Fontibacillus Phaseoli Sp. Nov. Isolated from Phaseolus Vulgaris Nodules
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Antonie van Leeuwenhoek (2014) 105:23–28 DOI 10.1007/s10482-013-0049-4 ORIGINAL PAPER Fontibacillus phaseoli sp. nov. isolated from Phaseolus vulgaris nodules Jose´ David Flores-Fe´lix • Rebeca Mulas • Martha-Helena Ramı´rez-Bahena • Marı´a Jose´ Cuesta • Rau´l Rivas • Javier Bran˜as • Daniel Mulas • Fernando Gonza´lez-Andre´s • Alvaro Peix • Encarna Vela´zquez Received: 26 July 2013 / Accepted: 3 October 2013 / Published online: 12 October 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract A bacterial strain, designated BAPVE7BT, Growth was observed to be supported by many carbo- was isolated from root nodules of Phaseolus vulgaris in hydrates and organic acids as carbon source. MK-7 was Spain. Phylogenetic analysis based on its 16S rRNA gene identified as the predominant menaquinone and the major sequence placed the isolate into the genus Fontibacillus fatty acid (43.7 %) as anteiso-C15:0, as occurs in the other with Fontibacillus panacisegetis KCTC 13564T its species of the genus Fontibacillus.StrainBAPVE7BT closest relative with 97.1 % identity. The isolate was displayed a complex lipid profile consisting of diphos- observed to be a Gram-positive, motile and sporulating phatidylglycerol, phosphatidylglycerol, four glycolipids, rod. The catalase test was negative and oxidase was four phospholipids, two lipids, two aminolipids and weak. The strain was found to reduce nitrate to nitrite and an aminophospholipid. Mesodiaminopimelic acid was to produce b-galactosidase but the production of gela- detected in the peptidoglycan. The G?Ccontentwas tinase, caseinase, urease, arginine dehydrolase, ornithine determined to be 45.6 mol% (Tm). Phylogenetic, che- or lysine decarboxylase was negative. Acetoin produc- motaxonomic and phenotypic analyses showed that tion and aesculin hydrolysis were found to be positive. strain BAPVE7BT should be considered a new species of genus Fontibacillus, for which the name Fontibacillus phaseoli sp. nov. is proposed (type strain, LMG 27589T, Electronic supplementary material The online version of CECT 8333T). this article (doi:10.1007/s10482-013-0049-4) contains supple- mentary material, which is available to authorized users. J. D. Flores-Fe´lix Á R. Rivas Á E. Vela´zquez (&) M.-H. Ramı´rez-Bahena Á J. Bran˜as Á D. Mulas Á A. Peix Departamento de Microbiologı´a y Gene´tica, Lab. 209, Unidad Asociada Grupo de Interaccio´n Planta- Universidad de Salamanca, Edificio Departamental de Microorganismo, Universidad de Salamanca-IRNASA- Biologı´a, Campus Miguel de Unamuno, CSIC, Salamanca, Spain 37007 Salamanca, Spain e-mail: [email protected] F. Gonza´lez-Andre´s Fertiberia SA, Madrid, Spain R. Mulas Á M. J. Cuesta Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de Leo´n, Leo´n, Spain M.-H. Ramı´rez-Bahena Á M. J. Cuesta Á A. Peix Á E. Vela´zquez Instituto de Recursos Naturales y Agrobiologı´ade Salamanca, Consejo Superior de Investigaciones Cientı´ficas (IRNASA-CSIC), Salamanca, Spain 123 24 Antonie van Leeuwenhoek (2014) 105:23–28 Keywords Fontibacillus Á Legume nodules Á was inoculated on modified yeast extract mannitol Endophyte Á Phaseolus vulgaris agar (YMA) as defined by Vincent (1970) (10 g L-1 -1 -1 mannitol, 1 g L yeast extract, 0.2 g L K2HPO4, -1 -1 -1 0.2 g L MgSO4.7H2O, 0.5 g L NaCl, 20 g L agar) and the plates were incubated at 28 °C for Introduction 4 days. In parallel, some of the disinfected entire nodules were incubated in the same medium in order Phaseolus is one of the most important legumes in to ensure their complete external disinfection and no human nutrition and is nodulated by several species of growth was observed around these nodules. The rhizobia that coexist with other endophytic bacteria, cultures used in further phenotypic and molecular some of them belonging to species of different genera studies were purified from a single colony after 2 days such as Herbaspirillum lusitanum and Phyllobacterium incubation at 28 °C on YMA. Subsequently, strain endophyticum, which were isolated from Phaseolus BAPVE7BT was grown on TSA medium (Difco, vulgaris nodules (Valverde et al. 2003; Flores-Fe´lix Becton–Dickinson, BBL). Reinfection experiments et al. 2013), and Cohnella phaseoli, isolated from were performed in Phaseolus vulgaris as was previ- nodules of Phaseolus coccineus (Garcı´a-Fraile et al. ously described (Ramı´rez-Bahena et al. 2009). 2008). In the present work we report a novel endophytic Phenotypic tests strain from the genus Fontibacillus,BAPVE7BT, isolated from root nodules of P. vulgaris. The genus Strain BAPVE7BT was grown on NA (nutrient agar) Fontibacillus was described by Saha et al. (2010)and for 48 h at 22 °C to check for motility by phase- currently contains two species: the type species Fonti- contrast microscopy using the hanging drop method. bacillus aquaticus, isolated from a warm spring, and Gram staining was carried out by the procedure Fontibacillus panacisegetis, isolated from soil (Lee described by Doetsch (1981) after 24 h incubation at et al. 2011). The genus Fontibacillus belongs to the 28 °C. The flagellation type was determined by family Paenibacillaceae and comprises Gram-variable, electron microscopy after 48 h incubation in nutrient motile and sporeforming rods with ellipsoidal terminal agar at 22 °C. The cells were gently suspended in or subterminal endospores. It is facultatively anaerobic sterile water and then stained with 0.2 % uranyl and has menaquinone MK-7 as the major mena- acetate and examined at 80 kV with a Zeiss EM 209 quinone. The polar lipid profile includes as major lipids transmission electron microscope. phosphatidylglycerol, diphosphatidylglycerol and The phenotypic characterization was performed phosphatidylethanolamine. The major fatty acids are according to the standard methods as described by anteiso-C15:0, C16:0, iso-C17:0 and anteiso-C17:0. Claus and Berkeley (1986) and Logan and Berkeley The members of genus Fontibacillus are catalase (1984). The API 20NE, API20E, API50CH and API negative and oxidase positive (Saha et al. 2010;Lee ZYM systems (bioMerieux, France) were used accord- et al. 2011). ing to the manufacturer’s instructions. The results were recorded after 48 h incubation at 30 °C. Anaer- Materials and methods obic growth was determined in fluid tetrathionate medium (Sigma, USA). Acetoin production, ability to Bacterial isolation and culture grow in presence of 2, 5 and 7 % NaCl, nitrate reduction, phenylalanine deaminase, catalase, gelatin- Strain BAPVE7BT was isolated from a nodule of ase, caseinase and oxidase were determined as P. vulgaris grown in Barco de A´ vila (Spain). To described by Claus and Berkeley (1986). Acid pro- sterilize the root nodules they were washed several duction from D-glucose, D-xylose, D-mannitol and times with sterile distilled water and were then surface L-arabinose and gas from glucose was determined in sterilized in HgCl2 2.5 % (w/v) for 2 min. The nodules liquid medium as described by Claus and Berkeley were rinsed five times with sterile distilled water and (1986). Growth was determined at temperatures of 4, then crushed using a sterile pestle in a sterile 15, 25, 37, 40 and 45 °C in TSA medium (Difco, eppendorff tube. The homogenized nodule tissue Becton–Dickinson, BBL). The growth at pH 5.7 and 123 Antonie van Leeuwenhoek (2014) 105:23–28 25 6.8 was tested as described Claus and Berkeley (1986) chromatography on cellulose plates using the solvent in Saboureaud broth without chloramphenicol (Difco, system of Rhuland et al. (1955). Becton–Dickinson, BBL) and Nutrient broth (Difco, Becton–Dickinson, BBL), respectively. Growth at pH 7–8 was tested in YMA medium containing 200 mM Results and discussion of Na2HPO4/NaH2PO4 and the growth at pH 9 and 10 was tested in the same medium containing 200 mM of Strain BAPVE7BT was observed to form white, NaHCO3/Na2CO3. mucoid, translucent and convex colonies on YMA The strains F. aquaticus DSM17643T and medium. Colonies on TSA medium were observed to F. panacisegetis CECT 7605T (obtained from DSMZ) be white cream, round, smooth and convex with were included in the phenotypic study as references, approximate diameters of 1– mm. The strain isolated grown under the same conditions. in this study was unable to produce nodules in P. vulgaris. Phylogenetic analysis Strain BAPVE7BT was observed to be Gram- positive, to form oval subterminal endospores in Amplification and sequencing of the 16S rRNA gene slightly or not swollen sporangia and was motile by were performed according to Rivas et al. (2007). The means of one polar flagellum (Supplementary Fig. S1). sequence obtained was compared with those from The comparison of the 16S rRNA gene sequence of GenBank using BLASTN (Altschul et al. 1990)andthe BAPVE7BT (1486 nucleotides; Genbank accession EzTaxon-e server (Kim et al. 2012). Sequences were number KF583881) with the sequences of type strains aligned using the Clustal_X software (Thompson et al. held in EzTaxon-e database indicated that this strain 1997) and distances were calculated according to Kim- belonged to the genus Fontibacillus since the most ura0s two-parameter model (Kimura, 1980). The phylo- closely related species were identified as F. panacise- genetic trees were inferred using the neighbour joining getis (97.1 % identity) and the type species of the genus, (NJ) and maximum likelihood (ML) models (Saitou and F. aquaticus(96.7 % identity). The NJ and ML analyses Nei, 1987; Rogers and Swofford, 1998). The MEGA5 showed similar results and are shown in Fig. 1. package (Tamura et al. 2011) was used for all analyses. The G?C content of the strain BAPVE7BT was 45.6 mol%, which is similar to the members of the Chemotaxonomic analyses genus Fontibacillus. Menaquinone 7 (MK7) was the only respiratory quinone detected. This menaquinone is For DNA base composition analysis, DNA was also predominant in the species of genus Fontibacillus prepared according to Chun and Goodfellow (1995).