Emendation of the Genus Sphingomonas Yabuuchi Et Al

International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1485–1496 DOI: 10.1099/ijs.0.01868-0

Emendation of the genus Sphingomonas Yabuuchi et al. 1990 and junior objective synonymy of the species of three genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastomonas ursincola

1 Department of Eiko Yabuuchi,1 Yoshimasa Kosako,2 Nagatoshi Fujiwara,3 Microbiology, Gifu 3,4 5 5 University School of Takashi Naka, Isamu Matsunaga, Hisashi Ogura and Medicine, Tsukasa-machi Kazuo Kobayashi3 40, Gifu 500-8705, Japan 2 Japan Collection of Microorganisms, Institute Author for correspondence: Kazuo Kobayashi. Tel: j81 6 6645 3745. Fax: j81 6 6646 3662. of Physical and Chemical e-mail: kobayak!med.osaka-cu.ac.jp Research (RIKEN), Wako- shi, Saitama 351-0198, Japan The 16S rDNA sequence similarities between the type strains of Sphingomonas 3 Department of Host paucimobilis and 32 other Sphingomonas species range from 902to996%. It Defense, Osaka City might be possible to divide the genus into several new genera according to a University Graduate School dendrogram drawn from 16S rDNA sequence similarity. However, the of Medicine, 1-4-3 Asahi- machi, Abeno-ku, Osaka phenotypic and biochemical information needed to deﬁne clusters of strains 545-8585, Japan representing distinct genera within this group of organisms was not 4 Institute of Skin Sciences, previously available. Although the cellular lipids of type strains of all 28 Club Cosmetics Co. Ltd, Sphingomonas species tested contained glucuronosyl-(1 ! 1)-ceramide Ikoma-shi, Nara 630-0222, together with 2-hydroxymyristic acid, other molecular species of Japan sphingoglycolipids were distributed randomly. Sphingomonas natatoria and 5 Department of Virology, Sphingomonas ursincola, bacteriochlorophyll a-containing, Gram-negative Osaka City University Graduate School of facultative phototrophs, belong to the cluster of the genus Sphingomonas. Medicine, 1-4-3 Asahi- Other phototrophic Porphyrobacter and Erythrobacter species in the machi, Abeno-ku, Osaka Sphingomonadaceae were classiﬁed into a cluster different from the genus 545-8585, Japan Sphingomonas, as reported previously. None of the physiological and biochemical characteristics considered, including cellular lipids and fatty acid composition, provided evidence for the division of the current genus Sphingomonas. It is therefore concluded that the genus Sphingomonas should remain undivided at this time. The species of three recently proposed genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastobacter ursincola, are junior objective synonyms of species of the genus Sphingomonas.

Keywords: glucuronosyl ceramide, 2-hydroxymyristic acid, bacteriochlorophyll a, alcapton

INTRODUCTION 2001). The recent detection of bacteriochlorophyll a from cells of Blastomonas natatoria DSM 3183T and Delineation of the genus Sphingomonas has been the puf genes encoding proteins of the photosynthetic subject of taxonomic discussion by various workers reactions detected by PCR (Hiraishi et al., 2000) has (Balkwill et al., 1997; Hiraishi et al., 2000; Ka$ mpfer et raised new interest. Based on the results of DNA– al., 1997; Stolz et al., 2000; Takeuchi et al., 1994, DNA hybridization and phylogenetic analysis of 16S rDNA sequences of B. natatoria DSM 3183T and T ...... Erythromonas ursincola DSM 9006 , the latter species Abbreviation: SGL, sphingoglycolipid. has been transferred to the genus Blastomonas in a

01868 # 2002 IUMS Printed in Great Britain 1485 E. Yabuuchi and others 3 g Bacto n ...... C)* m temperature ( 2 TSA 2 TSA, 30 2 TSA, 30 2 g Bacto yeast extract, n \ \ \ Tryptic soy agar, 28 Tryptic soy agar, 30 Tryptic soy agar,26 Tryptic soy agar, 30 Tryptic soy agar, 30 PYA, 26 5 g Bacto peptone, 0 n 0 g Bacto peptone, 0 n T T IFO 16084 IFO 16084 spp. PYA, 26 ) Tryptic soy agar, 28 ...... spp.spp. PYA, 26 1 Rosa Malus Malus Prunus persica 5 g agar in 100 ml), PYA (1 n Setaria viridis aromaticivorans aromaticivorans . . S S ‘Sterile’ water used beforeSea-water surgery sample from GulfNatural of mineral Alaska water, Taejon,Terrestrial Korea subsurface sedimentsRoots of Distilled waterPentachlorophenol-contaminated soilFreshwater sediment, Taejon, KoreaSewage-treatment-plant water sample, TokyoPlate contaminantSoil Natural mineral water, Taejon,Soil KoreaRoots Tryptic of soy Heart agar, infusion 30 agar,Freshwater 30 swimming poolUrine Tryptic soyHospital agar, mechanical 30 respirator Heart 1 infusion agar,Eutrophic 30 fountain water, Vienna,Roots Austria Difco of nutrient agar,Hairy 30 roots of Plant ( Blood HeartAs infusion agar, 26 DifcoBioﬁlm nutrient of agar, reactor 30 inoculatedCorky with root activated of sludge lettuce 1 As Natural mineral water, Taejon,Activated Korea sludgeSoil PYA, 26 Tryptic soyFreshwater agar, cyanobacterial 30 mat Heart infusion agar, 30 Water from the RiverWater Elbe from Tryptic the soy River agar,Clinical Elbe 28 specimen Heart infusion agar, 30 Difco nutrient agar ATCC medium 1700, 26 Heart infusion agar, 30 NA, 26 Tryptic soy agar TSA, 30 Tryptic soy agar, 28 Tryptic soy agar, 28 medium. Other media are abbreviated as NA (0 5 g agar in 100 ml). n species Rhizobium T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 15 g Bacto yeast extract and 1 n Received as Source of isolation Medium and incubation GIFU 11458 DSM 13593 KCTC 2881 IFO 16084 IFO 15499 GIFU 11526 ATCC 33790 JCM 11454 JCM 10874 JCM 10637 DSM 11019 JCM 11456 JCM 10192 IFO 15500 ATCC 35951 GIFU 11387 GIFU 2395 DSM 13101 IFO 15498 IFO 15208 JCM 99038 GIFU 2397 IFO 16085 DSM 10700 JCM 8521 IFO 16086 JCM 11457 IFO 15098 ATCC 12417 DSM 9006 DSM 6014 DSM 6383 GIFU 9882 Sphingomonas ...... 0 g Bacto tryptic soy broth and 1 n T 25 g Bacto peptone, 0 T n T T T T T T T T T T T 2 TSA (2 T T \ T T T T T T T T T T 2NA(0 T T \ . 1990 EY 4213 . 1993 EY 4304 . 1990) . 1996 EY 4219 . 1995 EY 4229 . 1997 EY 4296 . 1997 EY 4344 et al . 2001 EY 4374 . 1990 EY 4208 et al . 1977) et al et al et al . 1997 EY 4298 et al . 1990 EY 4215 . 1996 EY 4251 . 2000 EY 4375 . 1993 EY 2397 . 2001 EY 4224 et al . 1997) et al . 1997 EY 4218 et al et al . 2001 EY 4370 . 2000 EY 4343 et al . 1993 EY 4207 et al et al . 1995 EY 4228 . 2001 EY 4377 . 1997 EY 4297 et al et al . 2000 EY 4345 . 1995 EY 4341 . 1995 EY 4227 et al et al . 2001 EY 4376 et al . 2001 EY 4361 . 2001 KCTC 2881 et al Yabuuchi T T T T T et al Takeuchi et al Balkwill Zipper Nohynek et al Takeuchi et al et al et al et al et al T Kim et al et al et al (van Burgen (Holmes Balkwill Yabuuchi mpfer Lee Stolz $ Nohynek (Heumann 1962) Vancanneyt Yabuuchi Yun (Leifson 1962) Lee (Sly 1985) Takeuchi (Yurkov Denner Lee Yabuuchi Ka Fujii Balkwill Takeuchi Takeuchi Takeuchi Takeuchi 5 g agar in 100 ml), 1 . 1990 EY 2395 . 1990 EY 4216 . 1999 EY 4220 . 1999 EY 4250 . 1999 EY 4204 n 2 g agar in 100 ml) and 1 n . 1999 EY 4340 et al et al et al et al et al et al History and growth conditions of type strains of 33 Yabuuchi Yabuuchi Yabuuchi Yabuuchi Denner Yabuuchi Sphingomonas roseiﬂava Sphingomonas sanguinis Strain Sphingomonas adhaesiva Sphingomonas capsulata Sphingomonas chungbukensis Sphingomonas herbicidovorans Sphingomonas koreensis Sphingomonas mali Sphingomonas natatoria Sphingomonas ursincola Sphingomonas alaskensis Sphingomonas aquatilis Sphingomonas aromaticivorans Sphingomonas asaccharolytica Sphingomonas chlorophenolica Sphingomonas cloacae Sphingomonas echinoides Sphingomonas macrogoltabidus Sphingomonas parapaucimobilis Sphingomonas paucimobilis Sphingomonas pituitosa Sphingomonas pruni Sphingomonas stygia Sphingomonas suberifaciens Sphingomonas subterranea Sphingomonas terrae Sphingomonas trueperi Sphingomonas wittichii Sphingomonas xenophaga Sphingomonas yanoikuyae Sphingomonas subarctica Sphingomonas taejonensis Sphingomonas rosa 2 g NaCl and 1 n 0 Table 1...... Authorities are given for each name; these papers are not included in the reference list unless cited elsewhere. * Heart infusion agar and tryptic soy agar were from Difco. ATCC medium 1700 is yeast extract and 1

1486 International Journal of Systematic and Evolutionary Microbiology 52 Delineation of the genus Sphingomonas proposed new combination, Blastomonas ursincola unidentified base positions were excluded from the cal- (Yurkov et al. 1997) Hiraishi et al. 2000. culation. To evaluate the topology of the phylogenetic tree, a bootstrap analysis was performed (Yabuuchi et al., 1999; However, it had been proposed previously that B. Kosako et al., 2000). natatoria DSM 3183T and Erythromonas ursincola T Phenotypic characterization and carbon assimilation. Pheno- DSM 9006 belong to the genus Sphingomonas,as typic features were determined by the methods of Yabuuchi Sphingomonas natatoria (Sly 1985) Yabuuchi et al. et al. (1990, 1999). Production of oxidative acids from sugars 1999 and Sphingomonas ursincola (Yurkov et al. 1997) and polyalcohols was determined using OF basal medium Yabuuchi et al. 1999, by the study of polyphasic (Difco), except for three strains, Sphingomonas xenophaga taxonomy (Yabuuchi et al., 1999). The presence of EY 4343T, Sphingomonas herbicidovorans EY 4344T and T bacteriochlorophyll a and facultative phototrophic Sphingomonas roseiflava EY 4345 , that were unable to grow ability are unlikely to indicate a close phylogenetic in OF basal medium. To analyse these strains, cystine tryptic relationship in prokaryotes (Kondratieva et al., 1992). agar (Difco) was used. Assimilation tests were performed by Other phototrophic organisms, such as Erythrobacter using Biotype 100 and Biotype medium (bioMe! rieux) in accordance with the manufacturer’s instructions. API 20NE longus, Erythrobacter litoralis, Erythromicrobium (bioMe! rieux) was also used for characterization of ramosum and Porphyrobacter tepidarius (Hanada et organisms. al., 1997), are outside the genus Sphingomonas, but Production of homogentisic acid/alcapton/2,5-dihydroxy- included in Sphingomonadaceae Kosako et al. 2000, phenylacetic acid. Culture supernatants of all bacterial according to the presence of glucuronosyl ceramide in strains used in the present study, including Sphingomonas their cellular lipids and phylogenetic analysis of 16S herbicidovorans EY 4344T and Pseudomonas aeruginosa rDNA nucleotide sequences (Kosako et al., 2000). ATCC 23268, standard producers of homogentisic acid On the basis of phylogenetic analysis of 16S rDNA (Yabuuchi & Ohyama, 1972), were tested for production of homogentisic acid during the metabolism of phenylalanine sequences and polyamine profiles, Takeuchi et al. and tyrosine by using HPLC (Bory et al., 1990; Mathieu (2001) proposed that the genus Sphingomonas be et al., 1997). classified into four genera, Sphingomonas, Antimicrobial susceptibility testing. Except for Sphingo- Sphingobium, Novosphingobium and Sphingopyxis. monas suberifaciens EY 4204T, Sphingomonas pruni From a practical aspect, it is essential that any genus EY 4228T, Sphingomonas asaccharolytica EY 4229T and should not be delineated by phylogenetic analysis Sphingomonas mali EY 4341T, antimicrobial susceptibility alone, but should also be distinguished by a com- testing was performed using Sensi-discs and Mueller–Hinton bination of phenotypic tests that are performed easily II plates (Difco) (Kosako et al., 2000; Yabuuchi et al., 1999, in every microbiological laboratory. In this paper, we 2001). Sphingomonas suberifaciens EY 4204T was tested have examined the phenotypic characteristics of using ATCC medium 1700 supplemented with 1n5% agar species currently assigned to the genus Sphingomonas (Yabuuchi et al., 1999). Peptone\yeast extract agar was used for growth of Sphingomonas pruni EY 4228T and Sphingo- (Yabuuchi et al. 1990) in relation to their phylogeny T and found that there is no phenotypic evidence to monas asaccharolytica EY 4229 and a 50% concentration of nutrient broth supplemented with 1n5% agar was used support a proposal to split the species into four genera. for Sphingomonas mali EY 4341T. We thus conclude that the genus Sphingomonas should Analysis of cellular lipids and fatty acids. Bacterial cells remain undivided at this time. grown on agar media at optimal temperatures were harvested to analyse cellular lipids and fatty acids as METHODS described previously (Naka et al., 2000; Yabuuchi et al., 1990, 1999). Bacterial strains. The history and growth conditions of the type strains of 33 Sphingomonas species are listed in Table 1. Two of them were isolated from clinical specimens, two from RESULTS AND DISCUSSION a hospital environment, one from a diseased plant and the Phylogenetic analysis remaining 28 were of environmental origin. Growth media and incubation temperatures for each type strain are also Fig. 1 shows a dendrogram derived from 16S rDNA listed in Table 1. oligonucleotide sequence similarity among the type 16S rDNA sequences and phylogenetic analysis. Deposited strains of 33 Sphingomonas species and Acetobacter data on 16S rDNA sequences of the type strains of 33 aceti as an outgroup micro-organism. From Sphingo- Sphingomonas species, two subspecies of Zymomonas mobilis monas parapaucimobilis to Sphingomonas wittichii, as a representative member of Sphingomonadaceae and including Sphingomonas paucimobilis, the type species Acetobacter aceti as an outgroup organism were obtained of the genus, it seems to form a single large branch. from the DNA Database of Japan (DDBJ, Institute of From Sphingomonas chlorophenolica to Sphingomonas Genetics, Shizuoka, Japan) and were used for phylogenetic terrae, 17 species are divided into three branches. The analysis. Sequence accession numbers of bacterial strains type strain of Sphingomonas suberifaciens belongs to used in this study are shown in Fig. 1. Phylogenetic analysis the branch of Sphingomonas chlorophenolica. Both by 16S rDNA oligonucleotide sequences was performed as described previously (Kosako et al., 2000). In brief, nucleo- Sphingomonas ursincola and Sphingomonas natatoria are included in the Sphingomonas chlorophenolica– tide substitution rates (Knuc ; Kimura, 1980) were determined and a distance-matrix tree was constructed by the neighbour- Sphingomonas terrae branch. Numerical analysis of 27 joining method (Saitou & Nei, 1987) using the program type strains of Sphingomonas species demonstrates   (Thompson et al., 1994). Alignment gaps and that the members of three new genera proposed by http://ijs.sgmjournals.org 1487 E. Yabuuchi and others

...... Fig. 1. Dendrogram based on phylogenetic analysis of 16S rDNA sequence of type strains of 33 Sphingomonas species. Nucleotide substitution rates (Knuc) (Kimura, 1980) were determined using the program CLUSTAL W (Thompson et al., 1994). The neighbour-joining method (Saitou & Nei, 1987) was used to reconstruct a phylogenetic tree from the distance matrices by using NJPLOT. Alignment gaps and unidentiﬁed base positions were excluded from the calculation and ﬁnal analysis. To evaluate the topology of the phylogenetic tree, a bootstrap analysis was performed using 1000 bootstrapped trials. Bootstrap values are indicated at branching points.

Takeuchi et al. (2001) do not form solid clusters (Fig. herbicidovorans EY 4344T, but not other strains, 2). Our results do not support the proposal of the contained alcapton\homogentisic acid (Mann, 1979). genus Sphingomonas and three new genera, Sphingo- Because of its nutritional requirements, both API bium, Novosphingobium and Sphingopyxis, on the basis 20NE and Biotype 100 enabled us to estimate the of phylogenetic and chemotaxonomic analyses. assimilating ability of Sphingomonas suberifaciens EY 4204T. A combination of certain phenotypic features Phenotypic characteristics was useful to diﬀerentiate strains at the species level, T but not groups of strains at the generic level (Tables Colonies of Sphingomonas natatoria EY 4220 and 2–4 and Fig. 2). Sphingomonas ursincola EY 4250T, facultatively phototrophic organisms, were orange-coloured. At harvest, Distribution of sphingoglycolipids (SGLs) and major however, they gave a melted-chocolate-like colour. polyamines Sphingomonas herbicidovorans EY 4344T produced water-soluble melanin-like pigment on heart infusion Glucuronosyl-(1 ! 1)-ceramide (SGL-1), but not or tryptic soy agar plates. Grown slowly on Bacto- galacturonosyl-(1 ! 1)-ceramide (SGL-1h), was pres- phenylalanine agar slants, Sphingomonas herbicido- ent in cellular lipids of all 28 strains tested (Table 5). vorans EY 4344T turned the medium pink from the Other SGLs (SGL-2, -3 and -4) and fatty acids, such as surface. The culture supernatant of Sphingomonas 2-OH 15:0 and 2-OH 16:0, could not contribute to the

1488 International Journal of Systematic and Evolutionary Microbiology 52 Delineation of the genus Sphingomonas

...... Fig. 2. Numerical analysis of 27 type strains of Sphingomonas species. Numerical analysis was performed by using NTSYS-pc version 2 (Applied Biostatistics). The coefficient of simple matching was used for calculation and the dendrogram was depicted by UPGMA (Sneath & Sokal, 1973). S., Sphingomonas. Genera in parentheses follow Takeuchi et al. (2001). *, Not included in Takeuchi et al. (2001). division of the genus. Major polyamines detected were two phylogenetically related and facultatively photo- identified as spermidine and homospermidine trophic species, Sphingomonas natatoria and Sphingo- (Hamana & Matsuzaki, 1993; Segers et al., 1994; Stolz monas ursincola, from the genus Sphingomonas. et al., 2000; Takeuchi et al., 1995). The division of the genus Sphingomonas into four genera has been proposed on the basis of sequence Antimicrobial susceptibility of Sphingomonas species analysis of 16S rDNA, including signature sequences, and polyamine patterns (Busse & Auling, 1988; All strains tested were susceptible (S) or showed an Hamana & Matsuzaki, 1993; Takeuchi et al., 1995). intermediate response (IM) to antimicrobials such as More recently, similar methods were used to propose tetracycline, doxycycline and amikacin (Table 6). three new genera derived from the genus Sphingomonas Meropenem, panipenem, amoxicillin\clavulanic acid, (Takeuchi et al., 2001). In the present study, we have minocycline and imipenem exerted antimicrobial ac- examined 28 type strains of Sphingomonas species by tivity against most strains used in this study. Sphingo- T phenotypic, biochemical and genetic analyses. We monas suberifaciens EY 4204 , Sphingomonas natatoria have shown that there is no phenotypic and phylo- EY 4220T, Sphingomonas roseiflava EY 4345T and T genetic evidence to support a proposal to divide the Sphingomonas paucimobilis EY 2395 were each sus- species into new genera. Therefore, we have concluded ceptible to more than 30 of 36 discs. Although isolates that the genus Sphingomonas should remain undivided from the clinical specimen and from the hospital at this time. The species of three recently proposed environment were susceptible to fluoroquinolones, 12 genera, Sphingobium, Novosphingobium and Sphingo- strains originating from plants, soil and other sources pyxis, in conjunction with Blastobacter ursincola, are were resistant to certain or all new fluoroquinolones junior objective synonyms of species of the genus tested. The type strains of Sphingomonas pruni and Sphingomonas. Sphingomonas rosa were resistant to more than 50% of discs tested. Again, these results indicate that the pattern of antimicrobial susceptibility\resistance does Emended description of the genus Sphingomonas not provide any convenient basis for subdividing the Yabuuchi et al. 1990 genus. Gram-negative, asporogenous rods or ovoid cells. Phototrophic ability is distributed widely in pro- Soma size is 0n7 i 1n4 µm, 0n4 i 0n9 µm(Sphingomonas karyotes and does not necessarily indicate a close suberifaciens)or0n9 i 2n0 µm(Sphingomonas ursin- phylogenetic relationship (Kondratieva et al., 1992). cola). Budding or asymmetric division is visualized by Sphingomonas natatoria and Sphingomonas ursincola electron microscopy in two species (Sphingomonas are closely related phylogenetically within the genus natatoria and Sphingomonas ursincola). Bipolar stain- Sphingomonas (Yabuuchi et al., 1999). Both Sphingo- ing in Gram stain (Sphingomonas ursincola) and rosette monas ursincola and Sphingomonas natatoria have been formation in some species. Usually single polar identified as phototrophs by HPLC analysis of photo- flagellum when motile. Sphingomonas trueperi synthetic pigments and genetic studies (Hiraishi et al., is peritrichous. Sphingomonas chlorophenolica, 2000). In addition, flagella morphology is unlikely to Sphingomonas cloacae, Sphingomonas xenophaga, be a key feature to distinguish a genus from others Sphingomonas aromaticivorans, Sphingomonas sub- (Yabuuchi et al., 1995). The presence of bacterio- terranea, Sphingomonas capsulata, Sphingomonas chlorophyll a is not a reason, in isolation, to exclude stygia, Sphingomonas subarctica, Sphingomonas rosa, http://ijs.sgmjournals.org 1489 E. Yabuuchi and others ; ) – –  T j j j (  , Bacto . . –– S j j jj jj ...... S . EY 4345 ––– ––– – –– –– jjj jjj jjj jjj jjj S 1P 1P 1P ; 13, chlorophenolica ; 25, T . T roseiflava S ) ) . ;7, T j j j jjj j S ( ): 1, roseiflava ) )– – and . EY 4227 –( – –– – – ––– EY 4228 j j     S EY 4204 rosa –– – j j jj jj jj j j .      ; 18, pruni T S . S ) ) )– – xenophaga – – ––( jjj j j j j j jjj j j j . Sphingomonas ; 12, S T ., ; 24, , no growth. , )– T S . (1979). Data for the presence of EY 4213 – – –––––– – – – – – ––– ––––– suberifaciens j j   . S et al j j EY 4251 ;6, EY 4341 T jj jj j j j jj , no data; mali herbicidovorans .  . – jjj jjj j j j jjj S S subarctica parapaucimobilis EY 4343 . . ) S S –– ––– ––– ––––– – – ––– –( –– ––– –––– ––––– – ––– –( ––––( –– ––– –––– ––––– j ; 23, j T . For ...... T ; 11, ; 17, )– ––– –( T T ––– ––––– j j j jjj j j j j j jjj j j xenophaga . )– – ) EY 4218 S –( –– –––––––––(  EY 4224 jjjjjjjj jj j jj jjjjjjj j jjjjjjj ( species , negative reaction; EY 4297 EY 4220 ;5, )––––– T ) k –––( – – –( – –  jjj jjj j j jjj ( trueperi is the type species of the genus. Strains ( . wittichii stygia S ) . )( . – –  S j j j jj j jj jjj jjj j j S natatoria EY 4361 . ; 22, S )( ) T ; 28, – – – –– j j ; 10,  ( ( T T Sphingomonas ; 16, . (2000). Oxidative acid production was tested using Bacto OF basal medium. Characters are cloacae ) )–––––––––––– )– T . –– –––––––––––––––––– ––( –––– ––––– j j j j j jjjj ( ( S et al EY 4370 EY 4340 EY 4216 ;4, – –––––––––– –– – –– – j jj jj jj j  T EY 4250 ), yellowish after 3 or more days at room temperature. – – – –  j j j pituitosa . Sphingomonas paucimobilis S capsulata echinoides – –––––––– EY 4208 . . ursincola S S . ; 21, S –––– – – ––– –––  jjj jjjj jjjj j j j jjjj j j j jj jjjj j jjjj j j jj j jj j T ;9, ...... T ; 27, T . (1997) and Hiraishi ––– 678910111213141516171819202122232425262728 – –––––( ––––– ––– ; 15, j jjjjj           ), positive reaction after more than 4 days; T yanoikuyae , lemon-yellow; ( . et al j ) EY 2395 S  j j j j j j j jjj j EY 4298 EY 4215 ;3, ) T EY 4207 –– – –( –– – – – ––––– j   ( ) – – – ––––– –– ––  terrae j j j 1P 1P 1P 1P 1P 1P 1P 1P 1P Peri ( paucimobilis adhaesiva . subterranea . EY 4344 . , pink-yellow; S . S S )– – )– – – )– – – S – – ––– –– –––   jj j jj j j j ( ( ( ; 14, ;8, ; 20, ; 26, T T T T –––– –––– – 1234 5 –––– – –––– – –––– –– –––– –––– – jjj j j jjj j j jjjj j jj j j j                   were taken from Yurkov a , orange;  a herbicidovorans EY 4304 EY 4296 EY 2397 EY 4229 . S , positive reaction within 3 days; ( † j Physiological and biological characteristics of 28 type strains of ;2, T sanguinis -Ribose – –  Xylose Dulcitol MaltoseSucrose – Glucose Sorbitol Aesculin Gelatin Starch Mannitol Tween 80 Inositol Adonitol . Motility – Phenylalanine deaminase Oxidative acid from: Characteristic Colony colour* – Nostoxanthin Acylamidase Alcapton production – Citrate (Simmons) DNase Flagellation Bacteriochlorophyll Hydrolysis of: Oxidase (Kovacs) S , Deep yellow; 1P, Polar mono; Peri, peritrichous.  bacteriochlorophyll CTA medium was used rather than Bacto OF basal medium. Data for the presence of nostoxanthin were taken from Jenkins scored as: EY 4219 † asaccharolytica 19, macrogoltabidus Table 2...... Strains are arranged in the order of the dendrogram in Fig. 1. * aromaticivorans

1490 International Journal of Systematic and Evolutionary Microbiology 52 Delineation of the genus Sphingomonas - o j j j j j j j ...... –– – – –– –– –– ––– ––– j –––– ––– j j j jj j -sorbitol, adonitol,  j jj jjjjj – –– – )-arabitol, ––––––– –––– –– ––––––– j k (  –––– –––– –––––––– j j j j jjj jjjjj j j -valerate, histidine, ethanolamine, malonate, 3- )-arabitol, – n jjjjjjjj jjjj jjj j (  –– ––– ––––––––––– – ––––––––––– – –––––––––– – -amino- α - –––– j j j jj j jjjjjj j j j ......  )-sorbose, ––––––––– – j jjjjjjjjj jj j (  species obtained by Biotype 100 –––––– – –– – j j j j –––––––––––––––– ––– –––– –––– –– jj jj , substrate not assimilated. Final reading was performed after 7 days cultivation at –– –– – k j Sphingomonas –––––– –––––– j jj j jj – – – –– j j jjj -coumarate, trigonelline, histamine, m –––––––––––––––– –– j jjjj –––––––––––––– j jj j jjj jjjjj jjj jjj ...... – –– – –– – –– j j ––––––––––––– – ––– – ––– , Positive growth by substrate assimilation; j jjj jj j j )-tartrate, 3-phenylpropionate, j j (  ––––– – jj –––––––––––––– –––––––––––– ––––––––– ––––––––– ––––––––– –––––– ––––––– 12345678910111213141516171819202122232425262728 –––––––––––––––– ––––––––––––––––––– –––––––––––––––– ––––––––––––––––––– j jjj j -glucuronide, β -gluconate  Selected features of the assimilation proﬁles of type strains of 28 )-Tartrate )-Xylose – – )-Mannose – – )-Malate -glucopyranose, tryptophan. -Rhamnose –  k j j  k -Mannitol ( -Galacturonate ( ( -Glucosamine - -Alanine ( -Glutamate  Citrate – –  2-Oxoglutarate 2-Keto-  Benzoate  Phenylacetate  Substrate   α Succinate – –   Glutarate Lactulose Betaine C. The following compounds were not assimilated by any of the 28 type strains tested: dulcitol, m Table 3...... See Table 2 for30 identities of strains. methyl hydroxyquinoline http://ijs.sgmjournals.org 1491 E. Yabuuchi and others * – – – – – – – j ...... –– j jj jjj ryptophan, acid from – ––– ––– j – ––––––– ––––––– jj – –––––––– j jj j jj jjjjjjjjj – – –– -galactopyranosidase.  - ––– j jjjjjjjjjj jjjjj j j jjjjjj β ...... jjjjjjjjjj j jjjjjjjjjjjj j –– -Nitrophenyl ––– –– jjjjjjjjjjjjjj j p ––– – –––– ––––– –––– –– j jj j j species – – ––––––– -mannitol. PNPG,  j j – jjjjj jjj jjjjjjj j jjjjjj Sphingomonas –– – – ––––– ...... –– –– –––– –––––– –––––––––––––––– jjjjjjjjjjjjjjjjjjjjjjj jjjjjjjjj * – ––––––––––––– j j j jjj jj jj jjj j –– –– jjjj 12345678910111213141516171819202122232425262728 ––––––––––––––––––––––––––– jjj j jjj jjjjj j -Malic acid – Results of API 20NE tests on type strains of 28 -Acetyl glucosamine – – -Mannose -Arabinose – – -Capric acid   Glucose N n Gluconate – – Maltose – – Adipic acid – – – Citrate – –  Phenylacetate Substrate or test Aesculin PNPG Assimilation of: Table 4...... See Table 2 for identities of strains. All 28 type strains were positive for the Zn-dust test and negative for the nitrite test, indole production from t * Negative on API 20NE, but positive on aesculin agar slants and Biotype 100. glucose under mineral oil, arginine dihydrolase, urease and assimilation of

1492 International Journal of Systematic and Evolutionary Microbiology 52 Delineation of the genus Sphingomonas – tr j a  j ...... a 416 j a j 3-hydroxy fatty a   j a j a j c a j j , Hamana & Matsuzaki (1993). a e jjjjjjjjj j – . (1994); a j j et al a ...... j d , c , Segers – –––––––––––– d j species a d j j a –– – – jj j a j j Sphingomonas a – j , Busse & Auling (1988); a c j c a jj j j . (2000); a j et al a j ...... , Stolz a b 678910111213141516171819202122232425262728 j b , no data; CPA, cyclopropanoic acid. –––––––– j  . (2001); et al a jj j a j , not detected; , Takeuchi a a k 13236116799533 211 tr 5 3133 7 5143221316112186172949 –– j † c Selected cellular components characteristic for type strains of 28 h 9 ω 17:0 CPA tr 1 tr – tr 6 12 11 2 4 – tr tr 45 10 1 2 3 tr – 9 – 20 3 32 4 16:1 Homospermidine -H50–––4t3–1––tr––––––215––– 4tr–––––32–––– –––––243tr3–––13– ––tr––tr––––––––tr6–––––––––––– 132––tr––tr–7–4tr2 2-OH 14:02-OH15:0 2-OH16:1 2-OH16:0 10 14SGL-1 14 13 10 13 10 10 16 18 17 11 7 2 9 8 17 16 28 18 20 13 5 14 3 18 23 10 Other SGL(s)* – – – – – – – – Spermidine Data were taken from: Cellular component 1 2 3 4 5 Fatty acids (%): Lipids: Major polyamines: Table 5...... See Table 2 for identities of strains. All 28 type strains tested were positive for SGL-1 and ubiquinone 10. All 28 type strains tested were negative for * Other SGL(s) represents SGL-2,† -3 or -4. acids. tr, Less than 1%; http://ijs.sgmjournals.org 1493 E. Yabuuchi and others ...... ept for the combinations RRRRRS SIM RRRRRRRIM SSSSSSIMSIMIMRR ...... SSSSSIMRRRIMRRR SSSSSSIMRRSRRR RRIR SRRS SRRS RRRRRIMRS species determined by the Kirby–Bauer method RRRRRRRRRRSRRIMIMRSR RRRRIMIMRSRRSRRRSIMSS Sphingomonas ...... SSSSSSRRSSSRSRSRSSRSSSS SSSSSSRRSSSRSRSRSSRSSSS SSSSSSSSSSSSSSSSSSSSSSIM RRRSMMR SIMRRRRRRRR RRRRRRRSIMIMSRS SSSSSSIMRRSSSRSRSRIMRSSSSS SSSSSSIMRRRIMIMRSRSRRRIMRRSS SSSSSSIMRRIMSSSSSSRSSSIMSSS SSSSSSSSSSSSSSIMSRSSSSSSS SSSSSSSSSSRSSSSSSSSSSSSS SSSSSSSSSRSSSSSSIMSSRIMSSR RRRRRRRRSRRRRSRRSRRRRRRR 4939128813211699416516512141619912913 SSSSIMIMSSRSRIMSSRSRSIMSSRSSSS trimethoprim, discs contained a single agent. \ 2345678910111213141516171819202122232425262728 SSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSRSSRSSSS SSSSSSRIMRSRSSIMIMIMSRIM SSSSSSSSSSSRSSSRSRSSIMSIMSSSS SSSSSSSSSIMRSSSSSSSSSIMSSSSSS SSSSSSSSSSSSSRSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSRRRRRRRSSSSSSSIMIMRIMRIMSR SSSSSRRRRRRSSIMSSSSSSIMSRRIMSR SSSSSIMRSRRRSSIMSSSSSSSSSRSSR SSSSSRIMIMSRRSSIMSSSSSSSSSIMSRR RR ISRSS SRRRRRRRRSIMSS RRRRS S SIMSSRRS RRRRSRRSSRRRSRSRSRRRRRRRRS S SRSSRSIMRSRRRSRRSSSSSIMRSRSRIMR IM 28 25 27 22 16 33 27 24 28 28 23 15 20 27 27 32 20 31 20 31 24 22 20 17 27 24 27 23 j trimethoprim 0 SSSIMSSSSSSSSIMSSSSSSSSSSRSSSS 10) \ \ Antimicrobial susceptibility testing of 28 type strains of 25) CVC (20 n CVC and sulfamethoxazole \ 1 \ \ 8 n Number of agents resistedPercentage resistedNumber scored as S 8 11 22 9 31 1 25 39 25 8 25 33 22 22 36 58 44 25 25 11 44 14 44 14 33 39 44 53 25 33 25 36 (23 Antimicrobial agent (mg/disc)AMPC 1 Penicillin G (10)Ampicillin (10)Piperacillin (100)Cefoperazon (75) S S S R S R S R S R S R R R R S R S S R SDoxycycline (10) S SMinocycline (30)Polymyxin B S (300) IM S S S R S S R S R R S S S S S S R R R R R R S S S R R R S R S S Amoxicillin (25) S R S R R Moxalactam (30)Cefaclor (30)Ceftazidime (30)Flomoxef (30)Cefazolin (30)Cefmetazole (30)Cefotaxime (30) RMeropenem (10)Aztreonam (30) R S SImipenem (10)Carumonam (30) R S S R Panipenem (10) R SGentamicin (10) S S S Amikacin S (30) S SDibekacin (30) R RClarithromycin R R (15) R SRoxithromycin (15) S S R R IMErythromycin (15) STetracycline S S (30) R R S R S S R R S S R S R S RNorfloxacin S IM IM (10) S S R Trimethoprim R (5) S S S RCiprofloxacin R IM (5) S SOfloxacin S (5) S IM RTosufloxacin S R (5) R S IMLevofloxacin (5) S R RSparfloxacin S S (5) S S SSulfamethoxazole IM R R S S IM S S IMSummary: S R S R S IM IM S IM S R S S IM S S R S S S S IM IM R S IM IM R S S S S R R IM IM R IM IM S IM IM S R R R IM R R R IM R R R IM R S R R S R IM R IM S R R R R Table 6...... See Table 2 for identities of strains. Abbreviations: AMPC, amoxicillin; CVC, clavulanic acid; S, susceptible; IM, intermediate; R, resistant. Exc AMPC

1494 International Journal of Systematic and Evolutionary Microbiology 52 Delineation of the genus Sphingomonas

Sphingomonas ursincola, Sphingomonas natatoria, species with optimal growth temperatures of 26 or Sphingomonas roseiflava, Sphingomonas pruni and 28 mC are unlikely to be human pathogens, although Sphingomonas asaccharolytica are non-motile, since organisms in the natural environment that are resistant they do not show spreading growth on 0n3% semi-solid to various kinds of antimicrobials are potential human agar plates. Strictly aerobic. Metabolism is respiratory, pathogens. Strains of certain species metabolize but not fermentative. Cells of Sphingomonas ursincola dioxins (Wittich et al., 1992; Nohynek et al., 1995, and Sphingomonas natatoria contain bacteriochloro- 1996) and other chlorophenol derivatives (Wittich et phyll a and these species are facultative photo- al., 1992). The GjC content of DNA ranges from 59 organotrophs. Colony colour varies from species to to 67 mol% (HPLC). The type species is Sphingomonas species, for instance, deep-yellow, orange, lemon- paucimobilis (Holmes et al. 1977) Yabuuchi et al. 1990. yellow or non-pigmented. The deep-yellow pigment of Sphingomonas paucimobilis is reported to be a carotenoid, nostoxanthin, rather than a xanthomonadin. ACKNOWLEDGEMENTS This pigment was also found in Sphingomonas [Pseudo- This work was supported by grants from Ministry of Health, monas] echinoides (Denner et al., 1999) and Sphingo- Labour and Welfare (Research on Environmental Health, monas sanguinis [Pseudomonas paucimobilis] (Jenkins H11-Seikatsu-015), Research on Emerging and Re-emerging & Starr, 1985; Starr et al., 1977). In certain cases, Infectious Diseases (Health Sciences Research Grants), pigmentation differs depending on the medium used. Ministry of the Environment (Global Environment Re- Colonies of some non-pigmented strains such as search Fund), Ministry of Education, Culture, Sports, Sphingomonas yanoikuyae become lemon-yellow after Science and Technology and The United States–Japan Cooperative Medical Science Program against Tuberculosis more than 3 days incubation at room temperature. The and Leprosy. type strain of Sphingomonas herbicidovorans produces alcapton via accumulation of homogentisic acid, as observed in pyomelanogenic strains of Pseudomonas REFERENCES aeruginosa (Gessard, 1891; Mann, 1979; Yabuuchi & Balkwill, D. L., Drake, G. R., Reeves, R. H. & 7 other authors (1997). Ohyama, 1972). Taxonomic study of aromatic-degrading bacteria from deep-terrestrial- subsurface sediments and description of Sphingomonas aromaticivorans Catalase is produced. Oxidase-positive with a few sp. nov., Sphingomonas subterranea sp. nov., and Sphingomonas stygia exceptions. The type strain of Sphingomonas herbicido- sp. nov. 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