International Journal of Systematic and Evolutionary Microbiology (2015), 65, 3782–3787 DOI 10.1099/ijsem.0.000498

Pedobacter alpinus sp. nov., isolated from a plateau lake Ai-Hua Li, Hong-Can Liu and Yu-Guang Zhou

Correspondence China General Microbiological Culture Collection Center and State Key Laboratory of Microbial Yu-Guang Zhou Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China [email protected] Two Gram-staining-negative, aerobic, non-motile, red-pigment and rod-shaped bacterial strains, designated RSP19T and RSR28, were isolated from lake water and subjected to polyphasic taxonomic studies. Based on 16S rRNA gene sequence analysis, strains RSP19T and RSR28 exhibited 91.4–95.7 % similarity to the type strains of existing of the genus . The species most closely related phylogenetically were Pedobacter daechungensis (95.7 % sequence similarity), Pedobacter lentus (95.4 %), Pedobacter glucosidilyticus (94.5 %), Pedobacter panaciterrae (94.5 %) and Pedobacter terricola (94.2 %). MK-7 was the sole respiratory quinone. The major polar lipids were phosphatidylethanolamine, one unidentified

aminolipid and several unidentified polar lipids. The predominant fatty acids were C16 : 1v7c,

iso-C15 : 0 and iso-C17 : 0 3-OH. The genomic DNA G+C contents were 34.3 and 33.9 mol% for strains RSP19T and RSR28, respectively. Based on the phenotypic characteristics, biochemical properties and genetic analysis, strains RSP19T and RSR28 clearly represent a novel species of the genus Pedobacter, for which the name Pedobacter alpinus is proposed. The type strain is RSP19T (5CGMCC 1.14040T5KCTC 42456T5NBRC 110967T).

The genus Pedobacter, a member of the family Sphingobac- range from 33.3 to 47.4 mol% (Kang et al., 2014; Kook teriaceae, was first established by Steyn et al. (1998) with et al., 2014). the reclassification of two species of the genus Sphingobac- In this study, samples were collected from Ranwu Lake, a pla- terium as Pedobacter heparinus and Pedobacter piscium. teau lake in the west of China. They were serially diluted with At the time of writing, there are 48 recognized species 0.85 % saline solution and 0.2 ml of each dilution was spread with validly published names (www.bacterio.net/pedobac- on PYG (5.0 g Bacto peptone, 0.2 g yeast extract, 5.0 g ter.html). Members of the genus Pedobacter have been glucose, 3.0 g beef extract, 0.5 g NaCl, 1.5 g MgSO4 . found to inhabit a wide variety of environments, even the 7H2O, 1000 ml sterile water, 15 g agar, pH 7.0) and R2A Antarctic area. The first psychrotolerant species of the (BD, Difco) agar plates and incubated at 15 8C for 14 days. genus Pedobacter, , was isolated All single colonies were picked out and purified using repeated from alpine glacier cryoconite (Margesin et al., 2003). plate streaking. Strains RSP19T and RSR28 were red colonies Since then, several cold-adapted strains belonging to this isolated from PYG and R2A agar plates, respectively. genus have been identified in succession, such as Pedobacter T glacialis and Pedobacter huanghensis (from Arctic glacier Genomic DNA of strains RSP19 and RSR28 were foreland), Pedobacter arcticus (from arctic soil), Pedobacter extracted by using the Genomic DNA Rapid Isolation kit himalayensis (from glacial water) and Pedobacter nutrimenti for Bacterial Cells (BioDev-Tech) following the instruc- (from chilled food) (Derichs et al., 2014; Qiu et al., 2014; tions of the manufacturer. 16S rRNA gene sequences Shivaji et al., 2005; Zhou et al., 2012). The species of the were amplified by using universal primers 27F and 1492R genus Pedobacter are usually Gram-staining-negative, (Lane, 1991). The PCR products of 16S rRNA gene were aerobic, mostly non-motile rods, positive for catalase and cloned using the pGEM-T easy vector and sequenced. oxidase, with menaquinone-7 as the sole or major respirat- The full-length 16S rRNA gene sequences (1485 bp) ory quinone and phosphatidylethanolamine as the predo- obtained were aligned with available published sequences minant polar lipid. The genomic DNA G+C contents from GenBank and the EzTaxon-e server (Kim et al., 2012). 16S rRNA gene sequences of the most closely related taxa were retrieved and aligned with BioEdit (Hall, 1999). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA Phylogenetic analysis was performed by using MEGA 5.0 gene sequences of strains RSP19T and RSR28 are KP008109 and and phylogenetic trees were drawn by using neighbour- KP876467, respectively. joining (Saitou & Nei, 1987), maximum-likelihood Two supplementary figures are available with the online Supplementary (Felsenstein, 1981) and maximum-parsimony (Fitch, Material. 1971) methods. The topology of the neighbour-joining

Downloaded from www.microbiologyresearch.org by 3782 000498 G 2015 IUMS Printed in Great Britain IP: 124.16.145.98 On: Wed, 16 Dec 2015 01:50:50 Pedobacter alpinus sp. nov. phylogenetic tree was evaluated with 1000 replicates of tested with the API 50CH strip (bioMe´rieux). Oxidation bootstrap resampling. Strains RSP19T and RSR28 exhibited of substrates was evaluated with the GN3 MicroPlate 100 % 16S rRNA gene sequence similarity with each other. system (Biolog). Sensitivity to antibiotics was tested on They showed the highest 16S rRNA gene sequence PYG plates by using antibiotic discs (Beijing Tiantan Bio- similarities to Pedobacter daechungensis Dae 13T (95.7 %), logical Products) containing ampicillin (10 mg), amikacin followed by Pedobacter lentus DS-40T (95.4 %), Pedobacter (30 mg), azithromycin (15 mg), cefaclor (30 mg), cefazolin glucosidilyticus 1-2T (94.5 %), Pedobacter panaciterrae Gsoil (30 mg), cefoperazone (75 mg), cefotaxime (30 mg), ceftazi- 042T (94.5 %) and Pedobacter terricola DS-45T (94.2 %), dime (30 mg), ceftriaxone (30 mg), cefuroxime sodium respectively. Similarities with other recognized species of (30 mg), cefalotin (30 mg), chloramphenicol (30 mg), cipro- the genus Pedobacter were all less than 94.1 %. Three floxacin (5 mg), clarithromycin (15 mg), clindamycin other 16S rRNA gene sequences of high similarity are (2 mg), doxycycline (30 mg), erythromycin (15 mg), flerox- deposited in sequence databases. However, the respective acin (5 mg), lomefloxacin (10 mg), minocycline (30 mg), bacterial strains were not available from the sequence netilmicin (30 mg), nitrofurantoin (300 mg), oxacillin depositors for comparative studies. As shown in the neigh- (1 mg), penicillin G (10 IU), piperacillin (100 mg), rifampi- bour-joining tree, these two novel strains formed a separate cin (5 mg), sulfamethoxazole (1.25 mg), tetracycline branch in the distinct cluster comprising P. daechungensis (30 mg), tobramycin (10 mg) and vancomycin (30 mg). Dae 13T, P. lentus DS-40T, P. glucosidilyticus 1-2T, Detailed characteristics of strains RSP19T and RSR28 are P. panaciterrae Gsoil 042T, P. terricola DS-45T, P. arcticus provided in the species description, and differential charac- A12T, Pedobacter rivuli HME8457T and Pedobacter pituito- teristics with other species of the genus Pedobacter are sus MIC2002T (Fig. 1). The topology of the maximum- summarized in Table 1. likelihood and maximum-parsimony trees (Fig. S1 avail- Genomic DNA of strains RSP19T and RSR28 was extracted able in the online Supplementary Material) was essentially and DNA G+C contents were determined by HPLC identical with that of the neighbour-joining tree. (Mesbah et al., 1989); DNA of Lambda phage (Sigma) For physiological and chemotaxonomic research, the (49.8 mol%) and P. glucosidilyticus CCTCC AB 206110T following type strains were used as references: was used as references. P. glucosidilyticus CCTCC AB 206110T and P. arcticus For analysis of fatty acid profile, the two novel strains and five CCTCC AB 2010223T obtained from the China Center reference strains, P. daechungensis KCTC 12637T, P. lentus JCM for Type Culture Collection (CCTCC), P. daechungensis 14593T, P. glucosidilyticus CCTCC AB 206110T, P. terricola KCTC 12637T from the Korean Collection for Type Cul- JCM 14594T and P. arcticus CCTCC AB 2010223T,were tures (KCTC), P. lentus JCM 14593T and P. terricola JCM grown on trypticase soy agar (BD, Difco) at 20 8C for 72 h. 14594T from Japan Collection of Micro-organisms (JCM). Cell masses were harvested at the same exponential growth Cell morphology and flagella were observed by using light phase. Fatty acid methyl esters were separated and analysed microscopy and transmission electron microscopy according to the standard procedure of the Microbial Identi- (JEM1400, JEOL; Fig. 2). Gram staining was performed fication System (version 6.0, MIDI; GC model 6890, Agilent) according to the procedure of Collins et al. (1989). (Sasser, 1990). Peaks were identified using the TSBA6.0 data- Motility was determined with the hanging-drop technique base. Meanwhile, the fatty acid methyl esters were also ana- (Bernardet et al., 2002). Anaerobic growth was tested on lysed by the MIDI EUKARY method to determine the PYG agar at 20 8C for 2 weeks using Oxoid’s Atmosphere components of summed features 3, 4 and 9. Generation System. Growth at different temperatures Polar lipids were extracted by the procedure of Minnikin (4, 10, 15, 20, 25 and 30 8C) was determined on PYG et al. (1984) and separated by two-dimensional TLC agar and growth in presence of NaCl (0–5 %, w/v, at inter- (plates coated with silica gel, 10610 cm; Merck). The sep- vals of 0.5 %) was analysed in PYG broth at 20 8C. The pH arated spots were confirmed by spraying with 5 % ethano- range for growth was tested in PYG broth adjusted to lic molybdophosphoric acid (for total polar lipids), pH 4.0–11.0 (at intervals of 0.5 pH units), using 100 mM molybdenum blue (phospholipids), ninhydrin (aminol- acetate buffer (for pH 4.0–5.0), 100 mM phosphate ipids) and a-naphthol (glycolipids). Respiratory quinones buffer (pH 6.0–8.0) or 100 mM NaHCO /NaCO buffer 3 3 were extracted according to the protocol of Collins (pH 9.0–10.0) (Breznak & Costilow, 2007; Yumoto et al., (1985) and analysed by HPLC (Wu et al., 1989). 2004), respectively. Catalase and oxidase activities were determined by using 3 % (v/v) H2O2 and Bactident Oxi- From the results of MIDI EUKARY analysis, the major T dase Strips (Merck), respectively. DNase activity was fatty acids of strains RSP19 and RSR28 were C16 : 1v7c, assessed on DNase test agar (Merck). Hydrolysis of (28.6 % and 25.4 %, respectively), iso-C15 : 0 (27.4 % and Tween20, Tween60, Tween80, starch, aesculin, CM-cellu- 26.5 %) and iso-C17 : 0 3-OH (10.2 % and 14.0 %). lose, casein and gelatin was tested according to the methods These were in accordance with the closest phylogenetic of Smibert & Krieg (1994). Other enzyme activities were neighbours and the characteristics of species of the genus assessed using the commercial systems API 20E, 20NE Pedobacter. The fatty acid profiles of these two isolates and API ZYM (bioMe´rieux) following the manufacturer’s and the type strains of related species of the genus Pedobac- instructions. Acid production from carbohydrates was ter are shown in Table 2. Strains RSP19T and RSR28 could

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9 Pedobacter caeni LMG 22862T (AJ786798) 9 59 Pedobacter steynii WB 2.3-45T (AM491372) Pedobacter duraquae WB 2.1-25T (AM491368) Pedobacter metabolipauper WB 2.3-71T (AM491370) Pedobacter africanus DSM 12126T (AJ438171) 0.02 Pedobacter nutrimenti J22T (HF536497) Pedobacter panaciterrae Gsoil 042T (AB245368) 54 50 Pedobacter ginsengisoli Gsoil 104T (AB245371) Pedobacter heparinus DSM 2366T (CP001681) 76 T 51 Pedobacter nyackensis NWG-II14 (EU030686) DSM 15311T (FR733711) 99 Pedobacter antarcticus T 60 Pedobacter westerhofensis WB 3.3-22 (AM491369) 89 Pedobacter hartonius WB 3.3-3T (AM491371) 8-24T (KC569795) 61 Pedobacter glacialis Pedobacter boryungensis BR-9T (HM640986) T 99 74 Pedobacter insulae DS-139 (EF100697) Pedobacter koreensis WPCB189T (DQ092871) 90 Pedobacter jejuensis THG-DR3T (KC252614) 100 Pedobacter aquatilis AR107T (AM114396) DCY49T (HM776704) 74 Pedobacter ginsengiterrae Pedobacter rhizosphaerae 01-96T (AM279214) 94 Pedobacter alluvionis NWER-II11T (EU030688) 62 T Pedobacter roseus CL-GP80 (DQ112353) 75 Pedobacter soli 15-51T (AM279215) 56 Pedobacter sandarakinus DS-27T (DQ235228) 90 Pedobacter borealis G-1T (EU030687) Pedobacter ginsenosidimutans THG-45T (GU138374) 57 PB92T (AJLG01000244) 98 T Pedobacter suwonensis 15-52 (DQ097274) Pedobacter terrae DS-57T (DQ889723) 100 Pedobacter alpinus strain RSP19T (KP008109) Pedobacter alpinus strain RSR28 (KP876467) 57 62 Pedobacter daechungensis Dae 13T (AB267722) 62 T 63 Pedobacter lentus DS-40 (EF446146) 51 Pedobacter terricola DS-45T (EF446147) Pedobacter rivuli HME8457T (JQ911707) Pedobacter arcticus A12T (AKZJ01000016) Pedobacter glucosidilyticus 1-2T (EU585748) Pedobacter pituitosus MIC2002T (JX978785) 82 Pedobacter oryzae N7T (EU109726) Pedobacter huanghensis M1-27T (KC569794) 100 oryzae B9T (EU109722) Mucilaginibacter frigoritolerans FT22T (FN400860) Bacteroides fragilis DSM 2151T (AB050106)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strains RSP19T and RSR28 in relation to members of the genus Pedobacter. Bacteroides fragilis DSM 2151T was used as an outgroup. Only bootstrap values (expressed as percentages of 1000 replications) .50 % are shown at branch points. Filled circles indicate nodes also recovered in the maximum-likelihood tree. Bar, 0.02 substitutions per nucleotide position.

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phosphatidylethanolamine, one unidentified aminolipid and several unidentified polar lipids (L1, 4, 6, 7, 8 and 9 present in strain RSP19T; L1, 4, 8 and 9 in strain RSR28) (Fig. S2). This was consistent with the profiles of related species of the genus Pedobacter. However, the presence of unidentified polar lipids L6, 7 and 9 differentiated strain RSP19T from its phylogenetically related neighbours of the genus Pedobacter. The sole respiratory quinone of strains RSP19T and RSR28 was identified as MK-7, which is the major or only respiratory quinone ofall other members of the familySphingobacteriaceae (Margesin & Shivaji, 2011). The genomic DNA G+Ccontents of strains RSP19T and RSR28 were 34.3 and 33.9 mol%, respectively, which are in line with those of the other members of the genus Pedobacter. Based on the results of physiological, phylogenetic and Fig. 2. Transmission electron micrograph (TEM) of cells of strain chemotaxonomic analysis of the two isolates, it is con- T RSP19T grown on PYG agar plates at 20 8C for 3 days. firmed that strains RSP19 and RSR28 represent a novel species of the genus Pedobacter, for which the name Pedo- bacter alpinus is proposed. The type strain is RSP19T. be distinguished from the reference strains by their higher Description of Pedobacter alpinus sp. nov. content of C v5c. 16 : 1 Pedobacter alpinus (al.pi9nus. L. masc. adj. alpinus alpine, Results of polar lipids analysis revealed that the referring to the isolation of the type strain from an alpine major polar lipids of strains RSP19T and RSR28 were environment).

Table 1. Differential characteristics of strains RSP19T, RSR28 and the type strains of related species of the genus Pedobacter

Strains: 1, RSP19T; 2, RSR28; 3, P. daechungensis KCTC 12637T;4,P. lentus JCM 14593T;5,P. terricola JCM 14594T;6,P. glucosidilyticus CCTCC AB 206110T;7,P. arcticus CCTCC AB 2010223T. Data are all from this study except the DNA G+C contents of reference strains (An et al., 2009; Yoon et al., 2007; Luo et al., 2010 & Zhou et al., 2012). In API 20NE and 20E tests, all strains were positive for oxidase, catalase, ONPG and Voges-

Proskauer reactions, and negative for nitrate reduction, and H2S and indole production. +, Positive; W, weakly positive; 2, negative; (2), negative but positive in literature; (+), positive but negative in literature. DR, Dark red; R, red; RO: reddish orange; PL, pale yellow; PO, pale orange; C, cream; P, pink.

Characteristic 1 2 3 4 5 6 7

Isolation source Lake water Lake water Sediment Soil Soil Riverbed Arctic soil Colony colour DR R RO PY to PO PO C to PP Growth at 30 8C 22+++++ Growth with 4 % NaCl 222++ W 2 Growth at pH 5 22+ 2 ++ 2 Enzyme activities (API ZYM) Cystine arylamidase ++++++2 Trypsin 22+ 222 2 a-Fucosidase 22222+ (+) Oxidation of (GN3 MicroPlate): Dextrin 2 + 2 +++ + Sucrose +++22++ D-Mannose ++2 +++ 2 Trehalose 22+++++ Glycerol 22222++ L-Aspartic acid 2 ++++22 D-Galacturonic acid ++2 +++ 2 D-Glucuronic acid ++2 +++ + Acid produced from (API 50CH): D-Arabinose 22222++ Amygdalin 2 W + 2 ++ 2 L-Arabinose ++2 (2) ++ 2 DNA G+C content (mol%) 34.3 33.9 33.8 36.0 36.8 37.2 38.3

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Table 2. Cellular fatty acid compositions of strains RSP19T, is weakly positive for esterase (C4) and a-glucosidase. RSR28 and type strains of phylogenetically related species, In API 50CH, acids are produced from L-arabinose, D- which were determined by MIDI EUKARY method xylose, D-galactose, D-glucose, N-acetylglucosamine, aescu-

T T lin, lactose, sucrose and gentiobiose, but not from the other Strains: 1, RSP19 ; 2, RSR28; 3, P. daechungensis KCTC 12637 ;4, substrates. Strain SRP19T produces acid weakly from P. lentus JCM 14593T;5,P. terricola JCM 14594T;6,P. glucosidilyticus D-mannose and L-rhamnose. In Biolog GN3 detection, CCTCC AB 206110T;7,P. arcticus CCTCC AB 2010223T. Data are all gentiobiose, sucrose, a-lactose, N-acetyl-D-glucosamine, from this study and represent percentages of total fatty acids. –, Not a-D-glucose, D-mannose, D-galactose, L-rhamnose, L- detected. glutamic acid, pectin, D-galacturonic acid, D-glucuronic Fatty acid 1 2 3 4 5 6 7 acid, L-lactic acid and acetoacetic acid are oxidized; maltose, cellobiose, gelatin, methylpyruvate, D-lactic acid Saturated methyl ester and acetic acid are weakly oxidized. Strain

C14 : 0 1.7 1.3 – – – 0.9 0.8 RSR28 can oxidize dextrin and L-aspartic acid, but strain T iso-C15 : 0 27.4 26.5 19 28.6 31.0 25.5 30.0 RSP19 cannot. The other substrates are not oxidized. anteiso-C15 : 0 5.1 4.7 3.3 3.5 2.1 7.7 9.4 Resistant to amikacin, cefazolin, cefoperazone, ceftriaxone, iso-C15 : 0 3-OH 3.4 3.8 2.5 4.1 – 2.0 3.8 oxacillin, tobramycin and vancomycin; weakly resistant to C15 : 0 2-OH 2.3 – 2.5 – 0.4 1.9 – azithromycin, erythromycin and netilmicin; and sensitive C15 : 0 1.3 2.5 2.8 – – 3.9 – to ampicillin, cefaclor, cefotaxime, ceftazidime, cefuroxime C16 : 0 3.0 2.2 1.3 2.3 1.9 4.2 – sodium, cefalotin, chloramphenicol, ciprofloxacin, clari- iso-C16 : 0 2.0 2.9 5.0 2.6 2.0 3.4 3.8 thromycin, clindamycin, doxycycline, fleroxacin, C16 : 0 3-OH 2.8 1.4 – – – 2.3 3.2 lomefloxacin, minocycline, nitrofurantoin, penicillin G, iso-C17 : 0 3-OH 10.2 14 13.7 19.9 21.5 12.8 12.2 piperacillin, rifampicin, tetracycline and sulfamethoxazole/ Unsaturated v v trimethoprim. The predominant fatty acids are C16 : 1 7c, C15 : 1 6c 2.1 2.2 2.3 3.2 4.7 1.1 0.5 iso-C and iso-C 3-OH. The sole respiratory quinone iso-C H 1.3 2.0 3.2 – 0.2 0.6 – 15 : 0 17 : 0 16 : 1 is MK-7. The major polar lipids are phosphatidylethanola- C v7c 28.6 25.4 19.8 21.7 31.4 22.6 29.5 16 : 1 mine, one unidentified aminolipid and several unidentified C16 : 1v5c 5.0 4.9 4.2 2.9 – 2.1 2.7 C v8c – – 2.5 0.4 1.3 1.3 – polar lipids (6 unidentified polar lipids for strain RSP19T, 17 : 1 and 4 for strain RSR28). C17 : 1v6c – – 3.5 – – – – T T iso-C17 : 1 at 9* 2.7 3.0 8.6 5.8 0.3 2.3 – The type strain, RSP19 (5CGMCC 1.14040 5KCTC T T anteiso-C17 : 1 at 9* – 0.7 1.5 – – 2.7 – 42456 5NBRC 110967 ), was isolated from Ranwu Lake of Tibet province, in the west of China. The genomic *Unknown whether cis or trans. DNA G+C content of strain RSP19T is 34.3 mol%.

Acknowledgements Cells are Gram-staining-negative, aerobic, non-motile rods, 1.5–2.0 mm in length and 0.6–0.8 mm in width. Colonies on We appreciate JCM, CCTCC and KCTC for providing reference strains PYG agar are darker red to red, smooth, convex, for this study. We thank Dr Jing-Nan Liang for transmission electron microscopy. This work was supported by the National Science and Tech- 0.5–1.2 mm in diameter and circular with entire margins nology Foundation Project (2012FY111600, 31400106). after 4 days of incubation at 20 uC. Growth occurs at 4–25 uC (optimum, 20 uC), at pH 6.0–8.5 (optimum, pH 7.0–7.5) and with 0–2.5 % NaCl (optimum, 0–1.0 % NaCl). Positive for oxidase and catalase activities and nega- References tive for DNase activity. Aesculin is hydrolysed, Tween 60 An, D.-S., Kim, S.-G., Ten, L. N. & Cho, C.-H. (2009). Pedobacter and gelatin are weakly hydrolysed, but casein, starch, daechungensis sp. nov., from freshwater lake sediment in South CM-cellulose, Tween 20 and Tween 80 are not hydrolysed. Korea. Int J Syst Evol Microbiol 59, 69–72. Voges–Proskauer test is positive. H2S and indole pro- Bernardet, J. F., Nakagawa, Y. & Holmes, B. & Subcommittee on the duction are not present. Does not reduce nitrates to of Flavobacterium and Cytophaga-like of the nitrites. Negative for arginine dihydrolase, lysine decarbox- International Committee on Systematics of Prokaryotes (2002). ylase, ornithine decarboxylase, citrate utilization, urease, Proposed minimal standards for describing new taxa of the family tryptophan deaminase and fermentation of glucose. Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070. In API ZYM detection, positive for alkaline phosphatase, Breznak, J. & Costilow, R. N. (2007). esterase lipase (C8), lipase (C14), leucine arylamidase, Physicochemical factors in growth. In Methods for General and Molecular Microbiology, 3rd valine arylamidase, cystine arylamidase, acid phosphatase, edn, pp. 309–329. Edited by C. A. Reddy, T. J. Beveridge, naphthol-AS- BI-phosphohydrolase, b-galactosidase and J. A. Breznak, G. Marzluf, T. M. Schmidt & L. R. Snyder. N-acetyl-b-glucosaminidase, and negative for trypsin, Washington, DC: American Society for Microbiology. a-chymotrypsin, a-galactosidase, b-glucuronidase, b-glu- Collins, M. D. (1985). Isoprenoid quinone analysis in classification T cosidase, a-mannosidase and a-fucosidase; strain RSP19 and identification. In Chemical Methods in Bacterial Systematics,

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