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Pedobacter Lotistagni Sp. Nov. Isolated from Lotus Pond Water

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Pedobacter Lotistagni Sp. Nov. Isolated from Lotus Pond Water Antonie van Leeuwenhoek (2015) 107:951–959 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 / Published online: 23 January 2015 Ó Springer International Publishing Switzerland 2015 Abstract A light-yellow coloured, Gram-stain neg- 23344 T. The DNA G?C content was determined to be ative, rod-shaped, aerobic, non-motile bacterium, 36.8 mol%. In DNA–DNA hybridization tests, the designated THG-DN6.8T, was isolated from a lotus DNA relatedness between strain THG-DN6.8T and its pond near Donghaksa temple in Daejeon, South closest phylogenetic neighbour P. koreensis was Korea. The strain was found to grow well on nutrient found to be below 10 %. The predominant isoprenoid agar, optimally at pH 6.0–7.5, at temperature quinone was identified as menaquinone MK-7 and the 25–28 °C and in the presence of 0.5 % NaCl. Based major polar lipid as phosphatidylethanolamine. The on 16S rRNA gene sequence analysis, strain THG- major fatty acids of strain THG-DN6.8T were identi- T DN6.8 was found to share the highest sequence fied as iso-C15:0,C16:0,C18:0 and C16:1 x6c and/or T similarity with Pedobacter koreensis KCTC 12536 , C16:1 x7c (summed feature 3). On the basis of the followed by Pedobacter glacialis CCTCC AB phenotypic characteristics, genotypic analysis and 2012941T, Pedobacter kyungheensis KACC 16221T, chemotaxonomic characteristics, strain THG-DN6.8T Pedobacter caeni LMG 22862T, Pedobacter insulae is considered to represent a novel species of the genus KCTC 12820T and Pedobacter boryungensis KCTC Pedobacter, for which the name Pedobacter lotistagni sp. nov. is proposed. The type strain is THG-DN6.8T (= KCTC 42229T = JCM 30354T). Hina Singh and Juan Du have contributed equally to this work. Keywords Pedobacter lotistagni Á Gram-staining- Electronic supplementary material The online version of negative Á Menaquinone MK-7 Á 16S rRNA this article (doi:10.1007/s10482-015-0387-5) contains supple- mentary material, which is available to authorized users. H. Singh Á J. Du Á H. T. T. Ngo Á T.-H. Yi (&) Introduction Department of Oriental Medicinal Material & Processing, College of Life Science, Kyung Hee University Global The genus Pedobacter was first described by Steyn Campus, 1732 Deokyoungdaero, Giheung-gu, Yongin-si, et al. (1998) and belongs to the family Sphingobacte- Gyeonggi-do 446-701, Republic of Korea e-mail: [email protected] riaceae and order Sphingobacteriales. Mostly Pedob- acter species have been reported from soils (Yoon et al. K.-Y. Kim 2007; Luo et al. 2010; Zhou et al. 2012; Oh et al. 2013). Department of Genetic Engineering, College of Life Members of this genus are ubiquitous in nature and Science, Kyung Hee University Global Campus, 1732 Deokyoungdaero, Giheung-gu, Yongin-si, some species have been reported from various envi- Gyeonggi-do 446-701, Republic of Korea ronments such as a waterfall (Chung et al. 2014), 123 952 Antonie van Leeuwenhoek (2015) 107:951–959 glacial cryonite (Margesin et al. 2003), glacial water Suspended cells were placed on carbon- and formvar- (Qui et al. 2014), food (Derichs et al. 2014) and coated nickel grids for 30 s and grids were floated on compost (Lee et al. 2009). Currently, there are 45 one drop of 0.1 % (w/v) aqueous uranyl acetate, blotted species of the genus Pedobacter with validly published dry and then viewed with a transmission electron names (http://www.bacterio.net/pedobacter.html). microscope (Model JEM1010; JEOL) at 11,0009 Members of the genus Pedobacter are Gram-stain magnification under standard operating conditions. negative, rod-shaped bacteria, non-motile or motile by Gram-staining was determined using a bioMe´rieux gliding, with MK7 as the predominant isoprenoid Gram stain Kit according to the manufacturer’s quinone and G?C content of 36–45 mol% (Qui et al. instructions. Cells were grown in NB broth for 24 h 2014). More recently, the G?C content range of the at 28 °C and then tested for gliding motility by the genus has been emended by Kook et al. (2014)to hanging-drop technique (Skerman 1967). Growth on 36.0–47.5 mol%. The major fatty acids are iso-C15:0, Reasoner’s 2A agar (R2A; Difco), tryptone soya agar C16:0, iso-C15:1 G, iso-C17:0 C16:1 x6c, C16:1 x7c and (TSA, Oxoid), NA, Luria–Bertani agar (LB; Oxoid), iso-C17:0 3-OH. Pedobacter species mostly contain Marine agar (MB; Difco) and MacConkey agar phosphatidylethanolamine as the major polar lipid (Oxoid) was also assessed at 28 °C for 7 days. Growth (Zhou et al. 2012). In this study, we characterized by at different temperatures (4, 10, 15, 18, 25, 28, 30, 35, means of a polyphasic approach that a new isolate from 37 and 42 °C) and pH conditions (pH 4.0–10.0, at a lotus pond near Donghaksa temple in Daejeon, South intervals of 0.5 pH units) were determined using NB Korea belongs to the genus Pedobacter. The pheno- after 4 days of incubation at 28 °C. The following pH typic and genotypic characterization of the novel buffers were used (final concentration, 100 mM): strain, THG-DN6.8T, are described in this report. 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 Materials and methods was tested using of 0–5 % (w/v) NaCl (at 0.5 % intervals) in NB after 4 days of incubation at 28 °C. Isolation of the bacterial strain Growth was estimated by monitoring the optical density at 600 nm. Anaerobic growth was tested in A water sample was collected from a lotus pond near serum bottles containing NB supplemented with Donghaksa temple in Daejeon in a sterile falcon tube and thioglycolate (0.1 %) and in which the air was transferred to the laboratory. The water sample was substituted with nitrogen gas. Production of flexiru- diluted in 0.85 % (w/v) saline solution, serially diluted up bin-type pigments was determined by the reversible to 10-6 and spread on different media including nutrient colour shift to red, purple or brown when yellow or agar (NA, Difco). The plates were incubated at 28 °Cfor orange colonies are covered with aqueous 20 % KOH 1 week. Single colonies were purified by subculturing solution (Fautz and Reichenbach 1980). Methyl red them on to new NA plates. Firstly, the isolate was and Voges-Proskauer reaction were tested in Clark- routinely cultured on NA at 28 °C and stored as glycerol Lubs’ medium (Scharlau). Catalase activity was suspension 25 % (v/v) at -80 °C. Strain THG-DN6.8T determined by the production of bubbles from 3 % has been deposited in the Korean Collection for Type (v/v) H2O2 solution mixed with freshly grown cells and Cultures (KCTC 42229T) and Japan Collection of oxidase activity was determined by using of 1 % (w/v) Microorganisms (JCM 30354T). For comparative studies, N,N,N,N-tetramethyl-p-phenylenediamine reagent six reference Pedobacter type strains (Table 1)were (Sigma) according to the manufacturer’s instructions. obtained from various culture collection centers. These Tests for hydrolysis were performed on NA containing: reference strains were cultured under the same optimum casein [2 % skim milk (Oxoid)], starch [1 % starch conditions as strain THG-DN6.8T. (Difco)], esculin [0.1 % esculin and 0.02 % ferric citrate (Difco)], Tween 80 [0.01 % CaCl2Á2H2O and Morphological and physiological characterization 1 % Tween 80 (Sigma)], Tween 20 [0.01 % CaCl2- 2H2O and 1 % Tween 20 (Sigma)], chitin [1 % chitin After culturing for 3 days on NA at 28 °C, the from crab shell (Sigma)], L-tyrosine [0.5 % L-tyrosine morphology of the novel strain was examined. (Sigma)], carboxymethyl-cellulose (CMC) [0.1 % 123 Antonie van Leeuwenhoek (2015) 107:951–959 953 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 -----w - N-acetyl-glucosamine w ?????w D-maltose ??-???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 ---??-- DNA G?C Content (mol%) 38.8 38.0 39.4 42.4 42.7 39.4 38.5 All strains are positive for the following characteristics: hydrolysis of esculin and CMC; assimilation of D-Mannose and N-acetyl- glucosamine; 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 123 954 Antonie van Leeuwenhoek (2015) 107:951–959 CMC (Sigma)] and DNA [DNase agar, Scharlau system (Alliance 2690 system, Waters) as described (Spain); DNase activity revealed by flooding the plates previously (Mesbah et al.
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