Phylogenetic Evidence for Sphingomonas and Rhizomonas As Nonphotosynthetic Members of the Alpha-4 Subclass of the Proteobacteria

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1994, p. 308-314 Vol. 44, No. 2 0020-7713/94/$04.00 +0 Copyright 0 1994, International Union of Microbiological Societies

Phylogenetic Evidence for Sphingomonas and Rhizomonas as Nonphotosynthetic Members of the Alpha-4 Subclass of the Proteobacteria

MARIKO TAKEUCHI,' * HIROYUKI SAWADA,2t HIROSHI OYAIZU,3 AND AURA YOKOTA' Institute for Fermentation, Osaka, Yodogawa-ku, Osaka 532, Akitsu Branch, Fruit Tree Research Station, Ministq of Agriculture, Forestry and Fisheries, Akitsu, Hiroshima 729-24, and Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113,3 Japan

To clarify the taxonomic relationships of the genera Rhizomonas and Sphingomonas, the 16s rFWA sequence of Rhizornonas suberifaciens IF0 15211T (T = type strain) was determined. A phylogenetic analysis of aligned 16s rRNA gene sequences revealed that eight species of the genus Sphingomonas and R. suberifaciens are closely related to Erythrobacter longus and Porphyrobucter neustonensis and, therefore, belong in the alpha-4 subclass of the Proteobacteria. Within this subclass, Sphingomonus species and R. suberifaciens are phylogenetically interrelated and comprise several subgroups.-- Our findings show that the genus and species definitions of these organisms are in need of revision.

The genus Sphingomonas, whose type species, Sphingomonas of these organisms are similar to those reported for Sphin- paucimobilis, was previously named Pseudomonas paucimobilis gomonas paucimobilis (33, 42). Neither Rhizomonas suberifa- Holmes et al. (6), was proposed by Yabuuchi et al. (42) for ciens nor Sphingomonas paucimobilis produces acid on pep- yellow-pigmented, motile rods with single polar flagella and tone-glucose medium, but both organisms produce acid on nonmotile, nonfermentative, gram-negative rods. Sphingomo- ammonium or nitrate-glucose medium (6). Strains of these two nus paucimobilis contains large amounts of a unique sphingo- species have the same isoprenoid quinone (ubiquinone 10) and glycolipid with the long-chain base dihydrosphingosin, 2-hy- similar fatty acid compositions (8, 17, 33), and the results of droxymyristic acid (41,43), and isoprenoid quinone Q-10. Five grouping based on these two characteristics have been shown species, Sphingomonas paucimobilis, Sphingomonas parapauci- to coincide with the results of grouping based on rRNA-DNA mobilis, Sphingomonas yanoikuyae, Sphingomonas adhaesiva, homology data (33). and Sphingomonas capsulata, were described by Yabuuchi et On the basis of these findings, we suggested previously that al. (42). Recently, we described three new species of the genus species belonging to the genera Sphingomonas and Rhizomo- Sphingomonas, Sphingomonas sanguis, Sphingomonas macro- nus are closely related to each other (29). goltabidus, and Sphingomonas terrae (29). Sphingomonas sun- Recently, on the basis of sequencing data for 270 bases of guis is the new species name proposed for Sphingomonas 16s rRNA genes from eight strains of Rhizomonas species and genospecies 1 of Yabuuchi et al. (42). Sphingomonas macro- eight strains of Sphingomonas species, van Bruggen et al. (33) goltabidus and Sphingomonas terrae are polyethylene glycol- reported that Sphingomonas yanoikuyae is more closely related utilizing bacteria (11, 12) which were previously classified as to Rhizomonas suberifaciens than to other Sphingomonas spe- Flavobacterium species. Polyethylene glycol 4000 is utilized by cies and should be transferred to the genus Rhizomonas and a single bacterium, Sphingomonas terrae, but polyethylene that Sphingomonas capsulata should be removed from the glycol 6000 is utilized by symbiotic mixed cultures of two genus Sphingomonas and placed in a separate genus. strains (13), and the dominant bacterium in the mixed cultures In order to investigate the phylogenetic interrelationships of is Sphingomonas macrogoltabidus. these organisms, we determined the 16s rRNA gene sequence Recently, we compared the 16s rRNA sequences of the and chemotaxonomic characteristics of Rhizomonas suberifa- eight previously described and newly proposed species of the ciens and compared the resulting data with data for Sphin- genus Sphingomonas with the 16s rRNA sequences of 15 gomonas species, Erythrobacter longus, and other members of representative species belonging to the alpha subclass of the the alpha subclass of the Proteobacteria. Proteobacteria (29), and we found that the eight Sphingomonas In this paper we describe the 16s rRNA sequence of species form a large and heterogeneous cluster which is clearly Rhizomonas suberifaciens and phylogenetic evidence which separate from all other representatives of the alpha subclass of indicates that the genera Sphingomonas and Rhizomonas are the Proteobacteria (26,38) except Erythrobacter longus (23,24). nonphotosynthetic members of the alpha-4 subclass of the On the other hand, the genus Rhizomonas and the single Proteo bacteria. species Rhizomonas suberifaciens were proposed by van Brug- gen et al. (32) for the gram-negative, motile, rod-shaped bacteria that cause corky root of lettuce. Most strains of MATERIALS AND METHODS Rhizomonas suberifaciens are oligotrophic (31), but the mor- Cultures. Rhizomonas suberifaciens IF0 15211T (= ATCC phological, physiological, and chemotaxonomic characteristics 493ST = NCPPB 3629T) (T = type strain) and IF0 15212 (= ATCC 49382 = NCPPB 3631) were cultured at 28°C in shake * Corresponding author. Mailing address: Institute for Fermenta- flasks containing (per liter) 5.0 g of peptone, 2.5 g of glucose, tion, Osaka, 17-85, Juso-honmachi 2-chome7 Yodogawa-ku, Osaka 1.3 g of K,HPO,, 0.5 g of MgSO, * 7H,O, 0.5 g of KNO,, and 532, Japan. Phone: 06-300-6555. Fax: 06-300-6814. 0.06 g of Ca(N03),*4H,0 (pH 7.2) (PG medium). Eryth- ? Present address: National Institute of Agro-Environmental Sci- robacter longus IF0 14126* (= ATCC 33941T) was cultivated ences, 3-14, Kannondai, Tsukuba, Ibaraki 305, Japan. in seawater medium containing (per liter) 4.0 g of peptone, 2.0

308 VOL. 44, 1994 PHYLOGENETIC ANALYSIS OF RHZZOMONAS AND SPHINGOMONAS 309

TABLE 1. Comparison of morphological, physiological, and biochemical characteristics of the genera Sphingomonas and Rhizomonas

Characteristic Sphirigomonas" Rhizomonas Plant pathogenicity - h +' Oligotroph - v' Morphological characteristics Cell shape Rods Rods Color of colonies Yellow or whitish yellow Yellow or whitish yellow Motility V V Flagellum Single, polar Single, lateral, subpolar, or polar Physiological characteristics Oxidase + + Catalase + + Acid produced from glucose + + Arginine dihydrolase - - Nitrate reduction V + Bacteriochlorophyll a - - Chemical characteristics lsoprenoid quinone Q-10 Q- 10 Major cellular fatty acids Nonpolar fatty acid 18:1 18:l 2-Hydroxy fatty acids 14:0, 159, 16:O 14:0, 15:O 3-Hydroxy fatty acid - - Sphingolipids + (d-18:0, d-19:1, d-20:1, d-2111) + (d-18:0, d-20:l d-21~1) LPS - - d G+C content (mol%) 61.6-67.8 5 8.0-63.0 " Data from references 29 and 42. '' +, all strains are positive; -, all strains are ncgative; v, variable from strain to strain. ' Data from reference 33. All strains are positive according to van Bruggen et al. (33).

g of yeast extract, 10 mg of FeSO, * 7H,O, 750 ml of seawater, and analyzed as trimethylsilyl ethers by gas chromatography- and enough distilled water to bring the volume up to 1,000 ml mass spectrometry. (23, 24). Analysis of bacteriochlorophyll. Bacteriochlorophyll was Cellular lipids and fatty acid analysis. Cells were harvested extracted from dried cells with methanol by the method of Stal after they were cultured for 24 h in PG medium and freeze- et al. (28). Samples were extracted twice for 2 h, and the two dried, and then 50 mg of dried cells was mixed with 2 ml of 5% extracts were pooled in the dark at room temperature and HCl in methanol and the preparation was heated at 100°C for purified by thin-layer chromatography by using a petroleum 3 h. Fatty acid methyl esters were extracted with n-hexane and ether-acetone-methanol (82: 16:2, vol/vol) solvent system. were separated by thin-layer chromatography by using an In vitro amplification of rRNA genes. DNA was used for in n-hexane-diethyl ether (1 :1, vol/vol) solvent system. Nonpolar vitro amplification of the rRNA gene by the PCR technique acids and 2-hydroxy and 3-hydroxy fatty acids, visualized by (19) in combination with a 16s rRNA gene-specific primer spraying plates 0.02% dichlorofluorescein in ethanol, were pair, 5'-AGTTTGATCCTGGCTC OH-3' (identical to se- extracted with diethyl ether and analyzed by gas-liquid chro- quence positions 10 to 25 in the Escherichia coli numbering matography as described previously (29). The long-chain bases system [1]) and 5'-AAGGAGGTGATCCAGCC OH-3' (com- of the cellular sphingolipids were obtained from acid hydroly- plementary to positions 1541 to 1525), as described previously sates of dried cells as described by Yano et al. (44). The bases (29). Amplification was carried out by using a Taq polymerase were then subjected to thin-layer chromatography by using a of kit (Cetus Inc.). The PCR cycle parameters were as follows: chloroform-methanol-water (65:25:4, vol/vol) solvent system preheating for 2.5 min at 95"C, denaturation for 1 min at 94"C,

1 101 201 301 401 501 601 701 801 901 1001 1101 1201 1301 1401 FIG. 1. Partial 16s rRNA sequence of Rhizomonus suberifaciens. The first and last nucleotides are analogous to positions 10 and 1483 of the Escherichia coli sequence (1). w 0CI

? t,

a- 2 VOL.44, 1994 PHYLOGENETIC ANALYSIS OF RHIZOMONAS AND SPHZNGOMONAS 311

annealing for 2.5 min at 58”C, and extension for 2.5 min at crobium vannielii, M34127; Rhodopila globiforrnis, M59066; 72°C. After 30 cycles, the final step was incubation for 5 min at Rhodospirillum rubrum, M32030; Rickettsia prowazekii, 72°C. The lengths of the amplified fragments were determined M21789; Rochalimaea quintana, M11927; Roseobacter denitri- by agarose (1%, wt/vol) gel electrophoresis. ficans, M96746; Simonsiella rnuelleri, M59071; and Wolbachia Sequence determination and analysis. The amplified DNA pipientis, X61768. The nucleotide sequence data for Rhizomo- was purified by using Suprec-01 (Takara Co., Ltd., Shiga, nas suberifaciens have been deposited in the DNA Data Bank Japan) after electrophoresis on an agarose (l%, wt/vol) gel. of Japan data base under accession number D13737. The purified DNA was sequenced by using a Sequenase kit for ”S-dATP (United Biochemical Inc.) and the following prim- RESULTS AND DISCUSSION ers: 5’-AG?TTGATCCTGGCTC OH-3’ (identical to positions 10 to 25), 5’-GTGTTACTCACCCGT OH-3‘ (comple- Chemotaxonomic characteristics. Gas chromatography and mentary to positions 123 to 109), 5’-TACGGGAGGCAGC gas chromatography-mass spectrometry analyses of trimethyl- AG OH-3’ (identical to positions 343 to 357), 5‘-CTGCTGC silyl derivatives of long-chain bases from Rhizornonas suberi- CTCCCGTAG OH-3’ (complementary to positions 357 to faciens IF0 15211T and Erythrobacter longus IF0 14126T 342), 5’-GTGCCAGCAGCCGCGG OH-3’ (identical to posi- revealed that dihydrosphingosin d-20:1 was the major compo- tions 515 to 530), 5’-ACCGCGGCTGCTGGC OH-3’ (com- nent and that d-18:0, d-19:1, and d-21:l also were present. plementary to positions 531 to 517), 5’-TCTACGCATTTC Erythrobacter longus is a bacteriochlorophyll-containing bacte- ACC OH-3’ (complementary to positions 704 to 690), 5’-GTC rium, although it does not have photosynthetic activity. We AATTCCTTTGAGITT OH-3’ (Complementary to positions examined whether species of the genus Sphingomonas contain 924 to 907), 5’-AGGGTTGCGCTCGTTG OH-3’ (comple- bacteriochlorophyll and confirmed that bacteriochlorophyll mentary to positions 11 15 to 1loo), 5’-CCATTGTAGCACGT was absent in Sphingomonas paucimobilis IF0 14135T, Sphin- GT OH-3’ (complementary to positions 1242 to 1227), 5’-AC gomonas capsulata IF0 12533T, and Rhizomonas suberifaciens GGGCGGTGTGTAC OH-3’ (complementary to positions IF0 15211T. Thus, we suggest that the presence of bacterio- 1406 to 1392), and 5’-GGCTACCTTGTTACGA OH-3’ (com- chlorophyll is a distinctive characteristic which distinguishes plementary to positions 1510 to 1495). DNA sequences were the genus Erythrobacter from both the genus Sphingomonas aligned by using the ODEN system (7). Nucleotide substitution and the genus Rhizomonas. Table 1 shows the morphological, rates (K,,,, values) were calculated from all of the available physiological, biochemical, and chemotaxonomic characteris- sequence data after alignment (14), and a phylogenetic tree tics of the genera Sphingomonas and Rhizomonas, and on the was constructed by the neighbor-joining method (20). The basis of these data the two taxa are indistinguishable. All of the topology of the trees was evaluated by performing a bootstrap strains of both genera were characterized by having isoprenoid analysis of the sequence data, using Clustal V (5). quinone Q-10, by having 2-hydroxymyristic acid, and by lacking Nucleotide sequence accession numbers. The sequences 3-hydroxy fatty acid. The G+C content of the DNA of were aligned with previously published sequences obtained Rhizomonas suberifaciens IF0 15211T was 58.0 to 63.0 mol%, from the DNA Data Bank of Japan, GenBank, and EMBL and the G+C contents of strains of the genus Sphingomonas data bases and deposited under the following accession num- were 61.6 to 67.8 mol%. bers: Escherichia coli, M25588; Sphingomonas paucimobilis, In general, gram-negative bacteria contain lipopolysaccha- D13725; Sphingomonas parapaucimobilis, D13724; Sphingomo- ride (LPS) (endotoxin) and 3-hydroxy C,,, to C,, fatty acids in nas sanguis, D13726; Sphingomonas terrae, D13727; Sphin- their outer membranes. Although 3-OH fatty acids were not gomonas yanoikuyae, D 13728; Sphingomonas adhaesiva, detected in Rhizomonas suberifaciens, the presence of LPS in D13722; Sphingomonas capsulata, M59296; Sphingomonas this species has been reported by van Bruggen et al. (31, 32). macrogoltabidus, D 13723; Erythrobacter longus, M96744; Por- Kawahara et al. reported that Sphingomonas paucimobilis lacks phyrobacter neustonensis, M96745; Roseobacter denitrificans, the usual LPS in the outer membrane (10) but contains M96746; Afpia felis, M65248; Agrobacterium tumefaciens, amphiphilic glycosphingolipid, which might play a role similar M11223; Ancylobacter aquaticus, M62790; Magnetospirillum to the role of LPS in other gram-negative bacteria (9). In view magnetotacticurn, M58171; Azospirillum lipoferum, M59061; of these contradictory results, the presence of LPS in Rhizomo- Bartonella bacilliformis, M65249; Beijerinckia indica, M59060; nas species should be reexamined. Blastobacter denitrificans, X66025; Bradyrhizobiurn japonicum, Phylogenetic analyses. To clarify the phylogenetic relation- X66024; Brucella abortus, X13695; Caulobacter bacteroides, ships of the species belonging to the genera Sphingomonas and M83796; Cowdria ruminantium, X61659; Ehrlichia risticii, Rhizomonas and Erythrobacter longus, we determined the 16s M21290; Erythrobacter longus, M59062; Hirschia baltica, rRNA sequence of Rhizomonas suberifaciens IF0 1521lT (Fig. X52909; Hyphomicrobium vulgare, X53182; Hyphomonas jann- 1) and compared it with the sequences of eight species aschiana, M83806; Methylobacterium organophilum, M29028; belonging to the genus Sphingomonas (29), the sequences of Methylosinus trichosporium, M29024; Paracoccus denitrificans, the bacteriochlorophyll-containing bacteria Porphyrobacter X69159; Porphyrobacter neustonensis, M96745; Pseudomonas neustonensis (4) and Roseobacter denitriJicans (22), and the diminuta, M59064; Rhodobacter capsulatus, M34129; Rhodomi- sequences of 30 other species belonging to the alpha subclass

FIG. 2. Unrooted phylogenetic tree showing the relationships of Rhizomonas suberifaciens IF0 1521lT, Sphingomonas species, and other members of the alpha subclass of the Proteobacteria. The following reference microorganisms were included: alpha-1 group species Rhodopifa globiforrnis (30, 40), Magnetospirillum magnetotacticum (2), and Rhodospirillum rubrum (30, 40); alpha-2 group species Hyphomicrobium vulgare (25), Rhodomicrobium vanniefii (40), Ancyfobacter aquaticus (39), Afipia felis (37), Blastobacter denitrificans (37), Bradyrhizobium japonicum (37), Methylosinus trichosporium (30), Methylobacterium organophilum (30), Agrobacterium tumefaciens (30, 40), Bartonella bacilliformis (18), Rochali- maea quintana (36), Caulobacter bacteroides (27), and Pseudomonas diminuta (30); alpha-3 group species Hyphomonas jannaschiana (25), Hirschia baltica (21), Paracoccus denitr$cans (15,40), Rhodobacter capsufatus (30,40), and Roseobacter denitrificans (4); alpha4 group species Erythrobacter longus (40) and Porphyrobacter neustonensis (4); alpha group species Cowdria ruminantium (3), Ehrlichia risticii (34, 35), Rickettsia prowazekii (35), and Wolbachia pipientis (16); delta-3 group species Escherichia coli (30). Bar = 0.01 K,,, unit. TABLE 2. K,,, values and similarity values for the 16s rRNA sequences of Sphingornonasspecies, Rhizornonas suberifaciens,and other species belonging to the class Proteobacteria"

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21 21 22 23 24 25 26 27 28 2930 31 32 33 34 35 36 37 38 39 40 41

1 E.ml 0.216 0.2090.209 0.196 0.201 0.201 0.202 0.207 0.205 0.210 0.215 0.207 0.189 0.2020.201 0.209 0.222 0.215 0.1%0.210 0.233 0.225 0.217 0.224 0.195 0.193 0.231 0.223 0.221 0.2100.210 0.236 0.203 0.210 0.1910.230 0.212 0.2470.190 0.262 2 S.pau 81.3 0.015 0.0170.059 0.054 0.065 0.075 0.072 0.054 0.116 0.121 0.123 0.1260.129 0.1200.125 0.118 0.128 0.1150.130 0.171 0.196 0.084 0.147 0.132 0.129 0.136 0.156 0.1260.081 0.125 0.144 0.127 0.158 0.1390.150 0.126 0.165 0.145 0.245 3 S.wr 81.8 98.5 0.0760.059 0.055 0.065 0.0710.073 0.053 0.1100.119 0.104 0.128 0.123 0.1180,118 0.116 0.1240,110 0.129 0.174 0.1980.087 0.147 0.127 0.128 0.140 0.148 0.125 0.089 0.127 0.145 0.1220.158 0.136 0.156 0.123 0.164 0.143 0.245 4 S.san 81.8 98.3 99.3 0.057 0.054 0.065 0.070 0.070 0.050 0.110 0.1190.104 0.128 0.126 0.118 0.120 0.1160.121 0.123 0.129 0.174 0.1970.083 0.146 0.127 0.1250.140 0.148 0.126 0.086 0.1240.144 0.124 0.160 0.1360.154 0.1210.163 0.141 0.244 5 S.ter 82.8 94.4 94.4 94.6 0.047 0.107 0.057 0.2160.034 0.104 0.115 0.1090.105 0.116 0.096 0.112 0.115 0.1140.995 0.124 0.180 0.210 0.065 0.1330.114 0.106 0.130 0.144 0.125 0.067 0.1200.139 0.1170.153 0.129 0.147 0.099 0.151 0.1390.243 6 S.Y~81.8 94.8 94.7 94.8 95.4 0.045 0.067 0.053 0.0390.121 0.1210.122 0.130 0.129 0.099 0.121 0.135 0.128 0.101 0.116 0.183 0.213 0.077 0.143 0.133 0.131 0.1420.157 0.127 0.075 0.113 0.147 0.124 0.162 0.144 0.143 0.108 0.157 0.148 0.248 7 S.adh 82.5 93.8 93.8 93.8 98.9 95.6 0.054 0.019 0.030 0.103 0.115 0.106 0.109 0,1190.094 0.1100.116 0.113 0.095 0.122 0.1720.202 0.064 0.130 0.115 0.1080.130 0.141 0.120 0.066 0.119 0.134 0.1150.156 0.131 0.139 0.096 0.1440.141 0.239 8 S.=P 82.4 92.2 93.2 93.3 94.5 93.6 94.8 0.059 0.056 0.119 0.116 0.115 0.126 0.133 0.095 0.1240.125 0.1290.~196 0.139 0.168 0.204 0.069 0.133 0.124 0.118 0.129 0.1420.116 0.064 0.138 0.134 0.119 0.1580.139 0.1480.104 0.140 0.132 0.247 9 S.mc 81.9 93.2 93.1 93.3 97.9 94.9 98.1 94.4 0.041 0.106 0.116 0.120 0.112 0.129 0.1000.121 0.115 0.1160.099 0.125 0.173 0.200 0.062 0.131 0.122 0.113 0.125 0.147 0.131 0.063 0.120 0.144 0.120 0.158 0.134 0.145 0.1030.148 0.137 0.238 10 R.sub 82.1 94.8 94.9 95.2 96.7 96.2 97.0 94.6 96.1 0.108 0.123 0,106 0.111 0.1200.098 0.114 0.122 0.1150.095 0.121 0.181 0.213 0.079 0.1460.116 0.125 0.130 0.148 0.1270.073 0.1160.138 0.115 0.153 0.136 0.1470.101 0.157 0.147 0.242 11 A.fel 81.7 89.3 89.8 89.8 90.3 88.9 90.4 89.1 90.1 90.0 0.097 0.075 0.104 0.110 0.089 0.061 0.022 0.028 0.084 0.106 0.169 0.193 0.120 0.142 0.098 0.124 0.086 0.1090.114 0.120 0.114 0.128 0.092 0.161 0.1160.143 0.093 0.136 0.137 0.217 12 A.tum 81.4 88.8 89.1 89.1 89.4 88.9 89.4 89.3 89.3 88.7 91.0 0.084 0.124 0.111 0.060 0.103 0.102 0.0990.058 0.130 0.171 0.186 0.112 0.134 0.116 0.126 0,115 0.114 0.104 0.118 0.1140.120 0.104 0.153 0.129 0.166 0.061 0.130 0.135 0.211 13 A.wu 82.0 89.6 90.3 90.3 89.9 88.8 90.1 89.4 88.9 90.1 92.9 92.1 0.114 0.1020.094 0.082 0,088 0.077 0.082 0.1190.191 0.2170.123 0.153 0.106 0.135 0.101 0.111 0.1190.122 0.119 0.133 0.097 0.159 0.128 0.1630.087 0.157 0.120 0.223 14 M.w 83.3 88.4 88.3 88.3 90.2 88.1 89.9 88.4 89.6 89.7 90.3 88.6 89.5 0.091 0.114 0.115 0,119 0.1100.108 0.133 0.1780.191 0.130 0.147 0.122 0.117 0.1300.139 0.142 0.127 0.133 0.1470.127 0.138 0.099 0.148 0.111 0.167 0.135 0.226 15 A.lip 82.4 88.2 88.7 88.4 89.3 88.2 89.1 87.9 88.2 88.9 89.8 89.7 90.5 91.5 0.108 0.119 0.103 0.110 0.097 0.116 0.191 0.202 0.149 0.147 0.114 0.123 0.129 0.129 0.132 0.143 0.121 0.131 0.113 0.144 0.115 0.147 0.1000.163 0.1120.233 16 B.bac 82.5 88.9 89.2 89.2 91.0 90.8 91.2 91.1 90.6 90.9 91.7 94.391.2 89.5 90.0 0.095 0.099 0.101 0.346 0.127 0.1640.201 0.1100.126 0.102 0.112 0.104 0.122 0.0970.111 0.123 0.109 0.092 0.145 0.129 0.143 0.024 0.123 0.129 0.204 17 B.ind 81.8 88.5 89.2 88.9 89.6 88.8 89.8 88.6 88.8 89.5 94.2 90.4 92.2 89.4 90.4 91.2 0.073 0.0840.099 0.118 0.179 0.2060.137 0.141 0.095 0.128 0.086 0,089 0.114 0.131 0.119 0.1130.096 0.1610.131 0.163 0.098 0.1410.133 0.232 18 B.den 80.9 89.2 89.3 89.3 89.4 87.8 89.3 88.5 89.4 88.8 97.9 90.5 91.7 89.1 89.1 90.9 93.1 0.026 0.087 0.121 0.175 0.193 0.116 0.115 0.106 0.127 0.088 0.1140,112 0.119 0.124 0.118 0.098 0.160 0.123 0.1520.101 0.139 0.139 0.226 19 B.jw 81.4 88.3 88.6 88.8 89.5 88.3 89.6 88.2 89.3 89.4 97.2 90.8 92.7 89.8 89.8 90.6 92.1 97.5 0.087 0.104 0.178 0.200 0.123 0.1420.105 0.123 0.0970.112 0.119 0.123 0.1100.124 0.104 0.160 0.124 0.144 0.098 0.1380.146 0.222 20 B.ab 82.7 89.4 89.8 89.6 91.1 90.5 91.1 91.0 90.8 91.1 92.1 94.5 92.2 90.0 91.0 95.5 90.8 91.8 91.8 0.110 0.1840.185 0.105 0.124 0.103 0.110 0.116 0.118 0.089 0.109 0.109 0.0960.090 0.153 0.120 0.151 0.042 0.122 0.1300.219 21 C.bac 81.7 88.1 88.2 88.2 88.6 89.3 88.7 87.4 88.5 88.8 90.2 88.2 89.1 87.9 89.388.4 89.2 88.9 90.3 89.8 0.167 0.208 0.1320.132 0.128 0.122 0.149 0.1410.139 0.125 0.043 0.133 0.123 0.163 0.122 0.151 0.127 0.176 0.156 0.236 22 C.m 80.1 84.8 84.6 84.6 84.1 83.8 84.7 85.0 84.6 84.1 84.9 84.8 83.3 84.3 83.3 85.3 84.2 84.5 84.3 83.8 82.0 0.159 0.155 0.181 0.173 0.180 0.1990.205 0.193 0.161 0.168 0.193 0.166 0.2070.169 0.164 0.1740.184 0.196 0.148 23 E.ris 80.8 83.0 82.9 82.9 81.9 81.7 82.5 82.4 82.6 81.7 83.1 83.7 81.5 83.3 82.6 82.6 82.1 83.1 82.6 83.7 88.0 86.0 0.188 0.205 0.2190.206 0.213 0.225 0.203 0.185 0.200 0.214 0.194 0.222 0.181 0.1840.203 0.209 0.197 0.182 24 E.lon 81.3 92.1 91.8 92.2 93.8 92.7 93.9 93.4 94.0 92.6 88.9 89.7 88.7 88.1 86.6 89.8 87.6 89.3 88.7 90.2 88.0 86.1 83.5 0.1390.131 0.123 0.156 0.153 0.130 0.033 0.120 0.143 0.142 0.176 0.139 0.144 0.1180.150 0.144 0.223 25 H.bal 80.7 86.7 86.7 86.8 87.9 87.0 88.1 87.9 88.0 86.8 87.1 87.9 86.3 86.7 86.7 88.5 87.2 86.9 87.1 88.6 88.3 84.1 82.3 87.4 0.138 0.075 0.162 0.155 0.132 0.138 0.135 0.132 0.154 0.192 0.137 0.183 0.126 0.119 0.159 0.232 26 H.wl 82.9 87.9 88.3 88.3 89.5 87.9 89.4 88.6 88.7 89.3 90.9 89.3 90.1 88.7 89.5 90.5 91.1 90.1 90.2 90.4 88.8 84.7 81.3 88.0 87.5 0.121 0.134 0.119 0.1240.126 0.120 0.125 0.103 0.149 0.134 0.163 0.108 0.137 0.143 0.238 27 H.jan 83.0 88.2 88.3 88.5 90.1 88.1 90.0 89.2 89.6 88.588.6 88.5 87.8 89.2 88.7 89.788.3 88.4 88.7 89.8 86.6 84.1 82.3 88.7 32.9 88.8 0.1420.141 0.1200.121 0.113 0.116 0.132 0.1650.121 0.1630.117 0.190 0.147 0.233 28 M.org 80.1 87.6 87.2 87.2 88.1 87.1 88.1 88.2 88.5 88.1 91.9 89.4 90.5 88.1 88.2 90.3 91.9 91.7 91.0 89.3 87.2 82.7 81.6 86.0 55.587.8 87.1 0.116 0.137 0.143 0.141 0.144 0.110 0.164 0.147 0.1660.111 0.156 0.139 0.232 29 Ktri 80.8 86.0 86.6 86.6 86.9 85.9 87.1 87.1 86.7 86.6 89.9 89.5 89.7 87.4 88.2 88.8 91.6 89.5 89.7 89.2 87.4 82.1 80.8 86.2 86.1 89.1 87.2 89.3 0.151 0.147 0.1420.155 0.126 0.177 0.157 0.1880.129 0.159 0.158 0.256 30 P.den 81.0 88.4 88.5 88.4 88.5 88.3 88.9 89.3 88.0 88.3 89.5 90.3 89.1 87.1 88.0 91.0 89.5 89.7 89.1 91.6 88.5 83.1 82.4 88.1 88.0 88.6 88.9 86.4 86.4 0.135 0.126 0.054 0.116 0.168 0.140 0.1610.103 0.078 0.137 0.231 31 P.neu 81.8 92.3 91.6 91.9 93.6 92.9 93.7 93.8 93.9 93.1 88.9 89.2 88.8 88.3 87.0 89.7 88.0 89.1 88.7 89.9 95.9 85.5 83.7 96.8 87.5 88.4 88.8 87.0 86.7 87.7 0.1070.152 0.133 0.166 0.134 0.1510.119 0.149 0.140 0.227 32 P.dim 81.7 88.5 88.4 88.6 88.989.6 89.1 87.5 88.9 89.3 89.5 89.5 89.1 87.9 88.8 88.7 89.1 88.6 89.8 89.9 87.9 85.0 82.7 89.1 87.7 88.9 89.6 87.2 87.1 88.4 90.1 0.1350.131 0.165 0.123 0.1640.120 0.158 0,157 0.233 33 R.cap 79.8 86.9 86.8 86.9 87.4 86.7 87.8 87.8 86.9 87.5 88.3 88.9 87.9 86.7 88.0 89.9 89.6 89.2 88.6 91.0 88.7 83.181.5 87.0 88.0 88.5 89.3 86.9 86.1 94.8 86.3 87.7 0.134 0.180 0.137 0.1650.111 0.086 0.155 0.239 34 R.vm 82.3 88.3 88.7 88.6 89.2 88.6 89.4 89.1 88.9 89.4 91.4 90.3 91.0 88.3 89.6 91.4 91.0 90.9 90.3 91.5 85.4 85.2 83.1 87.1 86.2 90.4 88.0 89.8 88.4 89.3 87.9 88.0 87.8 0.149 0.131 0.142 0.0990.147 0.124 0.207 35 R.glo 81.8 85.8 85.8 85.7 86.2 85.4 86.0 85.8 85.8 86.2 85.5 86.2 85.8 87.586.9 86.8 85.5 85.7 85.7 86.2 88.7 82.1 81.1 84.4 83.186.5 85.2 85.3 84.3 85.0 85.2 85.2 84.1 86.5 0.141 0.189 0.151 0.197 0.122 0.260 36 R.rub 83.2 87.4 87.6 87.6 88.2 86.9 88.0 87.4 87.8 87.6 89.3 88.2 88.3 90.8 89.4 88.2 88.1 88.7 88.6 88.9 86.4 85.0 84.1 87.4 87.6 87.8 88.8 86.7 85.9 87.287.8 88.7 87.6 88.0 87.1 0.174 0.1300.163 0.125 0.218 37 Rmo 80.3 86.5 86.0 86.2 86.7 87.0 87.4 86.6 86.8 86.7 87.0 85.2 85.4 86.6 86.7 87.0 85.4 86.3 86.9 86.4 88.4 85.4 84.0 86.9 83.8 85.4 85.4 85.1 83.4 85.5 86.4 85.3 85.2 87.1 83.4 84.6 0.149 0.178 0.179 0.206 38 R.d82.4 88.4 88.7 88.8 90.8 90.0 91.0 90.3 90.4 90.5 91.3 94.2 91.8 89.7 90.6 97.7 90.9 90.6 90.9 96.0 84.5 84.6 82.5 89.2 88.5 90.0 89.3 89.7 88.2 90.4 89.1 88.9 89.7 90.8 86.4 88.1 86.6 0.131 0.1290.210 39 R.den 79.1 85.2 85.3 85.4 86.4 85.9 86.9 87.2 86.6 85.9 87.7 88.2 86.085.1 85.4 88.7 87.2 87.4 87.5 88.8 86.0 83.7 81.9 86.9 89.1 87.6 89.1 86.0 85.8 92.7 86.5 85.9 91.7 86.7 82.8 85.4 M.3 88.1 0.161 0.236 40 S.we 83.3 86.8 87.0 87.1 87.4 86.6 87.1 87.9 87.5 86.7 87.6 87.7 88.9 87.7 89.7 88.2 87.9 87.4 86.8 88.1 80.0 82.9 82.9 86.4 85.8 87.0 86.7 87.4 85.8 87.6 87.2 86.0 86.1 88.6 88.7 88.5 84.2 88.2 85.5 0.234 41 W.pip 78.0 79.4 79.4 79.4 79.4 79.1 79.1 79.2 79.7 79.5 81.3 81.8 81.1 80.7 80.2 82.3 80.2 80.7 81.0 81.2 80.2 86.7 84.1 80.9 80.2 79.9 80.1 80.2 78.580.3 80.6 80.2 79.8 82.1 78.3 81.3 82.3 81.8 79.9 80.1

a The values on the upper right are K,,, values, and the values on the lower left are sequence similarity values (expressed as percentages). Abbreviations for organisms: E. coli, Escherichia coli; S.pau, Sphingomonas paucimobilis; S.par, Sphingomonas parapaucimobilis; Ssan, Sphingomonas sanguis; S.ter, Sphingomonas terrae; S.yan, Sphingomonas yanoikuyae; S.adh, Sphingomonas udhaesiva; Sap,Sphingomonas capsulata; S.mac; Sphingomonas macrogoltabidus; Rsub, Rhizomonas suberifaciens; A.fel, AJipia fehs; A.tum, Agrobacterium tumefuciens; A.aqu, Ancylobacter aquaticus; M.mag, Magnetospirillurn magnetotacticurn; Alp, Azospirillum lipoferum; B.bac, Bartonella bacilliformis; Bind, BeiJerinckia indica; B.den, Blastobacter denitrijicans; B.jap, Bradyrhizobium japonicum; B.abo, Brucella abortus; C.bac, Caulobacter bacteroides; C.rum, Cowdria ruminantiurn; E.ris, Ehrlichia risticii; E.lon, Erythrobacter longus; H.ba1, Hirschia balticu; H.vul, Hyphomicrobiwn vulgare; H.jan, Hyphornonas jannaschiuna; M.org, Methylobacterium organophilum; M.tri, Methylosinus trichosporium; P.den, Paracoccus denitrificans; P.neu, Porphyrobacter neustonensis; P.dim, Pseudomonas dirninutu; R.cap, Rhodobacter capsulatus; R.van, Rhodomicrobium vannielii; R.glo, Rhodopila globiformis; R.rub, Rhodospirillum rubrum; R.pro, Rickettsia prowazekii; R.qui, Rochalimaea quintana; R.den, Roseobacter denitriJcans; S.mue, Simonsiella muelleri; W.pip, Wolbachia pipientis. VOL.44, 1994 PHYLOGENETIC ANALYSIS OF RHIZOMONAS AND SPHINGOMONAS 3 13

of the Proteobacteria. Figure 2 shows an unrooted phylogenetic rium from freshwater. Int. J. Syst. Bacteriol. 43:125-134. tree derived from KnUcvalues that were calculated by using 941 5. Higgins, D. G., A. J. Bleasby, and R. Fuchs. 1992. Clustal V: bases from 41 species. Table 2 shows the K,,, and sequence improved software for multiple sequence alignment. CABIOS similarity values for Sphingomonas species, Rhizomonas suberi- 8:189-190. 6. Holmes, B., R J. Owen, A. Evans, H. Malnick, and W. R. Willcox. faciens, and other members of the Proteobacteria. As shown in 1977. Pseudomonas paucimobilis, a new species isolated from Fig. 2, the branching pattern indicates that Rhizomonas suberi- human clinical specimens, the hospital environment, and other faciens and all of the Sphingomonas species belong in the sources. Int. J. Syst. Bacteriol. 27:133-146. alpha-4 subclass of the Proteobacteria. Moreover, an analysis of 7. ha, Y. 1991. Molecular evolutionary analysis system for DNA and our data confirmed the existence of four major subgroups. The amino acid sequences (ODEN), version 1.1. DNA Data Bank of first subgroup consists of Sphingomonas paucimobilis, Sphin- Japan, DNA Research Center, National Institute of Genetics, gomonas parapaucimobilis,and Sphingomonas sanguis (similar- Mishima, Japan. ity values, 98.3 to 99.3%); the second subgroup comprises the 8. Jantzen, E., and K. Bryn. 1985. Whole-cell and lipopolysaccharide polyethylene glycol-utilizing bacteria Sphingomonas macro- fatty acids and sugars of gram-negative bacteria. SOC. Appl. Bacteriol. Tech. Ser. 20:145-171. goltabidus, Sphingomonas terrae, and Sphingomonas adhaesiva 9. Kawahara, K., U. Seydel, M. Matsuura, H. Danbara, E. T. (similarity values, 97.9 to 98.9%); the third subgroup consists Rietschel, and U. Zahringer. 1991. Chemical structure of glyco- of Sphingomonas yanoikuyae and Rhizomonas suberifaciens sphingolipids isolated from Sphingomonas paucimobilis. FEBS (similarity value, 96.2%); and the last subgroup contains Lett. 292:107-110. Sphingomonas capsulata, which is clearly distinct from other 10. Kawahara, K., K. Uchida, and K. Aida. 1982. Isolation of an species belonging to the genus Sphingomonas (similarity val- unusual ‘lipid A’ type glycolipid from Pseudomonas paucimobilis. ues, 92.2 to 94.8%) and Rhizomonas suberifaciens (similarity Biochim. Biophys. Acta 712571-575. value, 94.6%). 11. Kawai, F., M. Fukaya, Y. Tani, and K. Ogata. 1977. Identification On the basis of the results described above, it is clear that all of polyethylene glycols (PEGS)-assimilable bacteria and culture characteristics of PEG 6,000. J. Ferment. Technol. 55429-534. of the species belonging to the genera Sphingomonas and 12. Kawai, F., T. Kimura, Y. Tani, and H. Yamada. 1984. Involvement Rhizomonas are nonphotosynthetic members of the alpha-4 of polyethylene glycol (PEG)-oxidizing enzyme in the bacterial subclass of the Proteobacteria, but these organisms are phylo- metabolism of PEG. Agric. Biol. Chem. 48:1349-1351. genetically interrelated and can be divided into several sub- 13. Kawai, F., and H. Yamanaka. 1986. Biodegradation of polyethyl- groups. The chemotaxonomic characteristics of the two genera ene glycol by symbiotic mixed culture (obligate mutualism). Arch. are rather similar, and there are few other remarkable differ- Microbiol. 146125-129. ences in morphological and physiological characteristics which 14. Kimura, M. 1980. A simple method for estimating evolutionary distinguish these subgroups. rates of base substitutions through comparative studies of nucle- van Bruggen et al. (33) suggested that Sphingomonas otide sequences. J. Mol. Evol. 16111-120. yanoikuyae should be placed in the genus Rhizomonas and that 15. Ludwig, W., G. 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