Description of Devosia Neptuniae Sp. Nov. That Nodulates and Fixes Nitrogen in Symbiosis with Neptunia Natans, an Aquatic Legume from India

System. Appl. Microbiol. 26, 47–53 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam

Description of Devosia neptuniae sp. nov. that Nodulates and Fixes Nitrogen in Symbiosis with Neptunia natans, an Aquatic Legume from India

Raúl Rivas1, Anne Willems2, Nanjappa S. Subba-Rao3, Pedro F. Mateos1, Frank B. Dazzo3, Reiner M. Kroppenstedt4, Eustoquio Martínez-Molina1, Monique Gillis2, and Encarna Velázquez1

1 Departamento de Microbiología y Genética, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, Salamanca, Spain 2 Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, Gent, Belgium 3 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA 4 DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany

Received: December 6, 2002

Summary

Neptunia natans is a unique aquatic legume indigenous to tropical and sub-tropical regions and is nodu- lated symbiotically by rhizobia using an unusual infection process unlike any previously described. Previ- ously, isolates of neptunia-nodulating rhizobia from Senegal were characterized as Allorhizobium undicola. Here we report on a different group of neptunia-nodulating rhizobia isolated from India. Sequenc- ing of the 16S rDNA gene from two of these Indian isolates (strains J1T and J2) show that they belong in the genus Devosia rather than Allorhizobium. Currently, the only described Devosia species is D. riboflavina (family Hyphomicrobiaceae, order Rhizobiales). The complete 16S rDNA sequences of strains J1T and J2 are 95.9% homologous to the type strain, D.riboflavina LMG 2277T, suggesting that these neptunia-nodulating strains from India belong to a new Devosia species. This hypothesis was confirmed by further studies of polyphasic taxonomy (DNA-DNA hybridisation, TP-RAPD patterns, SDS-PAGE of cellular proteins, 16S rDNA RFLP patterns, carbon source utilisation, cellular fatty acid analysis and other phenotypic characterisations), all of which support the proposal that these neptunia-nodulating strains constitute a new Devosia species, which we name Devosia neptuniae sp. nov. These Gram negative, strictly aerobic short rods are motile by a subpolar flagellum, positive for catalase, oxidase, urease and β-galactosidase, can utilise several carbohydrates (but not organic acids) as carbon sources and contain C18:0 3-OH, cis-7 C18:1 11-methyl and cis-7 C18:1 as their major cellular fatty acids. Unlike D. riboflavina, the longer-chain C24:1 3-OH and C26:1 3-OH hydroxy fatty acids are not detected. The type strain of D. neptuniae is LMG 21357T (CECT 5650T). Assignment of this new taxon represents the fourth example in the literature of a non-rhizobial genus of bacteria capable of forming a bonafide dinitrogen-fixing root-nodule symbiosis with legume plants.

Key words: Taxonomy – Devosia – symbiosis – Neptunia – legume

Introduction

The taxonomy of the family Rhizobiaceae has been the last year has been the isolation and characterization of subject of important changes in recent years. Currently, non-rhizobial species capable of nodulating and symbiot- most of the species nodulating legumes are included in ically fixing dinitrogen in legume plants. Members of the several families within the order Rhizobiales. In the early genus Methylobacterium [19] in the α-Proteobacteria and years of Rhizobium taxonomy, species were, in general, the genera Ralstonia and Burkholderia in the β-pro- described from legumes of agronomic importance, but a teobacteria can establish this type of symbiosis with trend in recent years to investigate the symbionts of exot- legumes [3, 12]. ic plants has resulted in important contributions to rhizo- Studies of the developmental morphology of the infec- bial taxonomy. One of the most interesting findings of tion process leading to formation of nitrogen-fixing root

0723-2020/03/26/01-047 $ 15.00/0 48 R. Rivas et al. nodules in Neptunia natans revealed several unique as- Physiological and biochemical tests pects of this specialised aquatic plant-bacterium symbiosis Catalase production was assayed by using 0.3% hydrogen that distinguish it from other legume root-nodule sym- peroxide with one colony taken from YED agar plates. Oxidase ′ ′ bioses [18]. The neptunia-nodulating strains J1T and J2 activity was detected by using N,N,N ,N -tetramethyl-1,4- phenylenediamine dihydrochloride. Utilisation of different car- used in these studies were isolated in India and since they bon sources and enzyme production were performed using API were fast-growers in media containing mannitol as carbon 20NE strips (Biomérieux) according to manufacturer’s instruc- source, they were provisionally included in the genus Rhi- tions. zobium. Later, taxonomic studies of various other isolates To test for intrinsic antibiotic resistance, cultures were inocu- from nodules on Neptunia natans growing in Senegal in- lated into 5 mL of YMB basal medium supplemented with 0.5% dicated that they belong to the new genus Allorhizobium yeast extract and containing a disc of ampicillin (2 µg), ery- [4]. A recent proposal to reclassify this genus as Rhizobi- thromycin (2 µg), ciprofloxacin (5 µg), penicillin (10 IU), poly- 3 1 µ 30 µ um [22] is premature in our opinion and we therefore will myxin ( 00 IU), cloxacillin ( g), oxytetracycline ( g), genta- 10 µ 30 µ 5 µ use the original valid genus name. More recently, we com- micin ( g), cefuroxime ( g), or neomicin ( g), (Becton Dikinson). Results were read after seven days of incubation. pleted the 16S rDNA sequence of strain J1T (AF469072), which showed that this strain belongs to the genus De- Extraction and analysis of fatty acids vosia rather than Allorhizobium. The homology between The analysis of cellular fatty acids content was performed on the 16S rRNA sequences of strains J1T and J2 and those of strain J1T and the type strain of D. riboflavina. Fatty acid Devosia riboflavina, the only species of the genus, was methyl esters were obtained from 40 mg of cells by saponifica- 95.9% suggesting that these Neptunia-nodulating isolates tion, methylation and extraction using minor modifications [9]. belong to a new species of Devosia [16]. of the method of Miller [11]. The fatty acid methyl esters mix- Genus Devosia was proposed by Nakagawa et al. [13] tures were separated using MIDI SHERLOCK Microbial Identi- to include a strain previously classified as Pseudomonas fication System (Microbial ID, Newark, DE 19711 U.S.A.) which consisted of a an Agilent model 7673A automatic sampler, riboflavina. The presence of long-fatty acids 3-OH C24:1 a 5% phenyl-methyl silicone capillary column (0.2 mm × 25 µm), and 3-OH C26:1 was considered an important character- a flame ionization detector, and a Hewlett-Packard model istic of this genus. According to the Second Edition KAYAK XA computer (Hewlett-Packard Co., Palo Alto, Califor- of Bergey’s Manual of Systematic Bacteriology whose nia, U.S.A.). Peaks of fatty acids up to 20 carbon atoms in guidelines have been included in the Web page length were automatically identified by retention time and their (http://server.mph.msu.edu/bergeys/) genus Devosia be- percentages calculated by the MIS Standard Software [17]. The longs to the family Hyphomicrobiaceae within the order gas chromatographic parameters were as follows: carrier gas, Rhizobiales in the α-Proteobacteria. This family also ultra-high-purity hydrogen; column head pressure 60 kPa; injec- includes the genus Azorhizobium that forms nodules in tion volume 2 µl; column split ratio, 100:1; septum purge 5 ml/min.; injection port temperature, 250 °C; detector tempera- adventitious roots protruding from stems of Sesbania ture, 300 °C. The oven temperature was increased from 170 to rostrata. Besides these two genera, the family Hypho- 270 °C at a rate of 5 °C/min. To detect longer chain hydroxy microbiaceae includes other genera whose relation with fatty acids (>C20), the samples were analysed by high-tempera- plants have not been established. ture gas chromatography system using a model 5890 II (Agilent) Based on a polyphasic taxonomy approach including GC with separation on a 12-m HT5 column (part no. 051385; phenotypic characteristics, fatty acid analysis, TP-RAPD SGE, Victoria, Australia), using H2 as carrier gas at a flow rate fingerprinting, 16S rDNA RFLP patterns, determination of 30 ml/min. The oven temperature was increased from of base composition and DNA-DNA hybridization we 210–400 °C at a rate of 10 °C/min. The injector temperature describe here Devosia neptuniae sp. nov. represented by was kept at 240 °C and the detector at 400 °C. Peaks of the hydroxy fatty acids were identified by comparing their retention strains from India that form nitrogen-fixing root nodules times to those of authentic standards. on floating stems of the unique aquatic legume, Neptunia natans (L.c.) Druce. TP-RAPD (two primers random amplified polymorphic DNA) fingerprinting Strains were grown for 24 h in TY medium (0.4% tryptone

Materials and Methods 0.3% yeast extract and 0.09% Ca2Cl). Cells (1.5 ml of each culture) were collected by centrifugation at room temperature in a Bacterial strains and culture media microspin centrifuge at 5000 g and then washed with 200 µl of Strains J1T (LMG 21357T) and J2 (LMG 21358), isolated a solution of 0.1% sarkosyl in water. The DNA was extracted from Neptunia natans nodules in a previous study [18] and type by adding 100 µl of 0.05M NaOH (DNA-free) and heating at strain of Devosia riboflavina LMG 2277T were maintained in 100 °C for 4 min. Samples were then placed in an ice bath and YMA medium [1] at 28 °C. 900 µl of water was added to each microtube and mixed thor- oughly. After an additional centrifugation at 4000 g for 3 min, Morphology 700 µl of the supernatants were harvested and frozen at –20 °C. Strain J1T was grown in nutrient broth during 48 h to check Crude DNA (2µl) was used as template for PCR amplifica- for motility by phase-contrast microscopy. The cells were also tion of 16S rDNA. PCR was performed using an AmpliTaq stained according to the classical Gram procedure described by reagent kit (Perkin-Elmer Biosystems, California, USA) follow- µ Doetsch [6]. For electron microscopy, the cells were grown on ing the manufacturer’s instructions (1.5 mM MgCl2, 200 M of nutrient agar for 2 days. The cells were gently suspended in ster- each dNTP and 2 U of Taq polymerase for 25 µl of final volume ile water and then stained with 0.2% uranyl acetate and exam- of reaction). The PCR primers used for amplification were the ined at 80kV with a Zeiss EM 209 transmission electron micro- forward primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3′, scope. Escherichia coli positions 8-27) and the reverse primer 1522R Devosia neptunia sp. nov. 49

′ ′ (5 -AAGGAGGTGATCCANCCRCA-3 , E. coli positions 1509– (Oxoid), 0.75; KH2PO4, 0.454; Na2HPO4 · 12 H2O, 2.388; CaCl2, 1522) at a final concentration of 2 µM. PCR conditions were as 1; agar, 20; pH 6.8–7) at 28 °C for 48 h. Whole-cell protein follows: pre-heating at 95 °C for 9 min; 35 cycles of denaturing extracts were prepared and separated by electrophoresis using at 95 °C for 1 min; annealing at 45 °C for 1 min and extension small modifications of the procedure of Laemmli [9] as at 72 °C for 2 min, and a final extension at 72 °C for 7 min. The described previously [4]. PCR products were stored at 4 °C. Eight microliters of PCR product were electrophoresed in DNA extraction, determination of %GC 1.5% agarose gel in TBE buffer (100 mM Tris, 83 mM boric and DNA-DNA hybridizations acid, 1 mM EDTA, pH: 8.5) at 6 V cm–1, stained in a solution DNA was extracted using the procedure of Pitcher [14], µ –1 containing 0.5 l ethidium bromide ml and photographed scaled up six-fold and including an extra chloroform extraction under UV light. Standard VI (Boehringer-Roche, USA) was used step to eliminate protein. To determine the DNA base composi- µ × as a size marker. To each sample 3 L of 6 loading solution tion, DNA was degraded enzymatically into nucleosides [10]. (30% glycerol, 0.25% xylene cyanol and 0.25% bromophenol The resulting nucleoside mixtures were separated by high-per- blue) were added. formance liquid chromatography using a Waters Symmetry

Shield C8 column at 37 °C. The solvent was 0.02 M NH4H2PO4 Restriction fragment analysis on 16S rDNA (pH 4.0) with 1.5% acetonitrille. Nonmethylated lambda phage The isolation of DNA was performed as described above. DNA (Sigma) was used as the calibration reference. The DNA concentration of the preparations was estimated by DNA-DNA hybridizations were performed using a mi- visually comparing the DNA samples with samples of known croplate method modified from Ezaki et al. [7] as described by concentrations of lambda-DNA in agarose gels stained with Willems et al. [21], with hybridizations carried out at 45 °C. ethidium bromide. The PCR primers used for amplification of 16S rDNA were the forward primer 8F (5′-AGAGTTTGATC CTGGCTCAG-3′, E. coli positions 8-27) and the reverse primer 1522R (5′-AAGGAGGTGATCCANCCRCA-3′, E. coli posi- Results and Discussion tions 1509-1522) at a final concentration of 0.2 µM. PCR conditions were as follows: pre-heating at 95 °C for 9 min; 35 cycles of denaturing at 95 °C for 1 min; annealing at 59 °C for 1min In previous studies we have reported the characteri- T and extension at 72 °C for 2 min, and a final extension at 72 °C zation of strains J1 and J2 from India that develop an for 7 min. The PCR products were stored at 4 °C. The PCRs unusual infection process in the aquatic legume Neptu- were performed in 25 µl reaction mixtures and the PCR prod- nia natans [16, 18]. Using LMW RNA profiling and ucts were digested with the restriction endonucleases, DdeI, 16S rDNA sequence analysis, we demonstrated that MspI, AluI and HinfI (Amersham-Pharmacia Biotech), as rec- these isolates are different from Senegal strains belong ommended by the manufacturers. The digests were separated by to Allorhizobium undicola, a species capable of nodu- electrophoresis in 2% agarose gels in Tris-borate-EDTA at lating the same legume host. From the 16S rDNA data, 6 Vcm–1 for approximately 2 h. A 100-bp ladder (Amersham- T and J2 are most closely related to Devosia ri- Pharmacia Biotech) was used as marker. strains J1 boflavina in GenBank as well as in the RDP-II database SDS-PAGE of whole-cell proteins (Michigan State University, Ribosomal Database Pro- Strains were grown on tryptone yeast extract (TY) agar ject-II), but they may constitute a distinct species. The (composition in g per liter: tryptone (Oxoid), 5; yeast extract 5S rRNA zone of the D. riboflavina type strain (data

Fig. 1. Transmission electron micrographs of strain J1T grown on nutrient agar for 48 h. Bars, 0.85 µm (A) and 0.57 µm (B). 50 R. Rivas et al. not shown) is the same as those already published for Morphology strains J1T and J2 [16], indicating that they all belong to Light microscopy of strain J1T grown on nutrient agar the same genus [2, 20]). We also demonstrated that indicated that it is a motile, Gram negative, non-sporu- both nifH and nodD genes are present on a plasmid in lating short rod. Figure 1 (A and B) show the cell mor- strains J1T and J2. These genes are similar to the equiva- phology of strain J1T in transmission electron micro- lent genes in Rhizobium tropici, suggesting that they graphs. The cells are ovoid to short rods (length of may have been acquired by horizontal transfer [16]. 1.1–1.4 µm, width of 0.7–0.9 µm) with a single subpolar The current polyphasic study was undertaken to further flagellum whose thickness is approximately 20 nm, and a characterise these neptunia-nodulating strains isolated wave periodicity with wavelength of approximately in India and provide a formal new species description of 1400 nm and a pitch of about 385 nm. Colonies are mu- them. coid and pearl white on YMA medium, typical of fast- growing rhizobia.

Table 1. Phenotypic characteristics of strain J1T LMG 21357T Physiological and biochemical characteristics (CECT 5650T and of D. riboflavina LMG 2277T (from Naka- gawa et al. [13] and from this study). Table 1 lists various physiological and biochemical characteristics of isolates J1Tand the type strain of D. ri- J1T LMG 2277T boflavina. The phenotypic characteristics of J2 were identical to J1T (not shown). The results indicate that strains Shape rod rod J1T and J2 are phenotypically similar to D. riboflavina, Size except that [unlike this latter type species] they do not Aerobic + + grow on N-acetylglucosamine and are susceptible to Fermentative – – ciprofloxacin. Oxidase + + Catalase + + Nitrate and nitrite reduction + + Arginine dehydrolase – – Table 2. Fatty acid composition (in percentage) of the species Urease + + D. neptuniae J1T sp. nov. and D. riboflavina LMG2277T Aesculin hydrolysis + + (DSM7230T). Gelatinase – – β-galactosidase + + Fatty acids D.neptuniae1 D.riboflavina1 D.riboflavina2

Assimilation of: non polar Glucose + + 14:1 – – 14.2 L-arabinose + + 14:0 0.9 – 1.0 D-mannose + + cis-7 16:1 2.6 2.7 3.5 Mannitol + + 16:0 24.8 19.1 18.2 Maltose + + 17:0 – – 0.7 N-acetyl-glucosamine – + Gentiobiose – – cyclo 17:0 3.4 0.7 – Caproate – – 3 Adipate – – cis-7 18:1 18.6 44.5 58.9 Malate – – 18:0 4.0 3.6 3.4 Citrate – – cis-7 18:1 11-methyl 36.4 25.7 – PAC (phenyl-acetate) – – 19:1 – 1.2 – Resistance to: cyclo 19:0 9.3 1.6 – Ampicillin + + cis-7 20:1 – 0.9 – Erythromycin + + Ciprofloxacin – + 3-OH acids Penicillin + + 8:0 3-OH 0.8 – Polymyxin w + 10:0 3-OH 1.0 – Cloxacillin + + 18:0 3-OH 98.2 present4 4.6 Oxitetracyclin + + 20:0 3-OH – 1.8 Gentamicin – – 20:1 3-OH – 1.8 Cefuroxime + + 22:0 3-OH – 6.1 Neomicine – – 22:1 3-OH – 8.0 24:1 3-OH – 41.0 Plasmid size 1600, 3100 3600 26:1 3-OH – 36.9 Presence of nifH and nodD +– 1own data, 2 data by Nakagawa et al. [13], 3diagnostic fatty acids +: positive. -: negative. ND: No data. w: weak. CECT, Colección in bold, 4long chain 3 hydroxy fatty acids to about 26 carbon Española de Cultivos Tipo, Spain. LMG, Collection of bacteria atoms could be detected, there exact chain length were not de- of the Laboratory of Microbiology, Gent, Belgium termined Devosia neptunia sp. nov. 51

Fatty acid composition The analysis of fatty acids of strain J1T showed that the main fatty acids were 11 methyl octadecanoic acid, cis-vaccenic acid and 3-hydroxy octadecanoic acid (Table 2). Basically, the pattern of fatty acids of strain J1T coincides with those of type strain of D. riboflavina although there are diagnostic quantitative and qualita- tive differences between them. As expected, D. riboflavina synthesized long-chain 3-hydroxy fatty acids, 3-OH C24:1 and 3-OH C26:1; these were not detected in strain J1T. Although these long chain 3-hydroxy fatty acids were considered characteristic of the genus De- vosia [13], our results indicate otherwise because they are not detected in the second species of Devosia reported here. For that an emendation of genus Devosia is proposed.

TP-RAPD fingerprinting TP-RAPD fingerprinting is a recently described method that uses the two primers employed to amplify the 16S rDNA molecule [15]. The patterns obtained are characteristic of each species and therefore this new procedure can be applied to studies of bacterial taxonomy and identification. Figure 2 shows that strains J1T and J2 (lanes 1 and 2) isolated from Neptunia natans display identical TP-RAPD pattern that differs from the pattern produced Fig. 2. TP-RAPD patterns of new isolates and the type strain of by D riboflavina LMG 2277T (lane 3). These results indi- Devosia riboflavina. MW, molecular weight marker: 2176, cate that strains J1T and J2 belong to the same species, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234 and 154 bp. which is different from D. riboflavina, the only previous- Lane 1, Devosia neptuniae J1T, lane 2, D. neptuniae J2, lane 3, ly described species of this genus. Devosia riboflavina LMG 2277T.

Restriction fragment analysis SDS-PAGE of whole-cell proteins PCR amplification of 16S rRNA genes resulted in a single fragment of about 1.5kb for the strains tested. The The protein profiles (Fig. 4) for strains J1T and J2 were PCR products were individually restricted with endonu- very similar, indicating that both strains most probably cleases DdeI, MspI, AluI and HinfI. All endonucleases belong to the same species. This protein profile was produced polymorphic restriction patterns. The different different from that of Devosia riboflavina LMG 2277T. types of RFLP patterns obtained for each enzyme are This result is in agreement with all the other tests in this shown in Fig. 3. Only fragments of about 100 bp or larg- study and further supports the conclusion that J1T and J2 er were considered. This analysis showed that strains J1T belong to a species different from Devosia riboflavina. and J2 have identical patterns with all restriction enzymes used and that the pattern obtained with each enzyme is DNA-DNA hybridizations and DNA base composition different from that obtained for the type strain of Devosia riboflavina. These results are in agreement with DNA-DNA hybridizations (Table 3) confirm that both those obtained using the 16S rRNA sequencing and new isolates represent a single species that is distinct from TP-RAPD fingerprinting. Devosia riboflavina.

Table 3. DNA-DNA hybridization data.

Labelled probe DNA from %GC Fixed DNA from strain ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– LMG 2277T LMG 21357T LMG 21358

D. riboflavina LMG 2277T 61.8 100 37.0 37.4 D. neptuniae J1T (LMG 21357T) 62.4 38.2 100 100 D. neptuniae J2 (LMG 21358) 62.0 30.4 98.9 100 52 R. Rivas et al.

Fig. 3. Restriction patterns of PCR-amplified 16S rRNA genes digested with (A) MspI, (B) DdeI, (C) AluI and (D) HinfI, obtained with strains used in this study. Lanes labeled MW contain the 100 bp ladder marker (Amersham-Pharmacia Biotech). Lanes 1, 4, 7 and 10 Devosia neptuniae J1T; lanes 2, 5, 8 and 11 D. neptuniae J2; lanes 3, 6, 9 and 12 Devosia riboflavina LMG 2277T.

Description of Devosia neptuniae sp. nov. Devosia neptuniae (neptuni’ae. N.L. gen. n. neptuniae of neptunia, named because the organism was isolated from Neptunia natans [L.c.] Druce). Gram negative, strictly aerobic, motile, non-spore forming rod-shaped cells (length of 1.1–1.4 µm, width of 0.7–0.9 µm). Colonies grown for 48 h at 28 °C on YMA medium are convex, smooth, pearl white and mucoid and usually 1 to 3 mm in diameter. The main cellular fatty acids were C18:0 3-OH, cis-7 C18:1 11-methyl and cis-7 Fig. 4. SDS-PAGE protein profiles of C18:1. The strains J1T and J2 of this species utilise glu- 2 strains of Devosia neptuniae and the cose, L-arabinose, mannose, mannitol and maltose as car- type strain of D. riboflavina. MWM, bon source. They produce oxidase, catalase, urease and molecular weight markers: lysozyme, β-galactosidase. Unlike D. riboflavina these strains do 14.5 kd; trypsin inhibitor, 20.1 kd; trypsinogen, 24 kd; carbonic anhy- not grow on N-acetyl-glucosamine. Aesculin is hydrol- drase, 29 kd; glyceraldehyde-3-phos- ysed. Nitrate and nitrite are reduced. They are sensitive to phate dehydrogenase, 36 kd; egg albu- gentamicin, neomicin and, in contrast with D. ribo- min, 45 kd; bovine albumin, 66 kd flavina, to ciprofloxacin. The G+C content of genomic and β-galactosidase, 116 kd. Lane 1, DNA is 62.0 to 62.4 mol%. The level of DNA-DNA hy- Devosia neptuniae J1T, lane 2, D. nep- bridization between J1T and D. riboflavina was 38.2%. tuniae J2, lane 3, Devosia riboflavina D. neptuniae was isolated from nitrogen-fixing nodules T LMG 2277 . of Neptunia natans in India and develops an unusual infection process in this unique aquatic legume. Strains J1T and J2 carry two plasmids (approx. 1600 and 3500 kb), Devosia Nakagawa et al. [13] emended one of which (1600 kb) carries nodD and nifH genes. The type strain is J1T (LMG 21357T, CECT 5650T). Its Cells are rod-shaped, 0.4 to 0.8 µm wide and up to 16S rDNA sequence is available from GenBank under 2.8 µm long. Motile by means of one or more polar or accession number AF469072. subpolar flagella. Gram negative and non-sporulated. Ob- ligately aerobic. Catalase and oxidase positive. Nitrate is reduced to nitrite. Urease and β-galactosidase positive. Aesculin hydrolysis is positive. The major cellular fatty Acknowledgements acids are C16:0, C18:1 and 11-methyl C18:1 in both This work was supported by Junta de Castilla y León to EV species described so far. C17:0 CYCLO and C19:0 and EMM. AW and MG are grateful to the Fund for Scientific Research–Flanders for a position as Postdoctoral Research fel- CYCLO fatty acids are also detected. The DNA G+C con- low and for research and personnel grants, respectively. We tent ranges between 61 and 63 mol%. Phylogenetically re- thank Jim Cole and Julia Bell of the Michigan State University lated to the members of family Hyphomicrobiaceae from Ribosomal Database Project-II and Bergey’s Manual Trust, re- α-Proteobacteria. Type species is Devosia riboflavina. spectively, for assistance and helpful advice. Devosia neptunia sp. nov. 53

References 14. Pitcher, D., Saunders, N., Owen, R.: Rapid extraction of bacterial genomic DNA with guanidinium thiocyanate. 1. Bergersen, F. J.: The growth of Rhizobium in synthetic Lett. Appl. Microbiol. 8, 151–156 (1989). media. Australian J. Biol. 14, 349–360 (1961). 15. Rivas, R., Velázquez, E., Valverde, A., Mateos, P. F., 2. Casamayor, E. O., Calderón-Paz, J. I., Pedrós-Alió, C.: Martínez-Molina, E.: A two primers random amplified 5S rRNA fingerprints of marine bacteria, halophilic archaea polymorphic DNA procedure to obtain polymerase chain and natural prokaryotic assemblages along a salinity gra- reaction fingerprints of bacterial species. Electrophoresis dients. FEMS Microbiol. Ecol. 34, 113–119 (2000). 22, 1086–1089 (2001). 3. Chen, W. M., Laevens, S., Lee, T. M., Coenye, T., De Vos, 16. Rivas, R., Velázquez, E., Willems A., Vizcaíno N., Subba- P., Mergeay, M., Vandamme, P.: Ralstonia taiwanensis sp. Rao N. S., Mateos P. F., Gillis M., Dazzo F. B., Martínez- nov., isolated from root nodules of Mimosa species and Molina E.: A new species of Devosia that forms a nitrogen- sputum of a cystic fibrosis patient. Int. J. Syst. Evol. Micro- fixing root-nodule symbiosis with the aquatic legume Nep- biol. 51, 1729–1735 (2001). tunia natans (L. f.) Druce. Appl. Environ. Microbiol. 68, 4. De Lajudie, P., Laurent-Fullele, E., Willems, A., Torck, U., 5217–5222 (2002). Coopman, R., Collins, M. D., Kersters, K., Dreyfus, B., 17. Sasser, M.: Identification of bacteria by gas chromatogra- Gillis, M.: Allorhizobium undicola gen. nov., sp. nov., phy of cellular fatty acids. USFCC Newsletter. 20, 1–6 nitrogen-fixing bacteria that efficiently nodulate Neptunia (1990). natans in Senegal. Int. J. Syst. Bacteriol. 48, 1277–1290 18. Subba-Rao, N. S., Mateos, P. F., Baker, D., Pankrazt, H., (1998). Palma, J., Dazzo, F. B., Sprent, J. I.: The unique root-nodule 5. De Lajudie, P., Willems, A., Pot, B., Dewettinck, D., symbiosis between Rhizobium and the aquatic legume Nep- Maestrojuan, G., Neyra, M., Collins, M. D., Dreyfus, B., tunia natans (L.f.) Druce. Planta. 196, 311–320 (1996). Kersters, K., Gillis, M.: Polyphasic taxonomy of rhizobia: 19. Sy, A., Giraud, E., Jourand, P., García, N., Willems, A., Emendation of the genus Sinorhizobium and description of De Lajudie, P., Prin, Y., Neyra, M., Gillis, M., Boivin-Mas- Sinorhizobium meliloti comb. nov., Sinorhizobium saheli son, C., Dreyfus, B.: Methylotrophic Methylobacterium sp. nov., and Sinorhizobium teranga sp. nov. Int. J. Syst. bacteria nodulate and fix nitrogen in symbiosis with Bacteriol. 44, 715–733 (1994). legumes. J. Bacteriol. 183, 214–220 (2001). 6. Doetsch, R. N.: Determinative Methods of Light Mi- 20. Velázquez, E., Trujillo M. E., Peix A., Palomo J. L., García- croscopy, pp. 21–33. In Manual of Methods for General Benavides P., Mateos P. F., Ventosa A., Martínez-Molina E.: Bacteriology (P. Gerhardt, R. G. E. Murray, R. N. Costilow, Stable low molecular weight RNA analyzed by staircase 7. Ezaki, T., Hashimoto, Y., Yabuuchi, E.: Fluorometric de- electrophoresis, a molecular signature for both prokaryotic oxyribonucleic acid – deoxyribonucleic acid hybridization and eukaryotic microorganisms. Syst. Appl. Microbiol. 24, in microdilution wells as an alternative to membrane filter 490–499 (2001). hybridization in which radioisotopes are used to determine 21. Willems, A., Doignon-Bourcier, F., Goris, J., Coopman, R., genetic relatedness among bacterial strains. Int. J. Syst. Bac- De Lajudie, P., Gillis, M.: DNA-DNA hybridization study teriol. 39, 224–229 (1989). of Bradyrhizobium strains. Int. J. Syst. Evol. Microbiol. 51, 8. Kuykendall, L. D., Roy, M. A., O’neill, J. J., Devine, T. E.: 1315–1322 (2001). Fatty acids, antibiotic resistance, and deoxyribonucleic acid 22. Young, J. M., Kuykendall, L. D., Martínez-Romero, E., homology groups of Bradyrhizobium japonicum. Int. J. Kerr, A., Sawada, H.: A revision of Rhizobium Frank 1889, Syst. Bacteriol. 38, 358–361 (1988). with an emended description of the genus, and the inclusion 9. Laemmli, U. K.: Cleavage of structural proteins during as- of all species of Agrobacterium Conn 1942 and Allorhizo- sembly of the head of bacteriophage T4. Nature 227, bium undicola de Lajudie et al. 1998 as new combinations: 680–685 (1970). Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola 10. Mesbah, M., Premachandran, U., Whitman, W. B.: Precise and R.vitis. Int. J. Syst. Evol. Microbiol. 51, 89–103 measurement of the G+C content of deoxyribonucleic acid (2001). by high-performance liquid chromatography. Int. J. Syst. Bacteriol. 39, 159–167 (1989). 11. Miller, L. T.: A single derivatization method for bacterial fatty acid methyl esters including hydroxy acids. J. Clin. Microbiol. 16, 584–586 (1982). 12. Moulin, L., Munive, A., Dreyfus, B., Boivin-Masson, C.: Nodulation of legumes by members of β-subclass of Pro- Corresponding author: teobacteria. Nature. 411, 948–950 (2001). Encarna Velázquez, Departamento de Microbiología y Genética, 13. Nakagawa, Y., Sakani, T., Yokota, A.: Transfer of Pseudo- Lab. 209, Edificio Departamental de Biología, Campus M. Una- monas riboflavina (Foster 1944) a Gram negative, motile muno, 37007 Salamanca, Spain rod with long chain 3-hydroxy fatty acids, to Devosia ri- Tel.: ++34-923 294532; Fax: ++34-923 224876; boflavina gen. nov., sp. nov. nom. rev. 46, 16–22 (1996). e-mail: [email protected]