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