Microbiol. Immunol., 44(7), 563-575, 2000 Proposal of Fam. Nov., Consisting of Yabuuchi et al. 1990, Erythrobacter Shiba and Shimidu 1982, Erythromicrobium Yurkov et al. 1994, Porphyrobacter Fuerst et al. 1993, Zymomonas Kluyver and van Niel 1936, and Sandaracinobacter Yurkov et al. 1997, with the Type Genus Sphingomonas Yabuuchi et al. 1990

Yoshimasa Kosako*°', Eiko Yabuuchi2, Takashi Naka3,4, Nagatoshi Fujiwara3, and Kazuo Kobayashi3

'JapanCollection of Microorganis ms,RIKEN (Institute of Physical and ChemicalResearch), Wako, Saitama 351-0198, Japan, 2Departmentof Microbiologyand Immunology , AichiMedical University, Aichi 480-1101, Japan, 'Departmentof Host Defense,Osaka City University, Graduate School of Medicine,Osaka, Osaka 545-8585, Japan, and Instituteof SkinSciences, ClubCosmetics Co., Ltd., Osaka,Osaka 550-0005, Japan

ReceivedJanuary 25, 2000; in revisedform, April 11, 2000. Accepted April 14, 2000

Abstract:Based on the results of phylogeneticanalysis of the 16SrDNA sequences and the presence of N- 2'-hydroxymyristoyldihydrosphingosine 1-glucuronic acid (SGL-1)and 2-hydroxymyristicacid (non- hydroxymyristicacid in Zymomonas)in cellular lipids,a new family,Sphingomonadaceae, for Group 4 of the alpha-subclassof the classProteobacteria is hereinproposed and a descriptionof the familyis given.The familyconsists of six genera, Sphingomonas,Erythrobacter, Erythromicrobium, Porphyrobacter, Sandara- cinobacterand Zymomonas.Thus, all the validlypublished and currently known genera in Group 4 of the alpha-subclassof Proteobacteriabelong to Sphingomonadaceaefam. nov.Among them, type strains of two species, Porphyrobacter and Erythrobacter, Sandaracinobactersibiricus and Sphingomonasursincola, respectively,are facultativelyphotosynthetic due to bacteriochlorophyll(Bchl)-a. The type strains of two subspeciesof Zymomonasmobilis are facultativeanaerobes. The presenceof SGL-1together with the results of a phylogeneticanalysis of the 16SrDNA sequences recommends a newcriteria by whichto definethe new family Sphingomonadaceae.The type genus is SphingomonasYabuuchi et al. 1990.

Keywords: Sphingomonadaceae fam. nov., New family,, Alpha-subclass, Sphingoglycolipid

*Address correspondence to Dr. Yoshimasa Kosako, Japan ismen and Zellkulturen, Braunschweig GmbH, Germany; EY, Collection of Microorganisms, RIKEN (The Institute of Physical Eiko Yabuuchi, Department of Microbiology and Immunology, and Chemical Research), Wako, Saitama 351-0198, Japan. Aichi Medical University, Aichi, Japan; GC, guanine plus cyto- Fax: x--81-48-462-4618. E-mail: [email protected] sine; GIFU, Department of Microbiology,Gifu University School of Medicine,Gifu, Japan; IAM, Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan; IFO, Institute for Fermenta- Abbreviations: ACM, Australian Collection of Microorgan- tion Osaka, Osaka, Japan; JCM, Japan Collection of Microor- isms, Department of Microbiology, University of Queensland, ganisms, RIKEN (The Institute of Physical and Chemical Queensland, Australia; AMPC, amoxicillin; ATCC, American Research), Saitama, Japan; LMG, Laboratorium voor Microbi- Type Culture Collection, Mananas, Virginia, U.S.A.; comb. nov., ologie, Universuteit Gent, Gent, Belgium; PYA, peptone (1%)- combinatio nova= new combination; CVC, clavulanic acid; yeast extract (1%)-NaCI (0.5%) agar; RDP, Ribosomal Data- DDBJ, DNA Data Bank of Japan, Institute of Genetics, Mishima, base Project, Center for Microbial Ecology, Michigan State Uni- Shizuoka, Japan; DSM, Deutsche Sammlung von Mikroorgan- versity, Michigan, U.S.A.; SGL, Sphingoglycolipid,

563 564 Y. KOSAKO ET AL

ATCC 10829, a type strain of Flavobacterium devo- the genus Sphingomonas. In addition to these, "Sphin- rans (Zimmermann 1890) Bergey et al. 1923"L, was a gomonas wittchii" was proposed by Yabuuchi et al for the high GC organism and found to have N-2'-hydrox- strain known as dibenzo-p-dioxin metabolizing strain ymyristoyl dihydrosphingosine 1-glucuronic acid (SGL- RW1 (personal communications). 1) in cellular lipids (35). From these and other fea- The class Proteobacteria Stackebrandt et al. 1988 tures, ATCC 10829 was reidentified as an strain of (24) was proposed for the phylogenetic taxon that Pseudomonas paucimobilis Holmes et al. 1977"L (9), includes the "purple and their relatives" which is listed in the approved list of bacterial names described by Woese et al (32). (22). Because strain ATCC 10829 was not a original On the basis of the results of the 16S rDNA sequence Zimmermann strain but a later misidentified organism, comparison, Takeuchi et al. reported in 1994 (27) that the the name F devorans could not be a senior synonym of genus Sphingomonas belongs to the alpha-subclass of the P paucimobilis. A similar component, SGL-1, was Proteobacteria and is a part of Group 4. Strains of fac- found in the cellular lipids of yellow-pigmented iso- ultatively photosynthetic species of four genera Por- lates originally identified as strains of Pseudomonas sp. phyrobacter, Erythrobacter, Sandaracinobacter and Eryth- including P paucimobilis, Flavobacterium sp. and Sphin- romicrobium, together with facultatively anaerobic gobacterium sp. In addition to this, the results of a Zyymomonasare also reported as belonging to Group 4 of phylogenetic analysis of partial nucleotide sequences the alpha-subclass of Proteobacteria. of the 16S rDNA led to the proposal of a new genus, In 1976, De Ley and Swing proposed an improved Sphingomonas Yabuuchi et al. 1990" with the type and nomenclature of the genus Zymomonas species Sphingomonas paucimobilis (Holmes et al. 1977) Kluyver and van Niel 1936" (4). From the phenotypic Yabuuchi et al. 1990" (34). In this new genus, three new and genetic data, they unified "Z. anaerobica" subsp. species, S. adhaesiva, S. parapaucimobilis and S. "anaerobica" and "Z. anaerobica" subsp. "immobilis," yanoikuyae, and a new combination, S. capsulata (Leif- and raised Zymomonas mobilis (Lindner) De Ley and son 1962") Yabuuchi et al. 1990" were named and Swing 1976" to a species. The proposal of Z. mobilis described. Two other strains containing SGL-1 in their subsp. pomaceae" by De Ley and Swing automatically cellular lipids were differentiated from the above-men- created Z. mobilis subsp. mobilis. tioned species; the authors were reluctant to name them The genus Erythrobacter is made up of two species. because each of them was a single strain. Erythrobacter longus Shiba and Simidu 1982" (21) and Since then, 14 new species and three new combina- Erythrobacter litoralis Yurkov et al. 1994" (36) were tions of Sphingomonas have been validly named and proposed for an containing bacteri- described. The 14 new species are: S. macrogoltabidus ochlorophyll a. Takeuchi et al. 1993"; S. sanguis Takeuchi et al. 1993' Erythromicrobium ramosum Yurkov et al. 1994" was and S. terrae Takeuchi et al. 1993' (25); 3-ketolactose- proposed for a strain isolated from a marine cyanobac- producing S. rosa Takeuchi et al. 1995"; S. pruni terial mat (36). A new genus, Porphyrobacter Fuerst et Takeuchi et al. 1995v'; S. asaccharolytica Takeuchi et al. al. 1993", with a single type species, P. neustonensis 1995" and S. mali Takeuchi et al. 1995" (26); S. Fuerst et al. 1993", was proposed for aerobic bacteri- chlorophenolica Nohynek et al. 1995".(17); S. herbici- ochlorophyll-synthesizing bacteria (7). Another species, dovorans Zipper et al. 1996" (38); S. subarctica P. tepidarius, was proposed by Hanada et al. 1997 (8). Nohynek et al. 1996" (18); S. trueperi Kaempfer et al. The former was isolated from fresh water and budding 1997' (10); an aromatic-degrading species, S. aromati- multiplication was observed by electron microscopy. civorans Balkwill et al. 1997"'; S. subterranea Balk- The latter was a moderately thermophilic aerobe iso- will et al. 1997"'; and S. stygia Balkwill et al. 1997" (1). lated from a hot spring. Three species, Blastobacter natatoria Sly 1985' (23), The type strain of Sandaracinobacter sibiricus Yurkov "Rhizomonas" suberifaciens van Bruggen et al. 1990" et al. 1997".(37) was isolated from a freshwater algo- (30) and Erythromonas ursincola Yurkov et al. 1997' bacterial mat near hydrothermal sulfide-containing vents (37) were transferred to the genus Sphingomonas, and S. along a river bottom. natatoria, S. suberifaciens and S. ursincola were pro- In this study, with the two exceptions of unavailable posed (33). Publication of these three new combinations type strains of Sandaracinobacter sibiricus and the was listed in Validation List No. 70. After a phylogenetic unsuccessfully revived lyophile of S. herbicidovorans , we analysis, Pseudomonas echinoides Heumann 1962" was confirmed the distribution of sphingoglycolipid (SGL-1), transferred to the genus Sphingomonas as Sphingomonas morphological, physiological and biochemical charac- echinoides (Heumann 1962) Denner et al. 1999' (5). At teristics. The phylogeny of the 16S rDNA sequence present, 23 species are validly published as members of among the species in the alpha-4 group, including San- SPHINGOMONADACEAE KOSAKO AND YABUUCHI FAM. NOV. 565 daracinobacter sibiricus, was analyzed in order to elu- Phenotypic characterization and antimicrobial sus- cidate the possibility of proposing a new family to Group ceptibility tests. Morphologies of the cells of each strain 4 of the alpha-subclass of Proteobacteria. were observed by optical microscopy of Gram-stained preparations. Motility was observed by microscopy Materials and Methods using a wet mount preparation. Owing to the condition of growth, 20 type strains of Sphingomonas species Bacterial strains. The strain designations and source were subjected to the phenotypic and antimicrobial tests. of isolation of type strains of each species used in this The phenotypic characteristics listed in Table 3 were study are shown in Table 1. Sphingomonas natatoria was determined by methods described previously (34). Sus- grown on peptone-yeast agar (PYA) medium and incu- ceptibilities to 36 antimicrobial agents were estimated by bated at 30 C. For Sphingomonas suberifaciens, ATCC the Kirby-Bauer method using ready-to-use Mueller- medium 1700 ("Rhizomonas" medium) was used at 26 C. Hinton medium II and Sensi Discs (Becton-Dickinson). The other strains of Sphingomonas species were grown Quinone analysis. Quinone systems of S. natatoria on Bacto-heart infusion agar and incubated at 30 C JCM 10396, S. paucimobilis JCM 7516 and S. suberi- unless otherwise stated. Two Zymomonas mobilis strains faciens JCM 8521 were determined by high-performance were grown at 30 C in a liquid medium composed of liquid chromatography as described by Karr et al (11). 10% glucose and 1% peptone. The type strain of Sphin- Cellular lipid and fatty acid analysis. The type strains gomonas herbicidovorans was reluctant to grow on the of all species except S. suberifaciens and two subspecies recommended DSM medium 457. The type strain of of Zymomonas mobilis were inoculated on Bacto-heart Sandaracinobacter sibiricus was unavailable because infusion agar and incubated aerobically at 30 C. S. it was not deposited in any permanently established cul- suberifaciens EY 4204 was grown on ATCC "Rhi- ture collection. zomonas" medium and incubated at 25 C. Two

Table 1. Strain designations and source of isolation of type strain-employed phenotypic characterization and chemical analyses in this study

EY: Eiko Yabuuchi, JCM: Japan Collection of Microorganisms, RIKEN, Wako, Saitama, Japan. 566 Y. KOSAKO ET AL

Zymomonas mobilis strains were grown at 30 C in a were harvested. The crude cellular lipids were extracted broth medium composed of 10% glucose and 1% pep- twice, first with a chloroform-methanol mixture (C-M) tone. Cells grown both on agar and in broth media 2:1 (v/v) and then C-M 1:3 (v/v) (secondly). The lipid

Table 2. Strain and sequence data accession numbers of the type strains of 45 species and 2 subspecies of 21 gen- era used in the phylogenetic analysis and levels of sequence similarity between Sphingomonas paucimobilis and other species employed in this study

" Sequence data received from Ribosomal Database Project. SPHINGOMONADACEAE KOSAKO AND YABUUCHI FAM. NOV. 567

Table 3. Morphological, physiological, and biochemical characteristics of 20 type strains in the genus Sphingomonas

°' EY strain number , Y: yellow, 0: orange. WG: weakly growth, NG: no growth, +: positive reaction within 3 days, -: negative reaction throughout 4 weeks, + *: positive reaction after 4 or more days, w: weakly positive reaction after 4 or more days. 568 Y. KOSAKO ET AL extracts were combined and washed using the method of hydrolyzed mixture with 6 N HCI, alkaline-stable lipids Folch et al (6). A portion of the crude lipids from each were extracted by C-M 2:1 (v/v). The crude and alka- strain was hydrolyzed by shaking in C-M 1:1 (v/v) with line-stable lipids were developed on thin-layer plates 0.5 N KOH at 30 C for 3 hr. After neutralization of the of silica gel G (Uniplate No. 01011, Analtech) with two

Table 4. Susceptibilities of type strain of 20 Sphingonionas species against 36 antibacterial agents

°' EY strain number , AMPC: amoxicillin, CVC: clavulanic acid. The No. 2-35 discs contain a single drug. The No. 1 and 36 discs con- tain 2 kinds of drugs combined. S: sensitive, M: moderate, R: resistant. SPHINGOMONADACEAE KOSAKO AND YABUUCHI FAM . NOV. 569

solvent systems; one of which was chloroform-methanol- Isolation of DNA and determination of GC contents. water 65:25:4 (v/v) and the other chloroform-methanol- DNAs were isolated by Marmur's method with minor acetic acid-water 100:20:12:5 (v/v). Crude and alkaline- modifications (15). The average guanine and cytosine stable lipids of S. paucimobilis EY 2395T were used as contents of the DNAs investigated were determined the control. A hydrogen sulfide solution (50%) was using the method of Tamaoka and Komagata (28). The used to detect spots of all kinds of lipids . Dittmer's Shimadzu high-performance liquid chromatography sys- reagent was used to detect the phospholipid, ninhydrine tem was used for quantification of nucleotides. Cali- solution for free amino base and Anthrone reagent for the bration was carried out with a standard mixture of four glycolipid. As for fatty acids, the extracted lipids were nucleosides (Yamasa). Sphingomonas paucimobilis hydrolyzed in methanol- 12 N HCl (5:1, v/v) at 100 C for JCM 7516, S. suberifaciens JCM 8521, S. natatoria 3 hr. After cooling, an equal volume of water was added JCM 10396 and S. ursincola JCM 10397 were subjected and the fatty acid methyl esters were extracted twice to the determination of GC contents. with n-hexane. Fatty acid analyses were performed The 16S rDNA sequence analysis. The 16S rDNA with a 5,890 series II gas chromatograph (Hewlett sequence data determined in this study have been Packard, Avondale, Pa., U.S.A.) equipped with a flame deposited in the DDBJ database (DNA Data Bank of ionization detector and SPB-1 (30 m X 0.25 mm fused Japan, National Institute of Genetics) under accession silica capillary column; Supelco Inc., Bellefonte, Penn., no. AB025012 for Sphingomonas aromaticivorans U.S.A.) at a temperature ranging from 150 C for 4 min to IFO 16084, AB025013 for S. stygia IFO 16085 and 250 C at an increase of 4 C/min. Each peak was identi- AB025014 for S. subterranea IFO 16086, respectively. fied by its retention time compared with those of a 22- The reference sequences were obtained from NCBI component bacterial acid methyl ester mixture (Supelco (The National Center of Biotechnology Information, Inc., Bellefonte, Pa., U.S.A.). http://www.ncbi.nlm.nih.gov/). Sequence accession

Fig. 1. Thin-layer chromatogram of cellular lipids of Sphingomonas paucimobilis EY 2395 (=JCM 7516), Erythrobacter longus EY 4203 (=JCM 6170), Erythromicrobium ramosum EY 4223, Porphyrobacter tepidarius EY 4231 and Zymomonas mobilis subsp. mobilis EY 4209 (=JCM 10190)[from left to right]: A. Solvent system: chloroform:methanol:acetic acid:H2O=100:20:12:5 (v/v); B. Solvent system: chloroform:methanol:H20=65:25:4 (v/v). Lipids sample a, crude lipids; b, alkali-stable lipids. The major spot from alkali-sta- ble lipids of the strains of type species of each genus with similar shape and R, value to that of sphingoglycolipid (SGL-1) from alka- li-stable lipids of S. paucimobilis is visualized by both solvent systems A and B. 570 Y. KOSAKO ET AL numbers for the reference strains used in this study are Morphological, physiological and biochemical charac- listed in Table 2. teristics are listed in Table 3 and antimicrobial profiles in Using the CLUSTAL W program (29), nucleotide Table 4. Except Gram reaction, lysine and ornithine substitution rates (K,,,) (12) were determined. The decarboxylation, arginine dehydration, gas from nitrate, neighbor-joining method (20) was used to reconstruct a and fermentation of glucose, no other common bio- phylogenetic tree from the distance matrices by NJPLOT chemical characteristics could be found even in the written by Manolo Gouy (Laboratoire de Biometrie, genus Sphingomonas. The results of antimicrobial sus- Univ. Lyon, Villeurbanne, France). Alignment gaps and ceptibility tests were also varied. unidentified base positions were not taken into consid- eration for the calculations. To evaluate the topology of Quinone Analysis the phylogenetic tree, a bootstrap analysis was per- S. natatoria and S. suberifaciens possess the same formed with 1,000 bootstrapped trials. respiratory quinone system. Major respiratory quinones of the type strains of the two species above were identi- Results cal to that of S. paucimobilis JCM 7516. They possess ubiquinone-10 (Q-10) as the major respiratory quinone. Phenotypic Characteristics From literature, we also confirmed that all type strains in It has been confirmed that none of the strains in Group 4 in the alpha-subclass of Proteobacteria possess Group 4 except those in the genus Sphingomonas could ubiquinone-10 (Q-10) as the major respiratory quinone grow in the medium for phenotypical characterization. (1, 7, 8, 10, 17, 18, 21, 25, 26, 33, 34, 36-38).

Fig. 2. Thin-layer chromatogram of cellular fatty acids of representative strains: EY 2395, Sphingomonas paucimobilis; EY 4201, 4202, 4208, 4226, S. yanoikuyae; EY 4203, Erythrobacter longus; EY 4204, S. suberifaciens; EY 4304, S. macrogoltabidus; EY 4341, S. mali; EY 4207, S. terrae; EY 4209, Zymomonas mobilis subsp. mobilis; EY 4210, Z mobilis subsp. pomaceae; EY 4218, S trueperi; EY 4219, S. chlorophenolica; EY 4220, S. natatoria; EY 4221, S. chlorophenolica; EY 4222, Erythrobacter litoralis; EY4223, Erythromicrobium ramosum; EY 4224, "S. wittichii"; EY4225, Sphingomonas sp. Strain SS3; EY 4227, S. rosa; EY 4228, S. pruni; EY 4229, S. asac- charolytica; EY 4230, S. trueperi; EY 4231, Porphyrobacter tepidarius; EY 4250, S. ursincola: Solvent system, n-hexane:diethyl ether= 80:20 (v/v). The pattern of cellular fatty acids from strains except for Zymomonas was composed of non-hydroxy and 2-hydroxy fatty acids. Only non-hydroxy fatty acid was detected from the two subspecies of Zymomonas mobilis. 3-Hydroxy fatty acid was not detect- ed in any of the strains. SPHINGOMONADACEAE KOSAKO AND YABUUCHI FAM. NOV. 571

except the Zymomonas strains was 2-hydroxy-myristic Cellular Lipid and Fatty Acid Analysis acid (20H 14:0). In the two Zymomonas strains, it was By using an acidic solvent system, the location of confirmed that there were no 2-hydroxy fatty acids in sphingoglycolipid (SGL-1) was detected in the alkaline their total extractable lipids. None of the test strains stable lipids of the strains of type species of Sphin- showed the detection of 3-hydroxy fatty acid. gomonas, Erythrobacter, Erythromicrobium, Porphyr- obacter and Zymomonas (Fig. IA). It was recognized as GC Contents and Phylogenetical Analysis being similar to that of S. paucimobilis EY 2395' because The GC contents of the DNA of Sphingomonas pauci- of a similar Rf value, negative color reaction for both mobilis JCM 7516, S. suberifaciens JCM 8521, S. nata- phosphor and free amino base and positive color reaction toria JCM 10396 and S. ursincola JCM 10397 were for carbohydrates. The similarity of major spots of 63.7, 59.0, 64.5 and 64.8%, respectively. We confirmed alkali-stable lipids (SGL-1) was confirmed in the chro- that the GC contents of the DNA of the type strains of the matogram taken from the neutral solvent system (Fig. species in the alpha-4 group ranged from 58.2 to 66 1B). As shown in Fig. 2 and Table 5, all strains of the mol% GC from literature (1, 7, 8, 10, 17, 18, 21, 25, 26, alpha-4 group had octadecenoic acid (18:1) as the major 33,34,36-38). fatty acid. The hydroxy fatty acid detected from all Nearly complete 16S rDNA sequences of each strain

Table 5. Cellular fatty acid composition (%) of total extractable lipids of type strains of 26 species and 2 subspecies of 5 genera

0 Number before the colon indicates the number of carbon atoms , and the number after refers to number of double bonds. tr: trace, less than 1%. b) Figures in parentheses indicate the position of the double bond . OH indicates a hydroxyl group at the 2nd carbon from the carboxyl end of the chain. 572 Y. KOSAKO ET AL

Fig. 3. Phylogenetic position of Sphingomonadaceae (alpha-4 group) among the Proteobacteria (alpha, beta, and gamma) obtained by the neighbor-joining analysis of 16S rDNA. Scale bar= 16 nucleotide substitutions per 1,000 nucleotides of thel6S rDNA sequence. Boot- strap values from 1,000 analyses are shown at the branch points. Escherichia coli JO-1 was used as an outgroup organism. SPHINGOMONADACEAE KOSAKO AND YABUUCHI FAM. NOV. 573 listed in Table 2 were utilized for the phylogenetical respiratory quinone system. Octadecenoic acid (18:1) analysis. Results are shown in Fig. 3. Levels of was the major fatty acid of the total extractable lipids sequence similarity between the 16S rDNA genes of S. (Table 5). paucimobilis (D16144) and that of other species Among the Gram-negative bacteria, the composition employed in this study are shown in Table 2. The 16S of hydroxy fatty acids is useful in classification. Hydroxy rDNA signature pattern for the alpha-4 group consists of fatty acids, usually 3-hydroxy fatty acids, are a compo- 105-116(C-C), 433-442(G-C), 750-754(T-C), 855- nent of lipopolysaccharides, which are distinctive com- 858(G-G), 1334-1336(G-C) and 1385(G). The numbers ponents of cell membranes of Gram-negative bacteria of the sequence position were referred to as the sequence (13). However, 2-hydroxymyristic (20H 14:0) acid was position number of Escherichia coli JO-1 (JO1695). detected in the strains of all the species in the alpha-4 According to the phylogenetic tree (Fig. 3), alpha-, group in the alpha-subclass of Proteobacteria except in beta- and gamma-subclasses are clearly separated as the type strains of two subspecies of Zymomonas mobilis mentioned by Woese (31). Within the alpha-subclass, the (Tables 5 and 6, Fig. 2). Moreover, none of the test strains alpha-4 group was also divided at the high bootstrap contained 3-hydroxy fatty acid. These above-described values at the branch points. Levels of sequence similarity characteristics are regarded as important chemical criteria between S. paucimobilis and other Sphingomonas species for determining the taxonomic position. range from 96.2 to 90.3%. Strains of Group 4 of the The phototrophic capacity of bacteria (i.e., ability to alpha-subclass of class Proteobacteria can be consistently use light as a source of energy) is now known to be categorized as members of the same phylogenetic lin- widely distributed throughout Prokaryotae and does not eage, revealing >90% 16S rDNA sequence similarity indicate a close phylogenetic relationship among those with S. paucimobilis (Table 2). taxa (14, 33). Two subspecies of Zymomonas mobilis As shown in Fig. 3, the alpha-4 group seemed to be are facultatively anaerobic bacteria and unique among divided into several clusters as mentioned by Takeuchi bacteria in fermenting sugar anaerobically by the Entner- elsewhere (personal communication). Further analyses Doudoroff pathway (19), whereas other species listed in are necessary to define the taxonomic position of each Table 1 are strictly aerobic and non-fermentative bacte- cluster. ria. Phototrophic capacity and growth condition (i.e., mode of metabolism), having previously been thought of Discussion as important characteristics in differentiating each genus, are now not recognized as useful for phylogenetic clas- It is quite obvious that phylogenetic data alone are sification above genus. insufficient to provide an adequate description of taxa of Without any description of phenotypic characteriza- any rank above species. The ad hoc committee on tion, Sandaracinobacter sibiricus was solely analyzed approaches to taxonomy within the Proteobacteria (16) phylogenetically by using the 16S rDNA sequence has stated that it is necessary for phylogenetic and phe- obtained from the DNA Data Bank, since neither sub- notypic characteristics, or polyphasic taxonomy (2), to be cultures nor lyophile of the type strain of the species were used in conjunction for the delineation of taxa at all available from any permanently established culture col- levels from kingdom to genus. Coherent phenotypic lection. Based on the above described results, we con- characteristics are essential for both description and cluded that the alpha-4 group of the class Proteobacteria recognition. should constitute a family, for which the name Sphin- In this study, the validated species in the alpha-4 gomonadaceae fam. nov. is proposed. group in the alpha-subclass of Proteobacteria have been Since Sandaracinobacter sibiricus is a member of confirmed to possess unique chemical features other the alpha-subclass (alpha-4 group) of the Proteobacteria than those in the alpha-subclass of Proteobacteria. A (36), it can be considered a member of the Sphin- novel sphingoglycolipid was firstly described by gomonadaceae fam. nov. Yamamoto et al (35). The structure of the sphingogly- colipid was identified as N-2'-hydroxymyristoyl dihy- Description of Sphingomonadaceae fam. nov.: drosphingosine 1-glucuronic acid. Yabuuchi et al (34) Sphingomonadaceae (Sphingo. mo. na. da. ce. ae. M. firstly reported the existence of the sphingoglycolipid L. fem. n., Sphingomonas, type genus of the family; among the bacterial species which were subsequently suff. -aceae, to denote family; M. L. fem. pl. n. Sphin- included in the new genus, Sphingomonas. All species in gomonadaceae, the Sphingomonas family) the alpha-4 group in the alpha-subclass of Proteobacte- The organisms of the family are Gram-negative, ria have been confirmed to possess SGL-1. Further- asporogenous rods or ovoid cells. They divide by bina- more, they possessed ubiquinone-10 (Q-10) as a major ry division, budding or asymmetric division, and can 574 Y. KOSAKO ET AL be observed by electron microscopy in S. natatoria and 47:191-201. S. ursincola. Aerobic or facultative anaerobic chemo- 2) Colwell, R.R. 1970. Polyphasic taxonomy of bacteria, p. organotrophs. The cells of some genus contain bacteri- 421-436. In lizuka, H., and Hasegawa, T. (eds), Culture ochlorophyll a (Bchl a) and are facultative photoorga- collections, University of Tokyo Press, Tokyo. 3) De Ley, J. 1978. Modem molecular methods in bacterial tax- notrophs. Colony color is different from species to onomy: evaluation, application, prospects, Proc. 4" Int. species such as deep yellow, orange, brown-orange, Conf. Plant. Path. Bact. Angers 1978, 347-357. faint yellow, creamy-yellow or non-pigmented. Motile 4) De Ley, J., and Swing, J. 1976. Phenotypic description, by means of polar flagella. numerical analysis, and proposal for an improved taxonomy The major fatty acids of cellular lipids are octade- and nomenclature of the genus Zymomonas Kluyver and cenoic acid (18:1) and 2-hydroxymyristic acid (20H van Niel 1936. Int. J. Syst. Bacteriol. 26: 146-157. 14:0) (with the exception of two subspecies of 5) Denner, E.B.M., Kampfer, P., Busse, H.-J., and Moore, Zymomonas mobilis). Ubiquinone-10 (Q-10) is the E.R.B. 1999. Reclassification of Pseudomonas echinoides Heumann 1962, 343', in the genus Sphingomonas as Sphin- major respiratory quinone. N-2'-hydroxymyristoyl gomonas echinoides comb. nov. Int. J. Syst. Bacteriol. 49: dihydrosphingosine 1-glucuronic acid (SGL-1) is the 1103-1109. major sphingoglycolipid in the cellular lipids. The GC 6) Folch, J., Lees, M., and Stanley, G.H.S. 1957. A simple content of the DNA ranges from 47.5 to 67.0 mol%. method for the isolation and purification of total lipids from The 16S rDNA signature pattern for the family consists animal tissues. J. Biol. Chem. 226: 497-509. of 105-116(C-C), 433-442(G-C), 750-754(T-C), 855- 7) Fuerst, J.A., Hawkins, J.A., Holmes, A., Sly, L.I., Moore, 858(G-G), 1334-1336(G-C) and 1385(G). The type C.J., and Stackebrandt, E. 1993. Porphyrobacter neusto- nensis gen. nov., sp. nov., and aerobic bacteriochlorophyll- genus Sphingomonas is Yabuuchi et al. 1990. Members synthesizing budding bacterium from fresh water. Int. 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