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System. Appl. Microbiol. 1,6,227138 (1993)@ Gustav Fischer Verlag, Stuttgart .Jena . New York
Taxonomic Study of Polyethylene Glycol-Utilizing Bacteria:Emended Description of the Genus Sphingomonasand New Descriptions of Sphingomonas macrogoltabidus sp. nov.,Sphingomonas sanguis sp. nov. vnd Sphingomonas terrae sp. nov.
M. TAKEUCHII, F. KANrAI2, y. SHIMAD.!r2, and A.YOKOTAI
1 Institute for Fermentation, Osaka, 17-85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532, Japan2 Department of Biology, Kobe University oi Commerce, Gakuen-nishimachi, Nishi-ku, Kobe 651-21, Japan
Received December 22, 1992
Summary
The taxonomic position of polyethylene glycol (PEG)-utilizing bacteria was examined. PEG 4,000 wasutilized by a single pure culture, but PEG 6,000 was u;tilized by mixed cultures of two strains. PEG 4,000-utilizing bacteria and the dominant bacteria in the PEG 6,000-utilizing mixed cultures which are respon-sible for the degradation of PEG were identified as new species of the genus Sphingomonas. based onchemotaxonomic and physiological characteristics together with DNA/DNA hybridization studies and165 rRNA sequence comparison, and we propose two new species, Spbingomonas rnacrogobabidus (typestrain: 203, IFO 15033) for the PEG 4,00O-utilizing bacteria and Sphingomonas teffde (type strain: E-1-A,IFO 15098) for the PEG 6,000-utilizing bacteria. And we also propose Sphingomonas sanguis for theremaining one strain, Sphingomonas genospecies 1 (type strain: lFO L3937). Emended description of thegerllts Sphingomonas is presented.
The other bacteria in the 6,000-utilizing mixed cultures, which support the growth of the dominantbacteria by degrading an inhibitory substance, glyoxylate, were tentatively identified as Rhizobium sp.,Agrobacterium sp. and Methylobacteriurn sp. Four isoiates which utilize PEGs having molecular weightslower than 4,000 were identified as Pseudomonas solanacearum, Alcaligenes xylosoxidans slbsp. denitri-ficans, Enterobacter diuersus and P sewdomonas uesicularis.
Key words: Sphingomonas rnacrogobabidus - Spbingomonds terrae - Polyethylene glycol (PEG)-utilizingbacteria - Sphingomonas sanguis
Introduction
Several bacteria capable of degrading polyethylene gly-col (PEG) have been reported in recent years: anaerobicdegradation by Deswlfouibrio desulfuricans, Bacteroidessp. (Dwyer and Tiedje, 1.986), Acetobacteriwm sp. (Schinkand Stieb, 1.983, Schramm and Schink, 199'J.), Pelobacteruenetianus (Schink and Stieb, 1,983; Schmid et a1.,1991)and aerobic degradation by Pseudomonas aeruginosa(Haines and Alexander, 1.975) and Pseudomonas stwtzeri(Obradors and Aguilar, 1991). Kawai and coworkers haveisolated some PEG-degrading bacteria which were tenta-tively identified as Flauobacterium sp. or Psewdornonas sp.(Katuai et al., 1977, 1.984; Yamanaka and Kauai, 1989).PEGs having molecular weights lower than 4,000 wereutilized by a single pure culture of Flauobacterium sp. or
Pseudomonas sp. On the other hand, PEG 6,000 wasutilized by a mixed culture of two strains, and single purecultures did not degrade PEGs. In the mixed culture,Flauobacteriwm sp. was found to be responsible for degra-dation of PEG, and the other strain identified as aPsewdomonas sp. was thought to play a role in supportinggrowth of Flauobacterium sp. by removing glyoxylic acid,an inhibitory substance liberated by Flauobacterium sp.(Kawai and Yamanake, 1986). Although the mechanismof PEG degradation and the characteristics of enzymesinvolved in the metabolism have been studied intensively(Katuai 1.990; Yamanaka and Katuai, 1.989), precise tax-onomic studies of these organisms have not been done.
In this paper, we report the reidentification of typical
228 M. Takeuchi, F. Kawai, Y. Shimada, and A. Yokota
strains of PEG 4,000- and PEG.6,000-utilizing bacteriapreviously classified as Pseudomonds sp. or Flauobacte-rium sp., and furthermore, propose a new species for theSphingomonas genospecies 1. of Yabwwchi et al. (L990).
Materials and Methods
Microorganisms and cwbure conditions. As shown in Table 1,21 strains of PEG-utilizing bacteria isolated by Ogata et al.(197 5) and Kawai et al. (1,977 , 1984) were used. PEG 5,000 wasdegraded only by mixed cultures consisting of two strains ofbacteria (Kawai andYamanaka, 1,986). PEG 6,000-utilizing bac-teria (A), which was tentatively identified as Flauobacterium sp.(Kawai et al. 1,977,1984), is responsible for the degradation ofPEG, and PEG 6,000-utilizing bacteria (B), which was tentativelyidentified as Pseudomonas sp. (Kawai et al. 1977, 1984), tsthought to support the growth of the dominant bacteria by de-grading an inhibitory substance, glyoxylate. Type strains of thespecies of the genus Spbingomonas were used for comparison oftaxonomic characteristics. All the strains used in this study werecultured at28'C with aerobic shaking in PY medium containing17o peptone, 0.2'h yeast extract, 0.2% NaCl and 0.2Y" glucose,pH7.0.
Determination of phenotypic characteristics. API (Appareiliset Proc6d6s d'Identification; La Balme les Grottes, MontalieuVercieu, France) 20NE and 50CH were used for physiologicaland biochemicai characteristics. API 50CH galleries were used to
determine the carbohydrate assimilation pattern of the organ-isms. Cultures grown on heart infusion agar for 20 h were sus-pended in st'erile saline. Each suspension was mixed with LRAassimilation medium (API) and distributed into 50 tubes of thegallery. The intensiry o{ turbidity was then measured after incu-bation at 28 "C for 1,2 and 4 days. DNase activity was examinedon DNase test agar (Catalog No.0632, Difco Lab., USA). Acidproduction from sugars and polyalcohols was tested in both OFbasal medium and PYP (peptone - yeast extract - phenol red)basal medium containing 0.05% peptone, 0.057" yeast extract,0.5% NaCl, 0.03% K2HPO4,0.03'h agar and 0.002'/, phenolred (Yabuucbi et al., 1,979).
Cellular lipids and fatty acid analysis. Cells harvested afterculturing 24 h in PY medium were freeze-dried, and 50 mg ofdried cells were mixed with 2 ml of 5% HCI in methanol andheated at 100"C for 3 h. Fatry acid methyl esters extracted withn-hexane were separated by thin-layer chromatography (TLC)using a solvent system of n-hexaneldiethyl ether (1 : 1, v/v). Non-polar, 2-hydroxy (2-OH) and 3-OH fatty acids, visualized byspraying 0.02% dichlorofluorescein in ethanol, were extractedwith diethyl ether and analyzed by gas-liquid chromatography(GLC).
The long-chain bases of the cellular sphingolipids were ob-tained from acid hydrolysates of dried cells as describedby Yanoet al. (1.982). The long-chain base was developed by TLC with asolvent system of chloroform,/methanoywater (65 :25 :4, vlv)and analyzed as trimethylsilyl ether by gas chromatography/massspectrometry (GC/MS).
Table l. Bacterial strains used and summary of identification of PEG-utilizing bacteria
Strain Substrate Formerly identified as(PEG)
Reference Identifiedas
42 40043 400c-1 1,000101 1,000L02 1,000103 4,000202 4,000203 4,000206 4,000D-1 4,000D-2 4,000[E-1] 6,000
tYwl 6,000l22al 6,000141,11 6,000l2l 6,000
Pseudomonas sp.Pseudomonas sp.Pseudomonas sp.Pseudomonas sp.Pseudomonas sp.Flauobacterium sp.Flauobacterium sp.Flauobacterium sp.Pseudomonas sp.Flauobacterium sp.Flauobacterium sp.Flauobacterium sp. (A) + Pseudomonas sp. (B)
Flauobacterium sp. (A) + Pseudomonas sp. (B)
Flauobacterium sp. (A) + Pseudomonas sp. (B)
Flauobacterium sp. (A) + Pseudomonas sp. (B)
Flauobacterium sp. (A) + Pseudomonas sp. (B)
P seudomonas solanacearumAl c ali gene s xy I o s o x i d an s srb sp. denitr ifi cansEnterobacter diuersusPseudomonas uesicularisGram-negative rod with Q-10Sp h ingomonas macr o gobabidusSp h ingomonas macr o goltabidwsSphingomonas macrogobabidrzs (IFO 15033r)Sp h in gom on as ma cr o go b a b i du s
Sp h in g om on as ma cr o go bab i du s
Sp h in gomonas macr o go ltabidusSiingomonas terrae (LFO 15098r)-f Rhizobium sp.Sphingomonas terrae + Rhizobium sp.Sphingomonas terrae + Rhizobium sp.Sphingomonas terrae + Agrobacterium sp.Sphingomonas terrae + Methylobacterium sp.
Sphingomonas sanguis (T)Sphingomonas terrae
(1)(1)(1)(1)(1)(1)(1)(1), (2)
(1)(1)(1), (3)
Sph ingomonas p aucimobilisS. parapawcimobilisS.yanoikuyaeS. adhaesiuaS. capsulataSpbingomonas genospecies 1
Sphingomonas genospecies 2
IFO 13935r (: JCM 75L6'r, GLFU 2395r)IFO 15100r (: JCM 7510r, GTFU t1387r)IFO t5 t02r (: JCM 737Lt , GIFIJ 9882'r)IFO 15099r (: ICM 7370r, GIFU 11458r)IFO 12533r (: JCM 7508r, GIFU r1526-t)lFO 13973 (: JCM 75t4,GIFU 2397)IFO 15103 (: JCM 7513, GIFU 11456)
[]: Mixed culture.Reference: (1"), Kawai et aL. (1,984); (2), Yamanaka et al. (1989), Kawai et aI. (1.977).
Abbreviation for culture collections: GIFU, Department of Microbiology, The Gifu University School of Medicine, Tsukasamachi,Gifu, Japan; IFO, Institute for Fermentation, Osaka, Japan; JCM, Japan Collection of Microorganism, The Physical and ChemicalInstitute (RIKEN), Wako, Saitama, Japan.
Gas-liquid chromatography (GLC). GLC analyses were car-ried out using a Shimadzu GC-9A gas chromatograph. Columnsemployed were 10% DEGS (diethylene glycol succinate) (2 m and5 m) at 180'C and 3% OV-1 (2 m) at 165'C for fatry acidanalysis and OV-1 (2 m) at 190'C for long-chain base analysis.Helium was used as the carrier gas at a flow rate of 50 m1/min.
DNA base composition. DNA was isolated by the method ofSaito and Miura (1,963). G+C content of DNA was determinedby reversed-phase high-performance liquid chromatography(HPLC) (Mesbah, 1.989; Tamaoka and Komagata, 1,984) akernuclease P1 and alkaline phosphatase treatment.
DNA/DNA hybridization. DNA/DNA hybridization was car-ried out by fluorometric hybridization in microdilution wells us-ing biotinylated DNA (Ezaki et al., 1989).
Isoprenoid quinone. Isoprenoid quinones were extracted twicewith chloroform/methanol (2:L, vlv) for 4 h, and purified byTLC using benzene as the solvent and then analyzed by HPLC.HPLC was carried out on a Shimadzu liquid chromatography LC6AD with a ZORBAX (4.6 mm x 15 cm) column. Ubiquinoneswere detected by their absorbance at 275 nm by means of aShimadzu Spectrophotometric Detector SPD-6A. Samples wereeluted with methanoi/isopropyl ether (4:1, vlv) at 1 ml/min at30'C. The elution time and area of each peak were calculatedwith Shimadzu Chromatopac C-R3A.
Sequencing of 165 ribosomal ribonucleic acid (rRNA). Thesequences of 165 rRNA were determined by polymerase chainreaction (PCR) (Sarft, et a1., 1988) according to the followingmethod. The 165 IRNA coding region of DNA was amplifiedfrom the total DNA by the use of cwo primers, one of them wasidentical to the sequence positions 10 to 25 (S'-AGTTTGA-TCCTGGCTC OH-3') (in Escberichia coli ntmbering system,Brosius et al., 1978) and the other complementary to the posi-tions 1541 to 1525 (5'-AAGGAGGTGATCCAGCC OH-3'),and of the Taq DNA polymerase (Cetus Inc., USA). The am-plified DNA was purified by using SuprecrM-01 (Takara Co.,Ltd., Japan) after electrophoresis on agarose gel. The purifiedDNA was sequenced by Sequenase I(it for 35-SdATP (UnitedBiochemical Inc., USA) with the primers 5'-AGTTTGATCCTGGCTC OH-3 (identical sequence with 1,0-25), 5'-GTGTTACTCACCCGT OH-3' (complementary to 1,23-1,09),5'-TACGGGAGGCAGCAG OH-3' (identical with 343-357), 5'-CTGCTGCCTCCCGTAG OH-3' (complementary to 3 57 -3 42),5'-GTGCCAGCAGCCGCGG OH-3' (identical with 515-530),5'-ACCGCGGCTGCTGGC OH-3' (complementary to 531-517), S'-TCTACGCATTTCACC OH-3' (complementary to7 04-690), 5'-GTCAATTCCTTTGAGTTT OH-3' (complemen-tary to 924-907), S'-AGGGTTGCGCTCGTTG OH-3' (com-plementary to 1 1 15- 1 1 00), 5'-CCATTGTAGCACGTGT OH-3'(complementary to 1.242-1,227), S'-ACGGGCGGTGTGTACOH-3' (complementary to 1,406-1,392), and 5'-GGCTACCTTG-TTACGA OH-3' (complementary to 1510-1495). V/hen the dou-ble stranded DNAs were sequenced with Sequenase, the DNApolymerase reaction stopped often at many locations of the se-quence. Therefore, we finaliy used the Mn buffer (Tabor andRichardson, 1989) for avoiding such problem. DNA sequenceswere aligned using ODEN system (Ina, 1991). Nucleotide sub-stitution rates (Knuc values) were calculated (Kimura, 1980), andthe phylogenetic tree was constructed by the neighbor-joiningmethod (Saitou and Nei, 1,987). The topology o{ the trees wereevaluated by bootstrap analysis of the sequence data using theClustal V (Higgins et al., 1992).
Nucleotide sequence accession nwmbers. The sequences werealigned to published sequences from GenBank and EMBL underthe following accession numbers: Sphingomonas capsulata,MS 9 29 6 ; Es ch erich ia coli, M25 5 8 8 ; P seudomonas a e r u gin o s a,M34133; Pseudomonas diminuta, M59064; Pseudomonas men-docina, M59 154; P seudomonas cep acid, M225 L8 ; Erythrobac-
15 System. Appl. Microbiol. Vol.1612
IdentificationofPEG-UtilizatingBacteria 229
ter longus, M59062; Erythrobacter sp., M59063; Rhodo-psewdomonas acidophila, M341,28; Ancylobacter aquaticus,M627 9 0 ; A gr o b act er ium tumef a ci. en s, M1, 1223 ; " R o c h a lim a e a
americana", M7 3229 ; Brucella melitensis, X1,3 69 5 ; Magneto'spirillum magnetotacticum, M58171; Beiierinckia indica,M59060; Azospirillwm lipoferum, M59061.
Results
1. Identification of PEG 4,000- and PEG 6,000-utilizing bacteria (A)
Morphological and physiological characteristics. All 11
strains tested were Gram-negative and rod-shaped organ-isms, and colonies on nutrient agat after 48 h were circu-lar, I to 3 mm in diameter, and they were low convex,smooth and opaque. The tests for which all 11 strains gavepositive results are catalase, oxidase, extracellular deoxy-ribonuclease activity and assimilation of cellobiose andtrehalose. On the other hand, hydrogen sulfide on TSIagar, alkali on Christensen's citrate, arginine dihydrolase,indole and urease are not produced, and nitrate reductionand phenylalanine deamination were negative. o-Fucose,L-xylose, gluconate, adonitol, dulcitol, inulin and glycogenwere not utilized. Strains except for 2-A were motile witha single polar flagellum. Deep-yellow pigments were pro-duced by strains E-1-A, 2-A,22a-A, 103 and D-2, but thecolor of colonies of the other 6 strains (YS7-A, 41,1,-4,202,203,206 and D-1) was creamy white.
Isoprenoid quinones. Isoprenoid quinones extractedfrom all 11 strains gaye a major peak corresponding toubiquinone 10 upon HPLC analysis.
Long-cbain bases of the cellular shingolipids. A11 11
strains contained relatively large amounts of long-chainbases, which were identified by GC/MS as 1,3-dihydroxy-2-aminooctadecane (dihydrosphingosin) (d-18:0), 1,3-di-hy dr oxy -2- aminononadecene (d- 1 9 : 1 ) and 1,3 -dihydroxy-2-aminoeicocene (d-20 : 1 ).
Cellular fatty acids.3-Hydroxy fatty acids were not de-tected in any of the strains. As shown in Table 2, L6:1,18 : 1, 2-OH 14: 0 and 2-OH 1,6: 0 fatty acids were themajor fatty acids in all the PEG 4,000-utilizing bacteria. Inthe PEG 6,00O-utilizing bacteria and Sphingomonasgenospecies 2 IFO 15103 ,17 :1,1.8 :1.,Z-OH L4 :0 and 2-OH 15 :0latty acids were the major ones. Al1 the strainsof the known species of the genus Sphingomonas had18:1 and z-OH 14:0 fatty acids as the major compo-nents of the cellular fatty acids.
G*C content of DNA and DNAIDNA similarity. Asshown in Table 3, the G+C content of 11 strains rangedfrom 63.0 to 65.0 mol% and high DNA/DNA similarityvalues were obtained among the six strains of PEG 4,000-utilizing bacteria. High similarity values were also ob-tained among five strains of PEG 6,000-utilizing bacteriaand Spbingomonds genospecies 2 IFO 15103. Thesestrains showed low DNA similarity values with the otherstrains of known species of the genus Sphingomonas.
Partial nucleotide sequence of 155 rRNA. We analyzedthe partial nucleotide sequences of 165 rRNA of one of sixstrains of 4,000-utilizing bacteria, IFO 15033 (: 293;,one of five strains of 6,000-utilizingbacteria, IFO 15098
230 M. Takeuchi, F. Kawai, Y. Shimada, and A. Yokota
(: E-1-A), and rhe known species of the genus Sphingo-monas, S. pawcinobills IFO li935r, S. piraporcimoiilisIFO 11100', 5. yanoikuyae IFO 15102r, S. adhaesiua IFO15099t, and Sphingomonds genospecies 1 (IFO 13937).
Table 2. Cellular fatty actd composirion of the isolates
Fig. 1 shows the partial 165 rRNA sequences for the teststrains, and K.,c values from 1181 bases are shown inTable 4. To ascertain the intragroup relationships, the se-quences of the species described above were compared
Strain IFO No. Non-polar' 2-OHb
15:01,4:0 L6:0 l6:1 l6:015:0L7:1.17:0 18:1 1,4:0
68-3253-4785-1566-3452-4856-443563340 48 1.2
31, 58 1.L
30 61, 936s9533 67
10010010082 18
100100
936551,9 32 451,1, 41. 4718 33 441,7 31 4217 36 4761364526823-293671,7*3933820-443171,5-284681.6-4432
13-871,1,7--7313 17 70187-1.263137--8017-65
4
,9
;
1;
;447
:
15033r
15098r
15 103'13935r15 100r151.02'l15099-r1.2533rl39i7rd
S. macrogobabidus 103
. 202203206D-1D_2
S.terrae E-1-A2AYW-A41.1-A22a-A
S.paucimobilisS. parapawcimobilisS.yanoikwyaeS. adhaesiuaS. capsulataS. sanguis
' Percentages of the totaI non-polar acids.o Percentages of rhe total 2-hydroxy acids.', Sphingomonas genospecies 2 oI Yabwuchi et al. (1990)." Sphingomonas genospecies 1. oI Yabuuchi et aL. (1990).
Table 3. DNA-DNA similarity among the strains of the six Sphingo?nonas species and PEG-utilizing bacteria
Strain IFO No. G+C(mol%)
% similarity wirh labeled DNA from
103 202 203 206 D-1 D-2 E-1-A 2-A Y!7-A
41,1,- IFO IFOA 15103 1393s
-; i zol -27 26 31. 27 33 1018---309-1418199--23262722100 - 101 1.4
- 100 79 91, 100
- 80 100 *90 86
- 86 88 100 899)
108 78 87 92 100 1.7
736613100106771323245471.1.9677121073669224353724
83100
375655
58101,2
58
64
t00- 100 83
78 104 100 103 10979 - 78 100
- 89 100
- 109 9923 28
-4745-3247-4447
-4e45 31 1.4 42 47-61,210-154410-6237-99s9-2523-34t6
731,1,6
S. macrogoltabidus 103202203206D-1D_2
S.terrae E-1-A2-AY!T,A41,1-A22a-A
S.paucimobilisS. parapaucimobilisS. yanoikuyaeS. adhaesiuaS. capsulataS. sangwis
65.054.3
15033r $.963.263.764.4
15098r $.064.964.1,
64.764.2
15103" 64.1]39351 A.715100r 65.1,
$rc2-r 61,.6l5o99r 67.8L2fi3r $.8B%7r'b 6L.8
^ Sphingomonas genospecies 2 of Yabwuchi et al. (1990).o Sphingomonas genospecies L oI Yabuuchi et al. (1990).
Identification of PEG-Utilizating Bacteria 231,
100SPAU AGUUUGAUCCUGGCUCAGAACGAACGCUGGCGGCAUGCCUAACACAUGCAAGUCGAACGAAGG. CUUCGGnnnUAGUGGCGCACGGGUGCGUAACGCGUGSPaT AGUUUGAUCCUGGCUCAGAACGAACGCUGGCGGCAUGccUAAcAcAUGcAAGUcGAACGAGGCcCUUCGGGAAUAGUGGCGCACGGGUGCGUAACGCGUGSSAN AGUUUGAUCCUGGCUCAGAACGAACGCUGGCGGCAUGCCUAACACAUGCAAGUCGAACGAAGG. CUUCGGNNNUAGUGGCGCACGGGUGCGUAACGCGUGSteT AGUUUGAUCCUGGCUCAGAACGAACGCUGGCGGCAUGCCUAACACAUGCAAGUCGAACGAGAU. CUUCGGAUCUAGUGGcGcAcGGGUGCGUAACGCGUGSyan AGUUUGAUCCUGGCUCAGAACGAACGCUGGCGGCAUGCCUAAUACAUGCAAGUCGAACGAGAU. CUUCGGAUeGAGUGGCCcAcGGGUGcGUAACGCGUGSAdh AGUUUGAUCCUGGCUCAGAACGAACGUUGGCGGCAUGCCUAACACAUGCAAGUCGAACGAGAU. CUUCGGAUCUAGUGGCCCACGGGUGCGUAACGCGUGSmac AGUUUGAUCCUGGCUCAGAACGAACGCUGGCGGCAUGCCUAACACAUGCAAGUCGAACGAAGU. cUUcGGACUUAGUGGCccAcGGGUGcGUAACGCGUG
200SPAU GGAAUCUGCCCUUGGGUUCGGAAUAACNNNNNGAAACGGCUGCUAAUACCUGAUGAUAUCCGUAGAUCAAAGAUUUAUCGCCUAAGGAUGAGCCCGCGUASPAT GGAAUCUGCCCUUGGGUUCGGAAUAACNNNNNGAAACGGCUGCUAAUACCUGAUGAUGACGAUAGUCCAAAGAUUUAUCGCCACAGGAUGAGCCCGCGUUSSAN GGAAUCUGCCCUUGGGUUCGGAAUAACNNNNNGAAACGGCUGCUAAUACCGGAUGAUGACGAAAGUCCAAAGAUUUAUCGCCUGAGGAUGAGCCCGCGUUSteT GGAAUCUGCCCUUGGGUUCGGAAUAACUCAGAGAAAUUUGUGCUAAUACCGUAUAAUGUCUUCGGACCAAAGAUUUAUcccCCAAGGAUGAGCccGcGUASYan GGAAUCUGCCCUUGGGUUCGGAAUAACUUCUGGAAACGGAAGCUAAUACCGGAUGAUGACGUAGGUCCAAAGAUUUAUCGccCAAGGAUGAGCccGCGUASAdh GGAAUCUGCCCUUGGGUACGGAAUAACUCAGAGAAAUUUGUGCUAAUACCGUAUAAUGUCUUCGGACCAAAGAUUUAUCGCCCAAGGAUGAGCCCGCGUASMAC GGAAUCUGCCCUUGGGUUCGGAAUAACUUAGAGAAAUNNGUGCUAAUACCGNAUAAUGACNNCGGUCCAAAGAUUUAUCGCCCAAGGAUGAGCCCGCGUA
300spau GGAUUAGGUAGUUGGUGGGGUAAAGGccUAccAAGGccAcGAUcCAUAGCUGGUCUGAGAGGAUGAUCAGCcAcAcUGGGAcUcAGAcAccccccAGAcUspar GGAUUAGGUAGUUGGUGGGGUAAAGGCcUAccAAcGcGAcGAUcCAUAGCUGGUCUGAGAGGAUGAUCAGCcAcAcUGGGAcUGAGAcAccccccAGAcUSSAN GGAUUAGGUAGUUGGUGGGGUAAAGGCCUACCAAGCcGAcGAUCCAUAGCUGGUCUGAGAGGAUGAUCAGCcAcAcUGGGAcUGAGAcAcGGcccAGAcUster GGAUUAGCUAGUUGGUGGGGUAAAGGCUcAccAAGGcGAccAUcCAUAGCUGGUCUGAGAGGAUGAUCAGCcAcAcUGGGAcUcAGAcAccccccAGAcUsyan GGAUUAGCUAGUUGGUGAGGUAAAGGCUCACCAAGGccAcGAUcCUUAGCUGGUCUGAGAGGAUGAUCAGCCACACUGGGACUGAGACACGGcccAGAcUSAdh GGAUUAGCUAGUUGGUGAGGUAAAAGCUCACCAAGGCGACGAUCCUUAGCUGGUCUGAGAGGAUGAUCAGCCACACUGGGACUGAGACACGGCCCAGACUsmac AGAUUAGCUAGUUGGUGGGGUAAAAGCUUACCAAGGCGACGAUCUUUAGCUGGUCUGAGAGGAUGAUCAGccACAcUcccAcUGAGAcAccccccAGAcU
400SPAU CCUACGGGAGGCAGCAGUGGGGAAUAUUGGACAAUGGGCGAAAGccUGAUccAGCAAUGccGCGUGAGUGAUGAAGGCCCUAGGGUUGUAAAGCUCUUUUSPaT CCUACGGGAGGCAGCAGUGGGGAAUAUUGGACAAUGGGCGCAAGccUGAUccAGCAAUGccccGUGAGUGAUGAAGGCccUAGGGUUGUAAAGCUCUUUUSsan CCUACGGGAGGCAGCAGUGGGGAAUAUUGGACAAUGGGCGAAAGCCUGAUCcAGCAAUGccccGUGAGUGAUGAAGGCcCUAGGGUUGUAAAGCUCUUUUSteT CCUACGGGAGGCAGCAGUGGGGAAUAUUGGACAAUGGGCGAAAGccUGAUccAGCAAUGccccGUGAGUGAUGAAGGCcCUAGGGUUGUAAAGCUCUUUUsyan ccUACGGGAGGcAccAcUcccGAAUAUUGGAcAAUGGGcGAAAGccUcAUccAGcAAUGccGcGUGAGUGAUGAAGGccuuAGGGUUGUAAAGcucuuuuSAdh CCUACGGGAGGCAGCAGUGGGGAAUAUUGGACAAUGGGCGAAAGCCUGAUCCAGCAAUGCCGCGUGAGUGAUGAAGGCCCUAGGGUUGUAAAGCUCUUUUsmac cCUACGGGAGGCAGCAGUGGGGAAUAUUGGACAAUGGGCGAAAGCcUcAUccAGcAAUGccGcGUGAGUGAUGAAGGccCUAGGGUUGUAAAGeUCUUUU
500SPAU ACCCGGGAAAAUAAUGACAGUACCGGGAGAAUAAGCCCCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGAGCUAGCGWGWCGGAAUUACUSPaT ACCCGGGAAGAUAAUGACUGUACCGGGAGAAUAAGCcccGGcUAAcUccGUGccAGcAGcCGCGGUAAUACGGAGGGGGCUAGCGITGUUCGGAAUUACUSsan ACCCGGGAAGAUAAUGACUGUACCGGGAGAAUAAGccccGGcUAAcUccGUGcCAGCAGCCGCGGUAAUACGAGGGGGGCUAGCGWGWCGGAAUUACUSteT ACCCGGGAUGAUAAUGACAGUACCGGGAGAAUAAGCUCCGGCUAACUUCGUGCcAGcAGccGCGGUAAUACGAGGGGAGCUAGCGWGUUCGGAAUUACUSyan ACCCGGGAUGAUAAUGACAGUACCGGGAGAAUAAGCUCCGGcUAAcUccGUGccAGcAGcCGCGGUAAUACGGAGGGAGCUAGCGUUGUUCGGAAUUACUSAdh ACCCGGGAUGAUAAUGACAGUACCGGGAGAAUAAGCUCCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGAGCUAGCGWGUUCGGAAWACU$NAC ACCCGGGAUGAUAAUGACAGUACCGGGAGAAUAAGCUCCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGAGCUAGCGUUGWCGGAAUUACU
500SPau GGGCGUAAAGCGCACGUAGGCGGCUUUGUAAGUCAGAGGUGAAAGCcUGGAGcUcAAcUccAGAAcUGcCIJWGAGACUGCAUCGCUUGAAUCCAGGAGASpaT GGGCGUAAAGCGCACGUAGGCGGCUUUGUAAGUCAGAGGUGAAAGCcUGGAGcUcAAcUccAGAAcUGcCUUUGAGACUGCAUCGCUUGAAUCCAGGAGASsan GGGCGUAAAGCGCACGUAGGCGGCUUUGUAAGUCAGAGGUGAAAGCcUGGAGcUcAAcUccAGAAcUGcCUWGAGACUGCAUCGCWGAAUCCAGGAGASteT GGGCGUAAAGCGCGCGUAGGCGGUUUUUUAAGUCAGAGGUGAAAGCccGGGGcUcAAccccGGAAUAGcCI]TTGAAACUGGAAAGCUAGAAUCUUGGAGASyan GGGCGUAAAGCGCACGUAGGCGGCUAUUCAAGUCAGAGGUGAAAGCccGGGGcUcAAccccGGAAcUGcCIJITGAAACUAGAUAGCWGAAUCCAGGAGASAdh GGGCGUAAAGCGCACGUAGGCGGCUAUUCAAGUCAGAGGUGAAAGCCCGGGGCUCAACCCCGGAAUAGCCWUGAAACUGGAAAACUAGAAUCUUGGAGASmac GGGCGUAAAGCGCGCGUAGGCGGUUUUUUAAGUeAGAGGUGAAAGCccGGGGcUcAAcccCGGAAUUGCCUWGAAACUGGAAAACWGAAUCUUGGAGA
700SPau GGUCAGUGGAAUUCCGAGUGUAGAGGUGAAAUUCGUAGAUAUUCGGAAGAACACCAGUUGCGAAGGCGGCUGACUGGACUGGUAUUGACGCUGAGGUGCGSPaT GGUCAGUGGAAUUCcGAGUGUAGAGGUGAAAUUCGUAGAUAUUCGGAAGAACACCAGUUGCGAAGGCGGCUGACUGGACUGGUAUUGACGCUGAGGUGCGSsan GGUCAGUGGAAUUCcGAGUGUAGAGGUGAAAUUCGUAGAUAUUCGGAAGAACACCAGUUGCGAAGGCGGCUGACUGGACUGGUAUUGACGCUGAGGUGCGStET GGUCAGUGGAAUUCCGAGUGUAGAGGUGAAAUUCGUAGAUAUUCGGAAGAACACCAGWGCGAAGGCGGCUGACUGGACUGGUAUUGACGCUGAGGUGCGSyan GGUGAGUGGAAUUCcGAGUGUAGAGGUGAAAUUCGUAGAUAUUCGGAAGAACACCAGUUGCGAAGGCGGCUCACUGGACUGGUAUUGACGCUGAGGUGCGSAdh GGUCAGUGGAAUUCCGAGUGUAGAGGUGAAAUUCGUAGAUAUUCGGAAGAACACCAGUGGCGAAGGCAGCUGACUGGACAAGUAUUGACGCUGAGGUGCGSmac GGUCAGUGGAAUUCcGAGUGUAGAGGUGAAAUUCGUAGAUAUUCGGAAGAACACcAGUGccGAAGGcGACUGACUGGACAAGUAUUGACGCUGAGGUGCG
800Spau AAAGCGUGGGGAGCAAACAGGAUUAGAUACccUGGUAGUccAcccCGUAAACGAUGAUAACUAGCUGUCCGGGCACUUGGUGCUUGGGUGGCGCACGUAASPAT AAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGAUAACUAGCUGUCCGGGCACWGGUGCUUGGGUGGCGCACGUAASSAN AAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGAUAACUAGCUGUCCGGGCACUUGGUGCWGGGUGGCGCAGCUAAStE! AAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGAUAACUAGCUGUCCGGGCUCAUAGAGCWGGGUGGCGCACGUAASYan AAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCcAcGccGUAAAcGAUGAUAACUAGCUGUCAGGGCACAUGGUGUWUGGUGGCGCACGUAASadh AAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCcAcGccGUAAAcGAUGAUAACUAGCUGUCCGGGCUCAUAGAGCUUGGGUGGCGCACGUAASmac AAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCcAcGccGUAAAcGAUGAUAACUAGCUGUCcGGGUUCAUAGAACUUGGGUGGCGCACGUAA*
,ooSPAU CGCAUUAAGUUAUCCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAGGAAUUGACGGGGGCcUGcAcAAGCGGUGGAGCAUGUGGUUUAAUUCGAAspar CGCAUUAAGUUAUCcGcCUGGGGAGUACGGcCGCAAGGUUAAMCUCAAAGGAAUUGACGGGGGcCUGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAAssan cGCAUUAAGUUAUcccccUGGGcAGUAccGccGcAAGGUUAAAAcUcAAAGGAAUUGACGGcccccuccAcAAGccGUGGAGcAUGUGGUUUAAUUccAAster cGCAUUAAGUUAUCcGccUGGccAGUAccccCGCAAGGUUAAAACUCAAAGGAAUUGACcccccccuccAcAAGcGGUGGAGCAUGUGGUUUAAUUCGAAsyan cGCAUUAAGUUAUCcGccUcccGAGUAcGGUccCAAGAUUAAAACUCAAAGGAAUUGACGGGGGCcUGCACAAGCGGUGGAGCAUGUGGWUAAUUCGAASAdh CGCAUUAAGUUAUCCGCCUGGGGAGUACGGUCGCAAGAUUAAAACUCAAAGGAAUUGACGGGGGCCUGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAAsmac CGCAUUAAGUUAUCccccUGGGGAGUACGGUCGCAAGGUUAAAACUCAAAGGAAUUGACcccccccUccAcAAGcGGUGGAGcAUcUccUUUAAUUcGAA
1000spau GcAAcGcGcAGAAccuuAccAGccuuucAcAUcGUAGGAccAcuu. CCAGAGAUGGAUUUCUUCC. uucccGGAAccuAcAcAcAGGUGcuccAUGGcucspar GcAAcGcGcAGAAccuuAccAGccuuuGAcAUGUccGGAccAuuu. ccAGAGAUGGAUcucuucc. uucccccA. CUGGAACACAGGUGCUGCAUGGCUGssan ccAAcGcccAGAAccuuAccAcccuuuGAcAUGUccccAccAuuu. ccAGAGAUGGAUcucuucc. uucGGGGA. cuccAAcAcAGGUGcuccAUGGcuGster GcAAcGcccAGAAccuuAcCAGCGUUUGACAUCcuGAccccccuuAcCAGAGAUGGUUUccuuuAGUUccccuccAUAGGUGAcAGGUGcuGCAUGGcucSYAN GCAACGCGCAGAACCUUACCAACGUUUGACAUCCCUAUCGCGGAUCGUGGAGACACUUUCCUUCAGUUCGGCUGGAUAGGUGACAGGUGCUGCAUGGCUGSAdh GCAACGCGCAGAACCUUACCAGCGUUUGACAUccUGAUcGcGGUUACoAGAGAUGGUUUcCUUCAGUUCGGCUGGAUCAGUGACAGGUGCUGCAUGGcUGSNAC GCAACGCGCAGAACCUUACCAGCGUUUGACAUCCUGAUCGCGGAUUAGAGAGAUCUUUUCcUUcAGUUcGGcUGGAUCAGUGACAGGUGCUGCAUGGcUG
232 M. Takeuchi, F. Kawai, Y. Shimada, and A. Yokota
110 0SP U UCGUCAGCUCGUGUCGUGAGAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUCGCCUUUAGUUACCAUCAUUUGGUUGGGUACUCUAAAGGAACCGCCSPAT UCGUCAGCUCGUGUCGUGAGAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUCGCCUUUAGUUACCAUCAWUGGUUGGGUACUCUAAAGGAACCGCCSSAN UCGUCAGCUCGUGUCGUGAGAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUCGCCUUUAGUUACCAUCAUUUGGUUGGGUACUCUAAAGGAACCGCCStET UCGUCAGCUCGUGUCGUGAGAUGUUGGGWMGUCCCGCAACGAGCGCAACCCUCAUCCCUAGUUGCCAUCAUUAAGUUGGGCACUCUAAGGAAACUGCCSYAN UCGUCAGCUCGUGUCGUGAGAUGWGGGUUMGUCCCGCAACGAGCGCAACCCUCGCCUUUAGUUGCCUGCAUUUAGUUGGGUACUCUAAAGGAACCGCCSAdh UCGUCAGCUCGUGUCGUGAGAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUCAUCCCUAGUUGCCAUCAUUAAGUUGGGCACUCUAAGGAAACUGCCSMAC UCGUCAGCUCGUGUCGUGAGAUGUUGGGUUMGUCCCGCAACGAGCGCAACCCUCAUCCCUAGUUGCCAUCAUUCAGUUGGGCACUCUAAGGAAACUGCC
1200SPAU GGUGAUAAGCCGGAGGAAGGUGGGGAUGACGUCAAGUCCUCAUGGCCCUUACGCGUUGGGCUACACACGUGCUACAAUGGCAACUACAGUGGGCAGCGACSPA! GGUGAUAAGCCGGAGGAAGGUGGGGAUGACGUCAAGUCCUCAUGGCCCWACGCGUUGGGCUACACACGUGCUACAAUGGCAACUACAGUGGGCAGCGACSSAN GGUGAUAAGCCGGAGGAAGGUGGGGAUGACGUCAAGUCCUCAUGGCCCUUACGCGCUGGGCUACACACGUGCUACAAUGGCAACUACAGUGGGCAGCGACStET GGUGAUAAGCCGGAGGAAGGUGGGGAUGACGUCAAGUCCUCAUGGCCCUUACGCGCUGGGCUACACACGUGCUACAAUGGCGGUGACAGUGGGCAGCAACSyan GGUGAUAAGCCGGAGGAAGGUGGGGAUGACGUCAAGUCcUcAUGGcccUUAcGcGUUGGGCUACACACGUGCUACAAUGGCGACUACAGUGGGCAGCCACSAdh GGUGAUAAGCCGGAGGAAGGUGGGGAUGACGUCAAGUCCUCAUGGCCCUUACGCGUUGGGCUACACACGUGCUACAAUGGCGGUGACAGUGGGCAGCAACSMAC GGUGAUAAGCCGGAGGAAGGUGGGGAUGACGUCAAGUCCUCAUGGCCCUUACGCGCUGGGCUACACACGUGCUACAAUGGCGGUGACAGUGGGCAGCAAC
1300SPAU CCUGCGAGGGCGAGCUAAUCCCCAAAAGUUGUCUCAGUUCGGAUUGUUCUCUGCAACUCGAGAGCAUGAAGGCGGAAUCGCUAGUAAUCGCGGAUCAGCASPaT CCUGCGAGGGCGAGCUAAUCCCCAAAAGUUGUCUCAGUUCGGAUUGUUCUCUGCAACUCGAGAGCAUGAAGGCGGAAUCGCUAGUAAUCGCGGAUCAGCASsaTT CCUGCGAGGGCGAGCUAAUCCCCAAAAGWGUCUCAGUUCGGAWGUUCUCUGCAACUCGAGAGCAUGAAGGCGGAAUCGCUAGUAAUCGCGGAUCAGCASteT CUCGCGAGAGGUAGCUAAUCUCCAAAAGCCGUCUCAGUUCGGAUUGUUCUCUGCAACUCGAGAGCAUGAAGGCGGAAUCGCUAGUAAUCGCGGAUCAGCASyan CUCGCGAGAGGGAGCUAAUCUCCAAAAGUCGUCUCAGUUCGGAUCGUUCUCUGCAACUCGAGAGCGUGAAGGCGGAAUCGCUAGUAAUCGcGGAUCAGCASAdh CUCGCGAGAGGUAGCUAAUCUCCAAAAGCCGUCUCAGUUCGGAUUGUUCUCUGCAACUCGAGAGCAUGAAGGCGGAAUCGCUAGUAAUCGCGGAUCAGCASmac CGGGCGAGGGCGAGCUAAUCUCCAAAAGCCGUCUCAGUUCGGAUUGUUCUCUGCAACUCGAGAGCAUGAAGGCGGAAUCGCUAGUAAUCGCGGAUCAGCA
1400SPau UGCCGCGGUGAAUACGUUCccAGGccUUGUAcAcAccGccCGUCACACCAUGGGAGUUGGAUUCACCCGMGGCGUUGCGCCAACCUAGCAAUAGGAAGCSpaT UGCCGCGGUGAAUACGUUCCCAGGCcUUGUAcAcAcccccCGUCACACCAUGGGAGUUGGAUUCACCCGAAGGCGUUGCGccAAccUAGCAAUAGGAAGCSsan UGCCGCGGUGAAUACGUUCCCAGGCcUUGUAcAcAccccccGUcAcAcCAUGGGAGUUGGAUUCACCCGAAGGCGUUGCGccAAccc. GCAAGGGMGGCStCT UGCCGCGGUGAAUACGUUCCCAGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUUGGUUUCACCCGMGGCCCAGAGCCAACCC. GCACGGGAGGAASYAN UGCCGCGGUGAAUACGUUCCCAGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUUGGAUUCACUCGAAGGCGUUGAGCUAACC . . GUAAGGAGGCA.SAdh UGCCGCGGUGAAUACGUUCCCAGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUUGGAUUCACCCGAAGGCAGUGCUCUAACCC. GCAAGGGAGGAASmac UGCCGCGGUGAAUAcGUUcccAGGccUUGUAcAcAccGcccGUcAcAccAUGGGAGITGGAUUcAcccGAAGGcAAUGcUcUAAccc. GCAAGGGAGGAA
1420SPAU AGGCGACCACGGUGGAUUNAGSPAT AGGCGAAGUCGUAGCUUAGGGSSAN AGGCGACCCACGGUGGGUUNNStET AGGUGAAUGCCUAGUUAUGNNsyan .GGCGACCACACAGUGGGUCnSAdh .GCUGACCACAGUGGGUCnnnSTNAC .GCUGACCACCGUGGGUCNNN
Fig. 1. 165 rRNA partial sequences ol S. paucimobilis (Spat), S. parapaucimobilis (Spar), S. sanguis (Ssan), S. terrae (Ster), S.
yanoihuyae (Syan), S. adhaesiua (Sadh) and S. macrogobabidws (Smac). The first and last nucleotides are analogous to positions 10 and1,483 o{ the E. coli sequence (Brosius et al. 1,978).Dot - a nucleotide gap; n - undetermined nucleotide.The nucleotide sequence data will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases with the followingaccession numbersf S. paucimobilislFo 13935r, Dt3725 s.parapaucimobilislFO 15100r, D13724;s.sanguis IFO 13937r, D1,3726;S. terrae IFO 15098r, D13727; S. yanoikuyae IFO 15 102r, D1,3728; S. adhaesiua IFO 15099r, D1,3722; S. maqogoltabidus IFO15033r, DL3723.
with those of S. capswlata and 15 previously studied bac-teria from 1.t genera. The phylogeneric tree based uponK,o. values is depicted in Fig.2. The branching patternindicated that the eight species of the genus Sphingomonasform a compact phylogenetic cluster very cleady separatedfrom other representatives of the cr-subclass of Proteobac-teria, except for Erytbrobacer longus, a bacteriochloro-phyll-containing bacterium.
As this arrangement (Fig.2) is in good agreement withthe available chemotaxonomic evidence, we consider it tobe a more accurate reflection of the phylogeny of thespecies of the genus Sphingomonas. All 11 strains of PEGutilizing bacteria are characterized by the presence ofsphingolipid as the cellular lipid, major long-chain bases inthe sphingolipid with d-18 : 0, d-1.9 :1 and d-20 :1, byhaving 18:1, L6 :1 and/or 17 :1. and z-OH 14: 0, 2-OH15 : 0 or 2-OH 16: 0 as major cellular fatty acids, by theabsence of 3-hydroxy fatty acids, by having isoprenoidquinones with Q-10, and by G+C content of DNA with63.0-65.0 molo/". On the basis of these physiological and
chemotaxonomic characteristics (Table 5) together withDNA/DNA hybridization studies and partial nucleotidesequence analysis of 165 rRNA, we believe that all 11
strains of PEG utilizing bacteria are species of the genus
Spbingomonas, and we propose Spbingomonds rnacrogol-tabi.dus sp. nov. for the PEG 4,0O0-utilizing bacteria, andSphingomonds terrae sp. nov. for the PEG 6,000-utllizingbacteria and the strain of Sphingomona.s genospecies 2,and Sphingomonas sanguis sp. nov. forthe strain of Sphin-gonnonas genospecies 1.
2. Identification of PEG 400-, PEG 1,000- and PEG6,000-utilizing bacteria (B )
PEG 400- and 1,OOO-utilizing bacteria and B strains inthe culture of E-1, YlW,22a,41,1. and 2, which are thoughtto support the growth of the dominant bacteria in the PEG6,000-utilizing mixed cultures, are all Gram-negative,nonsporeforming and rod shaped organisms. Colors ofcolonies are light brown or yellow, except for strain 2-B
Identification of PEG-Utilizating Bacteria 233
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which is pink. Physiological and chemotaxonomic charac-teristics examined are summarized in Table 6. From thedata mentioned here, these organisms were identified asdescribed in Table 1. Out of the five strains isolated frommixed cultures, three strains were tentatively identified asRhizobium sp., and strains 411-B and 2-B were tentativelyidentified as Agrobacterium and Methylobacterium spe-cies, respectively.
Discussion
Since the first report by Fincber and Payne (1.962) on aPEG-utilizing bacterium, there have been many studies onthe biodegradation of PEGs. Ogata et al. (1975) isolatedvarious fypes of PEG-utilizing bacteria based on their abil-ity to utilize PEGs with different molecular weights as thesole carbon and energy sources, and these PEG-utilizingbacteria were tentatively identified as Flauobacterium sp.or Pseudomonas sp. (Kawai et al.,'J,977,1984). However,further taxonomic studies on these bacteria have not beencarried out, and their taxonomic positions have remaineduncertain. Therefore, in this study, we attempted to deter-mine the taxonomic position of these PEG-utilizing bac-teria classified as Flauobacterium sp. or Pseudomonas sp.Based on the morphological, physiological and chemotax-
)Vagretospirilluil rugnetot@ticuilAuspirilltm lipolm
Sphingownas@guis
SphiLgoilonw poucimobilis
Sphingomonupsqpwiwbilis
Sphingomona yanoikuyu
Sphingomonu a.dhuiva
Sphingomon6 capsulara
ErYlrrub@ter lorgw"Ruhaliruuwricw"
Breella rulitcwisAgrob@terium tawt@icns
Beiieriwhillindia
Arcylobrcter aquaticw
Pseudomonu diminuta Rhodopseu.domnu rcidophila
Erythrobrcter sP'
Fig.2. Unrooted phylogenetic tree displaying the relationships ol Sphingomonas species and other reference organisms. Bar:0.02 K.,. unit.
-. Sphingomonu\ Mroeoltabid6Sphiagomo-nu tzm
onomic characteristics, PEG 400- and PEG 1,000-utilizingbacteria were identified as Pseudomonds solanacearum,Alcaligenes xylosoxidans subsp. denitrificans, Entero-b acter diuer sws and P seudomonas uesicularis, respectively(Table 1 and 6, data not shown).
On the other hand, all the strains of PEG 4,O00-utilizingbacteria and the dominant bacteria in the PEG 6,000-utilizing mixed cultures were found to belong to the genusSphingoruonas, and we proposed two new species. Theother kind of bacteria isolated from mixed cultures of PEG6,000 was tentatively identified as Rhizobium sp., Agro-bacterium sp. and Methylobacterium sp., respectively,based on their morphological, physiological and chemo-taxonomic characteristics (Tables 1 and 6). To determinewhether these strains E-1-B, YIV-W and 22a-B areRhizobiwm species, it is necessary to confirm nodule for-mation on roots of leguminous plants.
The genus name Spbingonnonas was proposed byYabuuchi et al. (1990). The type species of this genus isS.paucimobilrs, which was previously named Pseudo-monas paucimobilis by Holmes et aI. (1977). Recently,uan Bruggen et al. (1990) proposed the new genus nameRhizomonas for motile, Gram-negative bacteria whichcause corky root on lettuce. Since the whole-cell fatty acidprofile of the type strain of Rhizomonas swberifaciens re-sembles that of P. paucimobilis, they suggested that R.
Identification of PEG-Utilizating Bacteria 235
Table 5. Differential characteristics among the eight Sphingomonas species
Substrate or test S.macro- S. terraegolta-bidws(n:6) (n:5)
S.sanguis S.pauci S.para-mobiliso pauci-
mobilis'(n: 1) (n:7) (n:4)
S.yano- S.adhae- S.caPsu-ikuyaeu siua" lata"
(n: 1) (n: 1) (n: 1)
Deep-yellow colonyMotiliryH2S production on TSI agarDNase productionOxidative acid from glycerolOxidative acid from rhamnose
(API 20 NE system)Utilization o{ n-caprateHydrolysis of esculinHydrolysis of gelatin
(APU 50 CH system)Utilization of
Ribose, L-sorbose, sorbitolD-Lyxose, D-tagatoseGalactoseD-Mannose, D-fructoseEsculinB-GentiobioseMelezitose
Utilization of PEG 4,000Utilization of PEG 6,000PEG-400 dehydrogenase acivi ry
Major cellular fatty acidNon-polar fatty acid
2-Hydroxy fatty acid
216b
+
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ND
L6:L18:114:016:0
f
+
++
+
+++
++
++++3t4
+
18:1
1.4:0
++
+++
18:1
14:0
416
st6-+U61.t6
+++++
++
+
++
+++517
st7
+++++
3t6
st6s16516
st6+
516ND3
++
i+
+
+
++
+
++++
t,u irc1,t6
4t64t6++4t6
L7:L18:114:015:0
18:1
L4:0
18: 1
14:0
18: 1
l4:0
18: 1
l4:0
' Data lrom Yabuuchi et aL. (L990).b Number of strains showing positive results / number of strains tested.
" All strains positive.d All strains negatiYe.
' ND, Not determined.
suberifaciens is related to P. poucimobilis but not identi-cal. Our data on the chemotaxonomic characteristics of R.suberifaciens revealed the presence of sphingolipid and thepresence of 18:1, 16:0, L6:1 arrd z-OH 14:0 and theabsence of 3-hydroxy fatty acids as cellular fatry acids,which also indicated the resemblance of both genera. Todetermine the relationship between the genera Sphingo-monds and Rhizomonas, the analysis of complete nuc-leotide sequence of 165 rRNA is now undergoing in ourlaboratory, and the results will be published elswhere.
A bacteriochlorophyll-containing bacterium, E. longws(Shirnada et a1., 1985), was closely related to the genusSphingomon4s on the basis of its 165 rRNA sequence.Chemotaxonomic data (Urakami and Komagata, L988)indicates this organism shows some resemblance to thegents Sphingomonas, the presence of ubiquinon e 1.0, 60.7mol% G+C content of DNA, the presence of 18 : 1, 2-OHL4 :0, Z-OH 15 : 0 and 2-OH 16: 0 as the major cellularfattay acids. However, based on the presence of 3-OHfatty acids, bacteriochlorophyll a and carotenoids and
other taxonomic data, it can be differentiated from thegents Sphingomonas.
Emended descriptions of the genus Sphinomonas, andthe proposed new species S. macrogobabidws, S. terraeafid S. sanguls are given below.
Emendation of tbe genus Sphingomonas Ydbuuchi,Yano, Oyaizu, Hashimoto, Ezaki dnd Ydmamoto
sphin. go. monas, Gr. adj. sphingos of sphinx; Gr. n.monas a unit, monad; M. L. fem. n. Sphingornoncts, a
sphingosine-containing monad.The salient characteristics of the genus based on the
original description of Yabuucbi et al. (1.990) and our ownobservation are as follows.
The cells of species in this genus are Gram-negative,nonsporeforming, straight rods that have a single polarflagellum when motile. Catalase is produced. Colonies are
deep-yellow or whitish brown. Obligately aerobic. Acidwas produced oxidatively from pentoses, hexoses, disac-
236 M. Takeuchi, F. Kawai, Y. Shimada, and A. Yokota
Table 6. Taxonomic characteristics of polyethylene glycol-utilizing bacteria
Characteristic Strain
101c-14342 102 F,-1-B YStr-B 22a-B 411,-B 2-B
Substrate PEGGram-stainingMorphologyMotilityFlagellation
Color of cellsuSporeOxidaseCatalaseIsoprenoid quinone
G+C content (mol%)
Cellular fary acidbNon-polar
2-Hydroxy
3-Hydroxy
400 400
rod rod++polar peritri-single chouslt brown lt brown
++++Q-8 Q-8
63.8 62.9
1,000 6000
rod rod+-polarsingleIt brown lt brown
+++-Q-10 Q,10
52.9 62.s
18:1 18:1
18:0
14:0 l2:0L6:0 iL3:018:0 18:0
18:1
6000 6,000
rod rod++poiar polarsingle single1t brown pink
++++Q-10 Q-10
62.0 68.4
18:1 18:1
L4:0 1.4:0t6:0
L6:0 1.6:0 16:0L6:L 1.6:1. 16:118:1 18:1 18:116:0 'L4:0
L6:1.18:0L4:0 14:0 t4:0
L6:0
1,000
rod+peritrichousyellow
+Q-8,MK-854.9
1,000
rod+polarsinglelt brown
++Q-10
68.2
18:L'16:0
L4:0L7cyc18:012:0
iL3:014:0
6,000
'jd
lt brown
+
Q-10
63.2
18:1
t2:0iL3:018:018:1
5,000
rod
It brown
+
Q-10
61.9
18:1
12:0il3:018:018:1
Abbreviation: lt brown, light brown.For fatty acids, the number before the colon indicates the carbon chain length, and the number after the colon indicates the numberof double bonds in the chain; I designates lso branching at the end of the chain. cyc, cyclopropane.
charides, but not from polyalcohols and inulin. Respira-tory quinone is ubiquinone 10. Major cellular fatty acidsare 18 : 1., 16:1. and/or 1.7 :1., and one or more of 2-OH1.4:0, 2-OH 15:0 and 2-OH 16:0. Cellular lipid con-tains sphingolipid. ks major long-chain bases are d-18 : 0,d-L9 : L, d-20 : 'J. and d-21. : 1. The G* C content of DNA is61.6-67.8 molo/o. Type species is Sphingomonas pauci-mobilis (Holmes et al., L977) Yabuwchi et al., 1.990.
Description of Sphingomonds macrogoltabidus sp. nou.
macro.gol.ta'bi.dus. L. fem. n. macrogol, polyethyleneglycol; L. adj. tabidus, dissolving; M. L. fem. ad1. mauo-goltabidus, polyethylene glycol dissolving.
Six strains which utilize PEG 4,000 are included in thisspecies. They are Gram-negative and nonsporeformingrods and are motile with a single polar flagellum. Coloniesare circular, entire, low convex, smooth, opaque and whit-ish brown or yellow. Catalase, oxidase and DNase areproduced. Indole, urease, phenylalanine deaminase andhydrogen sulfide are not produced. PEG 4,000 is assimi-lated. Esculin, cellobiose, trehalose and B-gentiobiose areassimilated, but ribose, r-xylose, o-lyxose, D-tagatose, D-fucose, L-turanose, gluconate, 2-keto-gluconate, S-keto-gluconate, o-methyl-xyloside, adonitol, dulcitol, sorbitol,xylitol, D-arabitol, malate, n-captatq citrare, phenyl ace-tate, inulin and glycogen are not assimilated.
The G+C content of DNA is 63.2-65.0 mol%. Themajor isoprenoid quinone is Q-10. The major cellular fat-ty acids from whole cells are 1.6 : 1., L8 : L,2-OH 14 : 0 and2-OH 16:0. The major long-chain base composition ofsphingolipid is d-18 :0, d-1.9:1 and d-20:1.. The typestrain is IFO 15033 (:203).
Description of Sphingomonas terrae sp. nou.
ter'rae.L. r terra earth. M. L. gen. L terrae, of the earth.Five strains which utilize PEG 6,000 in a mixed culture ((A)strains of E-1, Yl y',22a, 41.1 and 2) and Sphingomonasgenospecies 2 (IFO 15103) are included in this species. Thecells are Gram-negative and nonsporeforming rods havinga regular shape with round ends. Four strains except forstrain 2-A are motile with a single polarJlagellum. Coloniesare circular, entire, low convex, smooth, opaque and light-or deep-yellow. Aerobic. Catalase, oxidase and DNase areproduced. Indole, urease, phenylalanine deaminase and hy-drogen sulfide are not produced. PEG 6,000 is assimilatedin a mixed culture. Cellobiose, maltose, trehalose, melezi-tose and starch are assimilated, but n-fucose, L-xylose,o-methyl-D-mannoside, B-methyl-xyloside, gluconate,2-keto-gluconate, 5-keto-gluconate, adonitol, dulcitol,xylitol, n-caprate, citrate, inulin and glycogen are not as-similated. Acids are usually not produced from glycerol,salicine and rhamnose.
The G+C content of DNA is 63.0-64.9 mol/". Majorisoprenoid quinone is Q-10. The major cellular fatty acidsfrom whole cells are 16:1., 17 : 1, 18 : L, 2-OH'1.4 :0 and2-OH 1.5: 0. The major long-chain base composition ofsphingolipid is d-18:0, d-19:1 and d-20:1. The typestrain is IFO 15098 (: E-1-A).
Description of Spbingomonas sanguis sp. nou.
san'guis. L. n. sanguis, blood.The cells are Gram-negative and nonsporeforming rods
and are motile with a single polar flagellum. Colonies arecircular, entire, low convex, smooth, opaque and deep-yellow. Catalase, oxidase, DNase and phenylalaninedeaminase are produced. Indole, urease and hydrogen sul-fide are not produced. PEG 4,000 and PEG 6,000 are notassimilated. Esculin and gelatin are hydrolyzed. r-Arabin-ose, t-xylose, galactose, D-fructose, melezitose, D-man-nose, salicine, maltose, lactose, cellobiose, trehalose, r-turanose and p-gentiobiose are assimilated, but ribose,sorbose, t-xylose, o-lyxose, D-tagatose, D-fucose, G-
methyl-xyloside, 2-keto-gluconate, 5-keto-gluconate,adonitol, o-arabitol, dulcitol, sorbitol, xylitol, malate, n-capratq phenyl acetatq gluconate, inulin and glycogen arenot assimilated. Acid was produced from glycerol andrhamnose.
The G+C content of DNA is 61.8 molh. Major iso-prenoid quinone is Q-10. The major cellular fatty acidsfrom whole cells are 18:1 and Z-OH 14:0. The majorlong-chain base composition of sphingolipid is d-18 : 0.
The type strain is IFO 1,3937 (: GIFU 2397).
Acknowledgments.The authors wish to express their thanks toDr. H. Oyaizer, Associate Prof. of Faculty of Agriculture, TheUniversiry of Tokyo, and Dr. H. Sawada, Akitsu Branch, FruitTree Research Station, Ministry of Agric. Forest. Fish., for theirhelp in partial nucleotide sequence anaiysis of 165 rRNA and alsoexpress their gratitude to Dr. T. Hasegawa, Director of this insti-tute, for his encouragement and support. This research was sup-ported by grant 03304017 hom Grant-in-Aid for Co-operativeResearch, the Ministry of Education, Science and Culture of Ja-pan.
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