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Home , Comamonadaceae, Comamonas, Delftia, Delftia acidovorans, Delftia lacustris, Delftia tsuruhatensis

TAXONOMIC DESCRIPTION Carro et al., Int J Syst Evol Microbiol 2017;67:1957–1960 DOI 10.1099/ijsem.0.001892

Delftia rhizosphaerae sp. nov. isolated from the rhizosphere of Cistus ladanifer

Lorena Carro,1† Rebeca Mulas,2 Raquel Pastor-Bueis,2 Daniel Blanco,3 Arsenio Terrón,4 Fernando Gonzalez-Andr es, 2 Alvaro Peix5,6 and Encarna Velazquez 1,6,*

Abstract A bacterial strain, designated RA6T, was isolated from the rhizosphere of Cistus ladanifer. Phylogenetic analyses based on 16S rRNA gene sequence placed the isolate into the genus within a cluster encompassing the type strains of , , and Delftia litopenaei, which presented greater than 97 % sequence similarity with respect to strain RA6T. DNA–DNA hybridization studies showed average relatedness ranging from of 11 to 18 % between these species of the genus Delftia and strain RA6T. and oxidase were positive. Casein was hydrolysed but gelatin and starch were not. Ubiquinone 8 was the major respiratory quinone detected in strain RA6T together with low amounts of ubiquinones 7

and 9. The major fatty acids were those from summed feature 3 (C16 : 1!7c/C16 : 1 !6c) and C16 : 0. The predominant polar were diphosphatidylglycerol, phosphatidylglycerol and . Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain RA6T should be considered as a representative of a novel species of genus Delftia, for which the name Delftia rhizosphaerae sp. nov. is proposed. The type strain is RA6T (=LMG 29737T= CECT 9171T).

The genus Delftia comprises Gram-stain-negative, non- The strain was grown on (NA; Sigma) for 48 h  sporulating, strictly aerobic rods, motile by polar or bipolar at 22 C to check for motility by phase-contrast microscopy flagella. Ubiquinone 8 (Q-8) is the main quinone, and minor using the hanging-drop method [8]. Gram staining and quinones are Q-7 and Q-9. The major fatty acids are hexade- staining of poly-b-hydroxybutyrate granules were carried out by the procedures described by Doetsch [8]. The flagel- canoic acid (C16 : 0), hexadecenoic acid (C16 : 1) and octadece- lation type was determined by electron microscopy after noic acid (C18 : 1), and 3-hydroxy fatty acids (C10 : 0 3-OH and  48 h of incubation of strain RA6T on NA at 22 C. The cells C8 : 0 3-OH) are present [1, 2]. The species of this genus have been isolated from soil [1], activated sludge [3] and fresh were gently suspended in sterile water and then stained with 2 % uranyl acetate and examined at 80 kV with a Tecnai water [4, 5], and some of them have been found in tissues of Spirit Twin transmission electron microscope. Strain RA6T plants [6, 7]. was Gram-stain-negative and motile by means of a polar In this work we characterized a strain, named RA6T, iso- flagellum (Fig. S1, available in the online Supplementary lated from rhizospheric soil of Cistus ladanifer and, based Material). on its genotypic, chemotaxonomic and phenotypic charac- Amplification and sequencing of the 16S rRNA gene were teristics, we propose its classification into a novel species performed according to the method of Rivas et al. [9]. The with the name Delftia rhizosphaerae sp. nov. sequence obtained was compared with those from the Gen- T Strain RA6 was isolated from rhizospheric soil of Cistus Bank database using the BLASTN [10] and EzTaxon-e server ladanifer plants growing in León (Spain) on TSA plates [11] programs. Sequences were aligned using the CLUSTAL X  (Sigma) incubated at 28 C for 48 h. The colonies of strain software [12], and distances were calculated according to RA6T were white–cream, round, smooth and convex with Kimura’s two-parameter model [13]. The phylogenetic trees approximate diameters of 1–3 mm. were inferred using the neighbour-joining and maximum-

Author affiliations: 1Departamento de Microbiología y Genetica and Instituto Hispanoluso de Investigaciones Agrarias (CIALE), Universidad de Salamanca, Salamanca, Spain; 2Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de León, León, Spain; 3Bioenergía y Desarrollo Tecnológico, S.L. (BYDT), León, Spain; 4Departamento de Biodiversidad y Gestión Ambiental, Universidad de León, León, Spain; 5Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de Investigaciones Científicas, IRNASA-CSIC, Salamanca, Spain; 6Unidad Asociada Grupo de Interacción Planta-Microorganismo Universidad de Salamanca-IRNASA-CSIC, Salamanca, Spain. *Correspondence: Encarna Velazquez, [email protected] Keywords: Delftia; Cistus ladanifer; rhizosphere. †Present address: School of Biology, Newcastle University, Newcastle upon Tyne, UK. The GenBank/EMBL/DDBJ accession number for 16S rRNA gene sequence of strain RA6T is KY075818. Two supplementary figures are available with the online Supplementary Material.

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likelihood models [14, 15]. The MEGA5 package [16] was DNA–DNA hybridization was performed by using the used for all analyses. The comparison of the 16S rRNA gene method of Ezaki et al. [20], following the recommendations sequence of strain RA6T (1487 nucleotides) against those of of Willems et al. [21]. DNA relatedness values between type strains held in the EzTaxon-e database indicated that strain RA6T and the type strains of species of the genus Delf- this strain belongs to the genus Delftia. Type strains of the tia with validly published names, D. acidovorans DSM 39T, remaining species of this genus, Delftia lacustris 332T, Delf- D. lacustris DSM 21246T, D. litopenaei DSM 27241T and D. T tia tsuruhatensis T7T, Delftia acidovorans IAM 12409T, Delf- tsuruhatensis DSM 17581 , were 13 % (±1 %), 13 % (±2 %), tia litopenaei wsw-7T and Delftia deserti YIM Y792T, 11 % (±1 %) and 18 (±1 %), respectively, confirming that the showed 98.7, 98.6, 98.5, 97.2% and 92.9 % 16S rRNA gene new isolate represents a novel species of the genus Delftia sequence similarity, respectively. The results of neighbour- according to the current species concept [22]. joining and maximum-likelihood analyses of the 16S rRNA The cellular fatty acids were analysed by using the Microbial gene sequences are shown in Fig. 1. They showed that, in Identification System (Microbial ID, MIDI) Sherlock 6.1 agreement with the 16S rRNA gene sequence similarity val- and the library RTSBA6 according to the technical instruc- T ues, strain RA6 grouped with the type species of the genus tions provided by this system [23]. Strains D. acidovorans Delftia, D. acidovorans, and that it therefore belongs to this DSM 39T, D. lacustris DSM 21246T, D. litopenaei DSM genus. The species D. deserti [17] formed a lineage phyloge- 27241T and D. tsuruhatensis DSM 17581T were included as netically divergent to the remaining species of the genus references. The strains were grown on TSA plates (Becton  Delftia and had 93.9 % 16S rRNA gene sequence similarity Dikinson, BBL) for 48 h at 28 C. Polar lipids were extracted, with respect to D. acidovorans, suggesting that D. deserti separated by two-dimensional TLC and identified according does not belong to the genus Delftia. to the method of Minnikin et al. [24] as modified by For DNA base composition analysis, DNA was prepared Kroppenstedt and Goodfellow [25]. The respiratory qui- nones, for extraction of which strain RA6T was cultivated according to the method of Chun and Goodfellow [18].  in TSB (Becton Dickinson, BBL) for 48 h at 28 C and The G+C content (mol%) of DNA was determined using 180 r.p.m. were analysed as described by Tindall [26]. Ubi- the thermal denaturation method [19]. The DNA G+C T quinone 8 was the major respiratory quinone (74 %) content of strain RA6 was 66.4 mol%. This value is simi- detected in strain RA6T together with lower amounts of lar to those of the most closely related species from the ubiquinones 7 (20 %) and 9 (6 %). The major fatty acids – genus Delftia, D. acidovorans (67 69 mol%), D. lacustris were those from summed in feature 3 (C16 : 1!7c/C16 : 1!6c) (65.3 mol%), D. tsuruhatensis (66.2 mol%) and D. litopenaei (44.3 %) and C16 : 0 (30.3 %). The profile of strain (67.6 mol%) [1, 3–5]. RA6Twas consistent with those found in the remaining

100 valerianellae DSM 16619T (KF931150) 66 CCUG 2113 T (AF078765) Simplicispira psychrophila DSM 11588T (JHYS01000032) azovorans KF46FT (NR_025114) 0.02 81 100 Xenophilus aerolatus 5516S-2T (EF660342) ‘ ruginosibacter ’ BN30 (KR051030) 51 100 Diaphorobacter aerolatus 8604S-37T (KC352658) composti YY287T (EF015884) 94 Comamonas koreensis KCTC 12005 T (AF275377) 100 Comamonas piscis CN1 T (KM263565) Delftia rhizosphaerae RA6T (KY075818) Delftia litopenaei wsw-7T (GU721027) T 56 Delftia acidovorans IAM 12409 (AB021417) 94 Delftia lacustris 332T (EU888308) 91 Delftia tsuruhatensis T7 T (AB075017) Delftia deserti YIM Y792T (KP300804) Caulobacter ginsengisoli Gsoil 317T (AB271055)

Fig. 1. Neighbour-joining based on nearly complete 16S rRNA gene sequences (1487 nucleotides) of Delftia rhizo- phaerae sp. nov. RA6T and closely related species of the genus Delftia. Caulobacter ginsengisoli Gsoil 317T was used as an outgroup. The significance of each branch is indicated by a bootstrap value calculated as a percentage for 1000 subsets (only values over 50 % are shown). Nodes marked with filled circles were also obtained with the maximum-likelihood algorithm. Bar, 0.02 substitutions per nucleotide position.

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Table 1. Cellular fatty acid composition of strain RA6T and related type description below, and the differences from the related species strains are given in Table 2. Strain RA6T differed from the most Strains: 1, D. rhizosphaerae sp. nov. RA6T; 2, D. lacustris DSM 21246T; closely related species in its growth at different temperatures, 3, D. tsuruhatensis DSM 17581T; 4, D. acidovorans DSM 39T; 5, D. litope- growth in presence of 5 % NaCl, the production of gelatinase, T naei DSM 27241 . Fatty acids present in all species in amounts lower caseinase, and b-glucosidase, and the assimilation of than 1 % are not shown. ND, Not detected. Data are from this study. some carbon and energy sources such as , caprate Fatty acid 1 2 3 4 5 and phenylacetate. These phenotypic differences support the assignment of strain RA6T to a novel species of the genus C12 : 0 2.6 2.4 2.4 2.8 2.6 Delftia. C14 : 0 1.1 0.7 0.7 0.9 0.8

C16 : 0 30.3 34.2 33.3 29.9 29.1 Collectively, the results of phylogenetic, chemotaxonomic and phenotypic analysis showed that strain RA6T represents C17 : 0 1.5 0.1 2.0 0.3 0.23 a novel species of the genus Delftia for which the name Delf- C10 : 0 3-OH 2.7 2.6 2.7 2.9 2.9 tia rhizosphaerae sp. nov. is proposed. C17 : 0 cyclo ND 3.4 3.6 4.4 0.6 Summed feature 3* 44.3 40.1 39.7 41.3 44.9 Summed feature 8† 13.6 14.6 12.6 16.1 18.1 DESCRIPTION OF DELFTIA RHIZOSPHAERAE SP. NOV. *Summed feature 3: C16 : 1!7c/C16 : 1!6c.

†Summed feature 8: C18 : 1!7c/C18 : 1!6c. Delftia rhizosphaerae (rhi.zo.sphae¢rae. Gr. n. rhiza a root; L. n. sphaera ball, sphere; N.L. fem. n. rhizosphaera the rhi- zosphere; N.L. gen. n. rhizosphaerae of the rhizosphere). species of the genus Delftia (Table 1). Strain RA6T displayed Cells are aerobic, Gram-stain-negative rods (width 0.8– a profile (Fig. S2) consisting of diphosphatidylglycerol, 1.0 µm, length 2.1–2.5 µm), motile by a polar flagellum. Poly- phosphatidylglycerol, phosphatidylethanolamine and three b-hydroxybutyrate accumulation is observed. Catalase- and unidentified phospholipids in low amounts. This profile is oxidase-positive. Colonies on nutrient agar medium are similar to those found in the remaining species of the genus white–cream-coloured. Grows from pH 6 to pH 10 (optimal Delftia, which have the same major polar lipids as those pH is 7). It can grow in the presence of 1 % NaCl. It grows at   found in strain RA6T [4]. Some differences were found in temperatures from 4 to 30 C (optimal temperature is 28 C). minor polar lipids since in strain RA6T, aminophospholi- pids were not detected and three unidentified phospholipids were found, whereas two unidentified phospholipids were Table 2. Differential phenotypic characteristics of strain RA6T and found in the species D. lacustris and D. tsuruhatensis, and related type strains three with different distribution in D. acidovorans and four Strains: 1, D. rhizosphaerae sp. nov. RA6T; 2, D. lacustris DSM 21246T; in D. litopenaei were identified [4]. 3, D. tsuruhatensis DSM 17581T; 4, D. acidovorans DSM 39T; 5, D. litope- T naei DSM 27241 . +, Positive reaction; À, negative reaction; W, weakly The phenotypic characterization was performed by using API positive reaction. Data are from this study. 20NE, API 32GN and API ZYM systems (bioMerieux) according to the manufacturer’s instructions. The strains Characteristic 1 2 3 4 5 examined in this study were grown on TSA plates (Difco, Bec- Growth at:  ton Dickinson, BBL) for 24 h for analysis of catalase and oxi- 37 C À + + + +  dase production. Catalase production was assayed by using 45 C À + ÀÀÀ 0.3 % hydrogen peroxide with one colony taken from TSA Growth in presence of: ¢ ¢ plates. Oxidase activity was detected by using N,N,N ,N -tetra- 5 % NaCl À + À + À methyl-1,4-phenylenediamine dihydrochloride. Abilities to  Production of: grow at different temperatures (from 4 to 50 C) were deter- Gelatinase À + + À W* mined on TSA plates (Difco, BBL). The ability to grow at dif- Caseinase + +† + + À ferent pH (from pH 4.5 to 8) was determined on TSA (Difco, Urease ÀÀ + + À BBL). PCA buffer (0.4 M Na HPO and 0.2 M citric acid) was 2 4 b-Glucosidase À + ÀÀÀ used to adjust the pH from 4.5 to 5.5, 0.2 M phosphate buffer to adjust the pH from 6.5 to 7.5 and 0.2 M TE buffer to adjust Assimilation of: À‡ À the pH to 8. The ability to grow in the presence of NaCl was Mannitol + + + § ÀÀ analysed on TSA (Difco, Becton Dickinson, BBL) supple- Caprate + + + À mented with NaCl to obtain final concentrations ranging Phenylacetate + + + + from 0.5 to 8 %. Caseinase and amylase were analysed as *This result was reported as negative by Chen et al. [4]. described by Claus and Berkeley [27]. The strains D. lacustris †This result was reported as weakly positive by Jørgensen et al. [5]. T T DSM 21246 , D. tsuruhatensis DSM 17581 , D. acidovorans ‡This result was reported as positive by Shigematsu et al. [3]. T T DSM 39 and D. litopenaei DSM 27241 were included in the §This result was reported as positive by Chen et al. [4]. phenotypic study as references. Results are given in the species

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