Flavobacterium lacus sp. nov., isolated from a high-altitude lake, and emended description of Flavobacterium filum

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1 International Journal of Systematic and Evolutionary Microbiology (2014), 64, DOI /ijs Flavobacterium lacus sp. nov., isolated from a high-altitude lake, and emended description of Flavobacterium filum Aihua Li, Hongcan Liu, Bingda Sun, Yuguang Zhou and Yuhua Xin Correspondence Yu-Guang Zhou zhouyg@im.ac.cn Yu-Hua Xin xinyh@im.ac.cn China General Microbiological Culture Collection Center and State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 101, PR China Two Gram-stain-negative, non-motile, rod-shaped bacterial strains, designated NP180 T and NR80, were isolated from water of Nam Co Lake, located in Tibet, China. Growth of strains NP180 T and NR80 occurred at C and at ph (optima, C and ph ). The 16S rrna gene sequence similarity to the phylogenetically closest related strains, Flavobacterium filum EMB 34 T, F. ponti GSW-R14 T and F. gelidilacus LMG T, was 95.1, 94.8 and 94.6 %, respectively. The predominant fatty acids were iso-c 15 : 0, iso-c 15 : 1 G, iso-c 17 : 0 3-OH and summed feature 9 (comprising iso-c 17 : 1 v9c and/or 10-methyl C 16 : 0 ). The major menaquinone of the two strains was menaquinone 6 (MK-6). Phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid were the major polar lipids in both strains. The G+C contents of the genomic DNA were 34.9 and 35.1 mol%, respectively, for strains NP180 T and NR80. DNA DNA relatedness between strains NP180 T and NR80 was 99 %, indicating that they belong to the same species. According to phylogenetic inference and phenotypic characteristics, a novel species, Flavobacterium lacus sp. nov., is proposed. The type strain is NP180 T (5CGMCC T 5NBRC T ). An emended description of Flavobacterium filum is also provided. The genus Flavobacterium, the type genus of the family Flavobacteriaceae, was proposed by Bergey et al. (1923) and its description has subsequently been emended repeatedly (Bernardet et al., 1996; Dong et al., 2013; Kang et al., 2013). The emended genus Flavobacterium contains bacteria that are Gram-stain-negative rods, motile by gliding or nonmotile, chemo-organotrophs and aerobes, generally catalase- and oxidase-positive, produce yellow colonies on agar and produce carotenoid or flexirubin-type pigments or both (Bernardet et al., 1996; Reichenbach, 1989). All species contain menaquinone 6 (MK-6) as their only or major respiratory quinone (Bernardet, 2011). The DNA G+C content ranges from 30 to 41 mol%, except Flavobacterium caeni, with a DNA G+C content of 52 mol% (Liu et al., 2010). The major polar lipid is phosphatidylethanolamine, and the predominant fatty acids are C 15 : 0, iso-c 15 : 0 and iso-c 15 : 1 G (Bernardet & Bowman, 2006, 2011). At the time of writing, 122 species were included in the genus Flavobacterium, with Flavobacterium aquatile as the type species ( The GenBank/EMBL/DDBJ accession numbers for the 16S rrna gene sequences of strains NP180 T and NR80 are KC and KC Three supplementary figures are available with the online version of this paper. html). Strains of the genus Flavobacterium occur in various habitats such as seawater (Yoon et al., 2011), marine sediment (Kaur et al., 2012), fresh water (Chun et al., 2013; Subhash et al., 2013), wastewater, soil (Lim et al., 2011; Liu et al., 2008), Antarctic lakes (McCammon et al., 1998; Van Trappen et al., 2003, 2004, 2005), glaciers (Dong et al., 2012; Xu et al., 2011; Zhang et al., 2006), salmonid fish (Kämpfer et al., 2012) and the guts of earthworms. Owing to the continuous exploration of extreme environments, the genus has expanded considerably, especially with psychrophilic and psychrotolerant species. Among published species of the genus Flavobacterium, 25 are coldadapted organisms, of which 14 were isolated from the Antarctic (Humphry et al., 2001; McCammon & Bowman, 2000; McCammon et al., 1998; Van Trappen et al., 2003, 2004, 2005; Yi & Chun, 2006; Yi et al., 2005) and eight from glaciers in China (Dong et al., 2012; Xin et al., 2009; Xu et al., 2011; Zhang et al., 2006; Zhu et al., 2003, 2013). Strains NP180 T and NR80 were isolated from Nam Co lake located in Tibet in the west of China, at an altitude of 4718 m (30u 309 N90u 169 E); the annual mean water temperature is uc. Samples of lake water were collected and 10-fold dilutions were plated on PYG agar (5.0 g Bacto peptone, 0.2 g yeast extract, 5.0 g glucose, 3.0 g beef extract, 0.5 g NaCl, 1.5 g MgSO 4.7H 2 O, G 2014 China General Microbiological Culture Collection Center Printed in Great Britain 933

2 A. Li and others 0 ml sterile water, 15 g agar, ph 7.0). The plates were incubated at 8 uc for 1 month. Single colonies were picked and purified by plating. All purified strains were preserved by lyophilization. Strains NP180 T and NR80 were selected for taxonomic studies. Genomic DNA of the two isolates was extracted using the Genomic DNA Rapid Isolation kit for bacterial cells (BioDev-Tech). 16S rrna gene sequences were amplified using the universal bacterial primers 27F and 1492R (Lane, 1991). PCR products were cloned using the pgem-t Easy vector and sequenced. The 16S rrna gene sequences (1477 bp for strains NP180 T and NR80) were submitted to GenBank ( and aligned with available sequences of related taxa in GenBank and the EzTaxon-e server ( Kim et al., 2012). Sequences of related strains were downloaded and multiple alignments were performed using BioEdit (Hall, 1999). Phylogenetic trees were reconstructed with MEGA 5.0 by using the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) algorithms. Bootstrap values were calculated based on 0 replications in order to evaluate confidence levels of the nodes. The topology of the neighbour-joining phylogenetic tree (Fig. 1) revealed an affiliation with the genus Flavobacterium, and strains NP180 T and NR80 constituted a cluster together with Flavobacterium filum EMB 34 T. The maximum-parsimony and maximumlikelihood trees showed the same topology. Strains NP180 T and NR80 exhibited % 16S rrna gene sequence similarity with each other and 95.1, 94.8 and 94.6 % sequence similarity with F. filum EMB 34 T, F. ponti GSW-R14 T and F. gelidilacus LMG T, respectively. Sequence similarity with other type strains of the genus Flavobacterium was,94 % (93.1 % with F. aquatile DSM 1132 T ). For morphological studies, strains NP180 T and NR 80 and F. filum KCTC T were grown on PYG at 20 uc for 3 days and observed by transmission electron microscopy (JEM1400; JEOL) (Fig. S1, available in the online Supplementary Material) and light microscopy. Colony morphology was examined after 7 days. Growth was tested on R2A, tryptic soy agar, nutrient agar, marine agar 2216 (Difco) and Anacker and Ordal s agar (Bernardet & Bowman, 2006). Gliding motility was tested by using the hanging drop technique (Bernardet et al., 2002). The presence of a capsule was assessed using wet negative staining (Fig. S2) and the Hiss staining method (Beveridge et al., 2007). The temperature range for growth was assessed on PYG slants at 4, 8, 15, 20, 25, 30, 35, 37 and 40 uc. Growth was assessed in PYG broth adjusted to ph using mm acetate buffer (for ph ), mm phosphate buffer (ph ) or mm NaHCO 3 /Na 2 CO 3 buffer (ph ) (Breznak & Costilow, 2007; Yumoto et al., 2004) and sterilized by filtration. NaCl tolerance was tested in PYG broth supplemented with 0 to 5.0 % (w/v) NaCl (at 0.5 % intervals). Strains were incubated in an anaerobic jar in order to test the requirement for oxygen using the Atmosphere Generation System (Oxoid). Catalase and oxidase activities were assessed using 3 % (v/v) H 2 O 2 and 1 % (w/v) N,N,N9,N9,-tetramethyl p-phenylenediamine, respectively. Production of flexirubintype pigments and Congo red absorption were assessed following the methods of Bernardet et al. (2002). Hydrolysis of casein, starch, chitin, tyrosine, gelatin, Tweens 20, 60 and 80, aesculin, urea, pectin and CM-cellulose was evaluated according to Smibert & Krieg (1994) and Reichenbach (1992). Hydrolysis of dextrin was investigated on PYG agar containing 1 % (w/v) dextrin. The DNase assay was carried out on the DNase test agar (Merck). Hydrolysis of egg yolk was tested on PYG agar supplemented with 50 % egg yolk emulsion. Acid production from carbohydrates, enzyme activities and additional biochemical characteristics were tested in API 50CH, API 20NE, API 20E and API ZYM strips (biomérieux), while oxidation of substrates was evaluated using the GN3 MicroPlate system (Biolog), both according to the manufacturers recommendations. Sensitivity to antibiotics was tested on PYG plates by using antibiotic discs (Beijing Tiantan Biological Products) containing ampicillin (10 mg), amikacin (30 mg), azithromycin (15 mg), cefaclor (30 mg), cefazolin (30 mg), cefoperazone (75 mg), cefotaxine (30 mg), ceftazidime (30 mg), ceftriazone (30 mg), cefuroxime sodium (30 mg), cephalotin (30 mg), chloramphenicol (30 mg), ciprofloxacin (5 mg), clarithromycin (15 mg), clindamycin (2 mg), doxycycline (30 mg), erythromycin (15 mg), fleroxacin (5 mg), lomefloxacin (10 mg), minocycline (30 mg), netilmicin (30 mg), nitrofurantoin (300 mg), oxacillin (1 mg), penicillin G (10 IU), piperacillin ( mg), rifampicin (5 mg), sulfamethoxazole (1.25 mg), tetracycline (30 mg), tobramycin (10 mg) and vancomycin (30 mg). The morphological, cultural and physiological properties of strain NP180 T are given in Table 1 and in the species description. For fatty acid analysis, strains NP180 T and NR80 and F. filum KCTC T were grown on TSA at 20 uc and cells were harvested at the exponential growth phase (48 h for the two former strains and 45 h for the latter). Cellular fatty acids were methylated, separated and identified according to the standard procedure of the Microbial Identification System (MIDI 6.0 version; Agilent 6890) (Sasser, 1990). Peaks were identified using the TSBA6.0 database. Polar lipids were extracted from the three strains and analysed by two-dimensional TLC following the procedures of Minnikin et al. (1984) and Komagata & Suzuki (1987); 5 % ethanolic molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol were sprayed onto the plates to detect total polar lipids, phospholipids, aminolipids and glycolipids, respectively. The quinones of strains NP180 T and NR80 and F. filum KCTC T were extracted and purified according to Collins (1985) and analysed by HPLC (Wu et al., 1989). The DNA G+C contents of the two isolates were determined by HPLC (Mesbah et al., 1989) using DNA of bacteriophage lambda as a reference (49.8 mol%). DNA DNA 934 International Journal of Systematic and Evolutionary Microbiology 64

3 Flavobacterium lacus sp. nov Flavobacterium fryxellicola LMG T (AJ811961) Flavobacterium omnivorum JCM T (AF433174) Flavobacterium xueshanense Sr22 T (HQ436466) Flavobacterium pectinovorum DSM 6368 T (AM230490) Flavobacterium micromati LMG T (AJ557888) Flavobacterium aquidurense WB T (AM177392) Flavobacterium soli DS-6 T (DQ178976) Flavobacterium anhuiense D3 T (EU046269) Flavobacterium defluvii EMB117 T (DQ372986) Flavobacterium compostarboris 15C3 T (GQ281769) Flavobacterium ginsengiterrae DCY55 T (HM776706) Flavobacterium glaciei 0499 T (DQ515962) Flavobacterium succinicans DSM 4002 T (AM230492) Flavobacterium psychrophilum IFO T (AB078060) Flavobacterium swingsii WB T (AM934651) Flavobacterium fluvii H7 T (EU109724) Flavobacterium psychrolimnae LMG T (AJ585428) Flavobacterium tiangeerense 0563 T (EU036219) Flavobacterium sinopsychrotolerans 0533 T (FJ654474) Flavobacterium xinjiangense ZF-6 T (AF433173) Flavobacterium frigoris LMG T (AJ557887) Flavobacterium degerlachei LMG T (AJ557886) Flavobacterium gillisiae ACAM 601 T (U85889) Flavobacterium flevense DSM 1076 T (AM230486) Flavobacterium antarcticum DSM T (FM163401) 98 Flavobacterium tegetincola ACAM 602 T (U85887) Flavobacterium myungsuense HMD1033 T (GQ148878) Flavobacterium segetis AT1048 T (AY581115) Flavobacterium weaverense AT1042 T (AY581114) Flavobacterium aquatile DSM 1132 T (AM230485) Flavobacterium filum EMB34 T (DQ372981) Flavobacterium lacus NP180 T (KC969641) Flavobacterium lacus NR80 (KC969642) Flavobacterium gelidilacus LMG T (AJ440996) Flavobacterium ponti GSW-R14 T (GQ370387) Flavobacterium aquaticum JC164 T (HE995762) Flavobacterium cheniae CGMCC T (EF407880) Flavobacterium dongtanense LW30 T (GU073293) Flavobacterium suncheonense GH29-5 T (DQ222428) Flavobacterium terrae R2A1-13 T (EF117329) Chryseobacterium gleum CCUG T (AM232812) Fig. 1. Neighbour-joining tree based on 16S rrna gene sequences, showing the phylogenetic relationship of strains NP180 T and NR80 and members of other Flavobacterium species. Chryseobacterium gleum CCUG T was used as an outgroup. Bootstrap values are shown at branching points (percentages of 0 resamplings; only values above 70 % are shown). Filled circles indicate nodes that were also recovered by using the maximum-likehood and maximum-parsimony methods. Bar, 0.02 substitutions per nucleotide position. hybridization of strains NP180 T and NR80 was carried out according to De Ley et al. (1970), using a Lambda 35 UV/ Vis spectrometer and a temperature programme controller (Perkin-Elmer). Strains NP180 T and NR80 contained mainly iso-c 15 : 0 (29.1 and 27.7 %, respectively), iso-c 15 : 1 G (19.8 and 17.3 %), iso-c 17 : 0 3-OH (8.1 and 8.4 %) and summed feature 9 (iso-c 17 : 1 v9c and/or 10-methyl C 16 : 0 ; 8.1 and 7.6 %). The fatty acid profiles of strains NP180 T and NR80 were nearly identical and similar to that of F. filum KCTC T, although containing smaller proportions of iso- C 15 : 0 and iso-c 15 : 1 G, and larger proportions of summed feature 9 (iso-c 17 : 1 v9c and/or 10-methyl C 16 : 0 ) (Table 2). MK-6 was the predominant respiratory quinone in strains NP180 T and NR80 (97 and 96 %, respectively); they also 935

4 A. Li and others Table 1. Characteristics that differentiate strain NP180 T from F. filum KCTC T All data shown were obtained in the present study. +, Positive; (+), weakly positive; 2, negative. The two strains are positive for hydrolysis of aesculin, dextrin, pectin, casein, Tween 60 and gelatin [an opposite result was reported for gelatin by Ryu et al. (2007)] and negative for hydrolysis of DNA, starch, urea, chitin, tyrosine and CM-cellulose, production of flexirubin-type pigments and absorption of Congo red. Characteristic NP180 T F. filum KCTC T Colony pigmentation on R2A agar Orange Pale yellow Gliding motility 2 + Presence of a capsule + 2 Growth at: 4 uc uc 2 + Optimum NaCl concentration (%, w/v) Nitrate reduction 2 + Citrate utilization + 2 Hydrolysis of Tweens 20 and Enzyme activities (API ZYM) Esterase (C4) + 2 N-Acetyl-b-D-glucosaminidase + (+) Acid phosphatase + (+) Arginine dihydrolase 2 + Acid production from: (API 50CH) Lactose 2 + Sucrose 2 + Oxidation of: (GN3 MicroPlate) Lactose 2 + Sucrose 2 + L-Glutamic acid + 2 D-Sorbitol 2 + contained minor amounts of MK-5 (3 and 4 %, respectively). These data are in line with those reported for all members of the family Flavobacteriaceae (Bernardet, 2011). TLC of total polar lipids is shown in Fig. S3. The major polar lipids of strains NP180 T and NR80 were phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. Minor amounts of several unidentified lipids were also present. The polar lipid profile of F. filum KCTC T was very similar. The G+C contents of the genomic DNA of strains NP180 T and NR80 were 34.9 and 35.1 mol%, respectively, in accordance with that of the reference strain and within the range reported for species of the genus Flavobacterium (Bernardet & Bowman, 2011). DNA relatedness between strains NP180 T and NR80 was 99 %, which indicated that the two strains belonged to the same genomic species. As the 16S rrna gene sequence similarity between strain NP180 T and strains of all species of the genus Flavobacterium with validly published names was well below 97.0 % (Stackebrandt & Ebers, 2006; Tindall et al., 2010), DNA DNA hybridization between them were not performed. The results of physiological, biochemical and genetic analyses support the recognition of a novel species within the genus Flavobacterium, for which the name Flavobacterium lacus sp. nov. is proposed. In addition, an emended description of Flavobacterium filum is also proposed on the basis of new data obtained in this study. Emended description of Flavobacterium filum Ryu et al The description is as given by Ryu et al. (2007) with the following amendments. Oxidase-positive. Gelatin is hydrolysed. The major polar lipids are phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid; several unidentified lipids are also present. MK-6 is the only respiratory quinone. Description of Flavobacterium lacus sp. nov. Flavobacterium lacus (la9cus. L. gen. n. lacus of a lake). Cells are Gram-stain-negative, strictly aerobic and nonmotile rods, approximately mm in diameter and mm long. A thick capsule is present. Colonies are orange, convex, circular, 1 4 mm in diameter with entire margins and a sticky consistency on PYG agar after 7 days at 15 uc. Growth also occurs on TSA, NA, R2A and Anacker and Ordal s agar, but not on marine agar Broth cultures are viscous. Growth does not occur under anaerobic conditions. Growth occurs at 4 25 uc (optimum, 20 uc), in PYG broth supplemented with % NaCl (optimum, 936 International Journal of Systematic and Evolutionary Microbiology 64

5 Flavobacterium lacus sp. nov. Table 2. Whole-cell fatty acid compositions of strain NP180 T and F. filum KCTC T Data are from this study. Fatty acids amounting to,1 % of the total fatty acids in both strains are not shown. TR, Trace (,1.0 %). Values for strains NP180 T and NR80 are means±sd. Fatty acid NP180 T and NR80 F. filum KCTC T Saturated iso-c 15 : ± anteiso-c 15 : 0 4.6± iso-c 16 : 0 2.3± C 16 : 0 1.2±0.1 TR iso-c 15 : 0 3-OH 5.7± iso-c 16 : 0 3-OH 1.8±0.1 TR iso-c 17 : 0 3-OH 8.3± Unsaturated iso-c 15 : 1 G 18.6± anteiso-c 15 : 1 A 1.2± C 15 : 1 v6c 3.0± iso-c 16 : 1 H 1.7± C 17 : 1 v8c 1.1±0.1 TR C 17 : 1 v6c 2.5±0.6 TR C 18 : 1 v5c 1.0±0.1 TR Summed features* 3 4.5± ± *As indicated by Montero-Calasanz et al. (2013), summed features are groups of two or three fatty acids that are treated together for the purpose of evaluation in the MIDI System and include both peaks with discrete ECLs as well as those where the ECLs are not reported separately. Summed feature 3 was listed as C 16 : 1 v7c and/or C 16 : 1 v6c; summed feature 9 was listed as iso-c 17 : 1 v9c and/or 10-methyl C 16 : %) and at ph (optimum, ph ). Catalase and oxidase activities are present. Nitrate is not reduced to nitrite. H 2 S and indole are not produced, but citrate is utilized. The Voges Proskauer test is weakly positive. Hydrolyses gelatin, casein, aesculin, dextrin, pectin and Tweens 20 and 60, but not tyrosine, DNA, Tween 80, starch, urea, chitin or CM-cellulose. No brown pigment is produced on tyrosine agar. Flexirubin-type pigments are not produced and Congo red is not absorbed by colonies. No precipitate is formed on egg yolk agar. The following activities are detected in the API ZYM strip: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, a-glucosidase, naphthol-as-bi-phosphohydrolase (weak) and N-acetyl-bglucosaminidase (weak). The following activities are not detected: a-galactosidase, b-galactosidase, b-glucuronidase, b-glucosidase, a-mannosidase and a-fucosidase. In the API 50CH strip, acid is produced from D-galactose, D-glucose, aesculin, starch and glycogen. Acid production from lactose is negative (type strain) or weakly positive, and acid is not produced from the other substrates in the API 50CH strip. In the GN3 MicroPlate, the following substrates are oxidized: acetoacetic acid, acetic acid, L-arginine, L-aspartic acid, citric acid, dextrin, D-galacturonic acid, gelatin, a- D-glucose, L-glutamic acid, glycyl L-proline, D-lactic acid methyl ester, D-malic acid, L-malic acid, maltose, mucic acid, pectin, L-serine, Tween 40, D-aspartic acid (weak), D- gluconic acid (weak) and methyl pyruvate (weak). The other substrates in the GN3 MicroPlate are not oxidized. Resistant to chloramphenicol (30 mg), fleroxacin (5 mg), oxacillin (1 mg), sulfamethoxazole (1.25 mg) and tobramycin (10 mg), but sensitive to amikacin (30 mg), ampicillin (10 mg), azithromycin (15 mg), cefaclor (30 mg), cefazolin (30 mg), cefoperazone (75 mg), cefotaxine (30 mg), ceftazidime (30 mg), ceftriazone (30 mg), cefuroxime sodium (30 mg), cephalotin (30 mg), ciprofloxacin (5 mg), clarithromycin (15 mg), clindamycin (2 mg), doxycycline (30 mg), erythromycin (15 mg), lomefloxacin (10 mg), minocycline (30 mg), netilmicin (30 mg), nitrofurantoin (300 mg), penicillin G (10IU), piperacillin ( mg), rifampicin (5 mg), tetracycline (30 mg) and vancomycin (30 mg). The predominant (.7 %) fatty acids are iso-c 15 : 0, iso-c 15 : 1 G, iso-c 17 : 0 3-OH and summed feature 9 (iso-c 17 : 1 v9c and/or 10-methyl C 16 : 0 ). MK-6 is the major respiratory quinone. Phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid are the major polar lipids. The type strain is NP180 T (5CGMCC T 5NBRC T ), isolated from water of Lake Nam Co in Tibet, China. The DNA G+C content of the type strain is 34.9 mol%. Acknowledgements We thank Professor J. P. Euzéby for kind assistance with Latin nomenclature. This work was supported by the National Natural Science Foundation of China (NSFC, no ), and the Knowledge Innovation Program of the Chinese Academy of Sciences (grant no. 188 KSCX2-YW-Z-0937). References Bergey, D. H., Harrison, F. C., Breed, R. S., Hammer, B. W. & Huntoon, F. M. (1923). Genus II. Flavobacterium gen. nov. In Bergey s Manual of Determinative Bacteriology, pp Baltimore: Williams & Wilkins. Bernardet, J.-F. (2011). Family I. Flavobacteriaceae Reichenbach In Bergey s Manual of Systematic Bacteriology, 2nd edn, vol. 4, pp Edited by N. R. Krieg, J. T. Staley, D. R. Brown, B. P. Hedlund, B. J. Paster, N. L. Ward, W. Ludwig & W. B. Whitman. New York: Springer. Bernardet, J.-F. & Bowman, J. P. (2006). 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