Spirosoma endophyticum sp. nov., isolated from Zn- and Cd-accumulating Salix caprea

A Gram-reaction-negative, yellow-pigmented strain, designated EX36T, was characterized using a polyphasic approach comprising phylogenetic, morphological and genotypic analyses. The endophytic strain was isolated from Zn/Cd-accumulating Salix caprea in Arnoldstein, Austria. Analysis of the 16S rRNA gene demonstrated that the novel strain is most closely related to members of the genus Spirosoma (95 % sequence similarity with Spirosoma linguale). The genomic DNA G+C content was 47.2 mol%. The predominant quinone was and the major cellular fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), C16 : 1ω5c, iso-C17 : 0 3-OH and iso-C15 : 0. On the basis of its phenotypic and genotypic properties, strain EX36T should be classified as a novel species of the genus Spirosoma, for which the name Spirosoma endophyticum sp. nov. is proposed. The type strain is EX36T ( = DSM 26130T = LMG 27272T).

The genus Spirosoma was first proposed by Larkin & Borrall (1984) and belongs to the family Flexibacteraceae in the phylum Bacteroidetes. At the time of writing the genus Spirosoma includes five species, the type species Spirosoma linguale (Larkin & Borrall, 1984), Spirosoma rigui (Baik et al., 2007), Spirosoma panaciterrae (Ten et al., 2009), Spirosoma spitsbergense and Spirosoma luteum (Finster et al., 2009). So far, Spirosoma strains have been isolated from various habitats, such as fresh water, permafrost soil or soil from a ginseng field. Strain EX36 T , which is proposed in this study to represent a novel species, was isolated in course of the analysis of bacteria associated with the heavy metal accumulating plant Salix caprea (Kuffner et al., 2010).
For the isolation of strain EX36 T , Salix caprea trees growing on a former Zn/Pb mining and processing site in Arnoldstein (Austria) were sampled (Kuffner et al., 2010). Xylem sap extract was directly plated on 10 % tryptic soy agar (TSA, Merck Darmstadt, Germany) and after 1 week of incubation single colonies were picked and streaked on phosphate-poor MOPS medium (Neidhardt et al., 1974) containing 0.1 % glucose and 1 mM ZnSO 4 . The strain was routinely cultured on 10 % TSA. For maintenance, the cell material was suspended in 10 % tryptic soy broth (TSB, Merck, Darmstadt, Germany) containing 15 % glycerol and stored at 280 u C. Endophytic colonization was confirmed by inoculating two maize and two potato cultivars, growing the plants under in vitro conditions and reisolating the strain from root and stem tissues.
For the extraction of bacterial DNA the Gen Elute Bacterial Genomic DNA kit (Sigma-Aldrich) was used. The 16S rRNA gene was amplified by PCR using the primers 8f (59-AGAGTTTGATCCTGGCTCAG-39) (Weisburg et al., 1991) and 1520r (59-AAGGAGGTGATCCAGCCGCA-39) (Edwards et al., 1989). Sequencing of the amplified PCR product was performed by LGC Genomics (Berlin, Germany). The obtained partial sequences were assembled using the programs BioEdit (Hall, 1999) and SEQMAN PRO (DNAstar). The consensus sequence was subjected to nucleotide BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to search the database of the National Center for Biotechnology Information (NCBI) for the closest relatives of the bacterial strains with validly published names. Sequence comparisons indicated that the isolate belonged to the family Flexibacteraceae.
Nearly complete 16S rRNA gene sequences of strain EX36 T and of all species of the genus Spirosoma with validly published names and of selected species of the family Cytophagaceae, which were downloaded from the NCBI GenBank sequence database, were imported into the ARB program package (Ludwig et al., 2004). Sequences were aligned into the SILVA SSURef 102 (Pruesse et al., 2007) database by using the option 'autosearch by PT_server' of the ARB editor. Alignments were manually corrected using the ARB editor. A maximum-likelihood phylogenetic tree was reconstructed using RAxML v. 7.4.2 (Stamatakis, 2006a) (Yang, 1994) and the CAT model (Stamatakis, 2006b), which was implemented in the rapid bootstrapping algorithm, (Stamatakis et al., 2008) was performed with 100 replicates and using general time reversible (GTR) as the substitution matrix. In Fig. 1 the position of EX36 T in the distinct cluster of the genus Spirosoma can be clearly recognized. The calculation of pairwise sequence similarity using a global alignment algorithm (Myers & Miller, 1988), which was implemented at the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012) showed highest sequence similarity values for strain EX36 T to Spirosoma linguale DSM 74 T (95.7 %), followed by S. luteum SPM-10 T (93.9 %), S. spitsbergense SPM-9 T (93.9 %), S. rigui KCTC 12531 T (93.8 %) and S. panaciterrae Gsoil 1519 T (92.5 %).
Growth of strain EX36 T was tested at various temperatures (4, 20, 23, 28, 37 and 41 u C) on 10 % TSA plates for up to 1 week. The pH range for growth (pH 4, 5, 6, 7, 8 and 9) was determined by measuring OD 600 changes in cultures incubated at 28 u C with shaking at 190 r.p.m. compared with an uninoculated control. Salt tolerance was determined by amending 10 % TSB with NaCl to final concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0 and 10.0 % NaCl (w/v). The Gram reaction of strain EX36 T was determined by using the non-staining method described by Buck (1982). Pigment analysis of cells grown on 10 % TSA was performed in triplicates by extraction with acetone according to the method described by Denner et al. (2001) using a U-2900 spectrophotometer (Hitachi). Minimal inhibition concentrations (MIC) for Zn and Cd were determined according to the method of Kuffner et al. (2008). Additionally cells were tested for flexirubin pigments using the method described by Bernardet et al. (2002). Oxidase and catalase activity were tested as outlined by Smibert & Krieg (1994). Additional biochemical tests were performed by the Identification Service of the DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) using API 20NE (bioMérieux) and GENIII plates (Biolog). Cell morphology after 4 days of growth at 28 u C was investigated using fluorescence and bright-field microscopy (IX81, Olympus; Axiovert 200 M, Zeiss). Antibiotic susceptibility was determined by the disc diffusion method on 10 % TSA plates.
Cells of strain EX36 T were rod-shaped, Gram-reactionnegative and 1.262217.5 mm in size (Fig S1, available in Spirosoma endophytica sp. nov. Table 1. Differential characteristics of strain EX36 T and recognized species of the genus Spirosoma Strains: 1, EX36 T (data from this study); 2, S. linguale DSM 74 T (Larkin & Borrall, 1984; and this study); 3, S. luteum DSM 19990 T (Finster et al., 2009); 4, S. spitsbergense DSM 19989 T (Finster et al., 2009); 5, S. rigui KCTC 12531 T (Baik et al., 2007); 6, S. panaciterrae DSM 21099 T (Ten et al., 2009). All strains are catalase-positive, Gram-reaction-negative and negative for nitrate reduction, utilization of gluconate, caprate, adipate and glycerol. +, Positive; 2, negative; W, weakly positive; ND, not determined; R, resistant; S, susceptible. IJSEM Online). Most cells were arranged in pairs, but filaments up to 55 mm were observed. EX36 T showed yellowish, opaque, semi-translucent colonies with a smooth and shiny surface and a circular and convex shape. The diameter of colonies grown on 10 % TSA at 28 uC for 1 week varied between 1.5 and 3.0 mm. The strain was positive for catalase and oxidase activity; detailed results of biochemical and physiological analyses are listed in Table 1 and in the species description. In contrast to other species of the genus Spirosoma, cells of EX36 T showed a length up to 17.5 mm, did not grow at 5 and 42 u C, did not tolerate NaCl concentrations higher than 0.6 % (w/v), had the lowest genomic G+C content and showed differences in antibiotic susceptibility. Low tolerance of Cd and Zn was observed (slow growth at 4 mM Zn and 1 mM Cd). The analysis of yellow pigments showed three absorption maxima at 428, 453 and 483 nm. EX36 T was negative for flexirubin-type pigments.
Analyses of cellular fatty acid composition, respiratory quinones, polar lipids and chromosomal G+C content were performed by the Identification Service of the DSMZ. The fatty acid profile was determined according to the protocol of the Microbial Identification System (MIDI). The major fatty acids of strain EX36 T were summed feature 3 (iso-C 15 : 0 2-OH and/or C 16 : 1 v7c; 49.3 %), C 16 : 1 v5c (23.8 %), iso-C 17 : 0 3-OH (6.2 %) and iso-C 15 : 0 (5.4 %). A detailed overview of the cellular fatty acid profiles of all species of the genus Spirosoma can be found in Table 2. Differences between the fatty acid profile of EX36 T and other species of the genus Spirosoma were found in the amounts of iso-C 15 : 0 , C 16 : 1 v5c and summed feature 3. In contrast to S. linguale DSM 74 T , the fatty acids C 15 : 0 and anteiso-C 15 : 0 were not detected.
The predominant menaquinone, in accordance with all other species of the genus Spirosoma, was MK-7. As polar lipids, phosphatidylethanolamine, two aminophospholipids, two aminolipids, a glycolipid and three unknown lipids were detected on the TLC plate. The DNA G+C content of strain EX36 T was 47.2 mol%, which is lower than reported values for all other species of the genus Spirosoma with validly published names.
The analysis of DNA2DNA similarity of strain EX36 T with its nearest phylogenetic neighbour S. linguale DSM 74 T was also carried out by the Identification Service of the DSMZ. The experiment was performed in duplicates. DNA2DNA hybridization showed a DNA2DNA similarity of 12.2 % (second measurement: 17.2 %), demonstrating that these two strains do not represent the same species.
The present data regarding 16S rRNA gene sequence analysis, physiological, chemotaxonomic and morphological properties indicates, that strain EX36 T represents a distinct species in the genus Spirosoma, for which the name Spirosoma endophyticum sp. nov. is proposed.
Spirosom endophyticum (en.do.phy9ti.cum. Gr. Pref. endo within; Gr. n. phyton plant; L. neut. suff. -icum adjectival suffix used with the sense of belonging to; N.L. neut. adj. endophyticum within plant, referring to the endophytic nature of the strain and its isolation from plant tissue).
The type strain, EX36 T (5DSM 26130 T 5LMG 27272 T ), was isolated from Zn/Cd-accumulating Salix caprea in Arnoldstein, Austria. The DNA G+C content of the type strain is 47.2 mol%.