Fontibacillus Phaseoli Sp. Nov. Isolated from Phaseolus Vulgaris Nodules

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 supplementary material, which is available to authorized users.

J. D. Flores-Fe´lix R. Rivas E. Vela´zquez (&) M.-H. Ramı´rez-Bahena J. Branãs D. Mulas A. Peix Departamento de Microbiologıá y Gene´tica, Lab. 209, Unidad Asociada Grupo de Interaccioń Planta- Universidad de Salamanca, Edificio Departamental de Microorganismo, Universidad de Salamanca-IRNASA- Biologıá, 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 M. J. Cuesta Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de Leoń, Leoń, Spain

M.-H. Ramı´rez-Bahena M. J. Cuesta A. Peix 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 Legume nodules was inoculated on modified yeast extract mannitol Endophyte 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 (Garcıá-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). (Saha et al. 2010; Lee et al. 2011). Mesodiaminopim- The mol% G?C content of DNA was determined elic acid (DAP) was detected in the peptidoglycan of using the thermal denaturation method (Mandel and strain BAPVE7BT, which presents peptidoglycan type Marmur, 1968). Chemotaxonomic analyses were A1c (Schumann, 2011). The major fatty acid (43.7 %) carried out by the Identification Service of DSMZ, identified was anteiso-C15:0, as occurs in the species of Dr. P. Schumman and Dr. B.J. Tindall (Braunschweig, Fontibacillus (Lee et al. 2011). The fatty acid profile T Germany). Isolate BAPVE7B was cultivated in TSA also contained C14:0 (2.7 %), C14:0 (2.2 %), C16:0 (Becton–Dickinson, BBL) for 48 h at 28 °C. The (9.1 %), iso-C15:0 (10.5 %), iso-C16:0 (8.2 %), iso- respiratory quinones and polar lipids were analysed as C17:0 (9.2 %), anteiso-C17:0 (10.2 %) and several fatty described by Tindall (1990) and the cellular fatty acids acids in amounts lower than 1 %. The fatty acid profile according to the instructions of the Microbial Identi- of strain BAPVE7BT is similar to those of the two fication System (MIDI; Microbial ID; TSBA40 4.0 Fontibacillus species and only slight differences were Library). For fatty acid analysis, reference Fontiba- found in the amounts of some fatty acids (Table 1). cillus type strains were analysed following growth Strain BAPVE7BT displayed a complex lipid profile under the same conditions. To perform the analysis of (Supplementary Fig. S2) consisting of diphosphatidyl- peptidoglycan, whole cells of strain BAPVE7BT glycerol, phosphatidylglycerol, four glycolipids, four were hydrolysed with HCl at 100 °C during 15 h. phospholipids, two lipids, two aminolipids and an The hydrolysates were subjected to thin-layer aminophospholipid. This profile is similar to those of 123 26 Antonie van Leeuwenhoek (2014) 105:23–28

59 Fontibacillus phaseoli BAPVE7BT (KF583881) 95 Fontibacillus panacisegetis KCTC 13564T (GQ303568) 88 Fontibacillus aquaticus GPTSA 19T (DQ023221) Paenibacillus polymyxa DSM 36T (AJ320493) Cohnella thermotolerans CCUG 47242T (AJ971483) 98 Thermobacillus xylanilyticus XET (AJ005795) 100 Ammoniphilus oxalaticus RAOx1T (Y14578) Oxalophagus oxalicus DSM 5503T (Y14581) 100 Aneurinibacillus aneurinolyticus DSM 5562T (X94194) 60 Brevibacillus brevis NBRC 15304T (AB271756)

0.02

Fig. 1 Maximun likelihood phylogenetic tree based on nearly [ 50 % (percentage of 1000 replications) are shown at the nodes. complete 16S rRNA gene sequence (1482 nucleotides) showing Filled circles indicate nodes that are also found with the the phylogenetic position of Fontibacillus phaseoli BAPVE7BT neighbour-joining algorithm. Bar, 2 nt substitutions per 100 nt within the Family Paenibacillaceae. Only bootstrap values

Table 1 Cellular fatty acid composition of strain BAPVE7BT The phylogenetic, chemotaxonomic and pheno- and related type strains of species from genus Fontibacillus typic analyses showed that strain BAPVE7BT repre- (Saha et al. 2010; Lee et al. 2011 and this study) sents a new species of the genus Fontibacillus for Fatty acid F. phaseoli F. panacisegetis F. aquaticus which the name Fontibacillus phaseoli sp. nov. is T T T BAPVE7B CECT 7605 DSM 17643 proposed. Saturated straight-chain C 1.6 0.7 0.7 12:0 Description of Fontibacillus phaseoli sp. nov. C14:0 3.8 3.2 4.8 C 2.9 3.8 1.9 15:0 Fontibacillus phaseoli (pha.seo0.li.i. N. L. masc. n. C 18.3 12.3 19.6 16:0 Phaseolus botanical genus name of the legume Saturated iso-branched Phaseolus vulgaris; N.L. gen. n. phaseoli of Phase- C 1.1 1.5 1.2 14:0 olus, referring to the isolation source of the strain, C 3.2 3.3 4.6 15:0 nodules of P. vulgaris). C 5.1 4.3 5.6 16:0 Cells are Gram positive rods (width 0.6–8.0 lm, C17:0 1.7 0.8 2.7 length 1.7–2.1 lm) and motile by means of a polar Saturated anteiso-branched flagellum. Oval subterminal endospores are formed in C13:0 nd 1.4 0.8 slightly or not swollen sporangia. Catalase negative C15:0 53.1 58.1 49.2 and weakly oxidase positive. Colonies on nutrient agar C17:0 9.3 6.2 9.1 medium are white cream, round, smooth and convex Unsaturated with approximate diameters of 1-3 mm. Anaerobic C16:1x11c nd 3.6 nd growth is positive. Grows at pH 9 but not at pH 5.7 and Fatty acids present in amounts lower than 1 % in all species are the optimal pH is 7. Grows in the presence of 2 % NaCl not shown. Data are from this study and weakly in 5 % NaCl. Growth in presence of 7 % NaCl is negative and the optimal NaCl concentration Fontibacillus species (Saha et al. 2010; Lee et al. 2011), for growth is 0.5–1 %. Grows from 10 °C–45 °C. although in strain BAPVE7BT phosphatidylethanol- Optimal temperature for growth is 30 °C. Menaquin- amine (PE) was not detected. one 7 (MK7) is the only respiratory quinone and The phenotypic characterization included the char- mesodiaminopimelic acid is present in the peptidogly- acteristics recommended in the minimal standards for can. The fatty acid profile consists of anteiso-C15:0 as aerobic endospore-forming bacteria (Logan et al. the major fatty acid with lesser amounts of C14:0,C14:0, 2009). The results are given in the species description C16:0, iso-C15:0, iso-C16:0, iso-C17:0 and anteiso-C17:0. below and the differences with the related species are The polar lipid profile consists of diphosphatidylglyc- summarised in Table 2. erol, phosphatidylglycerol, four glycolipids, four 123 Antonie van Leeuwenhoek (2014) 105:23–28 27

Table 2 Differential Characteristics F. phaseoli F. panacisegetis F. aquaticus phenotypic characteristics BAPVE7BT CECT 7605T DSM 17643T of Fontibacillus phaseoli sp. nov. and the other Gram reaction ? – ? Fontibacillus species Nitrate reduction ?? – Enzymatic activity (in API ZYM) Acid phosphatase ? –– Assimilation of (in API 20NE): N-acetylglucosamine – – ? Gluconate ? –– Acid from (in API 50CH): Glycerol – ?? D-Ribose – – ? Fructose – ?? D-mannose – ?? Melibiose ? –– ?, Positive reaction; –, Sucrose ? –– negative reaction; v, Trehalose ? –– variable; w, weakly positive Raffinose ? – ? reaction phospholipids, two lipids, two aminolipids and an phosphatases, a and b-glucosidases and a-galactosidase aminophospholipid. Nitrate is reduced to nitrite. Pro- are produced in addition to b-galactosidase. The duces b-galactosidase but does not produce gelatinase, production of phosphohidrolase is weak. Lipase, valine caseinase, indole, phenylalanine deaminase, urease, and cystine arylamidases, trypsin, chemotrypsin, glu- arginine dehydrolase, ornithine or lysine decarboxyl- curonidase, N-acetyl-glucosaminidase, a-mannosidase ase. H2S production is negative. Voges-Proskauer and and a-fucosidase production isnegative. The G?C aesculin hydrolysis are positive. In the API 20NE content of the type strain is 45.6 mol% (Tm). system the assimilation of glucose, L-arabinose, malt- The type strain BAPVE7BT (= LMG 27589T = ose and gluconate is positive and that of mannitol, CECT 8333T) was isolated from a root nodule of caprate, adipate, malate, citrate and phenylacetate is Phaseolus vulgaris in Spain. The GenBank/EMBL/ negative. The assimilation of mannose and N-acetyl- DDBJ accession number for the 16S rRNA gene glucosamine is weak. Acid but not gas is produced from sequence of strain BAPVE7BT is KF583881. glucose. In the API 50CH system the acid production from galactose, glucose, N-acetyl-glucosamine, salicin, Acknowledgments This work was supported by MINECO cellobiose, maltose, lactose, melibiose, sucrose, treha- (Spanish Central Government) Grant INNPACTO IPT-2011- 1283-060000. JDFF is recipient of a contract supported by this lose, raffinose, starch, glycogen, gentiobiose and project. MHRB is recipient of a JAE-Doc researcher contract L-lyxose is positive. The acid production from glycerol, from CSIC cofinanced by ERDF. erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, adonitol, methyl-a-D-xyloside, fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, References inositol, mannitol, sorbitol, methyl-a-D-mannoside, inulin, melezitose, xylitol, turanose, tagatose, D-fucose, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) L-fucose, D-arabinitol and L-arabinitol is negative. The Basic local alignment search tool. J Mol Biol 215:403–410 acid production from methyl-a-D-glucoside, amygdalin Chun J, Goodfellow M (1995) A phylogenetic analysis of the is weak. The hydrolysis of arbutin and aesculin is genus Nocardia with 16S rRNA sequences. Int J Syst Bacteriol 45:240–245 positive. Assimilation of gluconate and 2- and 5-Keto- Doetsch RN (1981) Determinative methods of light microscopy. gluconate is negative. In the API ZYM system, esterase, In: Gerdhardt P, Murray RGE, Costilow RN, Nester EW, esterase lipase, leucine arylamidase, acid and alkaline Wood WA, Krieg NR, Phillips GB (eds) Manual of 123 28 Antonie van Leeuwenhoek (2014) 105:23–28

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