Antonie van Leeuwenhoek DOI 10.1007/s10482-015-0387-5
ORIGINAL PAPER
Pedobacter lotistagni sp. nov. isolated from lotus pond water Hina Singh • Juan Du • Hien T. T. Ngo Ki-Young Kim • Tae-Hoo Yi
•
Received: 27 November 2014 / Accepted: 13 January 2015 Ó Springer International Publishing Switzerland 2015
Abstract A light-yellow coloured, Gram-stain negative, rod-shaped, aerobic, non-motile bacterium, designated THG-DN6.8T, was isolated from a lotus pond near Donghaksa temple in Daejeon, South Korea. The strain was found to grow well on nutrient agar, optimally at pH 6.0–7.5, at temperature 25–28 °C and in the presence of 0.5 % NaCl. Based on 16S rRNA gene sequence analysis, strain THGDN6.8T was found to share the highest sequence similarity with Pedobacter koreensis KCTC 12536T, followed by Pedobacter glacialis CCTCC AB 2012941T, Pedobacter kyungheensis KACC 16221T, Pedobacter caeni LMG 22862T, Pedobacter insulae KCTC 12820T and Pedobacter boryungensis KCTC
Hina Singh and Juan Du have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0387-5) contains supplementary material, which is available to authorized users. H. Singh J. Du H. T. T. Ngo T.-H. Yi (&) Department of Oriental Medicinal Material & Processing, College of Life Science, Kyung Hee University Global Campus, 1732 Deokyoungdaero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea e-mail: [email protected] K.-Y. Kim Department of Genetic Engineering, College of Life Science, Kyung Hee University Global Campus, 1732 Deokyoungdaero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea
23344 T. The DNA G?C content was determined to be 36.8 mol%. In DNA–DNA hybridization tests, the DNA relatedness between strain THG-DN6.8T and its closest phylogenetic neighbour P. koreensis was found to be below 10 %. The predominant isoprenoid quinone was identified as menaquinone MK-7 and the major polar lipid as phosphatidylethanolamine. The major fatty acids of strain THG-DN6.8T were identified as iso-C15:0, C16:0, C18:0 and C16:1 x6c and/or C16:1 x7c (summed feature 3). On the basis of the phenotypic characteristics, genotypic analysis and chemotaxonomic characteristics, strain THG-DN6.8T is considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter lotistagni sp. nov. is proposed. The type strain is THG-DN6.8T (= KCTC 42229T = JCM 30354T). Keywords Pedobacter lotistagni Gram-stainingnegative Menaquinone MK-7 16S rRNA
Introduction The genus Pedobacter was first described by Steyn et al. (1998) and belongs to the family Sphingobacteriaceae and order Sphingobacteriales. Mostly Pedobacter species have been reported from soils (Yoon et al. 2007; Luo et al. 2010; Zhou et al. 2012; Oh et al. 2013). Members of this genus are ubiquitous in nature and some species have been reported from various environments such as a waterfall (Chung et al. 2014),
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glacial cryonite (Margesin et al. 2003), glacial water (Qui et al. 2014), food (Derichs et al. 2014) and compost (Lee et al. 2009). Currently, there are 45 species of the genus Pedobacter with validly published names (http://www.bacterio.net/pedobacter.html). Members of the genus Pedobacter are Gram-stain negative, rod-shaped bacteria, non-motile or motile by gliding, with MK7 as the predominant isoprenoid quinone and G?C content of 36–45 mol% (Qui et al. 2014). More recently, the G?C content range of the genus has been emended by Kook et al. (2014) to 36.0–47.5 mol%. The major fatty acids are iso-C15:0, C16:0, iso-C15:1 G, iso-C17:0 C16:1 x6c, C16:1 x7c and iso-C17:0 3-OH. Pedobacter species mostly contain phosphatidylethanolamine as the major polar lipid (Zhou et al. 2012). In this study, we characterized by means of a polyphasic approach that a new isolate from a lotus pond near Donghaksa temple in Daejeon, South Korea belongs to the genus Pedobacter. The phenotypic and genotypic characterization of the novel strain, THG-DN6.8T, are described in this report.
Materials and methods Isolation of the bacterial strain A water sample was collected from a lotus pond near Donghaksa temple in Daejeon in a sterile falcon tube and transferred to the laboratory. The water sample was diluted in 0.85 % (w/v) saline solution, serially diluted up to 10-6 and spread on different media including nutrient agar (NA, Difco). The plates were incubated at 28 °C for 1 week. Single colonies were purified by subculturing them on to new NA plates. Firstly, the isolate was routinely cultured on NA at 28 °C and stored as glycerol suspension 25 % (v/v) at -80 °C. Strain THG-DN6.8T has been deposited in the Korean Collection for Type Cultures (KCTC 42229T) and Japan Collection of Microorganisms (JCM 30354T). For comparative studies, six reference Pedobacter type strains (Table 1) were obtained from various culture collection centers. These reference strains were cultured under the same optimum conditions as strain THG-DN6.8T. Morphological and physiological characterization After culturing for 3 days on NA at 28 °C, the morphology of the novel strain was examined.
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Suspended cells were placed on carbon- and formvarcoated nickel grids for 30 s and grids were floated on one drop of 0.1 % (w/v) aqueous uranyl acetate, blotted dry and then viewed with a transmission electron microscope (Model JEM1010; JEOL) at 11,0009 magnification under standard operating conditions. Gram-staining was determined using a bioMe´rieux Gram stain Kit according to the manufacturer’s instructions. Cells were grown in NB broth for 24 h at 28 °C and then tested for gliding motility by the hanging-drop technique (Skerman 1967). Growth on Reasoner’s 2A agar (R2A; Difco), tryptone soya agar (TSA, Oxoid), NA, Luria–Bertani agar (LB; Oxoid), Marine agar (MB; Difco) and MacConkey agar (Oxoid) was also assessed at 28 °C for 7 days. Growth at different temperatures (4, 10, 15, 18, 25, 28, 30, 35, 37 and 42 °C) and pH conditions (pH 4.0–10.0, at intervals of 0.5 pH units) were determined using NB after 4 days of incubation at 28 °C. The following pH buffers were used (final concentration, 100 mM): acetate buffer was used for pH 4.0–6.5 and phosphate buffer was used for pH 7.0–10.0. The pH of NB was confirmed after autoclaving. The requirement of NaCl was tested using of 0–5 % (w/v) NaCl (at 0.5 % intervals) in NB after 4 days of incubation at 28 °C. Growth was estimated by monitoring the optical density at 600 nm. Anaerobic growth was tested in serum bottles containing NB supplemented with thioglycolate (0.1 %) and in which the air was substituted with nitrogen gas. Production of flexirubin-type pigments was determined by the reversible colour shift to red, purple or brown when yellow or orange colonies are covered with aqueous 20 % KOH solution (Fautz and Reichenbach 1980). Methyl red and Voges-Proskauer reaction were tested in ClarkLubs’ medium (Scharlau). Catalase activity was determined by the production of bubbles from 3 % (v/v) H2O2 solution mixed with freshly grown cells and oxidase activity was determined by using of 1 % (w/v) N,N,N,N-tetramethyl-p-phenylenediamine reagent (Sigma) according to the manufacturer’s instructions. Tests for hydrolysis were performed on NA containing: casein [2 % skim milk (Oxoid)], starch [1 % starch (Difco)], esculin [0.1 % esculin and 0.02 % ferric citrate (Difco)], Tween 80 [0.01 % CaCl22H2O and 1 % Tween 80 (Sigma)], Tween 20 [0.01 % CaCl22H2O and 1 % Tween 20 (Sigma)], chitin [1 % chitin from crab shell (Sigma)], L-tyrosine [0.5 % L-tyrosine (Sigma)], carboxymethyl-cellulose (CMC) [0.1 %
Antonie van Leeuwenhoek Table 1 Differential phenotypic characteristics of strain THG-DN6.8T and the reference type strains of the genus Pedobacter Characteristics
1
2
3
4
5
6
7
Nitrate reduction
-
-
-
?
-
-
-
Indole production
-
-
-
-
?
-
-
Motility Hydrolysis of:
-
?
-
-
-
-
-
Tween 20
?
-
?
w
?
?
?
Tween 80
-
-
?
-
-
?
?
L-tyrosine
w
?
?
?
?
?
?
Casein
-
-
-
-
?
-
-
Starch
?
?
?
-
-
?
?
DNA
-
?
-
?
?
?
-
Gelatin
-
-
-
-
?
-
-
Assimilation of (API 20NE) b-galactosidase (PNPG)
?
?
?
?
?
-
-
D-glucose
?
?
-
?
?
w
?
L-arabinose
?
w
-
?
-
w
-
D-mannose
?
?
?
?
?
?
?
D-mannitol
N-acetyl-glucosamine D-maltose
-
-
-
-
-
w
-
w
?
?
?
?
?
w
?
?
-
?
?
?
w
Enzyme activities (API ZYM) Esterase (C4)
?
?
?
-
-
-
-
Esterase lipase (C8)
?
?
?
?
?
-
-
Lipase (C14)
?
?
-
-
-
-
?
Leucine arylamidase
?
?
?
?
?
?
?
Valine arylamidase
?
?
?
?
-
?
?
Cystine arylamidase
?
-
-
-
-
-
?
Trypsin
w
-
?
-
?
-
-
a-chymotrypsin
?
-
?
?
-
?
-
Naphtol-AS-BI-phosphohydrolase
?
?
?
?
-
?
?
a-galactosidase
?
-
?
?
-
-
?
b-galactosidase
?
?
?
?
?
-
-
a-glucosidase
?
-
?
?
-
?
?
b-glucosidase
?
-
?
?
-
-
-
N-Acetyl-b-glucosaminidase a-Mannosidase
? w
? -
? -
? ?
-
? -
? -
a-Fucosidase
-
-
-
?
?
-
-
38.8
38.0
39.4
42.4
42.7
39.4
38.5
DNA G?C Content (mol%)
All strains are positive for the following characteristics: hydrolysis of esculin and CMC; assimilation of D-Mannose and N-acetylglucosamine; oxidase, catalase, leucine arylamidase, alkaline phosphatase and acid phosphatase. All strains were negative for the following characteristics: hydrolysis of urea and chitin; assimilation of arginine dihydrolase, gluconate, caprate, adipate, malate, citrate and phenyl-acetate; glucose acidification and b-glucuronidase Strains: 1, THG-DN6.8T; 2, Pedobacter koreensis KCTC 12536T; 3, Pedobacter glacialis CCTCC AB 2012941T; 4, Pedobacter kyungheensis KACC 16221T; 5, Pedobacter caeni LMG 22862T; 6, Peobacter insulae KCTC 12820T; 7, Pedobacter boryungensis KCTC 23344 T ?, Positive; -, negative; w, weakly positive
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CMC (Sigma)] and DNA [DNase agar, Scharlau (Spain); DNase activity revealed by flooding the plates with 1 N HCl]. Plates were evaluated after 4 days of incubation at 28 °C. Basic chemical tests, carbonsource assimilation and enzyme activities for the novel isolate and all reference strains were conducted using API 20NE (bioMe´rieux) and API ZYM (bioMe´rieux), according to the manufacturer’s instructions. API 20NE were recorded after incubation for 48 h, under the optimal conditions for each strain, while API ZYM was recorded after incubation for 10 h. Molecular characterization and phylogenetic construction Genomic DNA was extracted and purified using a commercial Genomic DNA extraction kit (Solgent, Korea). The 16S rRNA gene was amplified with the universal bacterial primer pair 27F (50 -TACCAGGGTATCTAATCC-30 ) and 1492R (50 0 GGTTACCTTGTTACGACTT-3 ) (Weisburg et al. 1991) and the purified PCR products were sequenced by Solgent Co. Ltd (Daejeon, Korea). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database and EzTaxon e-server [http:// eztaxon-e.ezbiocloud.net/; Kim et al. (2012)]. SeqMan software version 4.1 (DNASTAR, Inc.) was used to compile the nearly complete (1,433 bp) 16S rRNA sequence of strain THG-DN6.8T. The multiple alignments were performed using the CLUSTAL_X program (Thompson et al. 1997) and gaps were edited using the BioEdit program (Hall 1999). The evolutionary distances were calculated using the Kimura two-parameter model (Kimura 1983). The phylogenetic trees were constructed using the neighbourjoining method (Saitou and Nei 1987), maximumparsimony (Fitch 1971) and maximum-likelihood method (Felsenstein 1981) in the MEGA5.2 Program (Tamura et al. 2011), with bootstrap values based on 1,000 replications (Felsenstein 1985). DNA G?C mol% content For determination of the DNA G?C content, genomic DNA of strain THG-DN6.8T was extracted and purified by the method of Moore and Dowhan (1995), then degraded enzymatically into nucleosides using nuclease P1 and alkaline phosphatase (Sigma). The nucleosides were analyzed using a reverse-phase HPLC
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system (Alliance 2690 system, Waters) as described previously (Mesbah et al. 1989) with reversed-phase column SunFireTM C18 (4.6 9 250 mm 9 5 lm), flow rate of 1.0 ml/min, solvent mixture of 200 mM (NH4)H2PO4/acetonitrile (97:3, v/v) as mobile phase and wavelength at 270 nm. The genomic DNA of Escherichia coli strain B (Sigma-Aldrich D4889) was used as a standard. DNA–DNA hybridization DNA–DNA hybridization was performed fluorometrically, according to the method developed by Ezaki et al. (1989) with modifications (Stabili et al. 2008), using photobiotin-labelled DNA probes and micro– dilution wells. DNA–DNA hybridization was carried out to determine levels of relatedness of the novel strain THG-DN6.8T with its closest relative Pedobacter koreensis KACC 15270T. The optimum renaturation temperature (30 °C) was calculated as [(0.51 9 G?C content) ? 47] - 36 (Gillis et al. 1970), where 36 °C is the correction for the presence of 50 % formamide (McConaughy et al. 1969). Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were converted to percentage DNA–DNA relatedness values. Chemotaxonomic characterization For quinone and polar lipids analysis, cells were grown in NA broth at 28 °C, shaken at 160 rpm for 2 day, centrifuged and freeze dried. Respiratory quinones were extracted from 300 mg freeze-dried cells with chloroform:methanol (2:1, v/v), separated using hexane and eluted with hexane:diethyl ether (90:10, v/v), then eluent was evaporated by rotatory evaporation and dissolved in acetone. Menaquinone purification was determined using a reverse-phase HPLC system (Waters, Alliance 2690 system) [wavelength 270 nm, solvent MeOH: isopropanol (7:5, v/v), flow rate; 1.0 ml/min] as previously described (Hiraishi et al. 1996; Collins and Jones 1981; Tamaoka et al. 1983). The polar lipids of strain THG-DN6.8T and its closest reference strain P. koreensis KACC 15270T were analyzed as described by (Minnikin et al. 1984). Two-dimensional thin layer chromatography (2DTLC) performed using TLC Kiesel gel 60 F254 plates
Antonie van Leeuwenhoek
(10 9 10 cm; Merck). The plates were first developed with chloroform:methanol:water (65:25:4, v/v/v) followed by a second development with chloroform: methanol:acetic acid:water (80:12:15:4, v/v/v/v). For the presence of total and specific lipids, the plates were developed using sprays of 5 % molybdatophosphoric acid (total lipids, Sigma), 0.2 % ninhydrin (aminolipids, Sigma) and 2.5 % a-naphthol-sulfuric acid (glycolipids, Sigma) and drying at 120 °C for 5–10 min. TLC plates were also sprayed with molybdenum blue reagent (Sigma) for detecting phospholipids. No heating step is needed for this reagent. For fatty acid analysis, strains THG-DN6.8T and all the reference strains were cultured on NA at 28 °C for 48 h. Cells in exponential growth phase were used. Fatty acids were extracted, methylated and saponified by the methods described for the Sherlock Microbial Identification system (MIDI) and analyzed by capillary GLC (Hewlet Packard 6890) using the TSBA library version 6.1 (Sasser 1990).
Results and discussion The 16S rRNA sequence of strain THG-DN6.8T (NCBI Genbank accession number KM035962) revealed that strain THG-DN6.8T belongs to the genus Pedobacter within the family Sphingobacteriaceae. The highest sequence similarity was found with species of the genus Pedobacter including P. koreensis KACC 15270T (97.3 %), Pedobacter glacialis KACC 11491T (96.7 %), Pedobacter kyungheensis NBRC 106274T (96.2 %), Pedobacter caeni (95.6 %), Pedobacter insulae (95.7 %) and Pedobacter boryungensis (95.6 %). A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed the position of strain THG-DN6.8T clustered within the genus Pedobacter (Fig. 1). A phylogenetic tree built using the maximum-likelihood method is presented as Supplementary Fig. S1. These results suggest that strain THG-DN6.8T represents a novel species within the genus Pedobacter. Colonies of strain THG-DN6.8T on NA agar were observed to be light-yellow, round, sticky and raised with approximate diameter 2–3 mm. Strain THGDN6.8T was found to grow on LB, R2A and NA but not to grow on TSA, MA and MacConkey agar. The strain was found to grows best on NA at temperatures
of 4–37 °C (optimum, 25–28 °C), at pH 6.0–8.5 (optimum, 6.0–7.0) and at 0.5–2.0 % NaCl (optimum, 0.5 %). Phenotypic analysis showed that strain THGDN6.8T cells are Gram-strain negative, non-motile, aerobic and rod shaped with size range approximately 0.3–0.5 9 1.3–2.0 lm (Supplementary Fig. S2). Tests for oxidase and catalase activity were found to be positive. Nitrate reduction and indole production were found to be negative. Strain THG-DN6.8T was found to be unable to hydrolyze Tween 80, casein, chitin, gelatin, urea and DNA but able to hydrolyze Ltyrosine, starch, Tween 20, CMC and esculin. The biochemical and physiological characteristics of strain THG-DN6.8T and the most closely related Pedobacter type strains are given in Table 1. The results suggest that the novel isolate represents a novel species of the genus Pedobacter. Strain THG-DN6.8T was found to contain MK-7 as the predominant isoprenoid quinone, in line with all other members of the genus Pedobacter. As shown in Supplementary Fig. S3, the major polar lipid was identified as phosphatidylethanolamine, which is similar to the polar lipid profile of the reference strain P. koreensis KACC 15270T. In addition, some unidentified aminophospholipids (APL1-3) and unidentified polar lipids (L1-2) were also detected in both the strains, whilst an additional unidentified polar lipid (L3) was observed in the reference strain only. An unidentified aminolipid (AL) was detected in the novel isolate but not in P. koreensis KACC 15270T. The previously reported polar lipid profile of Pedobacter jejuensis KACC 17172T (Kook et al. 2014) is similar in comparison with that of strain THG-DN6.8T as both strains contain phosphatidylethanolamine as the major polar lipid and significant amounts of unidentified aminophospholipids. Thus, the polar lipid profile of novel isolate is in line with those of members of the genus Pedobacter. The major fatty acids of strain THG-DN6.8T were identified as iso-C15:0 (17.4 %), C16:0 (16.6 %), C18:0 (10.2 %) and C16:1 x6c and/or C16:1 x7c (11.5 %) as was seen in the closest related reference type strains (Table 2). The proportions of C16:0 and C18:0 were found to be notably different from those in the closely related species. The DNA G?C content of novel isolate was determined to be 36.8 mol%, which lies within the range (36.0–47.5 mol%) for members of the genus Pedobacter (Kook et al. 2014). The DNA–DNA hybridization value of strain THG-DN6.8T with its
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Fig. 1 Neighbour-joining tree based on 16S rRNA gene sequence analysis, showing relationships between strain THGDN6.8T and members of the genus Pedobacter. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm.
Numbers at nodes indicate bootstrap percentages (based on 1,000 resampled datasets). Bootstrap values less than 80 % are not indicated. Mucilaginibacter auburnensis JM-1070T was used as an out group. Scale bar, 0.01 substitutions per nucleotide position
closest reference strain P. koreensis KACC 15270T was determined to be 9.5 ± 1.5. This low DNA–DNA relatedness suggests that strain THG-DN6.8T is a novel Pedobacter species, following the recommendations of Stackebrandt and Goebel (1994). The characteristics of the novel isolate are consistent with the description of the genus Pedobacter with regard to morphological, biochemical and chemotaxonomic properties. On the basis of the data from this polyphasic study, including analysis of 16S rRNA gene sequences, phylogenetic, phenotypic, biochemical and chemotaxonomic properties, strain THGDN6.8T (= KCTC 42229T = JCM 30354T) is
considered to represent a novel species of the genus Pedobacter, for which name Pedobacter lotistagni sp. nov. is proposed.
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Description of Pedobacter lotistagni sp. nov Pedobacter lotistagni (lo.ti.stag’ni. L. fem. n. lotos lotus; L. neut. n. stagnum pond; N.L. gen. n. lotistagni of a lotus pond). Cells are Gram-staining-negative, rod-shaped, aerobic and non-motile. Cell size is approximately 0.3–0.5 lm in width and 1.3–2.0 lm in length. Colonies are light-yellow, round, sticky, raised and
Antonie van Leeuwenhoek Table 2 Fatty acid profiles of strain THG-DN6.8T and reference type strains of the genus Pedobacter Fatty acid
1
2
3
4
5
6
7
C16:0
16.6
5.4
5.0
7.1
3.1
7.1
3.0
C18:0 Branched
10.2
3.2
tr
2.3
tr
4.6
tr
iso-C15:0
17.5
14.1
7
21.8
19.2
18.6
29.3
iso-C16:0
2.0
2.2
tr
tr
tr
1.3
tr
anteiso-C15:0
1.4
2.2
6.8
tr
2.5
1.9
2.1
iso-C16:1 H
1.4
2.2
tr
tr
tr
3.1
tr 4.2
Straight
Unsaturated C15:1 x6c
2.3
3.6
6.5
tr
ND
4.8
anteiso-C17:1 x9c
6.1
1.6
6.9
tr
ND
1.9
1.2
C18:3 x6c
2.7
tr
ND
tr
ND
tr
ND
iso-C15:0 3OH
2.1
1.8
1.5
3.1
3
2.5
2.5
iso-C16:0 3OH
4.1
9.6
tr
1.4
1.8
8.0
5.4
iso-C17:0 3OH
7.1
4.9
6.5
15.2
2.1
8.6
6.8
11.5
27.6
24.2
29.9
45.2
19.7
17.9
Hydroxy
Summed feature 3*
For fatty acid analysis, novel isolate and all reference strains were cultured on NA at 28 °C for 48 h and cells at exponential growth phase were used for analysis. Summed feature 3* could not be separated by the Microbial Identification System (MIDI). Fatty acids amounting to less than 1.0 % in all strains are not listed. Summed feature 3* consists of C16:1 x6c and/or C16:1 x7c Strains: 1, THG-DN6.8T; 2, Pedobacter koreensis KCTC 12536T; 3, Pedobacter glacialis CCTCC AB 2012941T; 4, Pedobacter kyungheensis KACC 16221T; 5, Pedobacter caeni LMG 22862T; 6, Peobacter insulae KCTC 12820T; 7, Pedobacter boryungensis KCTC 23344T ND, not detected; tr, trace amount (\0.5 %)
have a diameter of 2–3 mm. Positive for oxidase and catalase tests. Grows on R2A, NA and LB but does not grow on TSA, MA and MacConkey agar. Growth occurs at 4–37 °C and the optimum temperature is 25–28 °C. Growth occurs at pH 6.0–8.5 and optimum pH 6.0–7.0. Grows well in the presence of 0.5–2.0 % NaCl (w/v) and the optimum is 0.5 %. Unable to hydrolyze Tween 80, casein, chitin, gelatin, urea and DNA but able to hydrolyze L-tyrosine, starch, Tween 20, CMC and esculin. No flexirubin-type pigments. Negative in the Methyl Red-Voges-Proskauer test, for nitrate reduction and indole production. According to API 20NE testing, positive for assimilation of D-glucose, Larabinose, D-mannose, D-maltose, N-acetylglucosamine and D-maltose but negative for assimilation of Dmannitol, gluconate, caprate, adipate, malate, citrate and phenyl-acetate. According to API ZYM tests, positive for the following enzyme activities: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase,
a-chymotrypsin, acid phosphatase, Naphtol-AS-BIphosphohydrolase, a-galactosidase, a-glucosidase, bglucosidase and N-acetyl-b-glucosaminidase; weakly positive for leucine arylamidase, trypsin, b-galactosidase, a-mannosidase; and negative for b-glucuronidase and a-fucosidase. MK-7 menaquinone is the predominant isoprenoid quinone. The major polar lipid is phosphatidylethanolamine. The major fatty acids are iso-C15:0, C16:0, C18:0 and C16:1 x6c and/or C16:1 x7c (summed feature 3 as defined by MIDI). The DNA G?C content of the type strain is 36.8 mol%. The type strain, THG-DN6.8T (= KCTC 42229T = JCM 30354T), was isolated from a lotus pond in Daejeon, South Korea. The NCBI GenBank accession number for the 16S rRNA gene sequence of strain THG-DN6.8T is KM035962. Acknowledgments This work was conducted under the industrial infrastructure program (No. N0000888) for fundamental technologies which is funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).
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References Chung J, Kang YJ, Jahng KY (2014) Pedobacter pituitosus sp. nov. isolated from waterfall. Int J Syst Evol Microbiol 64:3838–3843 Collins MD, Jones D (1981) Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol Rev 45:316–354 Derichs J, Kampfer P, Lipski A (2014) Pedobacter nutrimenti sp. nov. isolated from chilled food. Int J Syst Evol Microbiol 64:1310–1316 Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39:224–229 Fautz E, Reichenbach H (1980) A simple test for flexirubin-type pigments. FEMS Microbiol Ecol 8:87–91 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791 Fitch WM (1971) Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20:406–416 Gillis M, De Ley J, De Cleene M (1970) The determination of molecular weight of bacterial genome DNA from renaturation rates. Eur J Biochem 12:143–153 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp Ser 41:95–98 Hiraishi A, Ueda Y, Ishihara J, Mori T (1996) Comparative lipoquinone analysis of influent sewage and activated sludge by high–performance liquid chromatography and photodiode array detection. J Gen Appl Microbiol 42:457–469 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721 Kimura M (1983) The neutral theory of molecular evolution. Cambridge University Press, Cambridge Kook M, Park Y, Yi TH (2014) Pedobacter jejuensis sp. nov., isolated from soil of a pine grove, and emended description of the genus Pedobacter. Int J Syst Evol Microbiol 64:1789–1794 Lee HG, Kim SG, Im WT, Oh HM, Lee ST (2009) Pedobacter composti sp. nov., isolated from compost. Int J Syst Evol Microbiol 59:345–349 Luo X, Wang Z, Dai J, Zhang L, Li J, Tang Y, Wang Y, Fang C (2010) Pedobacter glucosidilyticus sp. nov., isolated from dry riverbed soil. Int J Syst Evol Microbiol 60:229–233 Margesin R, Sproer C, Schumann P, Schinner F (2003) Pedobacter cryoconitis sp. nov. a facultative psychrophile from alpine glacier cryoconite. Int J Syst Evol Microbiol 53:1291–1296 McConaughy BL, Laird CD, McCarthy BJ (1969) Nucleic acid reassociation in formamide. Biochemistry 8:3289–3295
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Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G?C content of deoxyribonucleic acid by high–performance liquid chromatography. Int J Syst Bacteriol 39:159–167 Minnikin DE, O’Donnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, Parlett JH (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2:233–241 Moore DD, Dowhan D (1995) Preparation and Analysis of DNA. In: Ausubel FW, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current Protocols in Molecular Biology, Wiley, New York, pp 2–11 Oh HW, Kim BC, Park DS, Jeong WJ, Kim H, Lee KH, Kim SU (2013) Pedobacter luteus sp. nov., isolated from soil. Int J Syst Evol Microbiol 63:1304–1310 Qui X, Qu Z, Jiang F, Ren L, Chang X, Kan W, Fang C, Peng F (2014) Pedobacter huanghensis sp. nov. and Pedobacter glacialis sp. nov., isolated from Arctic glacier foreland. Int J Syst Evol Microbiol 64:2431–2436 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. MIDI Inc, Newark Skerman VBD (1967) A guide to the identification of the genera of bacteria, 2nd edn. Williams and Wilkins, Baltimore Stabili L, Gravili C, Tredici SM, Piraino S, Tala` A, Boero F, Alifano P (2008) Epibiotic Vibrio luminous bacteria isolated from some hydrozoa and bryozoa species. Microb Ecol 56:625–636 Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA–DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849 Steyn PL, Segers P, Vancanneyt M, Sandra P, Kersters K, Joubert JJ (1998) Classification of heparinolytic bacteria into a new genus, Pedobacter, comprising four species: Pedobacter heparinus comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. proposal of the family Sphingobacteriaceae fam. nov. Int J Syst Bacteriol 48:165–177 Tamaoka J, Katayama-Fujiruma A, Kuraishi H (1983) Analysis of bacterial menaquinone mixtures by high performance liquid chromatography. J Appl Bacieriol 54:31–36 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5.2: molecular evolutionary genetics analysis using maximum likelihood evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731– 2739 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703 Yoon MH, Ten LN, Im WT, Lee ST (2007) Pedobacter panaciterrae sp. nov., isolated from soil in South Korea. Int J Syst Evol Microbiol 57:381–386
Antonie van Leeuwenhoek Zhou Z, Jiang F, Wang S, Peng F, Dai J, Li W, Fang C (2012) Pedobacter arcticus sp. nov., a facultative psychrophile isolated from Arctic soil, and emended descriptions of the genus Pedobacter, Pedobacter heparinus, Pedobacter
daechungensis, Pedobacter terricola, Pedobacter glucosidilyticus and Pedobacter lentus. Int J Syst Evol Microbiol 62:1963–1969
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ORIGINAL PAPER
Pedobacter lotistagni sp. nov. isolated from lotus pond water Hina Singh • Juan Du • Hien T. T. Ngo Ki-Young Kim • Tae-Hoo Yi
•
Received: 27 November 2014 / Accepted: 13 January 2015 Ó Springer International Publishing Switzerland 2015
Abstract A light-yellow coloured, Gram-stain negative, rod-shaped, aerobic, non-motile bacterium, designated THG-DN6.8T, was isolated from a lotus pond near Donghaksa temple in Daejeon, South Korea. The strain was found to grow well on nutrient agar, optimally at pH 6.0–7.5, at temperature 25–28 °C and in the presence of 0.5 % NaCl. Based on 16S rRNA gene sequence analysis, strain THGDN6.8T was found to share the highest sequence similarity with Pedobacter koreensis KCTC 12536T, followed by Pedobacter glacialis CCTCC AB 2012941T, Pedobacter kyungheensis KACC 16221T, Pedobacter caeni LMG 22862T, Pedobacter insulae KCTC 12820T and Pedobacter boryungensis KCTC
Hina Singh and Juan Du have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0387-5) contains supplementary material, which is available to authorized users. H. Singh J. Du H. T. T. Ngo T.-H. Yi (&) Department of Oriental Medicinal Material & Processing, College of Life Science, Kyung Hee University Global Campus, 1732 Deokyoungdaero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea e-mail: [email protected] K.-Y. Kim Department of Genetic Engineering, College of Life Science, Kyung Hee University Global Campus, 1732 Deokyoungdaero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea
23344 T. The DNA G?C content was determined to be 36.8 mol%. In DNA–DNA hybridization tests, the DNA relatedness between strain THG-DN6.8T and its closest phylogenetic neighbour P. koreensis was found to be below 10 %. The predominant isoprenoid quinone was identified as menaquinone MK-7 and the major polar lipid as phosphatidylethanolamine. The major fatty acids of strain THG-DN6.8T were identified as iso-C15:0, C16:0, C18:0 and C16:1 x6c and/or C16:1 x7c (summed feature 3). On the basis of the phenotypic characteristics, genotypic analysis and chemotaxonomic characteristics, strain THG-DN6.8T is considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter lotistagni sp. nov. is proposed. The type strain is THG-DN6.8T (= KCTC 42229T = JCM 30354T). Keywords Pedobacter lotistagni Gram-stainingnegative Menaquinone MK-7 16S rRNA
Introduction The genus Pedobacter was first described by Steyn et al. (1998) and belongs to the family Sphingobacteriaceae and order Sphingobacteriales. Mostly Pedobacter species have been reported from soils (Yoon et al. 2007; Luo et al. 2010; Zhou et al. 2012; Oh et al. 2013). Members of this genus are ubiquitous in nature and some species have been reported from various environments such as a waterfall (Chung et al. 2014),
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Antonie van Leeuwenhoek
glacial cryonite (Margesin et al. 2003), glacial water (Qui et al. 2014), food (Derichs et al. 2014) and compost (Lee et al. 2009). Currently, there are 45 species of the genus Pedobacter with validly published names (http://www.bacterio.net/pedobacter.html). Members of the genus Pedobacter are Gram-stain negative, rod-shaped bacteria, non-motile or motile by gliding, with MK7 as the predominant isoprenoid quinone and G?C content of 36–45 mol% (Qui et al. 2014). More recently, the G?C content range of the genus has been emended by Kook et al. (2014) to 36.0–47.5 mol%. The major fatty acids are iso-C15:0, C16:0, iso-C15:1 G, iso-C17:0 C16:1 x6c, C16:1 x7c and iso-C17:0 3-OH. Pedobacter species mostly contain phosphatidylethanolamine as the major polar lipid (Zhou et al. 2012). In this study, we characterized by means of a polyphasic approach that a new isolate from a lotus pond near Donghaksa temple in Daejeon, South Korea belongs to the genus Pedobacter. The phenotypic and genotypic characterization of the novel strain, THG-DN6.8T, are described in this report.
Materials and methods Isolation of the bacterial strain A water sample was collected from a lotus pond near Donghaksa temple in Daejeon in a sterile falcon tube and transferred to the laboratory. The water sample was diluted in 0.85 % (w/v) saline solution, serially diluted up to 10-6 and spread on different media including nutrient agar (NA, Difco). The plates were incubated at 28 °C for 1 week. Single colonies were purified by subculturing them on to new NA plates. Firstly, the isolate was routinely cultured on NA at 28 °C and stored as glycerol suspension 25 % (v/v) at -80 °C. Strain THG-DN6.8T has been deposited in the Korean Collection for Type Cultures (KCTC 42229T) and Japan Collection of Microorganisms (JCM 30354T). For comparative studies, six reference Pedobacter type strains (Table 1) were obtained from various culture collection centers. These reference strains were cultured under the same optimum conditions as strain THG-DN6.8T. Morphological and physiological characterization After culturing for 3 days on NA at 28 °C, the morphology of the novel strain was examined.
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Suspended cells were placed on carbon- and formvarcoated nickel grids for 30 s and grids were floated on one drop of 0.1 % (w/v) aqueous uranyl acetate, blotted dry and then viewed with a transmission electron microscope (Model JEM1010; JEOL) at 11,0009 magnification under standard operating conditions. Gram-staining was determined using a bioMe´rieux Gram stain Kit according to the manufacturer’s instructions. Cells were grown in NB broth for 24 h at 28 °C and then tested for gliding motility by the hanging-drop technique (Skerman 1967). Growth on Reasoner’s 2A agar (R2A; Difco), tryptone soya agar (TSA, Oxoid), NA, Luria–Bertani agar (LB; Oxoid), Marine agar (MB; Difco) and MacConkey agar (Oxoid) was also assessed at 28 °C for 7 days. Growth at different temperatures (4, 10, 15, 18, 25, 28, 30, 35, 37 and 42 °C) and pH conditions (pH 4.0–10.0, at intervals of 0.5 pH units) were determined using NB after 4 days of incubation at 28 °C. The following pH buffers were used (final concentration, 100 mM): acetate buffer was used for pH 4.0–6.5 and phosphate buffer was used for pH 7.0–10.0. The pH of NB was confirmed after autoclaving. The requirement of NaCl was tested using of 0–5 % (w/v) NaCl (at 0.5 % intervals) in NB after 4 days of incubation at 28 °C. Growth was estimated by monitoring the optical density at 600 nm. Anaerobic growth was tested in serum bottles containing NB supplemented with thioglycolate (0.1 %) and in which the air was substituted with nitrogen gas. Production of flexirubin-type pigments was determined by the reversible colour shift to red, purple or brown when yellow or orange colonies are covered with aqueous 20 % KOH solution (Fautz and Reichenbach 1980). Methyl red and Voges-Proskauer reaction were tested in ClarkLubs’ medium (Scharlau). Catalase activity was determined by the production of bubbles from 3 % (v/v) H2O2 solution mixed with freshly grown cells and oxidase activity was determined by using of 1 % (w/v) N,N,N,N-tetramethyl-p-phenylenediamine reagent (Sigma) according to the manufacturer’s instructions. Tests for hydrolysis were performed on NA containing: casein [2 % skim milk (Oxoid)], starch [1 % starch (Difco)], esculin [0.1 % esculin and 0.02 % ferric citrate (Difco)], Tween 80 [0.01 % CaCl22H2O and 1 % Tween 80 (Sigma)], Tween 20 [0.01 % CaCl22H2O and 1 % Tween 20 (Sigma)], chitin [1 % chitin from crab shell (Sigma)], L-tyrosine [0.5 % L-tyrosine (Sigma)], carboxymethyl-cellulose (CMC) [0.1 %
Antonie van Leeuwenhoek Table 1 Differential phenotypic characteristics of strain THG-DN6.8T and the reference type strains of the genus Pedobacter Characteristics
1
2
3
4
5
6
7
Nitrate reduction
-
-
-
?
-
-
-
Indole production
-
-
-
-
?
-
-
Motility Hydrolysis of:
-
?
-
-
-
-
-
Tween 20
?
-
?
w
?
?
?
Tween 80
-
-
?
-
-
?
?
L-tyrosine
w
?
?
?
?
?
?
Casein
-
-
-
-
?
-
-
Starch
?
?
?
-
-
?
?
DNA
-
?
-
?
?
?
-
Gelatin
-
-
-
-
?
-
-
Assimilation of (API 20NE) b-galactosidase (PNPG)
?
?
?
?
?
-
-
D-glucose
?
?
-
?
?
w
?
L-arabinose
?
w
-
?
-
w
-
D-mannose
?
?
?
?
?
?
?
D-mannitol
N-acetyl-glucosamine D-maltose
-
-
-
-
-
w
-
w
?
?
?
?
?
w
?
?
-
?
?
?
w
Enzyme activities (API ZYM) Esterase (C4)
?
?
?
-
-
-
-
Esterase lipase (C8)
?
?
?
?
?
-
-
Lipase (C14)
?
?
-
-
-
-
?
Leucine arylamidase
?
?
?
?
?
?
?
Valine arylamidase
?
?
?
?
-
?
?
Cystine arylamidase
?
-
-
-
-
-
?
Trypsin
w
-
?
-
?
-
-
a-chymotrypsin
?
-
?
?
-
?
-
Naphtol-AS-BI-phosphohydrolase
?
?
?
?
-
?
?
a-galactosidase
?
-
?
?
-
-
?
b-galactosidase
?
?
?
?
?
-
-
a-glucosidase
?
-
?
?
-
?
?
b-glucosidase
?
-
?
?
-
-
-
N-Acetyl-b-glucosaminidase a-Mannosidase
? w
? -
? -
? ?
-
? -
? -
a-Fucosidase
-
-
-
?
?
-
-
38.8
38.0
39.4
42.4
42.7
39.4
38.5
DNA G?C Content (mol%)
All strains are positive for the following characteristics: hydrolysis of esculin and CMC; assimilation of D-Mannose and N-acetylglucosamine; oxidase, catalase, leucine arylamidase, alkaline phosphatase and acid phosphatase. All strains were negative for the following characteristics: hydrolysis of urea and chitin; assimilation of arginine dihydrolase, gluconate, caprate, adipate, malate, citrate and phenyl-acetate; glucose acidification and b-glucuronidase Strains: 1, THG-DN6.8T; 2, Pedobacter koreensis KCTC 12536T; 3, Pedobacter glacialis CCTCC AB 2012941T; 4, Pedobacter kyungheensis KACC 16221T; 5, Pedobacter caeni LMG 22862T; 6, Peobacter insulae KCTC 12820T; 7, Pedobacter boryungensis KCTC 23344 T ?, Positive; -, negative; w, weakly positive
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Antonie van Leeuwenhoek
CMC (Sigma)] and DNA [DNase agar, Scharlau (Spain); DNase activity revealed by flooding the plates with 1 N HCl]. Plates were evaluated after 4 days of incubation at 28 °C. Basic chemical tests, carbonsource assimilation and enzyme activities for the novel isolate and all reference strains were conducted using API 20NE (bioMe´rieux) and API ZYM (bioMe´rieux), according to the manufacturer’s instructions. API 20NE were recorded after incubation for 48 h, under the optimal conditions for each strain, while API ZYM was recorded after incubation for 10 h. Molecular characterization and phylogenetic construction Genomic DNA was extracted and purified using a commercial Genomic DNA extraction kit (Solgent, Korea). The 16S rRNA gene was amplified with the universal bacterial primer pair 27F (50 -TACCAGGGTATCTAATCC-30 ) and 1492R (50 0 GGTTACCTTGTTACGACTT-3 ) (Weisburg et al. 1991) and the purified PCR products were sequenced by Solgent Co. Ltd (Daejeon, Korea). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database and EzTaxon e-server [http:// eztaxon-e.ezbiocloud.net/; Kim et al. (2012)]. SeqMan software version 4.1 (DNASTAR, Inc.) was used to compile the nearly complete (1,433 bp) 16S rRNA sequence of strain THG-DN6.8T. The multiple alignments were performed using the CLUSTAL_X program (Thompson et al. 1997) and gaps were edited using the BioEdit program (Hall 1999). The evolutionary distances were calculated using the Kimura two-parameter model (Kimura 1983). The phylogenetic trees were constructed using the neighbourjoining method (Saitou and Nei 1987), maximumparsimony (Fitch 1971) and maximum-likelihood method (Felsenstein 1981) in the MEGA5.2 Program (Tamura et al. 2011), with bootstrap values based on 1,000 replications (Felsenstein 1985). DNA G?C mol% content For determination of the DNA G?C content, genomic DNA of strain THG-DN6.8T was extracted and purified by the method of Moore and Dowhan (1995), then degraded enzymatically into nucleosides using nuclease P1 and alkaline phosphatase (Sigma). The nucleosides were analyzed using a reverse-phase HPLC
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system (Alliance 2690 system, Waters) as described previously (Mesbah et al. 1989) with reversed-phase column SunFireTM C18 (4.6 9 250 mm 9 5 lm), flow rate of 1.0 ml/min, solvent mixture of 200 mM (NH4)H2PO4/acetonitrile (97:3, v/v) as mobile phase and wavelength at 270 nm. The genomic DNA of Escherichia coli strain B (Sigma-Aldrich D4889) was used as a standard. DNA–DNA hybridization DNA–DNA hybridization was performed fluorometrically, according to the method developed by Ezaki et al. (1989) with modifications (Stabili et al. 2008), using photobiotin-labelled DNA probes and micro– dilution wells. DNA–DNA hybridization was carried out to determine levels of relatedness of the novel strain THG-DN6.8T with its closest relative Pedobacter koreensis KACC 15270T. The optimum renaturation temperature (30 °C) was calculated as [(0.51 9 G?C content) ? 47] - 36 (Gillis et al. 1970), where 36 °C is the correction for the presence of 50 % formamide (McConaughy et al. 1969). Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were converted to percentage DNA–DNA relatedness values. Chemotaxonomic characterization For quinone and polar lipids analysis, cells were grown in NA broth at 28 °C, shaken at 160 rpm for 2 day, centrifuged and freeze dried. Respiratory quinones were extracted from 300 mg freeze-dried cells with chloroform:methanol (2:1, v/v), separated using hexane and eluted with hexane:diethyl ether (90:10, v/v), then eluent was evaporated by rotatory evaporation and dissolved in acetone. Menaquinone purification was determined using a reverse-phase HPLC system (Waters, Alliance 2690 system) [wavelength 270 nm, solvent MeOH: isopropanol (7:5, v/v), flow rate; 1.0 ml/min] as previously described (Hiraishi et al. 1996; Collins and Jones 1981; Tamaoka et al. 1983). The polar lipids of strain THG-DN6.8T and its closest reference strain P. koreensis KACC 15270T were analyzed as described by (Minnikin et al. 1984). Two-dimensional thin layer chromatography (2DTLC) performed using TLC Kiesel gel 60 F254 plates
Antonie van Leeuwenhoek
(10 9 10 cm; Merck). The plates were first developed with chloroform:methanol:water (65:25:4, v/v/v) followed by a second development with chloroform: methanol:acetic acid:water (80:12:15:4, v/v/v/v). For the presence of total and specific lipids, the plates were developed using sprays of 5 % molybdatophosphoric acid (total lipids, Sigma), 0.2 % ninhydrin (aminolipids, Sigma) and 2.5 % a-naphthol-sulfuric acid (glycolipids, Sigma) and drying at 120 °C for 5–10 min. TLC plates were also sprayed with molybdenum blue reagent (Sigma) for detecting phospholipids. No heating step is needed for this reagent. For fatty acid analysis, strains THG-DN6.8T and all the reference strains were cultured on NA at 28 °C for 48 h. Cells in exponential growth phase were used. Fatty acids were extracted, methylated and saponified by the methods described for the Sherlock Microbial Identification system (MIDI) and analyzed by capillary GLC (Hewlet Packard 6890) using the TSBA library version 6.1 (Sasser 1990).
Results and discussion The 16S rRNA sequence of strain THG-DN6.8T (NCBI Genbank accession number KM035962) revealed that strain THG-DN6.8T belongs to the genus Pedobacter within the family Sphingobacteriaceae. The highest sequence similarity was found with species of the genus Pedobacter including P. koreensis KACC 15270T (97.3 %), Pedobacter glacialis KACC 11491T (96.7 %), Pedobacter kyungheensis NBRC 106274T (96.2 %), Pedobacter caeni (95.6 %), Pedobacter insulae (95.7 %) and Pedobacter boryungensis (95.6 %). A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed the position of strain THG-DN6.8T clustered within the genus Pedobacter (Fig. 1). A phylogenetic tree built using the maximum-likelihood method is presented as Supplementary Fig. S1. These results suggest that strain THG-DN6.8T represents a novel species within the genus Pedobacter. Colonies of strain THG-DN6.8T on NA agar were observed to be light-yellow, round, sticky and raised with approximate diameter 2–3 mm. Strain THGDN6.8T was found to grow on LB, R2A and NA but not to grow on TSA, MA and MacConkey agar. The strain was found to grows best on NA at temperatures
of 4–37 °C (optimum, 25–28 °C), at pH 6.0–8.5 (optimum, 6.0–7.0) and at 0.5–2.0 % NaCl (optimum, 0.5 %). Phenotypic analysis showed that strain THGDN6.8T cells are Gram-strain negative, non-motile, aerobic and rod shaped with size range approximately 0.3–0.5 9 1.3–2.0 lm (Supplementary Fig. S2). Tests for oxidase and catalase activity were found to be positive. Nitrate reduction and indole production were found to be negative. Strain THG-DN6.8T was found to be unable to hydrolyze Tween 80, casein, chitin, gelatin, urea and DNA but able to hydrolyze Ltyrosine, starch, Tween 20, CMC and esculin. The biochemical and physiological characteristics of strain THG-DN6.8T and the most closely related Pedobacter type strains are given in Table 1. The results suggest that the novel isolate represents a novel species of the genus Pedobacter. Strain THG-DN6.8T was found to contain MK-7 as the predominant isoprenoid quinone, in line with all other members of the genus Pedobacter. As shown in Supplementary Fig. S3, the major polar lipid was identified as phosphatidylethanolamine, which is similar to the polar lipid profile of the reference strain P. koreensis KACC 15270T. In addition, some unidentified aminophospholipids (APL1-3) and unidentified polar lipids (L1-2) were also detected in both the strains, whilst an additional unidentified polar lipid (L3) was observed in the reference strain only. An unidentified aminolipid (AL) was detected in the novel isolate but not in P. koreensis KACC 15270T. The previously reported polar lipid profile of Pedobacter jejuensis KACC 17172T (Kook et al. 2014) is similar in comparison with that of strain THG-DN6.8T as both strains contain phosphatidylethanolamine as the major polar lipid and significant amounts of unidentified aminophospholipids. Thus, the polar lipid profile of novel isolate is in line with those of members of the genus Pedobacter. The major fatty acids of strain THG-DN6.8T were identified as iso-C15:0 (17.4 %), C16:0 (16.6 %), C18:0 (10.2 %) and C16:1 x6c and/or C16:1 x7c (11.5 %) as was seen in the closest related reference type strains (Table 2). The proportions of C16:0 and C18:0 were found to be notably different from those in the closely related species. The DNA G?C content of novel isolate was determined to be 36.8 mol%, which lies within the range (36.0–47.5 mol%) for members of the genus Pedobacter (Kook et al. 2014). The DNA–DNA hybridization value of strain THG-DN6.8T with its
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Antonie van Leeuwenhoek
Fig. 1 Neighbour-joining tree based on 16S rRNA gene sequence analysis, showing relationships between strain THGDN6.8T and members of the genus Pedobacter. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm.
Numbers at nodes indicate bootstrap percentages (based on 1,000 resampled datasets). Bootstrap values less than 80 % are not indicated. Mucilaginibacter auburnensis JM-1070T was used as an out group. Scale bar, 0.01 substitutions per nucleotide position
closest reference strain P. koreensis KACC 15270T was determined to be 9.5 ± 1.5. This low DNA–DNA relatedness suggests that strain THG-DN6.8T is a novel Pedobacter species, following the recommendations of Stackebrandt and Goebel (1994). The characteristics of the novel isolate are consistent with the description of the genus Pedobacter with regard to morphological, biochemical and chemotaxonomic properties. On the basis of the data from this polyphasic study, including analysis of 16S rRNA gene sequences, phylogenetic, phenotypic, biochemical and chemotaxonomic properties, strain THGDN6.8T (= KCTC 42229T = JCM 30354T) is
considered to represent a novel species of the genus Pedobacter, for which name Pedobacter lotistagni sp. nov. is proposed.
123
Description of Pedobacter lotistagni sp. nov Pedobacter lotistagni (lo.ti.stag’ni. L. fem. n. lotos lotus; L. neut. n. stagnum pond; N.L. gen. n. lotistagni of a lotus pond). Cells are Gram-staining-negative, rod-shaped, aerobic and non-motile. Cell size is approximately 0.3–0.5 lm in width and 1.3–2.0 lm in length. Colonies are light-yellow, round, sticky, raised and
Antonie van Leeuwenhoek Table 2 Fatty acid profiles of strain THG-DN6.8T and reference type strains of the genus Pedobacter Fatty acid
1
2
3
4
5
6
7
C16:0
16.6
5.4
5.0
7.1
3.1
7.1
3.0
C18:0 Branched
10.2
3.2
tr
2.3
tr
4.6
tr
iso-C15:0
17.5
14.1
7
21.8
19.2
18.6
29.3
iso-C16:0
2.0
2.2
tr
tr
tr
1.3
tr
anteiso-C15:0
1.4
2.2
6.8
tr
2.5
1.9
2.1
iso-C16:1 H
1.4
2.2
tr
tr
tr
3.1
tr 4.2
Straight
Unsaturated C15:1 x6c
2.3
3.6
6.5
tr
ND
4.8
anteiso-C17:1 x9c
6.1
1.6
6.9
tr
ND
1.9
1.2
C18:3 x6c
2.7
tr
ND
tr
ND
tr
ND
iso-C15:0 3OH
2.1
1.8
1.5
3.1
3
2.5
2.5
iso-C16:0 3OH
4.1
9.6
tr
1.4
1.8
8.0
5.4
iso-C17:0 3OH
7.1
4.9
6.5
15.2
2.1
8.6
6.8
11.5
27.6
24.2
29.9
45.2
19.7
17.9
Hydroxy
Summed feature 3*
For fatty acid analysis, novel isolate and all reference strains were cultured on NA at 28 °C for 48 h and cells at exponential growth phase were used for analysis. Summed feature 3* could not be separated by the Microbial Identification System (MIDI). Fatty acids amounting to less than 1.0 % in all strains are not listed. Summed feature 3* consists of C16:1 x6c and/or C16:1 x7c Strains: 1, THG-DN6.8T; 2, Pedobacter koreensis KCTC 12536T; 3, Pedobacter glacialis CCTCC AB 2012941T; 4, Pedobacter kyungheensis KACC 16221T; 5, Pedobacter caeni LMG 22862T; 6, Peobacter insulae KCTC 12820T; 7, Pedobacter boryungensis KCTC 23344T ND, not detected; tr, trace amount (\0.5 %)
have a diameter of 2–3 mm. Positive for oxidase and catalase tests. Grows on R2A, NA and LB but does not grow on TSA, MA and MacConkey agar. Growth occurs at 4–37 °C and the optimum temperature is 25–28 °C. Growth occurs at pH 6.0–8.5 and optimum pH 6.0–7.0. Grows well in the presence of 0.5–2.0 % NaCl (w/v) and the optimum is 0.5 %. Unable to hydrolyze Tween 80, casein, chitin, gelatin, urea and DNA but able to hydrolyze L-tyrosine, starch, Tween 20, CMC and esculin. No flexirubin-type pigments. Negative in the Methyl Red-Voges-Proskauer test, for nitrate reduction and indole production. According to API 20NE testing, positive for assimilation of D-glucose, Larabinose, D-mannose, D-maltose, N-acetylglucosamine and D-maltose but negative for assimilation of Dmannitol, gluconate, caprate, adipate, malate, citrate and phenyl-acetate. According to API ZYM tests, positive for the following enzyme activities: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase,
a-chymotrypsin, acid phosphatase, Naphtol-AS-BIphosphohydrolase, a-galactosidase, a-glucosidase, bglucosidase and N-acetyl-b-glucosaminidase; weakly positive for leucine arylamidase, trypsin, b-galactosidase, a-mannosidase; and negative for b-glucuronidase and a-fucosidase. MK-7 menaquinone is the predominant isoprenoid quinone. The major polar lipid is phosphatidylethanolamine. The major fatty acids are iso-C15:0, C16:0, C18:0 and C16:1 x6c and/or C16:1 x7c (summed feature 3 as defined by MIDI). The DNA G?C content of the type strain is 36.8 mol%. The type strain, THG-DN6.8T (= KCTC 42229T = JCM 30354T), was isolated from a lotus pond in Daejeon, South Korea. The NCBI GenBank accession number for the 16S rRNA gene sequence of strain THG-DN6.8T is KM035962. Acknowledgments This work was conducted under the industrial infrastructure program (No. N0000888) for fundamental technologies which is funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).
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