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Exiguobacterium Undae Sp. Nov. and Exiguobacterium Antarcticum Sp. Nov International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1171–1176 DOI: 10.1099/ijs.0.02185-0 Exiguobacterium undae sp. nov. and NOTE Exiguobacterium antarcticum sp. nov. DSMZ–Deutsche Sammlung Anja Fru$ hling, Peter Schumann, Hans Hippe, Bettina Stra$ ubler von Mikroorganismen und Zellkulturen GmbH, and Erko Stackebrandt Mascheroder Weg 1b, D-38124 Braunschweig, Germany Author for correspondence: Erko Stackebrandt. Tel: j49 531 2616352. Fax: j49 531 2616418. e-mail: Erko!DSMZ.de Four orange-pigmented strains from pond water (L1–L4) have been subjected to polyphasic taxonomic analyses. On the basis of ribotype analysis and Fourier-transform infrared spectroscopy, these strains form a genomically highly related group. 16S rDNA sequence analysis revealed 988% similarity between the 16S rDNA sequences of strains L2T and H2T, isolated previously from a microbial mat from Lake Fryxell, Antarctica. DNA–DNA reassociation values indicated the presence of two genomic clusters. While the DNA of strains L2T and L3 showed 100% DNA relatedness, strains L2T and H2T shared only 51% DNA relatedness. These two clusters differed in some phenotypic properties, e.g. utilization of melibiose, D-mannitol, adenosine 5'- monophosphate and uridine 5'-monophosphate, and in their fatty acid compositions. Based on the composition of isoprenoid quinones, peptidoglycan, polar lipids and fatty acids, these organisms are members of the genus Exiguobacterium. This is supported by 16S rDNA analyses, which revealed 97–98% similarity to Exiguobacterium acetylicum DSM 20416T and 932–938% similarity to Exiguobacterium aurantiacum DSM 6208T. E. acetylicum DSM 20416T, the closest phylogenetic neighbour, shows only 39% DNA similarity to strain L2T and 40% DNA similarity to strain H2T. Based on genomic distinctiveness and the clear differences in chemotaxonomy and physiology, two novel species are proposed, Exiguobacterium undae sp. nov. and Exiguobacterium antarcticum sp. nov. Keywords: Exiguobacterium undae sp. nov., Exiguobacterium antarcticum sp. nov., riboprinting, FT-IR, polyphasic taxonomy In a study applying modern, polyphasic approaches in were maintained in glycerol at k20 mC and preserved microbial taxonomy, surface water samples were taken by lyophilization. from a garden pond in Wolfenbuttel, Lower Saxony, $ Analysis of the 16S rDNA sequence of strain L2T and Germany, in June 2001 and streaked onto GS medium. phylogenetic comparison were done as described This medium (DSMZ catalogue no. 851; DSMZ, 1998) " previously (Rainey et al., 1996). The sequence of strain contained (l− )015 g glucose, 1 g yeast extract, 0 5g n n L2T is most similar (98 8% similarity) to that of the (NH ) SO ,01 g CaCO ,01 g Ca(NO ) ,005 g KCl, n % # % n $ n $ # n orange-pigmented strain H2T, originating from an 0 05 g K PO ,005 g MgSO .7H O, 0 20 g Na S.9H O n # % n % # n # # anaerobic enrichment of a microbial mat isolated from and 10 ml of a vitamin cocktail. Orange-pigmented Lake Fryxell, Antarctica (Brambilla et al., 2001). The colonies, developing within 48 h at room temperature, sequences of strains L2T and H2T were distantly related were isolated and purified on tryptone soy agar (Difco) to those of Exiguobacterium acetylicum DSM 20416T and four strains were designated strains L1–L4. Strains (97n2 and 97n9%, respectively) and Exiguobacterium T aurantiacum DSM 6208 (93n3 and 93n8%, respect- T T ................................................................................................................................................. ively). Strains L2 and H2 share 97n7% sequence Abbreviation: FT-IR, Fourier-transform infrared. similarity (Fig. 1a). The EMBL accession numbers for the 16S rDNA sequences of strains L2T (l DSM 14481T) and H2T (l DSM 14480T) are respectively AJ344151 and The genomic homogeneity of the pond isolates and AJ297437. relatives was analysed by riboprinting, using the 02185 # 2002 IUMS Printed in Great Britain 1171 A. Fru$ hling and others T T (a) Pond strain L2T (AJ344151) L3 showed 100% relatedness to strain L2 , strain H2 92 T Lake Fryxell strain H2T (AJ297437) was more distantly related to strain L2 (51%). The 100 heterologous DNA reassociation values of strains L2T T T 100 E. acetylicum DSM 20416 (X70313) and H2 were only 39 and 40% when compared with T E. acetylicum IFO 12146T (D55730) E. acetylicum DSM 20416 . 100 E. aurantiacum DSM 6208T (X70316) Fourier-transform infrared (FT-IR) spectroscopy E. aurantiacum Z8 (AF275717) (Tindall et al., 2000) was performed in order to verify 2% the phenotypic similarity of the isolates. The data clearly support the notion that the four isolates L1–L4 T (b) Pond strain L4 form a coherent cluster, while strains H2 and the type Pond strain L1 strains of E. aurantiacum and E. acetylicum branch Pond strain L2T more distantly (Fig. 1b). Pond strain L3 Lake Fryxell strain H2T In order to determine whether the new isolates are E. aurantiacum DSM 6208T members of the genus Exiguobacterium, chemotaxo- E. acetylicum DSM 20416T nomic markers were determined. Compounds ana- lysed included cell wall amino acids (Schleifer & 12·0 9·0 6·0 3·0 0 Kandler, 1972), isoprenoid quinones (Collins et al., Euclidian distance 1977; Groth et al., 1996), polar lipids (Minnikin et al., (c) Pond strain L4 1979; Collins & Jones, 1980) and fatty acids (Miller & Pond strain L1 Berger, 1984). Membership of strains L2T and H2T of Pond strain L2T Pond strain L3 the genus Exiguobacterium was verified by the presence Lake Fryxell strain H2T of lysine as the diagnostic amino acid of the pepti- E. acetylicum DSM 20416T doglycan (Lys–Gly peptidoglycan type) (Farrow et al., E. aurantiacum DSM 6208T 1994) and MK-7, MK-8 and MK-6 as the principal quinones (76:15:2 for strain L2T, 71:20:2 for strain 60 45 30 15 0 T Spectral distance H2 ). Polar lipids are phosphatidylglycerol, diphos- phatidylglycerol, phosphatidylethanolamine, phos- ................................................................................................................................................. phatidylserine, phosphatidylinositol and an unidenti- Fig. 1. Dendrograms of relatedness among the novel isolates fied phospholipid. Phosphatidylserine and phosphati- and type strains of Exiguobacterium species. (a) Phylogeny of 16S rDNA as derived from distance-matrix analysis. Bar, 2% dylinositol were not reported in the species descriptions nucleotide deviation. Numbers at branching points are boot- of E. aurantiacum and E. acetylicum; however, strains strap percentages (1000 resamplings). (b) Sorting bacterial strains of the latter species contain phosphatidylserine (N. according to their FT-IR spectral distance. Cluster analysis was Weiss and P. Schumann, unpublished). All the isolates performed using the first derivatives of the spectra considering − − contained moderate amounts of iso (i)C"$:!, anteiso the spectral ranges 1200–900 cm 1 (3i), 900–700 cm 1 (1i), − 3000–2800 cm 1 (1i), scaling to first range, applying Ward’s (ai)C"$:!,C"':! and C"':"ω""c fatty acids (Table 1), but some differences were observed between strains L1–L4 algorithm (see Helm et al., 1991). (c) Fatty acid relationships. T The dendrogram was generated by treating the Euclidian dis- and strain H2 . The latter strain contains significantly tances of the fatty acids with the unweighted pair group method larger amounts of C"':" (c and C"):" *c fatty acids, with arithmetic average algorithm. Numerical analyses were ω ω done using the standard MIS software (Microbial ID). Statistical lacks C"):"ω(c, aiC"(:! and iC"':! fatty acids and has a procedures are described by Eerolen & Lehtonen (1988) and smaller amount of iC"(:!. E. aurantiacum shares more O’Donnell (1985). fatty acid similarity with the novel isolates than does E. acetylicum (Fig. 1c), but both type strains display significant differences: E. aurantiacum lacks iC"&:! and C"':"ω(c fatty acids, while E. acetylicum possesses automated RiboPrinter (DuPont), with EcoRI as the significant amounts of iC"&:! (Jones & Keddie, 1986) restriction enzyme. The typing profiles of strains and C"%:! fatty acids. A dendrogram of fatty acid L1–L4 were very similar, while those of the Exiguo- similarities is depicted in Fig. 1(c). bacterium strains and strain H2T were more dissimilar (Fig. 2). Cultural and physiological characteristics (Gordon, 1973; Gregersen, 1978) (API 50CHE Biolog GP DNA–DNA reassociation was performed to deter- Microplate; aminopeptidase test according to Merck mine the relatedness between strain L2T and strains L1 1.13301.0001) of the novel isolates (Tables 2 and 3) and L3, strain H2T and E. acetylicum DSM 20416T, also confirm the generic affiliation. The isolates show which appeared to be the closest relative as judged genus-specific characters such as rod-shaped mor- from 16S rDNA analysis. DNA was isolated as phology, motility, peritrichous flagellation and ab- described by Cashion et al. (1977) and Escara & sence of spore formation; they are facultatively an- Hutton (1980) and DNA–DNA reassociation, per- aerobic and catalase- and oxidase-positive. Discount- formed under optimal conditions (2iSSC at 68 mC) ing variable and weak reactions, the pond isolates (Huß et al., 1983; Jahnke, 1992), was recorded with a share the highest phenotypic similarity with strain H2T Gilford 2600 spectrophotometer. While strains L1 and (five differences), while the numbers of differences 1172 International Journal of Systematic and Evolutionary Microbiology 52 Novel Exiguobacterium species kb 1·00 2·00 3·00 4·00 6·00 8·00 15·00 30·00 60·00 Pond strain
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