were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany and the Korean Culture Center of Microorganisms (KCCM), Seoul, South Korea, respectively. The cell morphology, Gram reaction, anaerobic growth and pH range for growth were determined as described by Park et al. (2014). Growth at 4, 10, 15, 20, 25, 30, 35, 37, 40 and 45 uC was measured on MA to measure the optimal temperature and temperature range for growth. Growth at various concentrations of NaCl [0, 0.5 and 1.0– 8.0 % (w/v) at increments of 1.0 %] was investigated by supplementing with appropriate concentrations of NaCl in marine broth 2216 (MB) prepared according to the formula of the Becton Dickinson medium except that NaCl was excluded. The requirement for Mg2+ ions was investigated by using MB, prepared according to the formula of the Becton Dickinson medium, with all of the constituents 141
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present except MgCl2 and MgSO4. Catalase and oxidase activities were determined as described by La´nyı´ (1987). Lipolytic activity was investigated by using tricaprylin (TCN)-MA 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. Hydrolysis of casein, starch, hypoxanthine, L-tyrosine 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 by La´nyı´ (1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea was 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 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 investigated by spreading a bacterial suspension on MA and applying antibiotic discs (Advantec) containing the following (mg per disc unless otherwise stated): ampicillin (10), carbenicillin (100), cefalotin (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); the plates were incubated for 2 days at 25 uC and susceptibility was revealed by the presence of a clear zone greater than 2.0 mm around the antibiotic disc. Enzyme activities were determined, after incubation for 8 h at 25 uC, by using the API ZYM system (bioMe´rieux); the strip was inoculated with cells suspended in artificial seawater from which CaCl2 was excluded.
column. The isoprenoid quinones were eluted with a mixture of methanol/2-propanol (2 : 1, v/v) using a flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 275 nm. For cellular fatty acid analysis, cell masses of strain HJTF-7T, S. marinus DSM 17750T, S. tropicus KCCM 90065T and Z. borealis KCCM 90094T were harvested from MA plates after cultivation for 5 days at 25 uC. The physiological age of the cell masses was standardized by observing the development of colonies on the agar 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 Sherlock Microbial Identification System (version 6.1, MIDI). The fatty acids were analysed by GC (Hewlett Packard 6890) 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 Embley & Wait (1994). 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 HPLC with a reverse-phase YMC ODS-A (25064.6 mm) column. The nucleotides were eluted with a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40 : 1, v/v), at a flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 270 nm.
Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using HPLC with a reverse-phase YMC ODS-A (25064.6 mm)
The morphological, cultural, physiological and biochemical characteristics of strain HJTF-7T are given in the genus and species descriptions (see below) or in Table 1 and Fig. S1 (available in the online Supplementary Material). The almostcomplete 16S rRNA gene sequence of strain HJTF-7T determined in this study comprised 1456 nt, representing approximately 95 % of the Escherichia coli 16S rRNA gene sequence. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain HJTF-7T joined the cluster comprising the type strains of two species of the genus Spongiibacter and four species of the genus Zhongshania with a bootstrap resampling value of 58.4 % (Fig. 1). This tree topology was also found in the trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain HJTF-7T exhibited 16S rRNA gene sequence similarity values of 92.5, 92.4, 91.5, 91.3, 91.3 and 90.4 % to S. marinus HAL40bT, S. tropicus CL-CB221T, Zhongshania guokunii ZS6-22T, Z. borealis CL-AS9T, Zhongshania antarctica ZS5-23T and ‘Zhongshania aliphaticivorans’ SM-2, respectively. It exhibited sequence similarity values of less than 91.5 % to the type strains of other recognized species.
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Cell biomass of strain HJTF-7T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 5 days in MB at 25 uC, and cell biomass of S. marinus DSM 17750T, S. tropicus KCCM 90065T and Z. borealis KCCM 90094T for polar lipid analysis was obtained from cultures under the same conditions. Chromosomal DNA was extracted and purified by the procedure described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize the contamination by 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 analyses were performed as described previously (Yoon et al., 2003).
Litorivivens lipolytica gen. nov., sp. nov.
The predominant isoprenoid quinone detected in strain HJTF-7T was ubiquinone-8 (Q-8), which is the same as that associated with the genus Spongiibacter (Jang et al., 2011). In Table 2, the cellular fatty acid profile of strain HJTF-7T is compared with those of the type strains of two species of the genus Spongiibacter, and Z. borealis, which were grown and analysed under identical conditions in this study. The major fatty acids (.10 % of the total fatty acids) detected in strain HJTF-7T were iso-C17 : 1v9c (24.2 %), iso-C15 : 0 (17.3 %), iso-C17 : 0 (13.8 %), iso-C11 : 0 3-OH (11.8 %) and C17 : 1v8c (11.1 %). The fatty acid profile of strain HJTF-7T was clearly
Table 1. Differential characteristics of strain HJTF-7T and the genera Spongiibacter and Zhongshania Taxa: 1, HJTF-7T; 2, Spongiibacter (data taken from this study unless indicated otherwise); 3, Zhongshania (LO et al., 2014). +, Positive reaction; 2, negative reaction; W, weakly positive reaction; V, variable reaction; ND, not described. All taxa were positive for alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and acid phosphatase activity. All taxa were negative for the utilization of citrate; and cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, a-mannosidase and a-fucosidase activity. Characteristic Growth at: 4 uC 40 uC NaCl requirement for growth Nitrate reduction Hydrolysis of aesculin Utilization of: D-Fructose D-Glucose Maltose D-Xylose Malate Pyruvate Susceptibility to: Ampicillin Carbenicillin Gentamicin Neomycin Oleandomycin Penicillin G Tetracycline Enzyme activity (API ZYM) Lipase (C 14), Valine arylamidase Naphthol-AS-BIphosphohydrolase N-Acetyl-b-glucosaminidase DNA G+C content (mol%)
1
2
2 + 2 2 2
2* +* +* 2* +*
2 + 2 2 2 +
2 2 2 2 2 +
V
+ + 2 2 + + +
2 2 + + 2 2 2
ND
2 2 2
2 2 +
V
2 55.9
3
V
2 V
+ +
V V V V V
ND ND
From the results of the phylogenetic analyses based on 16S rRNA gene sequences, it seems to be inappropriate to assign strain HJTF-7T into one of any recognized genera of the class Gammaproteobacteria (Fig. 1). The 16S rRNA gene sequence similarity values between strain HJTF-7T and other bacterial taxa are sufficient to differentiate this novel strain from previously known genera of the class Gammaproteobacteria. It has recently been proposed by LO et al. (2014) that Spongiibacter borealis should be reclassified as Zhongshania borealis. Strain HJTF-7T was differentiated from the genera Spongiibacter and Zhongshania by differences in phenotypic characteristics, including growth at 4 and 40 uC, NaCl requirements for growth, nitrate reduction, aesculin hydrolysis, utilization of some substrates, susceptibility to some antibiotics and the activity of some enzymes (Table 1). Unfortunately, the type strains of the species Z. antarctica and Z. guokunii were not available or were contaminated in two culture collections and so could not be used in this study. The phylogenetic data and differential chemotaxonomic and other phenotypic properties presented here suggest that strain HJTF-7T constitutes a novel genus and species within the class Gammaproteobacteria, for which the name Litorivivens lipolytica gen. nov., sp. nov. is proposed.
ND ND
Description of Litorivivens gen. nov.
ND
Litorivivens (Li.to.ri.viv9ens. L. n. litus -oris the seashore, coast; L. part. adj. vivens living; N.L. fem. adj. Litorivivens living in seashore).
ND
V
+ or
W
V 2 57.7–69.1* 51.5–53.6
*Data taken from Graeber et al. (2008), Hwang & Cho (2009) and Jang et al. (2011). http://ijs.sgmjournals.org
distinguishable from those of the type strains of two species of the genus Spongiibacter, and Z. borealis, by the differences in the proportions of branched and unsaturated fatty acids (Table 2). The fatty acid profile of strain HJTF-7T was also distinguishable from those of the type strains of the other species of the genus Zhongshania by the differences in the proportions of branched and unsaturated fatty acids (Li et al., 2011; LO et al., 2014). The major polar lipids detected in strain HJTF-7T were phosphatidylethanolamine and phosphatidylglycerol (Fig. S2). The polar lipid profile of strain HJTF-7T was similar to those of the type strains of species of the genera Spongiibacter and Zhongshania in that phosphatidylethanolamine and phosphatidylglycerol were major polar lipids, but distinguishable from them due to the absence of diphosphatidylglycerol (Fig. S2; LO et al., 2014). The DNA G+C content of strain HJTF-7T was 55.9 mol%, a value similar to those of the type strains of the species S. tropicus, Z. borealis, Z. guokunii, Z. antarctica and ‘Z. aliphaticivorans’, but lower than that of the type strain of S. marinus (Table 1; Graeber et al., 2008; Li et al., 2011; LO et al., 2014).
Cells are Gram-stain-negative, aerobic, flagellated and rodshaped or ovoid. Catalase- and oxidase-positive. Nitrate reduction is negative. The predominant ubiquinone is Q-8. The major fatty acids are iso-C17 : 1v9c, iso-C15 : 0, iso-C17 : 0, iso-C11 : 0 3-OH and C17 : 1v8c. The major polar lipids are phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content is 55.9 mol%. The type species is 143
S. Park and others
0.01
Marinimicrobium agarilyticum M18T (AY839870)
99.9
99.9
Marinimicrobium locisalis ISL-43T (EU874388)
50.3
Marinimicrobium koreense M9T (AY839869) Gilvimarinus chinensis QM42T (DQ822530)
53.5
Teredinibacter turnerae T7902T (AY028398)
63.5
Porticoccus litoralis IMCC2115T (EF468719) Saccharophagus degradans 2-40T (AF055269)
77.6
Umboniibacter marinipuniceus KMM 3891T (AB467279) Dasania marina KOPRI 20902T (AY771747) Chromatocurvus halotolerans EG19T (AM691086)
66.6 56.2 94.4
Pseudohaliea rubra CM41_15aT (EU161717) Haliea mediterranea 7SM29T (FN398053) Haliea salexigens 3X/A02/235T (AY576769)
100
Halioglobus pacificus S1-72T (AB602430)
89.7
Halioglobus japonicus S1-36T (AB602427) Zhongshania borealis CL-AS9T (HQ199599) 100
99.5
Zhongshania antarctica ZS5-23T (FJ889619) Zhongshania guokunii ZS6-22T (FJ889678) ‘Zhongshania aliphaticivorans’ SM-2 (KF982857)
Spongiibacter marinus HAL40bT (AM117932) 58.4
99.8
Spongiibacter tropicus CL-CB221T (EF988653) Litorivivens lipolytica HJTF-7T (KM017973) Neptunomonas naphthovorans NAG-2N-126T (AF053734) Thioprofundum lithotrophicum 106T (AB468957) 100
Thioprofundum hispidum gps61T (AB266389) Escherichia coli ATCC 11775T (X80725)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain HJTF-7T and representatives of some 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 generated with the maximum-likelihood and maximum-parsimony algorithms. E. coli ATCC 11775T (GenBank accession no. X80725) was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
Litorivivens lipolytica. A member of the class Gammaproteobacteria, according to 16S rRNA gene sequence analysis.
Cells are Gram-stain-negative, non-spore-forming and rodshaped or ovoid, approximately 0.1–0.3 mm in width and 0.3–1.5 mm in length. Motile by means of a single polar flagellum. Colonies on MA are circular, slightly convex, smooth, glistening, greyish-yellow and 0.5–1.0 mm in diameter after incubation for 5 days at 25 uC. Optimal temperature for growth is 25 uC; growth occurs at 10 and 40 uC, but not at 4 or 45 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 0–6.0 % (w/v) NaCl. Mg2+ ions are not required for growth. Growth does not occur
under anaerobic conditions on MA or on MA supplemented with nitrate. Catalase- and oxidase-positive. Nitrate is not reduced to nitrite. H2S is not produced. Tricaprylin is degraded. Tweens 20, 40, 60 and 80 are hydrolysed, but aesculin, casein, gelatin, hypoxanthine, starch, L-tyrosine, urea and xanthine are not hydrolysed. D-Glucose, acetate and pyruvate are utilized as sole carbon and energy sources, but L-arabinose, cellobiose, D-fructose, D-galactose, maltose, D-mannose, sucrose, trehalose, D-xylose, benzoate, citrate, formate, L-malate, succinate, salicin and L-glutamate are not. Susceptible to ampicillin, carbenicillin, chloramphenicol, oleandomycin, penicillin G, polymyxin B, streptomycin and tetracycline, but not to cefalotin, gentamicin, kanamycin, lincomycin, neomycin and novobiocin. In assays with the API ZYM system, activity of alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and acid phosphatase is present, but activity of lipase (C 14), valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase, a-mannosidase and a-fucosidase
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Description of Litorivivens lipolytica sp. nov. Litorivivens lipolytica [li.po.ly9ti.ca. Gr. n. lipos fat; N.L. fem adj. lytica (from Gr. adj. lytikos dissolving); N.L. fem. adj. lipolytica fat dissolving].
Litorivivens lipolytica gen. nov., sp. nov.
Table 2. Cellular fatty acid composition of strain HJTF-7T and the type strains of species of the genus Spongiibacter and of Zhongshania borealis Strains: 1, HJTF-7T; 2, Spongiibacter marinus DSM 17750T; 3, Spongiibacter tropicus KCCM 90065T; 4, Zhongshania borealis KCCM 90094T. All data obtained from this study. Fatty acids that represented ,0.5 % in all strains were omitted. tr, Trace (,0.5 %); –, not detected. Fatty acid Straight-chain C9 : 0 C10 : 0 C11 : 0 C12 : 0 C14 : 0 C16 : 0 C17 : 0 C18 : 0 Unsaturated C15 : 1v6c C15 : 1v8c C17 : 1v6c C17 : 1v8c C18 : 1v7c C18 : 1v9c Branched iso-C11 : 0 iso-C15 : 0 iso-C15 : 1 F iso-C17 : 0 iso-C17 : 1 v9c iso-C11 : 0 3-OH Hydroxy C10 : 0 3-OH C11 : 0 3-OH C12 : 0 3-OH C12 : 1 3-OH Summed features* 1 3 4
1
2
3
4
Acknowledgements This work was supported by Mid-career Researcher Program through NRF grant from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea (2012R1A2A2A01047369).
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is absent. The predominant ubiquinone is Q-8. The major fatty acids are iso-C17 : 1v9c, iso-C15 : 0, iso-C17 : 0, iso-C11 : 0 3-OH and C17 : 1v8c. The major polar lipids are phosphatidylethanolamine and phosphatidylglycerol. The type strain, HJTF-7T (5KCTC 42157T5CECT 8654T), was isolated from a tidal flat at Haenam, which is located at the southernmost point of South Korea. The genomic DNA G+C content of the type strain is 55.9 mol%. http://ijs.sgmjournals.org
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isolated from seashore sediment. Int J Syst Evol Microbiol 63, 1741–1745.
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romonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505. Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific
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Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali
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aquaemixtae sp. nov., isolated from the junction between the ocean and a freshwater spring. Int J Syst Evol Microbiol 64, 1378–1383. Rappe´, M. S., Vergin, K. & Giovannoni, S. J. (2000). Phylogenetic
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Litorisediminicola beolgyonensis gen. nov., sp. nov., isolated from a coastal sediment. Int J Syst Evol Microbiol 63, 2025–2031. Yoon, J.-H., Park, S. & Jung, Y.-T. (2013b). Aestuariihabitans
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Sasser, M. (1990). Identification of bacteria by gas chromatography of
DOI 10.1099/ijs.0.069500-0
Litorivivens lipolytica 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, flagellated, aerobic and rod-shaped or ovoid bacterial strain, designated HJTF-7T, was isolated from a tidal flat on the South Sea of South Korea, and its taxonomic position was investigated using a polyphasic approach. Strain HJTF-7T grew optimally at 25 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 HJTF-7T joined the cluster comprising the type strains of species of the genera Spongiibacter and Zhongshania. Strain HJTF-7T exhibited 16S rRNA gene sequence similarities of 90.4–92.5 % to the type strains of species of the genera Spongiibacter and Zhongshania and of less than 91.5 % to the type strains of other recognized species. Strain HJTF-7T contained Q-8 as the predominant ubiquinone. The major fatty acids were iso-C17 : 1v9c, iso-C15 : 0, iso-C17 : 0, iso-C11 : 0 3-OH and C17 : 1v8c and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The fatty acid and polar lipid profiles of strain HJTF-7T were distinct from those of members of the genera Spongiibacter and Zhongshania. The DNA G+C content of strain HJTF-7T was 55.9 mol%. The phylogenetic data and differential chemotaxonomic and other phenotypic properties revealed that strain HJTF-7T represents a novel genus and species within the class Gammaproteobacteria, for which the name Litorivivens lipolytica gen. nov., sp. nov. is proposed. The type strain of Litorivivens lipolytica is HJTF-7T (5KCTC 42157T5CECT 8654T).
From our studies aimed at discovering novel bacteria from marine environments of the South Sea of the Korean peninsula, many new bacterial taxa belonging to the class Proteobacteria have been described (Lee et al., 2013; Park et al., 2013; Park & Yoon, 2013; Yoon et al., 2013a, b). The majority of these novel taxa belong to the family Rhodobacteraceae within the class Alphaproteobacteria (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 HJTF-7T, isolated from a tidal flat at Haenam, which is located at the southernmost point of the Korean peninsula. Comparative 16S rRNA gene sequence analysis indicated that the novel strain forms a deep branch within the class Gammaproteobacteria, showing 16S rRNA gene sequence similarities of less than 92.5 % to any known bacterial taxa. The aim of the present work was to determine the exact taxonomic position of strain HJTF-7T by using polyphasic characterization, which included the determination of chemotaxonomic and other phenotypic properties as well as detailed phylogenetic investigations based on 16S rRNA gene sequences. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain HJTF-7T is KM017973. Two supplementary figures are available with the online Supplementary Material.
069500 G 2015 IUMS
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Tidal flat sediment was collected from Haenam, South Korea, and used as the source sample for the isolation of bacterial strains. Strain HJTF-7T was isolated by the standard dilution plating technique at 25 uC on marine agar 2216 (MA; Becton Dickinson and cultivated routinely under the same culture conditions. Spongiibacter marinus DSM 17750T, Spongiibacter tropicus KCCM 90065T and Zhongshania borealis KCCM 90094T, which were used as reference strains for the analyses of fatty acids and polar lipids and other phenotypic characterization, were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany and the Korean Culture Center of Microorganisms (KCCM), Seoul, South Korea, respectively. The cell morphology, Gram reaction, anaerobic growth and pH range for growth were determined as described by Park et al. (2014). Growth at 4, 10, 15, 20, 25, 30, 35, 37, 40 and 45 uC was measured on MA to measure the optimal temperature and temperature range for growth. Growth at various concentrations of NaCl [0, 0.5 and 1.0– 8.0 % (w/v) at increments of 1.0 %] was investigated by supplementing with appropriate concentrations of NaCl in marine broth 2216 (MB) prepared according to the formula of the Becton Dickinson medium except that NaCl was excluded. The requirement for Mg2+ ions was investigated by using MB, prepared according to the formula of the Becton Dickinson medium, with all of the constituents 141
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present except MgCl2 and MgSO4. Catalase and oxidase activities were determined as described by La´nyı´ (1987). Lipolytic activity was investigated by using tricaprylin (TCN)-MA 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. Hydrolysis of casein, starch, hypoxanthine, L-tyrosine 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 by La´nyı´ (1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea was 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 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 investigated by spreading a bacterial suspension on MA and applying antibiotic discs (Advantec) containing the following (mg per disc unless otherwise stated): ampicillin (10), carbenicillin (100), cefalotin (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); the plates were incubated for 2 days at 25 uC and susceptibility was revealed by the presence of a clear zone greater than 2.0 mm around the antibiotic disc. Enzyme activities were determined, after incubation for 8 h at 25 uC, by using the API ZYM system (bioMe´rieux); the strip was inoculated with cells suspended in artificial seawater from which CaCl2 was excluded.
column. The isoprenoid quinones were eluted with a mixture of methanol/2-propanol (2 : 1, v/v) using a flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 275 nm. For cellular fatty acid analysis, cell masses of strain HJTF-7T, S. marinus DSM 17750T, S. tropicus KCCM 90065T and Z. borealis KCCM 90094T were harvested from MA plates after cultivation for 5 days at 25 uC. The physiological age of the cell masses was standardized by observing the development of colonies on the agar 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 Sherlock Microbial Identification System (version 6.1, MIDI). The fatty acids were analysed by GC (Hewlett Packard 6890) 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 Embley & Wait (1994). 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 HPLC with a reverse-phase YMC ODS-A (25064.6 mm) column. The nucleotides were eluted with a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40 : 1, v/v), at a flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 270 nm.
Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using HPLC with a reverse-phase YMC ODS-A (25064.6 mm)
The morphological, cultural, physiological and biochemical characteristics of strain HJTF-7T are given in the genus and species descriptions (see below) or in Table 1 and Fig. S1 (available in the online Supplementary Material). The almostcomplete 16S rRNA gene sequence of strain HJTF-7T determined in this study comprised 1456 nt, representing approximately 95 % of the Escherichia coli 16S rRNA gene sequence. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain HJTF-7T joined the cluster comprising the type strains of two species of the genus Spongiibacter and four species of the genus Zhongshania with a bootstrap resampling value of 58.4 % (Fig. 1). This tree topology was also found in the trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain HJTF-7T exhibited 16S rRNA gene sequence similarity values of 92.5, 92.4, 91.5, 91.3, 91.3 and 90.4 % to S. marinus HAL40bT, S. tropicus CL-CB221T, Zhongshania guokunii ZS6-22T, Z. borealis CL-AS9T, Zhongshania antarctica ZS5-23T and ‘Zhongshania aliphaticivorans’ SM-2, respectively. It exhibited sequence similarity values of less than 91.5 % to the type strains of other recognized species.
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Cell biomass of strain HJTF-7T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 5 days in MB at 25 uC, and cell biomass of S. marinus DSM 17750T, S. tropicus KCCM 90065T and Z. borealis KCCM 90094T for polar lipid analysis was obtained from cultures under the same conditions. Chromosomal DNA was extracted and purified by the procedure described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize the contamination by 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 analyses were performed as described previously (Yoon et al., 2003).
Litorivivens lipolytica gen. nov., sp. nov.
The predominant isoprenoid quinone detected in strain HJTF-7T was ubiquinone-8 (Q-8), which is the same as that associated with the genus Spongiibacter (Jang et al., 2011). In Table 2, the cellular fatty acid profile of strain HJTF-7T is compared with those of the type strains of two species of the genus Spongiibacter, and Z. borealis, which were grown and analysed under identical conditions in this study. The major fatty acids (.10 % of the total fatty acids) detected in strain HJTF-7T were iso-C17 : 1v9c (24.2 %), iso-C15 : 0 (17.3 %), iso-C17 : 0 (13.8 %), iso-C11 : 0 3-OH (11.8 %) and C17 : 1v8c (11.1 %). The fatty acid profile of strain HJTF-7T was clearly
Table 1. Differential characteristics of strain HJTF-7T and the genera Spongiibacter and Zhongshania Taxa: 1, HJTF-7T; 2, Spongiibacter (data taken from this study unless indicated otherwise); 3, Zhongshania (LO et al., 2014). +, Positive reaction; 2, negative reaction; W, weakly positive reaction; V, variable reaction; ND, not described. All taxa were positive for alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and acid phosphatase activity. All taxa were negative for the utilization of citrate; and cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, a-mannosidase and a-fucosidase activity. Characteristic Growth at: 4 uC 40 uC NaCl requirement for growth Nitrate reduction Hydrolysis of aesculin Utilization of: D-Fructose D-Glucose Maltose D-Xylose Malate Pyruvate Susceptibility to: Ampicillin Carbenicillin Gentamicin Neomycin Oleandomycin Penicillin G Tetracycline Enzyme activity (API ZYM) Lipase (C 14), Valine arylamidase Naphthol-AS-BIphosphohydrolase N-Acetyl-b-glucosaminidase DNA G+C content (mol%)
1
2
2 + 2 2 2
2* +* +* 2* +*
2 + 2 2 2 +
2 2 2 2 2 +
V
+ + 2 2 + + +
2 2 + + 2 2 2
ND
2 2 2
2 2 +
V
2 55.9
3
V
2 V
+ +
V V V V V
ND ND
From the results of the phylogenetic analyses based on 16S rRNA gene sequences, it seems to be inappropriate to assign strain HJTF-7T into one of any recognized genera of the class Gammaproteobacteria (Fig. 1). The 16S rRNA gene sequence similarity values between strain HJTF-7T and other bacterial taxa are sufficient to differentiate this novel strain from previously known genera of the class Gammaproteobacteria. It has recently been proposed by LO et al. (2014) that Spongiibacter borealis should be reclassified as Zhongshania borealis. Strain HJTF-7T was differentiated from the genera Spongiibacter and Zhongshania by differences in phenotypic characteristics, including growth at 4 and 40 uC, NaCl requirements for growth, nitrate reduction, aesculin hydrolysis, utilization of some substrates, susceptibility to some antibiotics and the activity of some enzymes (Table 1). Unfortunately, the type strains of the species Z. antarctica and Z. guokunii were not available or were contaminated in two culture collections and so could not be used in this study. The phylogenetic data and differential chemotaxonomic and other phenotypic properties presented here suggest that strain HJTF-7T constitutes a novel genus and species within the class Gammaproteobacteria, for which the name Litorivivens lipolytica gen. nov., sp. nov. is proposed.
ND ND
Description of Litorivivens gen. nov.
ND
Litorivivens (Li.to.ri.viv9ens. L. n. litus -oris the seashore, coast; L. part. adj. vivens living; N.L. fem. adj. Litorivivens living in seashore).
ND
V
+ or
W
V 2 57.7–69.1* 51.5–53.6
*Data taken from Graeber et al. (2008), Hwang & Cho (2009) and Jang et al. (2011). http://ijs.sgmjournals.org
distinguishable from those of the type strains of two species of the genus Spongiibacter, and Z. borealis, by the differences in the proportions of branched and unsaturated fatty acids (Table 2). The fatty acid profile of strain HJTF-7T was also distinguishable from those of the type strains of the other species of the genus Zhongshania by the differences in the proportions of branched and unsaturated fatty acids (Li et al., 2011; LO et al., 2014). The major polar lipids detected in strain HJTF-7T were phosphatidylethanolamine and phosphatidylglycerol (Fig. S2). The polar lipid profile of strain HJTF-7T was similar to those of the type strains of species of the genera Spongiibacter and Zhongshania in that phosphatidylethanolamine and phosphatidylglycerol were major polar lipids, but distinguishable from them due to the absence of diphosphatidylglycerol (Fig. S2; LO et al., 2014). The DNA G+C content of strain HJTF-7T was 55.9 mol%, a value similar to those of the type strains of the species S. tropicus, Z. borealis, Z. guokunii, Z. antarctica and ‘Z. aliphaticivorans’, but lower than that of the type strain of S. marinus (Table 1; Graeber et al., 2008; Li et al., 2011; LO et al., 2014).
Cells are Gram-stain-negative, aerobic, flagellated and rodshaped or ovoid. Catalase- and oxidase-positive. Nitrate reduction is negative. The predominant ubiquinone is Q-8. The major fatty acids are iso-C17 : 1v9c, iso-C15 : 0, iso-C17 : 0, iso-C11 : 0 3-OH and C17 : 1v8c. The major polar lipids are phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content is 55.9 mol%. The type species is 143
S. Park and others
0.01
Marinimicrobium agarilyticum M18T (AY839870)
99.9
99.9
Marinimicrobium locisalis ISL-43T (EU874388)
50.3
Marinimicrobium koreense M9T (AY839869) Gilvimarinus chinensis QM42T (DQ822530)
53.5
Teredinibacter turnerae T7902T (AY028398)
63.5
Porticoccus litoralis IMCC2115T (EF468719) Saccharophagus degradans 2-40T (AF055269)
77.6
Umboniibacter marinipuniceus KMM 3891T (AB467279) Dasania marina KOPRI 20902T (AY771747) Chromatocurvus halotolerans EG19T (AM691086)
66.6 56.2 94.4
Pseudohaliea rubra CM41_15aT (EU161717) Haliea mediterranea 7SM29T (FN398053) Haliea salexigens 3X/A02/235T (AY576769)
100
Halioglobus pacificus S1-72T (AB602430)
89.7
Halioglobus japonicus S1-36T (AB602427) Zhongshania borealis CL-AS9T (HQ199599) 100
99.5
Zhongshania antarctica ZS5-23T (FJ889619) Zhongshania guokunii ZS6-22T (FJ889678) ‘Zhongshania aliphaticivorans’ SM-2 (KF982857)
Spongiibacter marinus HAL40bT (AM117932) 58.4
99.8
Spongiibacter tropicus CL-CB221T (EF988653) Litorivivens lipolytica HJTF-7T (KM017973) Neptunomonas naphthovorans NAG-2N-126T (AF053734) Thioprofundum lithotrophicum 106T (AB468957) 100
Thioprofundum hispidum gps61T (AB266389) Escherichia coli ATCC 11775T (X80725)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain HJTF-7T and representatives of some 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 generated with the maximum-likelihood and maximum-parsimony algorithms. E. coli ATCC 11775T (GenBank accession no. X80725) was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
Litorivivens lipolytica. A member of the class Gammaproteobacteria, according to 16S rRNA gene sequence analysis.
Cells are Gram-stain-negative, non-spore-forming and rodshaped or ovoid, approximately 0.1–0.3 mm in width and 0.3–1.5 mm in length. Motile by means of a single polar flagellum. Colonies on MA are circular, slightly convex, smooth, glistening, greyish-yellow and 0.5–1.0 mm in diameter after incubation for 5 days at 25 uC. Optimal temperature for growth is 25 uC; growth occurs at 10 and 40 uC, but not at 4 or 45 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 0–6.0 % (w/v) NaCl. Mg2+ ions are not required for growth. Growth does not occur
under anaerobic conditions on MA or on MA supplemented with nitrate. Catalase- and oxidase-positive. Nitrate is not reduced to nitrite. H2S is not produced. Tricaprylin is degraded. Tweens 20, 40, 60 and 80 are hydrolysed, but aesculin, casein, gelatin, hypoxanthine, starch, L-tyrosine, urea and xanthine are not hydrolysed. D-Glucose, acetate and pyruvate are utilized as sole carbon and energy sources, but L-arabinose, cellobiose, D-fructose, D-galactose, maltose, D-mannose, sucrose, trehalose, D-xylose, benzoate, citrate, formate, L-malate, succinate, salicin and L-glutamate are not. Susceptible to ampicillin, carbenicillin, chloramphenicol, oleandomycin, penicillin G, polymyxin B, streptomycin and tetracycline, but not to cefalotin, gentamicin, kanamycin, lincomycin, neomycin and novobiocin. In assays with the API ZYM system, activity of alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and acid phosphatase is present, but activity of lipase (C 14), valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase, a-mannosidase and a-fucosidase
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Description of Litorivivens lipolytica sp. nov. Litorivivens lipolytica [li.po.ly9ti.ca. Gr. n. lipos fat; N.L. fem adj. lytica (from Gr. adj. lytikos dissolving); N.L. fem. adj. lipolytica fat dissolving].
Litorivivens lipolytica gen. nov., sp. nov.
Table 2. Cellular fatty acid composition of strain HJTF-7T and the type strains of species of the genus Spongiibacter and of Zhongshania borealis Strains: 1, HJTF-7T; 2, Spongiibacter marinus DSM 17750T; 3, Spongiibacter tropicus KCCM 90065T; 4, Zhongshania borealis KCCM 90094T. All data obtained from this study. Fatty acids that represented ,0.5 % in all strains were omitted. tr, Trace (,0.5 %); –, not detected. Fatty acid Straight-chain C9 : 0 C10 : 0 C11 : 0 C12 : 0 C14 : 0 C16 : 0 C17 : 0 C18 : 0 Unsaturated C15 : 1v6c C15 : 1v8c C17 : 1v6c C17 : 1v8c C18 : 1v7c C18 : 1v9c Branched iso-C11 : 0 iso-C15 : 0 iso-C15 : 1 F iso-C17 : 0 iso-C17 : 1 v9c iso-C11 : 0 3-OH Hydroxy C10 : 0 3-OH C11 : 0 3-OH C12 : 0 3-OH C12 : 1 3-OH Summed features* 1 3 4
1
2
3
4
Acknowledgements This work was supported by Mid-career Researcher Program through NRF grant from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea (2012R1A2A2A01047369).
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